2024-03-28T18:17:26Zhttps:/www.ncbi.nlm.nih.gov/pmc/oai/oai.cgioai:pubmedcentral.nih.gov:290982001-03-22bmcnephpmc-openBMC NephrolBMC Nephrology1471-2369BioMed CentralLondonPMC29098PMC2909829098110382661471-2369-1-111038266Survey/Cross Sectional StudyEarly peri-operative hyperglycaemia and renal allograft rejection in patients without diabetesThomasMerlin C1mdorbell@hotmail.comMoranJohn2john.moran@nwahs.sa.gov.auMathewTimothy H1tim.mathew@nwahs.sa.gov.auRussGraeme R1grame.russ@nwahs.sa.gov.auMohan RaoM1mohan.rao@nwahs.sa.gov.auRenal Unit, The Queen Elizabeth Hospital, Adelaide, South Australia.Intensive Care Unit, The Queen Elizabeth Hospital, Adelaide, South Australia.2000410200011119720001892000Background
Patients with diabetes have an increased risk for allograft rejection, possibly related to peri-operative hyperglycaemia. Hyperglycaemia is also common following transplantation in patients without diabetes. We hypothesise that exposure of allograft tissue to hyperglycaemia could influence the risk for rejection in any patient with high sugars. To investigate the relationship of peri-operative glucose control to acute rejection in renal transplant patients without diabetes, all patients receiving their first cadaveric graft in a single center were surveyed and patients without diabetes receiving cyclosporin-based immunosuppression were reviewed (n = 230). Records of the plasma blood glucose concentration following surgery and transplant variables pertaining to allograft rejection were obtained. All variables suggestive of association were entered into multivariate logistic regression analysis, their significance analysed and modeled.
Results
Hyperglycaemia (>8.0 mmol/L) occurs in over 73% of non-diabetic patients following surgery. Glycaemic control immediately following renal transplantation independently predicted acute rejection (Odds ratio=1.08). 42% of patients with a glucose < 8.0 mmol/L following surgery developed rejection compared to 71% of patients who had a serum glucose above this level. Hyperglycaemia was not associated with any delay of graft function.
Conclusion
Hyperglycaemia is associated with an increased risk for allograft rejection. This is consistent with similar findings in patients with diabetes. We hypothesise a causal link concordant with epidemiological and in vitro evidence and propose further clinical research.
Background
Hyperglycaemia is common following renal transplantation [1]. Aside from patients with diabetes, many dialysis patients have impaired glucose tolerance [2] and much of the standard post-transplant management is diabetogenic. Recent data has demonstrated that patients with diabetes are at increased risk for allograft rejection [3]. We have recently described how glycaemic control correlates with allograft rejection in patients with diabetes, raising the possibility of a causal association between peri-operative hyperglycaemia and allograft rejection [4]. Transplantation is a unique situation where naive tissue may be suddenly subjected to a hostile hyperglycaemic environment. Acute rejection is thought to be initiated in the early postoperative period by antigen presentation, possibly in response to allograft inflammation and injury. Acute hyperglycaemia is known to enhance ischaemic injury [5], antigen presentation [6], apoptosis [7], and augment the inflammatory response [8]. We hypothesise that exposure of allograft tissue to hyperglycaemia could influence the risk for rejection, not only in diabetes, but in any patient with an elevated glucose. This study investigates the relationship of peri-operative hyperglycaemia to acute rejection in patients without diabetes.
Methods
A retrospective review was made of the records for each of the 365 patients who underwent their first cadaveric renal transplant (CD1) at The Queen Elizabeth Hospital, Adelaide, Australia between January 1990 and January 2000. Patients with primary graft failure, death without graft function or graft loss due to technical complications were excluded (n=25). All established diabetic patients were also excluded (n = 50) and are studied elsewhere [4]. Patients who did not carry the preoperative diagnosis of diabetes but who required insulin in the postoperative period or subsequently developed de novo or post-transplant diabetes mellitus were not designated as diabetic for the purposes of this study. All CD-1 patients commencing Cyclosporin A, mycophenolate or azathioprine, and/or prednisolone as their starting immunosuppression on an intention-to-treat basis were identified (n=230) and formed the primary study group. In this protocol [9], cyclosporin A (5 mg/kg/d with diltiazem or 8 mg/kg/d without diltiazem), mycophenolate (2 g/d) or azathioprine (2 mg/kg/d) were first given orally 6-8 hours after transplantation. Every patient received an intravenous bolus dose of 1 g of methylprednisolone prior to surgery and 500 mg on the following morning. Only CD1 patients sensitised to panel reactive lymphocytotoxic antigens (peak PRA > 50%) or with a positive T or B-cell cross-match received oral prednisolone (30 mg/d), commenced from day two after morning blood testing.
The serum blood glucose immediately following surgery (while still in theatre recovery) and fasting results from the following two mornings were obtained from laboratory records. Transplant records were obtained from a common database including donor age and gender, recipient age, gender, and race, body mass index (kg/m2), type of dialysis, ischaemic time, duration of operation, PRA, peak and current, HLA-A, -B, and -DR matching. Post-transplantation records of all (230) patients were examined for the presence of allograft rejection. Acute rejection was said to occur if biopsy-proven or clinical rejection occurred within 30 days of transplantation. If a biopsy was not performed, clinical rejection was retrospectively identified by a sustained rise by more than 10% from the predicted serum creatinine, responsive to adjunctive immunosuppressive therapy.
Statistical tests
All variables considered as possible predictors of acute rejection were entered into a multivariate logistic regression; initial bi-variable screening was not used. The full model incorporated seven categorical variables (donor gender, recipient gender, race, type of dialysis, sensitization/use of oral prednisolone, the use of azathioprine or mycophenolate, the presence of delayed graft function requiring dialysis) scored one, zero and ten continuous variables (donor age, recipient age, body mass index (kg/m2), warm and cold ischaemic times, duration of operation and post operative glucose concentrations immediately following and on the two mornings after surgery). HLA matching was also considered as a continuous variable (scored 0-6). No collinearity was present in the full data set. Variables were sequentially removed from the model using the likelihood ratio test with the significance level set at p=0.05. Functional form (in particular, non-linearity) of continuous variables in the final model was checked (i) graphically, using partial residual plots and (ii)formally, by both parametric and non-parametric means. Categorical ("cut-point") analysis of continuous variables was not primarily utilized except when testing for first order interactions. Model performance was assessed using indices of calibration: Hosmer-Lemershow χ2 test (p > 0.1) and discrimination: area under the receiver operating curve (ROC) curve (acceptable discrimination area > 0.7). Confidence intervals for the final model were computed using the bootstrap method (BCa, bias corrected and accelerated). The parameters of the final model were estimated as risk ratios (generalized linear model with binomial family and log link) to correct the odds ratio interpretation under conditions of common prevalence (>10%) in the study population. Uni-variate results are expressed as ± 95% confidence interval. Stata® statistical software, version 6.0 (1999) was used.
ResultsDemographics
Two hundred and thirty patients were both not diabetic at the time of admission and received cyclosporin-based immunosuppression following successful transplantation. All (230) patients were reviewed in this study. Mean age of the recipient was 44.7 ± 1.7 years, and 60% of patients were men. 7 % of patients were Australian aboriginal. Mean donor age was 36.3 ± 2.2 years. Mean HLA match was 2.4 ± 0.2 and mismatch 2.9 ± 0.2 antigens. 18 % of patients were sensitised and therefore received oral prednisolone from day two. 33% received mycophenolate and 67% received azathioprine.
Hyperglycaemia
The mean blood glucose concentration immediately after surgery was 10.75 ± 0.56 mmol/L (median 9.90 mmol/L, range 5.0-25.6 mmol/L). The mean blood glucose concentration taken the following morning (mean = 12.3 ± 1.5 hours, range 7-16 hours after surgery) was 9.34 ± 0.43 mmol/L (median 8.60) and 8.0 ± 0.31 mmol/L on the morning of day-2. A glucose concentration of greater than 8.0 mmol/L was present in 73 % of patients in our study with more than 31% having a glucose greater than 11.2 mmol/L immediately after surgery. 51% of all patients had glucose levels greater than 8.0 mmol/L both after surgery and on the following morning. The incidence of hyperglycaemia had no relationship to pre-operative glucose levels, ischaemic time, duration of operation, donor weight or age. In addition, the amount of intra-operative or postoperative dextrose solutions received by patients did not influence the incidence of hyperglycaemia. Glycaemic control immediately following surgery correlated with control on subsequent days (R2=0.076).
Acute rejection
Acute rejection occurred in 147 patients (64% overall) at a mean of 8.2 ± 0.8 days after transplantation. 88 patients (60%) had biopsy-proven rejection and had 59 patients (40%) had clinical rejection without biopsy confirmation. Six variables independently predicted allograft rejection including donor youth, the use of azathioprine (over mycophenolate), HLA-matching and the presence of delayed graft function (table 1). Sensitisation was inversely correlated with rejection in this study, probably as a result of the exclusive use of oral steroids in this group. In addition, serum glucose immediately following renal transplantation was independently associated with acute rejection. (Full model: Hosmer-Lemeshow χ2 = 4.93, p=0.76, area under the ROC curve = 0.75, Bootstrap 95% confidence interval 0.71-0.81). The initial relationship between serum glucose and probability of rejection was assumed to be linear. However an examination of partial residual plots suggested a non-linear relationship. The power of this relationship was identified using a non-linear power (Box-Tidwell) available in Stata statistical software [10]. This power function was subsequently used to generate a graph of probability of rejection versus glucose concentration using a fractional polynomial routine (fig 1). The only significant interaction, between glucose levels and the use of prednisolone/sensitisation (p=0.01, likelihood ratio test), was incorporated into this model (fig 2). This interaction was not able to be demonstrated using a different estimator (BinReg). The mean glucose levels immediately following surgery were 9.8 ± 0.78 mmol/L in patients without rejection and 10.8 ± 0.57 mmol/L in patients with rejection. 42% of patients with a glucose < 8.0 mmol/L following surgery developed rejection compared to 71% of patients who had a serum glucose above this level. While persistent hyperglycaemia on the morning after surgery also predicted rejection, it did not offer better discriminating power compared to that immediately following surgery.
Discussion
Hyperglycaemia is common following transplantation in patients without diabetes. 73 % of patients in our study developed hyperglycaemia > 8.0 mmol/L following surgery which persisted beyond 12-hours in 51% of patients. Many patients with renal failure have so called "uraemic pre-diabetes" characterised by impaired glucose tolerance, insulin resistance and hyperinsulinism [2]. In addition, standard peri-transplant management is diabetogenic. Non-diabetic transplant recipients who are treated with steroids show insulin resistance comparable to diabetic patients [11]. Although all patients in our study received the same large doses of methylprednisolone, there was a wide range of glycaemic responses. Certainly, even a slow infusion of 5% dextrose can cause hyperglycaemia in a non-diabetic patient on steroids [12], but the use of intra-operative or peri-operative dextrose did not correlate with blood sugar levels in our study. Cyclosporin A may also affect insulin resistance [13], although it seems unlikely that any patient in this study would have received sufficient cyclosporin to produce hyperglycaemia the morning following transplantation.
Some have suggested hyperglycaemia is simply an epiphenomenon, acting as a marker for more extensive peri-operative insult [14]. However, blood glucose levels in our study did not correlate with intra-operative blood loss, duration of operation or cold ischaemic times. It is also possible that more extensive renal damage may lead to a greater rise in `stress' hormones. Some studies in myocardial infarction have shown a correlation between extent of ischaemic injury and glucose levels [14]. However immediacy of allograft function did not correlate with sugar levels in our study.
The Australia and New Zealand Dialysis and Transplant registry (ANZDATA) has reported that renal transplant patients with diabetes have more acute rejection [3]. We have previously demonstrated that amongst patients with diabetes, those with hyperglycaemia have enhanced rejection rates [4]. We report here for the first time, that peri-operative hyperglycaemia is also associated with an increased risk of allograft rejection in patients without diabetes. Patients with peri-operative hyperglycaemia had significantly more rejection than those who remained euglycaemic. Acute rejection is thought to be initiated in the early post-operative period by allograft injury and the inflammatory response to that injury. We hypothesise that early hyperglycaemia may directly increase the risk of allograft rejection by one of three broad mechanisms.
First, high sugar levels may exacerbate warm ischaemic damage, with the resulting tissue injury acting as trigger for rejection. It is known that hyperglycaemia, worsens renal ischaemic injury in experimental models [5], suggesting a direct role for glucose. The generation of lactate and reactive oxygen species are augmented by acute hyperglycaemia and re-perfusion injury may also be increased [15]. High sugars have a direct vasoconstrictor effect in non-diabetic renal vessels [16]. and result in endothelial dysfunction through hyper-osmolarity, oxidant formation, and protein kinase C (PKC) activation [17]. In addition, high sugars may also have a pro-coagulant effect [7]. Although no relationship between glucose levels and delayed graft function was observed in our study, some studies have shown the incidence of delayed graft function to be increased in patients with diabetes [18]. A state of relative insulin deficiency (ie. hyperglycaemia) may also result in reduced glucose uptake and increased lipolysis in ischaemic tissue, leading to the generation of toxic fatty acids [14]. This has prompted the use of insulin in hyperglycaemic patients to prevent ischaemic injury following myocardial infarction. However most patients with renal failure are hyperinsulinemic. It seems unlikely that this level of hyperinsulinism, regardless of hyperglycaemia, would be inadequate to suppress lipolysis in graft tissue.
Secondly, antigen presentation and co-stimulation are increased in hyperglycaemia. The expression of MHC class I and class II antigens on allograft cells are up-regulated by glucose-induced ischaemia/reperfusion injury and oxidative stress [19]. The production of chemokines that induce expression of MHC antigens are increased [8] and the tissue response to interferon-gamma is enhanced by the presence of high glucose concentrations [6]. Reactive oxygen species, potentiated by hyperglycaemia, are capable of activating peripheral dendritic cells [20]. Apoptosis, also enhanced by hyperglycaemia [7], can initiate re-perfusion-induced inflammation and tissue injury [21] as well as enhance antigen presentation [22] Expression of co-stimulatory molecules are up-regulated directly by hyperglycaemia [8,23] and indirectly, by glucose-enhanced ischaemia [24], and oxidative stress [25].
Thirdly, hyperglycaemia induces an exaggerated inflammatory response to ischaemia/reperfusion and rejection [8]. The acute phase response is increased by insulin deficiency [14]. Production of Nuclear factor kappa B (NFkB) is enhanced in the presence of high glucose levels [24], leading to up-regulation of both cellular and humoral effectors of inflammation. Expression of adhesion molecules including ICAM-1 and VGEF are increased by hyperglycaemia [26]. In addition, increased expression of CD18, VCAM-1, E-selectin [27] and the phosphorylation of PCAM-1 [28] combine to enhance the adhesion and trans-endothelial migration of monocytes. Reactive oxygen species, generated in hyperglycaemia, lead to the induction of proinflammatory cytokines [29] and both the production and activity of these cytokines including TNF-α and interferon-gamma [6] may be enhanced by high glucose levels. Augmented production of TGFβ-1 in hyperglycaemia also suppresses the production of IL-10 [30].
Marked hyperglycaemia following steroid and surgery also suggests the presence of the insulin resistance syndrome. This metabolic milieu (to which graft tissue would be newly exposed) is characterised by hypertension, dyslipidaemia, hyperinsulinism and increased levels of circulating advanced glycation end-products (AGE), leptin, TNF-alpha, IL-1, IL-6, and IL-12.[31,32] These may act, by themselves or in combination with hyperglycaemia, to up-regulate allograft injury or rejection. Patients with the insulin resistance syndrome also possess abnormalities of the innate immune system including an augmented cytokine responsiveness that may predispose to rejection [32]. A recent study has shown that pre-transplant serum C-reactive protein (CRP), a recognised marker of inflammatory responsiveness also independently predicts allograft rejection [33]. Peri-operative hyperglycaemia could therefore identify such allo-responsive patients without being causal. At the same time, it would enable better targeting of immuno-therapy. However, the fact that better sugar control reduced rejection rates in patients with diabetes (who presumably all have this milieu) suggests that hyperglycaemia is more than just an marker of an occult diabetic state. In addition, short-term intensive glycaemic control rapidly results in normalization of immune function and markers of inflammation [34].
Patients on triple therapy in this study had substantially less rejection than patients on double therapy. This is consistent with other published work [9]. The reduced probability of rejection in patients who received oral steroids patients appeared to be greatest in those with very high glucose levels (Fig 2). This may be an effect both of the relatively small numbers of patients with very high glucose levels (at the tail) and of our modeling strategy. The interaction was not demonstrated using a different estimator. However, if real, this may also the underscore the need for more potent immunotherapy in hyperglycaemic patients who have an increased risk for allograft rejection.
Conclusions
This, and previous studies by us, have shown that patients with early hyperglycaemia, whether diabetic or not, have an increased risk for allograft rejection. While hyperglycaemia is not the only risk for allograft rejection, it is both common and eminently susceptible to intervention. There are sound reasons why sugars should be tightly controlled following transplantation in patients with diabetes. Further, we believe the prevention of early hyperglycaemia and attention to insulin resistance may also serve to reduce allograft injury and decrease rejection episodes in patients without diabetes. Further research is needed to determine if such interventions can improve transplant outcomes.
Pre-publication history
The pre-publication history for this paper can be accessed here:
PourmandGEbrahimiMRMehrsaiARTaheriMPatient blood glucose levels before and after kidney transplantation200032 (3)56656810812115FliserDPaciniGEngelleiterRKautzky-WillerAPragerRFranekERitzEInsulin resistance and hyperinsulinemia are already present in patients with incipient renal disease.1998 May53(5)13431347Australia and New Zealand Dialysis and Transplant Registry.ANZDATA Registry Report 1999. (Edited by Disney APS, Russ GR, Walker R, Sheil AGR, Collins J, Herbert K, Kerr P). Adelaide, South Australia. 1999ThomasMCMathewTHRussGRRaoMMPeri-operative hyperglycaemia and increased allograft rejection following renal transplantation in patients with diabetes (abstract)2000PodrazikRMNataleJEZelenockGBD'AlecyLGHyperglycaemia exacerbates and insulin fails to protect in acute renal ischaemia in the rat.1989 Jun46(6)572578PavlovicDvan de WinkelMvan der AuweraBChenMCSchuitFBouwensLPipeleersDEffect of interferon-gamma and glucose on major histo-compatibility complex class I and class II expression by pancreatic beta- and non-beta-cells1997 Jul82(7)23292336MinCKangEYuSHShinnSHKimYSAdvanced glycation end products induce apoptosis and pro-coagulant activity in cultured human umbilical vein endothelial cells.1999 Dec46(3)197202PanesJKuroseIRodrigez-VacaDAndersonDCMiyasakaMTsoPGrangerDNDiabetes exacerbates inflammatory responses to ischaemia-reperfusion.1996931611678616923EliasTJBannisterKMClarksonARRussGRMathewTHBarrattLJFaullRJExcellent long-term graft survival in low risk, primary renal allografts treated with prednisolone-avoidance immunosuppression.2000 Apr14(2)157161RoystenPAmblerGGSg112: Non-linear regression models involving power or exponential functions of co-variates. 1999173180EkstrandAVEffect of steroid therapy on insulin sensitivity in insulin-dependent diabetic patients after kidney transplantation.1991 Oct-Dec5(4)244248CheeYCBlood glucose levels in non-diabetics on intravenous dextrose infusions.1985 Apr14(2)294296KutkuhnBHollenbeckMHeeringPKochMVoiculescuAReinhardTGrabenseeBDevelopment of insulin resistance and elevated blood pressure during therapy with cyclosporine A.1997 Jan6(1)1317CapesSEHuntDMalmbergKGersteinHCStress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: a systematic overview.2000 Mar 4355(9206)773778CerielloAAcute hyperglycaemia and oxidative stress generation.199714(suppl3)S4599272613WalczykMHPulliamJBennettWMEffects of hyperglycaemia and mannitol infusions on renal hemodynamics in normal subjects.1990 Oct300(4)218224AkbariCMSaouafRBarnhillDFNewmanPALoGerfoFWVevesEndothelium-dependent vasodilatation is impaired in both microcirculation and macrocirculation during acute hyperglycaemia.1998 Oct28(4)687694TroppmannCGillinghamKJBenedettiEAlmondPSGruessnerRWNajarianJSMatasAJDelayed graft function, acute rejection, and outcome after cadaver renal transplantation. The multivariate analysis.1995 Apr 1559(7)962968GoesNUrmsonJRamassarVHalloranPFIschaemic acute tubular necrosis induces an extensive local cytokine response. Evidence for induction of interferon-gamma, transforming growth factor-beta 1, granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-10.1995 Feb 2759(4)565572RataultKAldermanCChainBMKatzDRReactive oxygen species activate human peripheral blood dendritic cells.199926(1-2)2322389890657DaemenMARCVeerCVDeneckerGHeemskerkVHWolfsTGAMInhibition of apoptosis by ischaemia-reperfusion prevents inflammation199910454154910487768RoverePVallinotoCBondanzaACrostiMCRescignoMRicciardi-Castagnoli P, Rugarli C, Manfredi AA Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function.1998 Nov 1161(9)44674471MoriatsuTakadaAnilChandrakerKariC NadeauMohamedH SayeghNicholasLTilney The role of the B7 co-stimulatory Pathway in Experimental Cold ischaemia/reperfusion injury.1997100(5)119912039276737YerneniKKBaiWKhanBVMedfordRMNatarajanRHyperglycaemia-induced activation of nuclear transcription factor kappaB in vascular smooth muscle cells.1999 Apr48(4)855864TemaruRUrakazeMSatouAYamazakiKNakamuraNKobayashiMHigh glucose enhances the gene expression of interleukin-8 in human endothelial cells, but not in smooth muscle cells: possible role of interleukin-8 in diabetic macroangiopathy.1997 May40(5)610616MorigiMAngiolettiSImbertiBDonadelliRMichelettiGFigliuzziMRemuzziAZojaCRemuzziGLeucocyte-endothelial interaction is augmented by high glucose in an NF-kB-dependent fashion.1998101190519159576755TakamiSYamashitaSKiharaSKameda-TakemuraKMatsuzawaYHigh concentration of glucose induces expression of intercellular adhesion molecules-1 in human umbilical vein endothelial cells.1998138(1)35419678769RattanVShenYSultanaCKumarDKalraVKGlucose-induced transmigration of monocytes is linked to phosphorylation of PECAM-1 in cultured endothelial cells.1996 Oct271(4 Pt 1)E7117ManduteanuIVoineaMSerbanGSimionescuMHigh glucose induces enhanced monocyte adhesion to valvular endothelial cells via a mechanism involving ICAM-1, VCAM-1 and CD1819996(4)31532410475094ReinholdDAnsorgeSSchleicherEDElevated glucose levels stimulate transforming growth factor-beta 1 (TGF-beta 1), suppress interleukin IL-2, IL-6 and IL-10 production and DNA synthesis in peripheral blood mononuclear cells.1996 Jun28(6)267270HansonLAPadyukovLStrandvikBWramnerLThe immune system of the hunter-gatherer meets poverty and excess.2000 Apr 1297(15)18231826Fernandez-RealJMRicartWInsulin resistance and inflammation in an evolutionary perspective: the contribution of cytokine genotype/phenotype to thriftiness.1999 Nov42(11)13671374PerezRVBrownDJKatznelsonSADubinJAMullerHGChangTRudichSMMcVicarJPKaysenGAPre-transplant systemic inflammation and acute rejection after renal transplantation.2000 Mar 1569(5)869874AlbertiniJPValensiPLormeauBAurousseauMHFerriereFAttaliJRGattegnoLElevated concentrations of soluble E-selectin and vascular cell adhesion molecule-1 in NIDDM. Effect of intensive insulin treatment.1998 Jun21(6)10081013Figures and Tables
Fractional polynomial showing the probability of rejection in sensitised and un-sensitised CD1 patients versus the immediate post operative glucose level.
Fractional polynomial showing the probability of rejection in sensitised and un-sensitised CD1 patients versus the immediate post operative glucose level incorporating the interaction term.
It is well established that ACE-inhibitors should be avoided in patients with renal artery stenosis. In recent years it has also been recommended that caution should be demonstrated when angiotensin II blockers are used in the same type of patients but the evidence is based only on few cases.
Results
We describe a case where use of the angiotensin II antagonist candesartan (Atacand) induced renal failure in a patient with bilateral renal artery stenosis. The course of the case is enlighted by results from sequential renography, selective renal vein catheterisation for measurement of renin, and angiographic findings.
Conclusions
In patients with renal artery stenosis the angiotensin II antagonist candesartan should be avoided.
Background
ACE-inhibitors are well-established in treatment of hypertension. In recent years selective angiotensin II AT1-receptor antagonists have been introduced as an alternative. Although the same system is manipulated, the two types of medication differ in several ways. In brief, the major differences are: 1) whereas blockade of angiotensin II formation by ACE-inhibition is incomplete due to alternative synthesis pathways, e.g. chymase pathway, angiotensin II antagonists block the receptors at the target organ, 2) the relative effect on AT1- and AT2-receptors, and 3) differential effect on bradykinin metabolism since ACE inhibition inhibits ACE inactivation of bradykinin. The latter is thought to be the major reason for the higher rate of side-effects seen with ACE inhibitors compared to angiotensin II antagonists. Whereas the evidence that ACE-inhibitors should be avoided in patients with renal artery stenosis is substantial, the evidence is more sparse with regard to angiotensin II antagonists and restricted to losartan.
Below, we present a case of reversible deterioration in renal function following treatment with the angiotensin II antagonist candesartan.
Case
A 60 years old male with previous alcohol abuse and known hypertension for the last 5 years was admitted to our hospital with a diagnosis of hypertension. At the time of admission the blood pressure (BP) was 230/140 mmHg despite treatment with metoprolol (Selozok), amiloride/hydrochlorthiazide (Sparkal Mite) and candesartan (Atacand). The patient was hyperkalemic and had an increased serum-creatinine (237 μmol/l). Antihypertensive treatment was intensified including addition of loop-diuretic and hydration.
Following normalization of BP, serum-potassium and serum-creatinine the patient was discharged with an appointment for ambulatory renography. 99mTc-DTPA renography (Fig. 1), performed after the patient had been on candesartan treatment for 4 months, showed symmetrical renal function, however, the absolute renal function was almost abolished (total estimated GFR 4 ml/min).
Renography during candesartan treatment.
At the time the patient had an elevated serum-creatinine (817 μmol/l) and urea (48 mmol/l) and was anuric. Diuresis was re-established following use of intravenous loop-diuretic and hydration. Renal biochemistry then improved. Atacand was discontinued and 3 weeks later renography (Fig. 2) demonstrated improved renal function: total estimated GFR 47 ml/min. The left kidney was responsible for only 1/3 of the total renal function.
Renography 3 weeks after discontinuation of candesartan treatment.
Later, a selective catheterisation of the renal veins was performed for measurement of renin. The renin measurements showed increased left sided renin production (Table 1). Arteriography showed an arteriosclerotic abdominal aorta with significant bilateral stenosis of the renal arteries, more prominent on the left side.
Levels of renin in the renal veins demonstrating increased levels on the left side. Values are in mIU/l.
Right renal vein
Left renal vein
Reference (antecubital vein)
Without loop-diuretic
92
148
92
stimulation
With loop-diuretic
105
230
103
stimulation
The patient was referred to PTA of the left renal artery stenosis. Under the procedure for PTA, which was unsuccessful bilateral renal occlusion occurred. The patient then underwent bilateral vein graft renal arterial by-passes.
Two months after this operation renography showed improved absolute and relative function of the left kidney whereas the absolute function of the right kidney was unchanged (Fig. 3). Furthermore, pre- and post-captopril renography appearances were similar (Figs. 3-4), indicating no functionally important stenosis. A 24-hour ambulatory BP measurement showed well-regulated BP under treatment with amlodipine (Norvasc), doxazosin (Carduran) and bendroflumethiazide (Centyl).
Renography following bilateral renal by-pass surgery.
Captopril renography following bilateral renal by-pass surgery.
Discussion
It is well established that ACE inhibitors should be avoided in patients with critical renal artery stenosis. In contrast, this is not as well established with respect to angiotensin II antagonists. Over the last years a few cases have shown that renal function may be impaired if the angiotensin II antagonist losartan is used in patients with renal artery stenosis [1,2,3,4] but two of these cases were in the special situation of kidney transplants [3,4]. To our knowledge we are the first to report a case of renal impairment induced by the angiotensin II antagonist candesartan. Subsequent to the introduction of losartan, several other angiotensin II blockers have been marketed over the last couple of years and differences in receptor affinity and kinetics are reported. Therefore, our case supports the thought that caution should be shown using other types of angiotensin II antagonists. As mentioned in the introduction several differences exist between ACE inhibitors and angiotensin II, AT1-receptor antagonists. Therefore, differences with regard to interference with renal function and thereby when the compound should be avoided could exist. From a theoretical point of view however, one would expect that both principles of blockade should be avoided in renal artery stenosis. The mechanism of the renin-angiotensin system in regulation of renal function is believed primarily to be due to the effect of angiotensin II on the efferent arteriolar tone keeping the pressure relatively constant in the glomerulus and thereby keeping GFR constant over a wide range of perfusion pressures, i.e. systemic BP. However, the differential effect on e.g. bradykinin, which is a vasodilator and also have other actions, could in theory make the two types of blockade clinically different. At present, the relative risk of precipitating renal failure by using the different compounds is unsettled. Thus in one case renal function deteriorated following both the ACE inhibitor enalapril and losartan [1]. In contrast, another case-report observed deterioration of renal function during enalapril treatment but no effect of subsequent losartan treatment [5]. Conversely, in a study comparing the usefulness of the ACE inhibitor captopril and losartan renography for detection of renovascular hypertension it was in a single case found that losartan but not captopril induced a fall in renal function in a kidney with more than 80% renal artery stenosis [6].
Our case also demonstrates that renography is an easy way to examine and follow patients with deterioration of renal function during treatment with angiotensin II antagonists or ACE inhibitors. When of relevance, the additional use of angiography and selective renin measurements add further evidence for the reason for renal impairment.
We conclude, that further studies are needed to demonstrate potential differences in the use of ACE inhibitors and angiotensin II antagonists in patients with suspected renal artery stenosis. Until then, both compounds should be avoided in this category of patients.
Note
Written consent for publication of the case was obtained from the patient.
Competing interests
None declared
Pre-publication history
The pre-publication history for this paper can be accessed here:
Icodextrin is a high molecular weight, starch-derived glucose polymer, which is capable of inducing sustained ultrafiltration over prolonged (12–16 hour) peritoneal dialysis (PD) dwells. The aim of this study was to evaluate the ability of icodextrin to alleviate refractory, symptomatic fluid overload and prolong technique survival in PD patients.
Methods
A prospective, open-label, pre-test/post-test study was conducted in 17 PD patients (8 females/9 males, mean age 56.8 ± 2.9 years) who were on the verge of being transferred to haemodialysis because of symptomatic fluid retention that was refractory to fluid restriction, loop diuretic therapy, hypertonic glucose exchanges and dwell time optimisation. One icodextrin exchange (2.5 L 7.5%, 12-hour dwell) was substituted for a long-dwell glucose exchange each day.
Results
Icodextrin significantly increased peritoneal ultrafiltration (885 ± 210 ml to 1454 ± 215 ml, p < 0.05) and reduced mean arterial pressure (106 ± 4 to 96 ± 4 mmHg, p < 0.05), but did not affect weight, plasma albumin concentration, haemoglobin levels or dialysate:plasma creatinine ratio. Diabetic patients (n = 12) also experienced improved glycaemic control (haemoglobin Alc decreased from 8.9 ± 0.7% to 7.9 ± 0.7%, p < 0.05). Overall PD technique survival was prolonged by a mean of 11.6 months (95% CI 6.0–17.3 months). On multivariate Cox proportional hazards analysis, extension of technique survival by icodextrin was only significantly predicted by baseline net daily peritoneal ultrafiltration (adjusted HR 2.52, 95% CI 1.13–5.62, p < 0.05).
Conclusions
Icodextrin significantly improved peritoneal ultrafiltration and extended technique survival in PD patients with symptomatic fluid overload, especially those who had substantially impaired peritoneal ultrafiltration.
Background
Icodextrin is an isosmolar, starch-derived, high molecular weight glucose polymer, which is minimally absorbed across the peritoneal membrane. Two previous randomised, controlled trials have shown that this agent promotes sustained ultrafiltration and small solute clearances equivalent to that achieved with hypertonic (3.86%/4.25%) glucose exchanges [1,2]. Moreover, in subjects with higher peritoneal membrane transport characteristics, icodextrin appears to achieve superior fluid removal compared with glucose-based dialysates [3,4]. Ultrafiltration is produced by colloidal, rather than crystalline, osmotic pressure and is sustained over prolonged (12 to 16 hour) dwells [5]. There is also emerging evidence that this isosmolar solution may be less damaging to the peritoneal membrane than glucose-based dialysates [6].
Two small, retrospective studies in CAPD patients [7][8]have suggested that substitution of icodextrin for a long-dwell glucose exchange may result in respective extensions of technique survival by median values of approximately 8 months [7] and 22 months [8]. However, these studies were potentially limited by recall bias and by the lack of a clear definition of "ultrafiltration failure," thereby introducing uncertainty as to the true extent of PD prolongation. Furthermore, neither of these studies attempted to examine which patient characteristics predicted a satisfactory ultrafiltration response to icodextrin and subsequent enhancement of technique survival. The aim of the present study was to prospectively evaluate the ability of icodextrin to alleviate fluid overload and extend technique survival in PD patients on the verge of being transferred to haemodialysis because of refractory, symptomatic fluid retention. Subsequent analyses were also performed to determine which patients were most likely to respond to icodextrin therapy.
MethodsPatients
All patients over the age of 18 years who were receiving PD at the Princess Alexandra Hospital between 30 January 1999 and 31 May 2001 were included in the study if they had pulmonary or peripheral oedema that was refractory to a combination of (i) fluid restriction (≤ 800 ml/day), (ii) frusemide therapy (≥ 250 mg daily), (iii) dwell time optimisation according to transport status, and, (iv) hypertonic glucose exchanges (consisting of at least one daily 4.25% exchange). All patients were considered by their treating physicians to be at the point of transfer to haemodialysis because of refractory fluid overload. CAPD patients had received a minimum of 4 exchanges and 8 litres of dialysis fluid per day. Those individuals on automated PD (APD) were treated with a continuous cycling PD (CCPD) regimen consisting of at least 12.5 L of dialysis fluid per day. Informed consent was obtained from all patients prior to their participation in the trial and the study protocol was reviewed and approved by the Princess Alexandra Hospital Research Ethics Committee.
Study protocol
The study followed a prospective, open label, pre-test/post-test design. One 12-hour 7.5% icodextrin exchange (2.5 L Extraneal®, Baxter Healthcare, Castlebar, Ireland) was substituted for either one overnight (CAPD patients) or daytime (APD patients) 4.25% glucose exchange. The remaining glucose exchanges (all 2.5% glucose exchanges except for 2 patients who each had an additional 4.25% glucose exchange) were not altered at any stage. Clinical and laboratory indices of fluid retention (weight, blood pressure, serum albumin concentration, plasma sodium concentration, haemoglobin concentration), peritoneal membrane transport status (peritoneal equilibration test [9]) and daily net peritoneal ultrafiltration were recorded prior to, and 1 month after, commencing icodextrin. Glycated hamoglobin (HbA1c) levels were additionally measured in diabetic patients prior to, and 3 months after, commencing icodextrin. The decision regarding whether subjects remained on PD or were transferred to haemodialysis was left to the discretion of their treating nephrologist. Patients were followed at monthly intervals until the end of the study (31 May 2001), at which point data were censored.
The primary outcome measure was extension of PD technique survival, which was regarded as the time interval between commencement of icodextrin and either PD completion or study termination. Secondary outcome measures included changes in daily ultrafiltration volume, indices of fluid retention, dialysate:plasma creatinine ratio at 4 hours (D:P Cr 4 h), and HbA1c during the initial month (or 3 months for HbA1c) of icodextrin therapy.
Statistical analysis
Normality of data was evaluated by the Kolmogorov-Smirnov test with Lilliefor's correction. Results are expressed as mean ± standard error (SEM) for continuous parametric data, median (interquartile range) for continuous non-parametric data, and frequencies and percentages for categorical data. PD technique survival curves, survival probabilities and estimated mean survival times were generated according to the Kaplan-Meier method. Data were not censored for death. Differences in the survival curves between the subgroups were evaluated using the log rank test. In order to ascertain the patient characteristics that independently predicted extension of PD technique survival, a multivariate Cox proportional hazards model regression analysis was subsequently performed, which included age, gender, racial status, diabetes, ultrafiltration and high transporter status as covariates. A backward elimination procedure was carried out with removal testing based on the probability of the Wald statistic until the most parsimonious model was identified. Adjusted survival curves were estimated using the Cox average covariate method, which calculates predicted survival probabilities at the mean levels of the covariates. Pre- and post-icodextrin changes in the secondary outcome measures of daily ultrafiltration volume, fluid retention indices, D:P Cr 4 h and HbA1c were assessed by paired t-test or Wilcoxon signed rank tests, depending on data distribution. Data were analysed using the using the statistical software package, SPSS release version 10.0.5 (SPSS Inc., Chicago, IL). P values less than 0.05 were considered significant.
ResultsPatient characteristics
Seventeen patients out of a total PD population of 263 met the inclusion criteria for the study during the 28-month recruitment period. Their characteristics are shown in Table 1. Compared with the general PD population at the Princess Alexandra Hospital, patients with refractory, symptomatic fluid overload had significantly higher weights (80.8 ± 4.3 versus 72.5 ± 1.5 kg, p < 0.05), lower daily net ultrafiltration rates (855 ± 210 versus 1310 ± 100 ml, p < 0.05) and greater frequencies of diabetes mellitus (71% versus 26%, p < 0.001), ischaemic heart disease (71% versus 37%, p = 0.01) and either high or high-average peritoneal transport characteristics (89% versus 58%, p < 0.05). All 17 eligible subjects agreed to participate in the study and none were lost to follow-up. The total follow-up time on icodextrin was 98.7 patient-months.
Baseline patient characteristics. Results are presented as mean ± SEM, percentage or median (range), depending on data type.
Characteristic
Value
Demographic
Age (years)
56.8 ± 2.9
Male
9 (53%)
Caucasian
9 (53%)
Weight (kg)
80.8 ± 4.3
Diabetes Mellitus
12 (71%)
Ischaemic Heart Disease
12 (71%)
Dialysis
Dialysis Duration (months)
18.6 ± 3.5
APD
5 (29%)
Prescribed Dialysate Volume (L/day)
12(8–21)
Ultrafiltration (mL/day)
855 ± 210
Residual Renal Creatinine Clearance (L/wk)
17.1 ± 5.1
Transport Status (H / HA / LA / L)
18%/71%/12%/0%
ESRF Cause
Diabetic Nephropathy
11(65%)
Chronic Glomerulonephritis
2 (12%)
Diffuse Cortical Necrosis
1 (6%)
Renovascular Nephrosclerosis
1 (6%)
Systemic Lupus Erythematosus
1 (6%)
Bladder Cancer
1 (6%)
Extension of PD technique survival
Substitution of an icodextrin exchange for a glucose exchange was associated with a significant prolongation of technique survival (mean 11.6 months, 95% CI 6.0–17.3 months) in patients who were otherwise about to be transferred to haemodialysis for refractory, symptomatic fluid overload (Fig. 1). The causes of technique failure included fluid overload (n = 4), peritonitis (n = 1) and death (peritonitis n = 1, acute myocardial infarction n = 1). No individuals underwent renal transplantation. Patients who had low daily ultrafiltration, defined as a net daily ultrafiltration of less than 1 L [10], remained on PD for a considerably longer time period following icodextrin administration than those who had normal daily ultrafiltration (Fig. 2). Using a multivariate Cox proportional hazards model analysis, baseline daily ultrafiltration (L/day) was the only independent predictor of technique survival on icodextrin (adjusted hazard ratio 2.52, 95% CI 1.13–5.62, p < 0.05). Extension of time on PD was not significantly associated with age, gender, ethnicity, diabetes mellitus or high transporter status.
Kaplan-Meier technique survival curve in 17 fluid-overloaded PD patients following commencement of icodextrin.
Relataive effects of low daily ultrafiltration (LDUF, peritoneal ultrafiltration < 1000 mL/day) and normal daily ultrafiltration (NDUF) on icodextrin technique survival (log rank 7.2, p < 0.01).
Effect of icodextrin on ultrafiltration, fluid status indices and glycaemic control
Prescription of icodextrin for 1 month resulted in a significant 599 ml increase in mean daily ultrafiltration rate and a 10 mmHg reduction in mean arterial pressure (Table 2). Patient weight decreased from 80.8 ± 4.3 to 79.6 ± 4.2 kg, but the difference did not achieve statistical significance (p = 0.14). No significant changes were observed in haemoglobin levels or serum concentrations of sodium, chloride, osmolality, urea, creatinine and glucose.
Effect of prescription of icodextrin for 1 month on ultrafiltration, fluid retention indices and selected biochemical parameters in 17 PD patients with refractory fluid overload. * p < 0.05 versus baseline
Parameter
Baseline
1 month Post-Icodextrin
Net daily ultrafiltration (ml)
855 ± 210
1454 ± 215*
Mean arterial pressure (mmHg)
106 ± 4
96 ± 4*
Weight (kg)
80.8 ± 4.3
79.6 ± 4.2
Haemoglobin (g/L)
107 ± 3
104 ± 4
Serum albumin (g/L)
33.6 ± 1.0
32.1 ± 0.9
Serum sodium (mmol/L)
137 ± 1
136 ± 1
Serum chloride (mmol/L)
94 ± 1
93 ± 1
Serum osmolalitv (mOsm/kg)
290 ± 3
288 ± 3
Serum urea (mmol/L)
16.1 ± 1.6
16.7 ± 2.2
Serum creatinine (mmol/L)
0.80 ± 0.07
0.71 ± 0.06
Serum glucose (mmol/L)
9.8 ± 1.6
7.8 ± 1.2
In the 12 patients with diabetes mellitus, substitution of icodextrin for glucose in one exchange daily led to a significant fall in HbA1c from 8.9 ± 0.7 to 7.9 ± 0.7% (p < 0.05). Seven out of 12 patients required a reduction in insulin prescription, whilst dosages in the remaining patients were not changed.
Adverse effects
Icodextrin therapy was not associated with a significant alteration in D:P Cr 4 h (pre-icodextrin 0.72 ± 0.05 versus post-icodextrin 0.74 ± 0.05, p = 0.55). Peritonitis rates following the institution of icodextrin were also not significantly different from antecedent rates (pre-icodextrin 0.20 versus post-icodextrin 0.21 episodes per patient-year, p = 1.00). No skin rashes or other adverse effects were observed.
Discussion
The results of the present study suggest that icodextrin may play a useful role in alleviating symptomatic fluid overload and extending technique survival in patients who have failed PD because of intractable hypervolaemia. Within the first month of substituting one icodextrin exchange for a glucose exchange, net daily peritoneal ultrafiltration was increased by nearly 600 ml and mean arterial pressure fell back towards normotensive levels by an average of 10 mmHg. The degree of symptomatic improvement obtained was sufficient to prolong time on PD by an average of 1 year.
These results support the previous findings of an analysis by Peers et al [7] of 56 ultrafiltration failure patients entered into a compassionate-use programme, in which icodextrin therapy extended median technique survival by approximately 8 months. Based on the cost differential between icodextrin-based PD and in-centre haemodialysis, this translated into a saving of approximately £1500 per year of extended PD life. A subsequent report by Wilkie and colleagues [8] similarly demonstrated that icodextrin extended median PD technique survival by 22 months in 33 patients with ultrafiltration failure. However, both of these retrospective studies were significantly limited by the fact that there had been no a priori well-defined indications or protocols for icodextrin administration. Thus, the true degree of prolongation of PD technique survival was uncertain. Moreover, the univariate survival analysis performed by Wilkie and associates [8] was censored for mortality, even though it is conceivable that fluid overload potentially contributed to some or all of the 8 deaths that occurred in the study. Both of these limitations were addressed in the present study by not censoring PD technique survival for patient death and by prospectively and precisely defining refractory, symptomatic fluid overload as a study inclusion criterion.
The extension of PD life in our study appears to have been related to a substantial increase in peritoneal fluid removal and was most marked in those patients who had poor initial daily ultrafiltration (<1 L/day). For each additional litre/day of peritoneal ultrafiltration prior to icodextrin commencement, the adjusted risk of subsequent early technique failure was increased by approximately 150%. These results are supported by Imholz et al [11], who demonstrated that icodextrin promoted a greater net ultrafiltration in 5 CAPD patients with low ultrafiltration than those with normal ultrafiltration (918 ± 85 vs 657 ± 104 ml, respectively, p = 0.06).
In keeping with previous studies of patients with refractory fluid overload [12-14], most (89%) of the patients in this investigation were high or high-average transporters. Their generally favourable response to icodextrin is consistent with the reported observation in stable peritoneal dialysis patients that the mass transfer area coefficient of creatinine was positively correlated with transcapillary ultrafiltration induced by icodextrin, but not glucose dialysate [3]. Similarly, Woodrow et al [4] noted in euvolaemic CCPD patients that the difference in daytime ultrafiltration between icodextrin and 3.86% glucose was positively correlated with the dialysate:plasma creatinine ratio, suggesting that icodextrin achieves superior fluid removal compared with glucose-based dialysates in subjects with higher peritoneal membrane transport characteristics. Such a correlation was unable to be demonstrated in our patients with symptomatic fluid overload, possibly because of the great preponderance of high and high-average transporters and the consequent narrowing of the range of dialysate:plasma creatinine ratios.
Inadequate ultrafiltration contributes directly to 8% of PD technique failure in Australia and New Zealand [15] and probably indirectly to an even larger number of patients. The risk of ultrafiltration failure increases progressively with time on dialysis and has been reported to be as high as 31% by 6 years [12]. It is possible to extend time on PD to a limited extent by increasing the number of hypertonic (3.86%/4.25%) glucose exchanges, but this is believed to cause further peritoneal damage [16]. Icodextrin on the other hand, has been shown to promote equivalent or superior ultrafiltration to glucose PD solutions, whilst avoiding the local and systemic sequelae of peritoneal glucose exposure [1,2,6,17-19]. The improvement in peritoneal ultrafiltration per gram of carbohydrate absorbed seen with icodextrin may be particularly advantageous to diabetic PD patients, who have an increased risk of type I ultrafiltration failure [20-24] and who frequently experience destabilisation of their glycaemic control following the use of hypertonic glucose dialysis solutions. Our study indicated that exchanging icodextrin for glucose dialysate resulted in a reduction in both insulin requirements and HbA1c levels in hypervolaemic diabetic PD patients. This issue has not been previously formally evaluated in icodextrin trials.
One of the weaknesses of our study is the lack of a parallel control group, which makes it difficult to be precise about the degree of extra PD time that was afforded by commencing icodextrin therapy. However, there clearly was a pronounced extension of PD technique survival in this well-defined group of patients with refractory fluid overload who had failed glucose-based dialysis. The important question that remains unanswered by this study is whether patient outcomes are optimised by giving such individuals a trial of icodextrin therapy or immediately transferring them to haemodialysis.
Conclusions
Icodextrin can significantly augment peritoneal ultrafiltration, alleviate fluid overload, improve diabetic glycaemic control and extend technique survival in PD patients with refractory, symptomatic fluid overload. Those individuals with low net daily ultrafiltration volumes (< 1 L/day) appear to be more likely to derive benefit from incorporation of icodextrin into their PD regimens than patients with higher ultrafiltration volumes.
List of abbreviations
APD Automated peritoneal dialysis
CAPD Continuous ambulatory peritoneal dialysis
CCPD Continuous cycling peritoneal dialysis
D:P Cr 4 h Dialysate:plasma creatinine ratio at 4 hours
HbA1c Glycated haemoglobin
LDUF Low daily ultrafiltration
NDUF Normal Daily ultrafiltration
PD Peritoneal dialysis
Declaration of competing interests
Dr Johnson has previously received consultancy fees from Baxter Healthcare Pty Ltd.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors are grateful to the Princess Alexandra Hospital renal physicians for their assistance with the execution of this study.
Hypertensive nephrosclerosis is the second most common cause of end-stage renal failure in the United States. The mechanism by which hypertension produces renal failure is incompletely understood. Recent evidence demonstrated that an unscheduled and inappropriate increase in apoptosis occurred in the Dahl/Rapp rat, an inbred strain of rat that uniformly develops hypertension and hypertensive nephrosclerosis; early correction of the hypertension prevents the renal injury. The present study examined the role of the Fas/FasL pathway in this process.
Methods
Young male Dahl/Rapp salt-sensitive (S) and Sprague-Dawley rats were fed diets that contained 0.3% or 8.0% NaCl diets. Kidneys were examined at days 7 and 21 of the study.
Results
An increase in Fas and FasL expression was observed in glomerular and tubular compartments of kidneys of hypertensive S rats, whereas dietary salt did not change expression of either of these molecules in normotensive Sprague-Dawley rats. Associated with this increase was cleavage of Bid and activation of caspase-8, the initiator caspase in this apoptotic pathway, by day 21 of the study.
Conclusions
Augmented expression of apoptotic signaling by the Fas/FasL pathway occurred during development of end-stage renal failure in this model of hypertensive nephrosclerosis.
Background
Hypertension is a common medical problem in the United States, occurring in as many as 43 million individuals [1]. While end-stage renal failure represents the second most common cause of end-stage renal disease [2], perhaps only 1 in 2,500 hypertensive patients develops clinically important hypertensive nephrosclerosis. This low frequency of end-organ renal damage suggests a potential predisposition to this complication. The inbred Dahl/Rapp rat is a genetic model of salt-sensitive hypertension [3,4]. On a diet containing 8.0% NaCl, young Dahl/Rapp salt-sensitive (S) rats rapidly and uniformly manifest low-renin hypertension and die from hypertensive nephrosclerosis within weeks of institution of the high-salt diet [5]. The pathology is identical to human hypertensive renal disease and consists of arteriolosclerosis, glomerulosclerosis, and interstitial scarring with tubular cell dropout. While other animal models of hypertension can be employed to study the pathogenesis of hypertension, because of the rapid and reproducible development of renal failure, S rats provide a unique means to investigate the pathogenesis of hypertensive nephrosclerosis. In this respect, this model is very useful in understanding hypertensive nephrosclerosis that occurs in human low-renin hypertension, especially in defined, more homogeneous populations such as the black patients described by Grim and associates [6].
Recent studies have shown that apoptosis was accentuated in kidneys of S rats during development of nephrosclerosis [7]. These studies demonstrated increased numbers of TUNEL-positive cells, increased cytoplasmic nucleosome content and caspase-3 activation in kidneys of hypertensive S rats, compared to normotensive Sprague-Dawley control rats on the same high-salt diet. An increase in apoptosis rates in glomeruli and tubules was observed, confirming a process that involved several different cell types in the kidney. Apoptotic bodies were noted particularly in areas of glomerular sclerosis and in dilated tubules. An increase in the number of PCNA-stained cells was also demonstrated during the 21-day time frame, but because renal function deteriorated, renal failure correlated therefore positively with apoptosis and inversely with proliferation rates. Thus, the apoptotic process appeared to be a strong determinant of outcome in this model.
The Fas/FasL system transmits apoptotic signals from the surrounding environment into the cell. Fas contains a single transmembrane domain and belongs to the tumor necrosis factor (TNF)/nerve growth factor family [8]. FasL contains a single transmembrane domain and is also a member of the same TNF family [9]. A soluble form of FasL has been described, but appears to be less capable of inducing apoptosis, when compared with the bound form [10,11]. The binding of FasL with Fas initiates receptor oligomerization, which recruits Fas-associated death domain (FADD) [12]. FADD binds procaspase-8 and permits activation of caspase-8 through self-cleavage [13]. Caspase-8 activates the effector caspases, which commits the cell to the orderly process of apoptosis [14,15]. In addition, caspase-8 cleaves Bcl-2-interactive-death-agonist (Bid) [16]. Truncated Bid localizes to the mitochondria and promotes cytochrome c release; this process also serves as a major apoptotic-signaling pathway for Fas [16-18]. Depending upon the amount of caspase-8 that is activated, the predominant pathway can be either Bid cleavage with subsequent mitochondrial release of cytochrome c or activation of the effector caspase pathway [18]. Resident cells of the kidney express both Fas and FasL and Fas/FasL signaling is functional in these cells [19-24]. A recent review by Ortiz and associates [25] noted that the combined literature demonstrating participation of Fas/FasL pathway in renal injury essentially fulfilled Koch's postulates.
In kidneys of S rats, associated with apoptosis was a temporal increase in expression of Fas, which was demonstrated using Western analysis of lysates of kidney cortex [7]. The purpose of the present study was to demonstrate the site of expression and potential functional significance of Fas and FasL in the kidneys of S rats during development of hypertensive nephrosclerosis.
Materials and MethodsAnimal preparation
Studies were conducted using 40 male Dahl/Rapp salt-sensitive (SS/Jr, termed S) and 40 male Sprague-Dawley (SD) rats, initially 28 days of age, obtained from Harlan Sprague Dawley, Indianapolis, IN. The rats were housed under standard conditions and given a formulated diet (AIN-76A, Dyets, Inc., Bethlehem, PA) that contained either 0.3% or 8.0% NaCl. These diets were prepared specifically to be identical in protein and electrolyte composition and differed only in NaCl and sucrose content. Groups of S and SD rats on 0.3% and 8.0% NaCl were maintained contemporaneously for each of the experiments performed. On days 7 and 21 of study, rats from all the groups were anesthetized by intraperitoneal injection of sodium pentobarbital (Abbott Laboratories, North Chicago, IL), 50 mg/kg. Laparotomy was performed and the kidneys were perfused in situ through the aorta with cold heparinized 0.9% NaCl solution until blanching occurred, which generally required infusion of 50 to 60 ml saline over 2 min. Both kidneys were harvested under sterile conditions to obtain protein for Western analysis and for light microscopy and histochemistry after fixation in 4% paraformaldehyde in PBS.
In situ detection of DNA fragmentation using TUNEL
In situ detection of DNA fragmentation was performed by incorporation of fluorescein-12-dUTP at the 3'-OH ends of DNA using Terminal deoxynucleotidyl Transferase (TUNEL assay) (Apoptosis Detection System, Fluorescein, Promega, Madison, WI), as performed previously by this laboratory [7,26]. Nuclei were counterstained using propidium iodide (Sigma Chemical Co., St. Louis, MO). For fluorescein, the excitation and barrier filters were set at 450–490 and 515–560 nm, respectively. Red fluorescence of propidium iodide was observed using excitation and barrier filters of 515–560 and 580 nm, respectively.
Immunohistochemistry
After embedding in paraffin, sections 5 μm in thickness were immersed twice into xylene for 5 min each, followed by immersion twice for 3 min each in 100% ethanol and then 95% ethanol. Slides were rinsed for 30 sec using deionized water and then immersed twice in deionized water for 5 min. To detect Fas, tissue sections were initially treated with 1% SDS in TBS (100 mM Tris-HCl, pH 7.4, 138 mM NaCl and 27 mM KCl) for 5 min at room temperature, followed by three 5-min washes in TBS alone. Slides were covered in 0.1–1.0% H2O2 for 5 min at room temperature and incubated in goat serum for 30 minutes at room temperature. Slides were incubated for 30 minutes at room temperature in PBS containing monoclonal mouse anti-human Fas/CD95/APO-1 (Transduction Laboratories, Lexington, Kentucky), 0.5–1.0 μg/ml, and 10% goat serum. Slides were rinsed and covered with TBS containing 10% rat serum and peroxidase-labeled polymer conjugated to goat anti-mouse IgG (DAKO EnVision™+ System, DAKO Corporation, Carpinteria, CA) for 30 minutes at room temperature and color was developed using 3,3'-diaminobenzidine (DAB) Chromogen solution (DAKO). Cells were counterstained using hematoxylin and the slides were mounted in standard fashion. As a negative control, the primary antibody was omitted from the reaction. As an additional control, the anti-Fas antibody was pre-incubated with recombinant soluble human Fas (PharMingen, San Diego, CA), 0.5 μg/ml, for 1 h at room temperature prior to use as the primary antibody to stain the specimens. To detect FasL, paraffin-embedded sections were initially heated twice at 95°C for 5 min in 10-mM sodium citrate buffer, pH 6.0. Staining then proceeded in standard fashion using goat polyclonal anti-FasL antibody (goat polyclonal antibody IgG raised against FasL(N-20), Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1–4 μg/ml, followed by biotinylated donkey anti-goat IgG. Color was developed using the avidin-biotin detection system (ImmunoCruz Staining Systems, Santa Cruz Biotechnology, Inc.).
Immunoenzyme double staining of kidney tissue was performed using DAKO EnVision Doublestain System (DAKO). The sections were initially heated twice at 95°C for 5 min in 10-mM sodium citrate buffer, pH 6.0, and then incubated for 30 min with the mouse anti-Fas IgG (Transduction Laboratories), 1:500 dilution in PBS containing 1% BSA. The sections were incubated in horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse immunoglobulins for 30 min and developed with 3,3'-diaminobenzidine (DAB) Chromogen solution (DAKO). After incubation for 10 min in the Doublestain Block (DAKO) at room temperature, the specimens were incubated for 30 min with the goat anti-FasL IgG (Santa Cruz Biotechnology, Inc.), 1:50 dilution in PBS containing 1% BSA. The samples were then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG for 30 min. The slides were developed using alkaline-phosphatase substrate to produce the red color. The sections were then counterstained using hematoxylin before study.
The specimens were examined using a light microscope (Leica, Germany) equipped with a digital camera (Model C5810, Hamamatsu Photonics K.K.). Semi-quantification of stained tubular epithelial cells was determined at ×20 magnification by projecting the image and counting the number of positive cells; a total of six different fields were analyzed for each kidney and the results were averaged to produce a final count for each kidney. Quantification of staining of glomerular compartment was determined at ×20 magnification by counting the number of stained cells in twenty-five glomeruli.
Immunoblot analysis
Samples of renal cortex from S and SD rats on the 0.3% and 8.0% NaCl diets for 7 and 21 days were diced into small pieces and chilled in 3 ml of ice cold RIPA buffer per gram of tissue, then homogenized (Omni-Mixer 17105; Omni, Waterbury, CT) in standard fashion. Reagents were from Sigma Chemical Co. The solutions were mixed gently and incubated on ice for 30 min, then homogenized by passage through 21-gauge needle. Thirty μl of phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml, was added per gram of tissue and incubated on ice for 30 min. Samples were centrifuged at 15,000 ×g for 20 min at 4°C and the supernatant was harvested. Total protein of each sample was determined using a kit (Micro BCA protein assay reagent kit, Pierce, Rockford, IL.). For each experiment that examined a protein of interest, all samples were processed simultaneously. Samples containing 60 μg of total protein were mixed with an equal volume of 2x SDS-Laemmli sample buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 200 mM dithiothreitol, 4% SDS, and 0.2% bromphenol blue) and boiled for 5 min, then resolved using either 12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred on to nitrocellulose membrane using an electroblotting apparatus (Bio-Rad, Richmond, CA). Following incubation in blocking buffer (10 mM Tris-HCl, pH 7.5, containing 10% non-fat dry milk, 100 mM NaCl, and 0.1% Tween 20), the membranes were probed with an affinity-purified rabbit polyclonal IgG directed against caspase-8 (Caspase-8 p20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:200 dilution in blocking buffer, overnight at 4°C. The membranes were then washed five times with TBS-Tween and were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Bio-Rad, Hercules, CA), 1:5,000 dilution. After three additional washes using TBS-Tween, the membrane was developed using ECL Western blotting system and Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ). The antibody used in these studies recognized bands of the expected size for pro-caspase-8 (about 55 kDa) and active caspase-8 (about 20 kDa). In other experiments, samples containing 80 μg total protein underwent SDS-PAGE using 15% polyacrylamide gels. The samples were transferred on to nitrocellulose membranes, which were incubated in blocking buffer (TBS, pH 7.5, containing 10% dried milk and 0.1% Tween-20) and then rabbit anti-Bid antiserum (BD PharMingen, San Diego, CA), 1:2000 dilution in the same blocking buffer, at 4°C, overnight. Following incubation with donkey anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology Inc. Santa Cruz, CA), 1:6000 dilution, for 1 h at room temperature, the membrane was developed as described above.
Statistical analysis
All data were presented as mean ± standard error. Significant differences were determined using one-way analysis of variance with standard post-hoc testing (Statview, version 5.0, SAS Institute, Cary, NC). A P value of < 0.05 assigned statistical significance.
Results
Kidneys of S and SD rats on both 0.3% and 8.0% NaCl diets were examined on days 7 and 21. The blood pressure responses to dietary salt intake of the Dahl/Rapp S rat, an experimental model of hypertensive nephrosclerosis, and of the SD strain of rat have been published. After seven days on the high salt diet, S rats were hypertensive (146 ± 2 mm Hg) but had preserved renal morphology [5]. By day 21 on the 8.0% NaCl diet, the mean blood pressure increased to 169 ± 7 mm Hg [7,27,28]. Mean blood pressures of SD rats were not influenced by intake of salt over this time frame of study (118 ± 4 mm Hg on the 8.0% NaCl diet, compared to 115 ± 4 mm Hg on the 0.3% NaCl diet). In addition, SD animals were healthy, the intake of either diet was similar, and they demonstrated no renal abnormalities [27-29]. In the present study, by day 21 the kidneys of S rats demonstrated significant vascular, glomerular and tubulo-interstitial injury. TUNEL demonstrated apoptosis in glomerular and tubular compartments (Fig 1). SD rats on both diets and S rats on 0.3% NaCl diet demonstrated by light microscopy no renal morphological alterations over the same period of study. These findings were similar to previous publications from this laboratory [5,7].
Representative light micrograph (top panel) of a kidney from an S rat that had been on 8.0% NaCl diet for three weeks. The figure shows typical hypertensive renal lesions, which include thickening of the arteriolar wall (arrow), glomerular sclerosis, and dilatation of tubules with tubular atrophy (asterisks) and presence of cast material (C) in some tubules. Expansion of the interstitium, indicated by an increase in the distance between the tubules, was also evident. H/E stain. Black bar represented 50 μm. The dual immunofluorescent studies at the bottom show how exuberant the apoptotic process can be in this model. The bottom left panel represents a section that was counterstained with propidium iodide to label the nuclei (red color). The middle panel is the same section that was stained using the TUNEL assay, which fluorescein-labels the 3'-OH termini of DNA. Several nuclei in the tubular and glomerular compartments demonstrate the green fluorescence. The bottom right panel is a combined image; the yellow color is the result of overlapping propidium iodide and fluorescein labeling. White bar represented 50 μm. Arrowheads denote glomerulus.
Using immunohistochemistry, expression of Fas was increased specifically in kidneys of S rats on 8.0% NaCl diet by day 7 (Fig. 2). Staining was inhibited when the anti-Fas antibody was pre-incubated with recombinant soluble Fas (Fig. 2E and 2J). Quantification analysis comparing the four groups of rats in the study showed that expression of Fas in glomerular and tubular cells was greatest (P < 0.05) in kidneys of S rats exposed to the 8.0% NaCl diet for 7 and 21 days (Fig. 3). Comparing animals on the two diets for the same duration, dietary salt did not alter Fas expression in kidneys of SD rats. The increase in Fas expression in kidneys of S rats on the 8.0% NaCl diet was observed in cells in the glomeruli, tubules and arteries (Fig. 4A,B,C). Cytoplasmic staining was particularly apparent in these samples.
Representative images of immunohistochemical stains of kidney sections from each of the group of rats in the study. While Fas staining was evident in kidneys from SD rats on both diets (panels A, B, F, and G) and S rats on 0.3% NaCl (panels C and H), an increase in staining was observed in kidneys from S rats on 8.0% NaCl diet (panels D and I). Staining was diminished when the antibody was pre-incubated with soluble Fas (panels E and J). The bar represented 50 μm.
Semi-quantitative analysis of Fas staining showed an increase (P < 0.05) in expression of Fas by glomerular and tubular cells from rats on 8.0% NaCl diet for 7 and 21 days (n = 6 rats in each group), compared to the other groups of rats examined at the same time. Asterisks denote the group that was different (P < 0.05) than the other three groups from the same day of study. The increase was prominent on day 21 of the diet.
Representative higher magnification images of immunohistochemical stain using antibodies to Fas and FasL. Fas stain was noted in cells in glomeruli (arrowheads, panel A), tubules (panel B) and arterial smooth muscle (arrows, panel C). White bar represented 20 μm; L, lumen. FasL expression was observed in glomerular (panel D) and tubular (panel E) cells, where expression was prominent particularly on the lumenal side. Black bars represented 50 and 10 μm, respectively. Co-expression of both Fas (brown color) and FasL (red color) was identified in some tubular cells (panels F, G and H). Bar in panel F represented 100 μm and represented 50 μm in panels G and H.
Over the same time period, FasL expression was also examined (Fig. 5A,B,C,D,E,F,G,H,I,J). The anti-FasL antibody labeled cells in both glomerular and tubular compartments, but expression was more subtle than Fas, particularly in the glomeruli. Quantification of these observations showed an increase in expression of FasL in both glomerular and tubular compartments of kidneys of S rats on the 8.0% NaCl diet, compared with the other three groups (Fig. 6). Again, dietary salt did not alter glomerular and tubular expression of FasL in kidneys of SD rats. Tubular epithelial cell expression of FasL appeared to be predominantly apical (Fig. 4D and 4E), although cytoplasmic staining was also observed, particularly in cells of S rats on the 8.0% NaCl diet. In addition, some tubular epithelial cells of kidneys from S rats on 8.0% NaCl diet for 21 days appeared to express both Fas and FasL (Fig. 4F,G and 4H).
Representative images of immunohistochemical stains using antibody to FasL. Panels A and F represent negative controls, where the primary antibody was omitted from the reaction. Expression of FasL did not appear to be altered by dietary salt in the SD rats, but increased in S rats on the 8.0% NaCl. The effect was pronounced by the third week of study. Expression was observed in both glomerular and tubular compartments of the kidney. Black bar represented 50 μm.
Semi-quantitative analysis of FasL expression in the kidney (n = 6 rats in each group). Compared with the other three groups examined at the same time in the study, an increase (P < 0.05) in FasL staining was observed in glomerular and tubular cells of S rats on the 8.0% NaCl diet. Asterisks denote the group that was different (P < 0.05) than the other three groups from the same day of study. The increase was prominent by the third week of study.
To determine whether the Fas/FasL apoptotic pathway was active, Western blotting of renal cortical lysates was performed using an antibody that recognized both procaspase-8 and the active form of caspase-8. In these experiments, active caspase-8 was demonstrated in lysates from renal cortex of S rats on 8.0% NaCl diet for three weeks (Fig. 7). Active caspase-8 was not demonstrated in samples from rats on the diet for 7 days (data not shown). Tissue was also examined for cleavage of Bid, which was identified specifically in lysates from renal cortex obtained from S rats on 8.0% NaCl diet for three weeks (Fig. 7).
The top panel showed a Western blot using an anti-caspase-8 polyclonal IgG, which recognized both procaspase-8 and the active enzyme. Active caspase-8 (about 20 kDa) was observed only in kidney cortex of S rats on the 8.0% NaCl diet for 21 days. The bottom panel showed a Western blot that used an anti-Bid polyclonal IgG, which also recognized the 13 and 15 kDa cleaved fragments of Bid (tBid). Fragments of Bid (tBid) were apparent in lysates of renal cortex of S rats on the 8.0% NaCl diet for 21 days. Each lane represented a single rat (n = 4 rats in each group).
Discussion
Recent evidence suggests that apoptosis is involved in several pathological processes in the lung [30-32] and kidney [33-37]. Using the Dahl/Rapp salt-sensitive (S) rat, which develops hypertensive nephrosclerosis that resembles the human disease process [5], this laboratory demonstrated an early and inappropriate apoptotic process that occurred in both glomerular and tubular compartments of the kidneys and correlated with the decline in glomerular filtration rate. Apoptosis can be significant particularly three weeks into the course of hypertensive nephrosclerosis in this model (Figure 1). A previous study used RNase protection assay and Western blotting to demonstrate an increase in Fas expression in kidney cortex of hypertensive S rats. These findings were observed by day 21 of study [7]. The present series of experiments demonstrated the diffuse sites of expression of Fas and FasL in the kidney and explored further the possibility that the Fas/FasL pathway was active during the development of hypertensive renal disease in this rodent model of hypertension. Expression of both Fas and FasL was observed in kidneys of both SD and S rats, but specifically increased over time in hypertensive S rats. Associated with the increased expression was cleavage of procaspase-8 to produce the active enzyme and cleavage of Bid, indicating activation of the Fas/FasL apoptotic pathway in the course of progressive renal failure. These data, along with the previous studies that confirmed morphological evidence of apoptosis and biochemical evidence of caspase-3 activation in these kidneys [7], showed a role for the Fas/FasL pathway in promoting the loss of kidney function that develops in the setting of hypertensive nephrosclerosis in this model.
The mechanism of augmented Fas and FasL expression in kidneys of hypertensive S rats is uncertain. In addition to apoptotic signaling, the Fas/FasL system may also be involved in proliferation. Hueber, et al., showed that Fas might promote T-cell proliferation by modulating release of calcium from intracellular stores [38]. FasL is anti-proliferative by regulating cell-cycle progression [39]. Previous studies demonstrated an increase in cell proliferation rates in kidneys of S rats on the high-salt diet [7], so it is possible that Fas and FasL may play a role in control of proliferation. Another potential explanation is tissue hypoxia, which has been suggested to upregulate Fas in the kidney [36,37]. MDCK cells in culture demonstrated increased expression of Fas, FasL and FADD in response to partial ATP depletion [40]. Fas and FasL expression in the kidney may serve as indicators of cellular stress. Tissue hypoxia therefore remains a possible mechanism of increased expression of Fas and FasL, although it seems less likely that the arteriolosclerosis that developed in S kidneys was severe enough to produce hypoxic conditions sufficient to up-regulate Fas, until the third week of study.
Increased expression of Fas and FasL appeared to increase the propensity to undergo apoptosis, but was not the sole determinant of this process. Despite the increase in expression of Fas and FasL at day 7 of study, caspase-8 activation and Bid cleavage were not observed. Fas/FasL-mediated apoptotic signaling is modulated by other intracellular factors. Induction of inhibitor of apoptosis proteins (IAP) is one such mechanism [41-43]. In vascular smooth muscle cells, surface expression of Fas [44] and molecules involved in signaling the apoptotic process, such as caspase-8 and caspase-3, appear to be regulated and provide another level of cellular control of apoptosis [45]. However, the potential anti-apoptotic mechanisms present early in the course were ineffective by day 21, when caspase-8 activation was evident. A previous study suggested that the apoptotic process occurred early in the development of hypertension in this model [26], so the data suggested that an apoptotic mechanism in addition to activation of the Fas/FasL pathway is also involved in development of hypertensive nephrosclerosis in S rats.
In conclusion, kidneys of S rats have been shown to be very sensitive to hypertensive injury, which is mediated at least in part by the apoptotic mechanism controlled by Fas. Augmented expression of both Fas and FasL by glomerular and tubular cells resulted in caspase-8 activation. The pronounced increases in Fas and FasL expression and activation of this pathway by the third week of study also correlated with previous evidence that documented irreversibility of the renal injury if the hypertension was not corrected by this time [5]. These data supported the view that the Fas/FasL pathway promoted nephrosclerosis in this model. Activation of the Fas/FasL pathway may also serve as a mechanism of tubular epithelial cell loss and interstitial fibrosis, which are also prominent renal features of hypertension. The role of the Fas/FasL pathway in human hypertensive nephrosclerosis remains to be determined.
Abbreviations
SD, Sprague-Dawley rat
S, Dahl/Rapp salt-sensitive rat
Fas, Fas antigen (or CD95 and APO-1)
FasL, Fas antigen ligand (or CD95L and APO-1L)
PBS, phosphate-buffered saline
RIPA, radioimmunoprecipitation assay
SDS, sodium dodecyl sulfate
TBS, Tris-buffered saline
Bid, Bcl-2-interactive-death-agonist.
Competing interests
None declared
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
National Institutes of Health grant (R01 DK46199) and the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, supported this work. The authors thank Ms. Karen Lewis for the excellent technical support.
Angiotensin converting enzyme inhibitors are routinely prescribed to patients with chronic kidney disease because of their known renoprotective effects. We evaluated the effect of short-term therapy with the angiotensin converting enzyme inhibitor, enalapril, in early Alport syndrome, defined as disease duration less than 10 years and a normal glomerular filtration rate.
Methods
11 children with early Alport syndrome were investigated. Two consecutive early morning urine specimens were collected at the start of the study for measurement of urinary creatinine, total protein, albumin, TGF-β, and nitrite excretion. Patients were treated with enalapril, ≅ 0.2 mg/kg/day, once a day for 14 days. Two early morning urine specimens were collected on days 13 and 14 of enalapril treatment and two weeks later for measurement of urinary creatinine, total protein, albumin, TGF-β, and nitrite excretion.
Results
Prior to treatment, urinary excretion of transforming growth factor-β and nitrite, the major metabolite of nitric oxide, was within normal limits in all patients. Administration of enalapril for 2 weeks did not alter urinary albumin, transforming growth factor-β, or nitrite excretion.
Conclusion
These findings suggest that early Alport syndrome represents a disease involving exclusively intrinsic glomerular barrier dysfunction. At this stage of the illness, there is no evidence of angiotensin II-mediated proteinuria or increased production of transforming growth factor-β and, therefore, routine treatment with an angiotensin converting enzyme inhibitor may not be warranted.
Alport syndrome (AS) is a glomerulopathy caused by genetic mutations in type IV collagen, the main collagenous constituent of the glomerular basement membrane (GBM) [1]. The predominant form of AS is inherited as an X-linked dominant disease. Hematuria is the initial finding and proteinuria develops later secondary to the defective composition and ultrastructure of the GBM [1]. Proteinuria increases with age and is a useful marker of disease progression. End stage renal disease (ESRD) eventually ensues in affected males, while the disease course is more benign in females. There is no specific therapy for AS and renal transplantation remains the definitive treatment [1].
Transforming growth factor-β (TGF-β) and nitric oxide (NO) are important mediators of renal disease progression. TGF-β is a fibrogenic cytokine involved in normal wound healing and pathological fibrosis (). Intra-renal production of TGF-β is enhanced in patients with IgA nephropathy, diabetic nephropathy, lupus nephritis, and focal segmental glomerulosclerosis (FSGS) when they develop glomerular scarring [3]. The factors that control TGF-β overexpression in these renal diseases are not well understood. NO is a short-acting signaling molecule that is involved in the regulation of glomerular hemodynamics, mesangial proliferation, and net mesangial matrix production [4]. Diminished renal production in chronic renal disease may contribute to kidney fibrosis [5]. The role of TGF-β and NO in promoting progressive disease in AS is not known.
Treatment with angiotensin converting enzyme inhibitors (ACEI) is renoprotective in virtually all proteinuric forms of kidney disease [6]. By reducing intraglomerular hypertension, ACEI prevent glomerular hyperfiltration. However, because angiotensin II is a potent inducer of TGF-β release, ACEI may also diminish renal fibrosis by a non-hemodynamic mechanism [7,8]. Both ACEI and NO retard the progression to kidney failure and their renoprotective effects correlate with suppression of TGF-β [9-12].
In view of these observations, we performed the following study to determine urinary protein, TGF-β, and nitrite (the stable metabolite of NO) excretion in children with early AS and to evaluate the impact of short-term ACEI therapy on these measurements.
Materials and MethodsPatients
11 children (5M:6F), mean age 8.6 ± 1.1 years, with renal early biopsy-proven AS were included in this study. The duration of disease from the date of diagnostic biopsy until participation in this study was 56 ± 12 months. The estimated glomerular filtration rate (GFR) was calculated using the age and sex-appropriate length-serum creatinine formulas [13,14]. Normal levels of blood pressure were defined based upon the updated Task Force Report on Blood Pressure Control in Children and Adolescents [15].
Clinical protocol
Two consecutive early morning urine specimens were collected at the start of the study for measurement of urinary creatinine, albumin, TGF-β, and nitrite excretion. Patients were then given enalapril (EN), ≅ 0.2 mg/kg/day, once a day for 14 days. The daily dose of enalapril was between 5–10 mg. The amount of drug and duration of treatment were the same as in a previous evaluation of ACEI therapy in sickle cell nephropathy [16]. Two early morning urine specimens were collected on days 13 and 14 of EN treatment. Administration of the ACEI was then stopped and two early morning urine specimens were collected again two weeks later. This study was approved by the Institutional Review Board of the Schneider Children's Hospital of the North Shore-Long Island Jewish Health System and informed consent was signed prior to patient enrollment.
Analytical methods
Urine creatinine was measured using standard methods. Urinary albumin and TGF-β were determined using commercially available EIA kits (R & D Systems, Minneapolis, MN). Urinary albumin excretion was expressed as the urine albumin: creatinine ratio (UAC) (mg/mg). The Greiss reaction was used to measure urinary nitrite excretion, which was expressed as nmol/mg creatinine.
Statistical analysis
Data are presented as mean ± SE. Differences between groups were analyzed using the ANOVA and t-test and results were considered significant if P < 0.05.
Results
In the 11 patients who enrolled in this clinical study, the estimated GFR was 146 ± 14 ml/min/1.73 m2 . Blood was not drawn at the end of the 14-day EN treatment period and, as a consequence, the effect of ACEl therapy on GFR could not be determined. All of the children had a normal blood pressure without antihypertensive medications. Prior to EN treatment, the mean UAC was 1.2 ± 0.9 (range: 0.01–9.6). In addition, the level of TGF-β in the urine was 60 ± 11 pg/mg creatinine, within the range observed in healthy subjects (50–300 pg/mg creatinine) [17]. Finally, the baseline urinary nitrite excretion was 1466 ± 330 versus 836 ± 274 nmol/mg creatinine in normal control children (P = 0.16).
At the end of two weeks of EN therapy, UAC was 130 ± 33% of pre-treatment levels, and remained elevated 14 days after treatment was discontinued, namely 134 ± 31% of the baseline value. A decline in UAC >33% was observed in only 2 patients, both of whose initial urine albumin concentration was >1000 mg/L (the urinary albumin: creatinine ratio (mg/mg) was 1.4 and 9.6 in these 2 children). In the urine samples collected after two weeks of ACEl therapy and after withdrawing EN two weeks later, the urinary TGF-β excretion was 55 ± 11 and 57 ± 10 pg/mg creatinine, respectively. Compared to pre-treatment levels, urinary TGF-β levels were 110 ± 23 and 112 ± 30%, respectively, of the baseline value at the completion of the 2-week course of ACEI therapy and 2 weeks after discontinuation of the medication (P > 0.4). Similarly, short-term ACEI therapy had no effect on urinary nitrite excretion, 92 ± 20 and 84 ± 7% of baseline level, after 2 wk of EN therapy, and 2 wk after discontinuing the ACEI, respectively (P > 0.3). The changes in urinary TGF-β and nitrite excretion were not different in the two patients with significant albuminuria compared to the other 9 children.
All of the urinary measurements, i.e., albuminuria, TGF-β, and nitrite excretion, were comparable in boys and girls at baseline and in response to ACEI therapy. Although there was no correlation between the effect of EN therapy on albuminuria and the change in urinary TGF-β or nitrite excretion, the later two variables were significantly related to one another (P < 0.04).
Discussion
The children with biopsy-proven AS who were enrolled in this study had early disease, based upon a normal GFR, normal blood pressure, and sub-nephrotic range proteinuria. The urinary abnormalities observed in these patients were probably a consequence of genetic mutations in the α5 chains of type IV collagen and defective GBM ultrastructure [1]. Although the long-term risk of disease progression differs in male versus female patients, based on the similar level of urinary protein excretion, it is likely that at the time of these investigations glomerular barrier function and the mediators of proteinuria were similar in all of the study participants.
In other progressive glomerulopathies, alterations in TGF-β and NO have been implicated in the steady decline in renal function, glomerulosclerosis, and tubulointerstitial fibrosis. The source of the fibrogenic cytokines and vasoactive mediators varies in different disease entities. For example, in IgA nephropathy, immune complex nephritis, and FSGS increased intra-renal levels of TGF-β and NO are the consequence of release by resident and/or infiltrating immunoeffector cells [3]. In contrast, in diabetic nephropathy, metabolic disturbances, namely hyperglycemia, directly modulate TGF-β and NO synthesis by glomerular mesangial and renal tubular epithelial cells [3,7]. In diabetic nephropathy, alterations in intra-renal TGF-β production have been assessed by measuring urinary excretion of this cytokine [17].
Measurements of urinary nitrite excretion are less interpretable than determinations of urinary nitrite + nitrate excretion because a variable portion (50–90%) of NO-related end products in the urine may represent nitrate [18]. However, nitrate excretion is primarily dependent on dietary intake and there is a close correlation between urinary nitrite and nitrate excretion in healthy subjects [18]. Therefore, because this was a short-term study and patients did not modify their diet for the duration of the 4-week experimental period, it is likely that the urinary nitrite excretion accurately reflects renal NO synthesis. The small non-significant decrease in urinary nitrite excretion after ACEI therapy, despite the known effect of these drugs to stimulate endothelial NO release, may be the consequence of a decline in GFR. A blood sample was not obtained at the conclusion of ACEI treatment to enable measurement of renal function. However, most pediatric patients with a GFR in the range noted for the children in this study do not develop a decline in renal function in response to EN therapy.
Our data indicate that in early AS, hematuria and sub-nephrotic range proteinuria are exclusively the result of intrinsic glomerular barrier dysfunction and are not the consequence of acutely reversible alterations in glomerular hemodynamics. In contrast to children with type I diabetes mellitus [17], there is no increase in renal TGF-β or NO production at this stage of the disease. The clinical data are in agreement with recent findings in a transgenic model of autosomal recessive Alport syndrome in which TGF-β was only involved in the late progression of the nephropathy [19,20]. Although male patients with AS are at greater risk than females of developing progressive kidney failure, our findings suggest that differences in renal production of TGF-β in the early phase of the disease cannot explain the impact of gender on prognosis. Although higher levels of proteinuria may stimulate release of these and other inflammatory mediators, this pathogenetic mechanism is not activated in early AS.
In virtually all forms of glomerular disease, administration of ACEl to patients with nephrotic range proteinuria results in a significant and reproducible reduction in urinary protein excretion and presumably an improvement in the long-term prognosis [6]. However, early in the course of AS, when our patients had sub-nephrotic range proteinuria there was no evidence of an angiotensin II-sensitive alteration in glomerular hemodynamics that manifested as a decrease in albuminuria. The more pronounced antiproteinuric response in the 2 children whose initial urine albumin concentration exceeded 1000 mg/L is consistent with a previous report of enalapril use in pediatric patients with AS [21].
It is important to note that our protocol to determine the effect of short-term administration of ACEl on urinary protein excretion has been effective in other forms of glomerular disease in which hemodynamic alterations have been linked to perturbations in glomerular permselectivity, namely sickle cell nephropathy [16]. We cannot exclude the possibility that once-a-day administration of EN was ineffective because of different pharmacokinetics and a shorter drug half-life in children. The response to short-term ACEI was significantly lower in our patients with AS (2/11 children had >33% reduction in albuminuria) versus published reports in adults with sickle cell nephropathy (10/10 had a comparable decrease in albuminuria) (P < 0.0005) [16]. The 95% confidence interval on this difference indicates that at most 40% of children with AS are likely to demonstrate a meaningful reduction in albuminuria following 2 weeks of EN therapy. Thus, despite the small sample size in this study, it is reasonable to conclude that ACEI treatment will be ineffective in lowering urinary protein excretion in children with early AS.
Whether long-term ACEI therapy is effective in patients with AS who have developed heavy proteinuria is uncertain. Additional studies will be needed to determine whether ACEI treatment at later stages of the disease reduces proteinuria, precisely when in the disease course ACEI responsiveness supervenes, and whether this change is paralleled by alterations in urinary TGF-β and nitrite excretion. Experimental protocols designed to evaluate the impact of ACEI therapy in transgenic models of AS will help clarify the role of this treatment at various stages in the course of the disease.
Conclusion
Our results suggest that early AS represents an isolated defect in GBM composition and ultrastructure resulting in a pure glomerular protein leak. At this stage of the disease, there is no stimulus to increase TGF-β or NO production nor is there evidence of an angiotensin II-sensitive alteration in glomerular hemodynamics. Short-term administration of an ACEI to patients with early AS does not cause a reversible decrease in proteinuria. We speculate that with longer duration of disease and increasing urinary protein excretion, the pathophysiology of AS becomes similar to other glomerulopathies with involvement of altered glomerular hemodynamics, inflammatory mediators, chemokines, and fibrogenic cytokines. At this advanced stage, aggressive ACEI therapy may have a renoprotective role. Further studies are needed to determine the exact time point in the course of AS when administration of an ACEI is a useful intervention to effectively lower urinary protein excretion and stabilize renal function.
Competing Interests
None
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgement
This work was supported in part by a grant from Merck Inc. (West Point, PA).
Although previous research has demonstrated that referral to pre-dialysis clinics is associated with favourable objective outcomes, the benefit of a pre-dialysis clinic from the perspective of patient-perceived subjective outcomes, such as quality of life (QOL), is less well defined.
Methods
A retrospective incident cohort study was conducted to determine if pre-dialysis clinic attendance was a predictor of better QOL scores measured within the first six months of hemodialysis (HD) initiation. Inclusion criteria were HD initiation from January 1 1998 to January 1 2000, diagnosis of chronic renal failure, and completion of the QOL questionnaire within six months of HD initiation. Patients receiving HD for less than four weeks were excluded. An incident cohort of 120 dialysis patients was identified, including 74 patients who attended at least one pre-dialysis clinic and 46 patients who did not. QOL was measured using the SF 36-Item Health Survey. Independent variables included age, sex, diabetes, pre-dialysis clinic attendance and length of attendance, history of ischemic heart disease, stroke, peripheral vascular disease, heart failure, malignancy, and chronic lung disease, residual creatinine clearance at dialysis initiation, and kt/v, albumin and hemoglobin at the time of QOL assessment. Bivariate and multivariate linear regression analyses were used to identify predictors of QOL scores.
Results
Multivariate analysis suggested that pre-dialysis clinic attendance was an independent predictor of higher QOL scores in four of eight health domains (physical function, p < 0.01; emotional role limitation, p = 0.01; social function, p = 0.01; and general health, p = 0.03), even after statistical adjustment for age, sex, residual renal function, kt/v, albumin, and co-morbid disease. Pre-dialysis clinic attendance was also an independent predictor of the physical component summary score (p = 0.03).
Conclusions
We conclude that pre-dialysis clinic attendance favourably influences patient-perceived quality of life within six months of dialysis initiation.
Background
The National Institutes of Health have recommended that patients with chronic progressive renal insufficiency be referred to a multidisciplinary pre-dialysis team in order to minimize patient morbidity and ensure a smooth transition to dialysis therapy. [1] Referral to a pre-dialysis clinic is associated with many desirable outcomes including longer dialysis-free time intervals, [2] enhanced employment opportunities, [3] better patient education, [4,5] and greater participation in the selection of dialysis modality. [4,5] Patients referred to a multi-disciplinary pre-dialysis program also demonstrate better metabolic profiles, are less likely to require central venous catheter insertion, and require fewer in-hospital emergency dialysis starts and hospital admission days compared to patients who receive standard care. [6,7]
However, research exploring the benefits of pre-dialysis care from a patient-oriented perspective using self-reported quality of life measurements is limited. In a single Brazilian study, quality of life was compared between patients diagnosed with chronic renal failure less than one month before dialysis initiation (n = 53) and those patients diagnosed more than six months before starting dialysis (n = 60) using the Kidney Disease Questionnaire (KDQ). [8] Compared to patients diagnosed earlier, quality of life scores were significantly worse in the late-diagnosis group – a finding that was particularly evident among elderly patients. However, statistical adjustment for covariates other that age and time of diagnosis was not performed. As well, only 28% of patients were over age 60, suggesting that the association between early diagnosis and improved quality of life may have been underestimated. Further study using a more representative sample reflecting the demographics of an aging Canadian population is therefore warranted.
The present study hypothesized that attendance at a pre-dialysis clinic is an independent predictor of improved quality of life scores measured within the first six months of dialysis initiation in an incident cohort of chronic hemodialysis patients. Quality of life, measured using the SF 36-item Health Survey, was compared between patients who attended a pre-dialysis clinic and those who did not, adjusted for clinical and biochemical covariates.
MethodsPatient population
Kingston General Hospital is a tertiary care university teaching centre in Southeastern Ontario, Canada, providing pre-dialysis, hemodialysis, peritoneal dialysis and transplantation programs. The hospital is the only centre offering nephrology consultation in this region, serving a population of about 450,000 people. Referral patterns are therefore very centralized. Patients receive hemodialysis at either Kingston General Hospital or one of several affiliated satellite units that offer both self-care and full nursing care services.
Patients with renal failure are referred by primary care physicians to a nephrologist for initial assessment. The care of patients diagnosed with chronic and progressive renal failure who do not require imminent dialysis initiation is subsequently transferred by the nephrologist to the pre-dialysis clinic. Direct referral to the pre-dialysis clinic by a primary care physician is not permitted. The pre-dialysis clinic is scheduled weekly and staffed by nurse clinicians, dieticians, social workers, pharmacists, and nephrologists. The nephrologists at the hospital operate a group practice and staff the pre-dialysis clinic according to a rotating schedule.
The medical records of all patients beginning chronic hemodialysis at Kingston General Hospital or affiliated satellite dialysis units were reviewed by the primary investigator for study eligibility. Study inclusion criteria were first hemodialysis initiation from January 1 1998 to January 1 2000, history of chronic irreversible renal failure, and completion of the QOL survey within six months of dialysis initiation. Exclusion criteria included receipt of dialysis for less than four weeks and previous kidney transplantation. Reasons for non-completion of the SF-36 questionnaire within six months of dialysis initiation included death (16 patients from the pre-dialysis group and nine patients from the non-pre-dialysis group) and transfer to peritoneal dialysis (two patients from the non-pre-dialysis group). Five patients were excluded due to a significant number of missing data points. An incident cohort of 120 adult hemodialysis patients satisfying the above inclusion and exclusion criteria was identified, of whom 74 patients had attended the pre-dialysis clinic on at least one occasion, while 46 patients began dialysis without previous pre-dialysis clinic attendance.
Independent variables
Possible predictors of outcome included age (at dialysis initiation), sex, diabetes (yes/no), pre-dialysis clinic attendance (yes/no), length of pre-dialysis clinic attendance, date of QOL assessment, history of ischemic heart disease, congestive heart failure, peripheral vascular disease, stroke, malignancy, and chronic lung disease, Kt/V (measured within three months of QOL assessment), residual creatinine clearance (at dialysis initiation), albumin and hemoglobin levels (measured within one month of QOL assessment). Positive diabetic status included a history of either Type I or II diabetes. Patients were classified as having attended the pre-dialysis clinic if they presented on at least one occasion. Length of pre-dialysis clinic attendance was measured from the date of first attendance to date of dialysis initiation. The date at which the QOL assessment was performed was included as an independent variable (calculated from the date of dialysis initiation to QOL assessment) in order to account for any possible confounding effect due to the different times at which the Health Survey was administered. Kt/V was estimated based on a single-pool model using the urea reduction ratio method. Residual creatinine clearance at the time of dialysis initiation was estimated using the Gault-Cockroft method and the serum creatinine level at the start of dialysis initiation. History of co-morbid disease was extracted from medical records using local chart review and the Canadian Organ Replacement Registry (a national dialysis database). Angina was defined as any previous or present history of ischemic symptoms (possibly but not necessarily requiring anti-anginal therapy) or any past history of coronary artery angioplasty or bypass surgery. Congestive heart failure was defined as any previous or present history of dyspnea, orthopnea or paroxysmal nocturnal dyspnea due to cardiac etiologies and not solely attributable to renal failure and volume expansion alone. Echocardiographic documentation of systolic dysfunction was not required. Peripheral vascular disease was defined as either a typical history of exertional leg discomfort or previous history of vascular angioplasty or bypass surgery. Stroke was defined clinically and did not require radiographic confirmation. Patients were primarily of Caucasian race, reflecting the demographics of the southeastern Ontario region. Race was therefore not included as an independent variable.
Dependent variables
QOL was assessed using the SF 36-Item Health Survey. [9,10] This health survey is routinely administered every three months to all dialysis patients at Kingston General Hospital as standard care in order to monitor patient well-being. The survey is a well-validated self-report questionnaire that assesses QOL in eight domains: physical function (phy), physical role limitation (rlp), emotional role limitation (rle), social function (soc), body pain (pai), emotional well being (ewb), energy (eng), and general health (gh). The maximum score in each domain is 100 – higher scores being desirable and indicative of better well-being or less pain. Changes in score of 5 units have been shown to be clinically relevant. [9] Additionally, the final multivariate model was used to explore the association between selected independent predictors of outcome and two composite scores that summarize physical and emotional QOL. Composite physical (praw) and emotional (eraw) scores were calculated based on an aggregation of the eight separate domain scores using standard published equations (where each of the domain scores is standardized using age-stratified Canadian national means and standard deviations). [11,12]
Statistical analysis
The distribution of independent and dependent variables was examined using univariate analyses. Comparison of continuous and categorical independent variables was performed between patients who attended the pre-dialysis clinic and those who did not using t-test, Mann-Whitney, and chi-square analyses as appropriate (Tables 1 and 2; see Additional file Table 1 and Additional file Table 2). QOL scores were compared between patients who attended the pre-dialysis clinic and those who did not using t-test and Mann-Whitney analyses as appropriate (Table 3; see Additional file Table 3). The association between independent variables and QOL scores in each of the eight health domains was examined using bivariate linear regression analysis (Table 4; see Additional file Table 4). Based on the results of bivariate analyses, clinically relevant and statistically significant independent variables were selected for multivariate linear regression analysis (Table 5; see Additional file Table 5). The final model was examined for adherence to linear model assumptions. In the situation where linear model assumptions were compromised due to the non-parametric distribution of outcome variables, non-parametric bivariate and multivariate analyses were also performed using Spearman correlation and logistic regression analyses respectively. As such, non-parametric analyses were used to examine physical and emotional role limitation scores due to the non-parametric distribution of these two dependent variables. The non-parametric distribution of these two domains is attributed to the relatively small number of questions devoted to these two domains in the SF 36-item Health Survey and is therefore not unexpected. Recognizing that earlier research using the health survey has traditionally reported results using parametric analyses despite the non-parametric distribution of the data, the present paper will present both parametric and non-parametric results in order to conform to previous methodologies and permit comparisons.
Analyses were preformed using the SAS statistical analysis package for personal computers (version 6.12, Cary, NC). The study protocol was approved by the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board.
Results
The distribution of categorical independent variables within the entire cohort and according to pre-dialysis clinic attendance is presented in Table 1 (see Additional file Table 1). Overall, 55.8% of patients were male, while diabetes was identified in 43.6% of patients. Almost 40% of patients had a history of ischemic heart disease. The distribution of categorical independent variables was compared between the group of patients who attended the pre-dialysis clinic and those who did not. No statistically significant between-group differences were identified.
Table 2 (see Additional file Table 2) shows the distribution of continuous independent variables within the entire cohort and according to pre-dialysis clinic attendance. Mean age for the entire cohort was 62.7 years. Mean residual creatinine clearance clearance at dialysis initiation was 0.18 ml/s. A comparison of continuous independent variables according to pre-dialysis clinic attendance demonstrated no statistically significant differences between the two groups, with the exception of residual creatinine clearance. Residual creatinine clearance at dialysis initiation was significantly higher among the group of patients attending the pre-dialysis clinic (p = 0.05).
Physical function, physical role limitation, emotional role limitation, social function, and general health scores were statistically higher among patients who attended the pre-dialysis clinic compared to patients who did not (Table 3; see Additional file Table 3).
Results of a series of bivariate linear regression analyses examining the association between each independent variable and each of the eight QOL domains are presented in Table 4 (see Additional file Table 4). A positive parameter estimate denotes a positive correlation between independent variable and QOL score, while a negative parameter estimate indicates an inverse relationship. Younger age, male gender, pre-dialysis clinic attendance, longer attendance at the pre-dialysis clinic, higher residual creatinine clearance at dialysis initiation, absence of ischemic heart disease, stroke, and peripheral vascular disease, and higher albumin level were associated with a significantly higher physical function score. Higher scores in the physical role limitation domain were associated with younger age, pre-dialysis clinic attendance, and higher albumin level. Because physical role limitation scores did not follow a normal distribution, the bivariate analysis was re-examined using a nonparametric Spearman correlation analysis. Using a nonparametric analysis, pre-dialysis clinic attendance still predicted higher QOL score. Younger age (p = 0.02), pre-dialysis clinic attendance (p = 0.03), absence of ischemic heart disease (p = 0.04) and stroke (p = 0.03), and higher albumin level (p = 0.03) were associated with higher physical role limitation scores. Linear regression analysis suggested that higher emotional role limitation score was predicted by longer pre-dialysis clinic attendance. Due to the non-parametric distribution of the emotional role limitation score, a non-parametric analysis was also preformed, which demonstrated that pre-dialysis clinic attendance and longer pre-dialysis clinic attendance were the only predictors of better emotional role limitation score (p = 0.04 and p = 0.01 respectively). Significant predictors of better social function were pre-dialysis clinic attendance, absence of lung disease, and higher albumin level. Male gender, absence of ischemic heart disease, peripheral vascular disease and malignancy, and higher albumin level were significant predictors of higher pain scores (where higher score is indicative of less pain). Better emotional well-being was associated with male gender alone. Younger age, higher residual creatinine clearance at dialysis initiation, absence of peripheral vascular disease and lung disease, and higher albumin level predicted better energy level. Male gender, pre-dialysis clinic attendance, absence of ischemic heart disease, peripheral vascular disease and lung disease, and higher albumin level were significant predictors of higher general health scores.
A total of nine independent variables were selected for inclusion in the final multivariate analysis: pre-dialysis clinic attendance, residual creatinine clearance, Kt/V, age, albumin, sex, ischemic heart disease, peripheral vascular disease, and diabetes (Table 5; see Additional file Table 5). Age, sex, pre-dialysis clinic attendance, ischemic heart disease, peripheral vascular disease, and albumin were included as covariates because of their significant associations with QOL scores in bivariate analyses. Despite the absence of statistically significant associations with QOL scores, diabetes and Kt/V were included as covariates in the final multivariate model because of their perceived clinical relevance. Since residual renal function at dialysis initiation was significantly higher among patients attending the pre-dialysis clinic compared to those who did not, residual creatinine clearance was also included in the final model in order to permit statistical adjustment. The final multivariate model suggested that pre-dialysis clinic attendance was an independent predictor of four of eight health domains: higher physical function, emotional role limitation, social function, and general health scores. Physical function score was predicted by pre-dialysis clinic attendance, younger age, and absence of ischemic heart disease and peripheral vascular disease, while emotional role limitation and social function scores were predicted by pre-dialysis clinic attendance alone. Male gender and absence of peripheral vascular disease were independent predictors of higher pain score (reflecting less pain). Higher emotional well-being score was predicted by male gender. Pre-dialysis clinic attendance, male gender, and absence of peripheral vascular disease were independent predictors of higher general health score. Because of the non-parametric distribution of the physical and emotional role limitation scores, repeat analyses were performed for these two QOL domains using multivariate logistic regression analyses. The conclusions of logistic and linear regression analyses were identical.
Multivariate analysis using the final model suggested that pre-dialysis clinic attendance, younger age, and absence of ischemic heart disease and peripheral vascular disease were independent predictors of the composite physical health score (Table 5; see Additional file Table 5). Pre-dialysis clinic attendance was not an independent predictor of the mental health composite score.
Adjusted r-squared values (Table 5; Additional file Table 5) indicate the extent to which the variability of each health survey domain score can be explained by the final multivariate model. The final model accounts for 42% and 28% of the physical function score and composite physical score variability respectively, suggesting that the model is most effective in predicting physical function scores.
Discussion
Although previous research has suggested that pre-dialysis clinic attendance is associated with better objective outcomes compared to standard care, research examining the benefits of pre-dialysis care with respect to patient-oriented outcomes such as QOL is limited. The results of the present study suggest that attendance at a predialysis clinic is an independent predictor of improved quality of life measured within the first six months of dialysis initiation in an incident cohort of chronic hemodialysis patients. The benefits of pre-dialysis clinic attendance are therefore not limited to objective measures of patient health such as decreased hospitalization and better biochemical profiles at the time of dialysis initiation. Rather, these results suggest that the benefits of pre-dialysis clinic attendance can be extended to include more subjective measures of patient health.
The evaluation of medical therapies using patient-oriented outcomes has acquired a larger research profile in recent years. Nonetheless, to our knowledge, only one study has formally evaluated the benefits of pre-dialysis clinic attendance using patient-administered QOL questionnaires. [8] The present study confirms the conclusions of Sesso et al, who suggested that pre-dialysis clinic attendance improved QOL as measured by the Kidney Disease Questionnaire. However, only 28% of patients were over age 60 in the Brazilian study. Recognizing that the benefits of pre-dialysis clinic attendance were particularly pronounced among elderly patients, the value of pre-dialysis clinic attendance may have been underestimated by this earlier study. The mean age of 62.7 years in the present cohort is a closer representation of North American dialysis populations. The study is also benefited by a publicly funded and universally accessible Canadian healthcare system in which the importance of confounding variables such as socioeconomic status has been minimized. Furthermore, whereas the results of the Brazilian study were adjusted for age and time of diagnosis, differences in residual renal function and other covariates were not included. The present study has controlled for several covariates including age, sex, ischemic heart disease, peripheral vascular disease, diabetes, albumin, residual renal function, and Kt/V.
Interestingly, pre-dialysis clinic attendance was associated with improved QOL scores in a wide range of domains that included both physical and emotional outcomes. Attendance was an independent predictor of better QOL score in four of eight domains: higher physical function, emotional role limitation, social function, and general health. Indeed, pre-dialysis clinic attendance was the only independent predictor of QOL score in the case of emotional role limitation and social function. Importantly, the difference in QOL scores between patients who did and patients who did not attend the pre-dialysis clinic is not only statistically significant but clinically significant as well. Previous research has suggested that a difference in SF-36 scores of five or more points is considered clinically relevant. [9,11]
The means by which a pre-dialysis clinic leads to better physical and emotional QOL is likely multi-factorial, resulting from opportunities for patient education, dietary counseling, modality selection, dialysis access creation, and management of co-morbid illnesses. The importance of early pre-dialysis correction of anemia using erythropoietin has also been emphasized in recent years. Several studies have suggested that earlier initiation of erythropoietin may improve patient morbidity and mortality by retarding or preventing the development of cardiomyopathy secondary to anemia. [13]
The association between several covariates and QOL score seems intuitive in many cases. For example, in addition to pre-dialysis clinic attendance, younger age and absence of ischemic heart disease and peripheral vascular disease were independent predictors of better physical function score. Absence of peripheral vascular disease was also an independent predictor of better pain and general health scores. Although bivariate analysis suggested that higher serum albumin level predicted better QOL in many domains (including physical function, physical role limitation, social function, pain, energy, and general health), a statistically independent relationship between serum albumin and QOL was not demonstrated by multivariate analysis.
Both pre-dialysis clinic attendance and increasing length of time attending the pre-dialysis clinic were predictors of better physical function and emotional role limitation scores in univariate analysis, but not multivariate analysis, thus suggesting statistical redundancy between these two covariates. The absence of an independent association between length of pre-dialysis clinic attendance (and presumably severity of chronic renal failure) and QOL outcome parallels the conclusions of Harris and colleagues, who found that, in a cohort of 360 non-dialysis patients with chronic renal failure, functional status was correlated with socioeconomic status and comorbid illness, but not severity of renal function. [14]
Interestingly, multivariate analysis suggested that male gender was an independent predictor of better pain, emotional well-being, and general health scores. Although a significant association between male gender and QOL scores may seem doubtful, this finding has in fact been demonstrated previously in non-dialysis populations in which males consistently score slightly higher than females in all QOL domains. [12]
QOL scores in the present study are comparable to earlier reports and underscore the fact that QOL is severely compromised among dialysis populations. [15,16] In a prospective Dutch study involving 13 dialysis centres, QOL measured three months after dialysis initiation using the SF-36 questionnaire was significantly impaired compared to a general non-dialysis population, particularly with respect to physical role limitation and general health status. [15] Mean physical role limitation and general health scores for hemodialysis patients corresponded to the lowest 8% and 12% of the reference group respectively. Independent predictors of poor quality of life included a higher number of co-morbid illnesses, lower hemoglobin level, and lower residual renal function. Pre-dialysis attendance was not included as a covariate.
Compared to more objective measures of patient well-being, the importance of QOL, not only as an outcome, but also as a predictor of patient morbidity and mortality, is probably under-estimated. For example, DeOreo has demonstrated that dialysis patients with a physical component summary score below the cohort median were twice as likely to die and 1.5 times more likely to be hospitalized compared to patients with scores above the median. These findings suggest that patient-reported QOL is as much a significant predictor of mortality as more well-established markers of patient care such as normalized protein catabolic rate and Kt/V. [17] Patient morbidity and mortality is significant even prior to dialysis initiation. [14,18] Therefore, interventions that can potentially improve QOL should be pursued early and aggressively during the pre-dialysis phase of chronic renal insufficiency. The present study suggests that pre-dialysis care can improve QOL measured six months after dialysis initiation, while the conclusions of DeOreo argue that such improvement could lead to lower patient mortality.
The inability of the present study to identify any predictors of the mental health composite score may be due to small sample size causing a Type II statistical error. However, other researchers have demonstrated that, while hemodialysis patients report low physical scores, mental and emotional scores are less affected, suggesting that the absence of any statistically significant predictors of poor mental health composite score in the present study may be valid. [15,16]
Surprisingly, diabetes did not predict QOL scores, even in bivariate analysis. This also could be due to effect of small sample size. Alternatively, failure to identify an association between diabetes and QOL scores may be due to short follow-up time in which the more long-term complications of diabetes could not be realized. Furthermore, the results of studies that have reported an association between diabetes and functional status are possibly limited by the use of cross-sectional study designs and prevalent cohort populations. [19] In a prospective cohort of dialysis patients over age 60, Kutner and colleagues showed that diabetic nephropathy was not an independent predictor of survival when controlled for functional status in multivariate analysis. [16]
Kt/V was not a predictor of QOL outcomes in this study or previous investigations. [15] Some investigators have suggested that the absence of an association between Kt/V and QOL may be due to the effect of residual renal function and the possibility that clearance achieved by native kidneys is superior to that obtained by dialysis. [15] However, residual creatinine clearance was not a predictor of QOL outcomes in the present study. The absence of a statistically significant association between Kt/V and QOL scores could also be attributed to short follow-up time in which QOL assessments were performed soon after dialysis initiation. Alternatively, since Kt/V values were confined to a relatively narrow range in which the majority of patients achieved adequate dialysis dosing, a Type II statistical error simply due to lack of variability of the independent variable may account for the absence of any statistically significant association.
The present study is limited by the retrospective design in which the possibility of selection bias cannot be definitively excluded. Indeed, previous research has demonstrated that referral of patients by primary care physicians to nephrology programs is influenced, either consciously or unconsciously, by patient-related factors including age, gender, diabetes, and other co-morbid illnesses. [20,21] Consequently, referral bias is currently a feature of primary care practice and is likely unavoidable regardless of study design. Not surprisingly, the existence of referral bias in the present study is suggested by the lower residual renal function at the time of dialysis initiation seen in patients who did not attend the pre-dialysis clinic compared to those patients who did attend the clinic. Patient self-selection is also a consideration. For example, it is possible that non-compliant patients are less willing to attend the pre-dialysis clinic, and as such, the poorer QOL scores reported by patients who did not attend the pre-dialysis clinic may be related to issues of self-selection and compliance rather than pre-dialysis clinic attendance itself. However, other than residual renal function, no statistically significant demographic or clinical differences between those patients who did attend the pre-dialysis clinic and those who did not were identified in the present study. Notwithstanding the possibilities of referral and self-selection biases, the decision to examine an incident population restricted to new dialysis patients has avoided the other biases associated with earlier investigations using cross-sectional study designs and prevalent populations. [19]
The retrospective study design also causes difficulty in isolating the independent influences of confounding covariates. For example, pre-dialysis clinic attendance and early dialysis initiation are separate yet potentially confounding variables, both of which are likely related to improved QOL scores. In the absence of randomized prospective studies, the effect of each covariate is difficult to examine independently since patients referred to a pre-dialysis clinic are probably less likely to require emergent dialysis initiation due to more regular follow-up. Consequently, it can be argued that the benefits of pre-dialysis clinic attendance may be the result of either selection biases (related to patient age and co-morbid illness) or confounding variables (such as timing of dialysis initiation and residual renal function) rather than pre-dialysis clinic interventions in and of themselves. However, a comparison of continuous and categorical independent variables between patients who did and did not attend the pre-dialysis clinic failed to demonstrate any statistically significant difference between the two groups apart from residual creatinine clearance at the time of dialysis initiation (which was therefore included as a covariate in multivariate analysis).
The results of the present study support the NIH recommendation suggesting that patients with chronic progressive renal insufficiency should be referred to a pre-dialysis team in order to minimize patient morbidity and mortality. While previous research has demonstrated that pre-dialysis clinic attendance is associated with favourable objective outcomes (including better biochemical profiles and decreased hospitalization at the time of dialysis initiation), the results of the present study suggest that the benefits of a pre-dialysis program can be extended to include more subjective measures of health status. The importance of this finding is underscored by earlier research demonstrating that QOL scores are themselves independent predictors of patient mortality. However, it remains unclear to what extent the benefits of pre-dialysis clinic attendance are sustainable over time. Longitudinal studies evaluating the effects of pre-dialysis care with respect to prospective repeated-measure QOL assessments and objective outcomes are therefore required.
Competing interests
None declared.
Authors' contributions
Authors 1 (CW) and 2 (RP) designed the study and carried out data entry. Authors 3 (ML) and 4 (DH) performed the statistical analyses. Author 4 (DH) drafted the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional file 1
Click here for file
Additional file 2
Click here for file
Additional file 3
Click here for file
Additional file 4
Click here for file
Additional file 5
Click here for file
Acknowledgements
The authors grateful acknowledge the assistance of the Canadian Organ Replacement Registry and medical record staff of Kingston General Hospital. We also appreciate the assistance of Ms. Wilma Hopman, Mckenzie Health services Research Group, Queen's University, Kingston, Ontario, Canada.
Bilateral emphysematous pyelonephritis is a life threatening condition usually occurring in diabetics. Management of this condition has traditionally been aggressive and surgery is considered mandatory. However, this is itself a hazardous intervention in a septic, unstable patient with circulatory or liver failure. When bilateral disease is present, the need for long-term dialysis is obviously unavoidable.
Case presentation
We herein report one of the few cases of bilateral emphysematous pyelonephritis successfully managed by non-surgical treatment.
Background
Emphysematous pyelonephritis (EPN) is a rare, life-threatening condition, usually occurring in diabetic patients [1]. Patients with emphysematous pyelonephritis are typically very ill with circulatory/liver failure caused by sepsis. In most cases, a normal native kidney is involved unilaterally, but in 10% of cases, the condition is bilateral [2]. Management of this condition has traditionally been aggressive and surgery has been considered mandatory. Many of the earlier series have stressed the very high mortality rate (75%) and the need for urgent nephrectomy [3-6]. However, this is itself a hazardous intervention in a septic patient with unstable circulatory or liver failure. In the case of bilateral renal disease, the patient requires long-term dialysis. With the advent of CT scanning, more powerful antibiotics, and better access to life support, an alternative medical approach to radical surgery has emerged [7].
Here we report one of the few cases of bilateral empysematous pyelonephritis successfully managed by non-surgical treatment.
Case report
A 41-year-old non-insulin dependent diabetic female with transverse myelitis was referred after a 7-day history of fever, generalized malaise, vomiting, increasing breathlessness and oliguria. She was a type 2 diabetic (onset 14 year before), requiring insulin for glucose control. She had been treated with high dose of steroids for transverse myelitis.
Upon admission, the patient appeared confused and obtunded, was febrile (39.0 C), tachycardic (110 beats/min) and hypotensive (90/60 mm Hg). She was conscious but not completely oriented in time and space. Cardiac and respiratory examinations were unremarkable.
Relevant laboratory data were as follows: glucose 31.5 mmol/L, urea of 89 mmol/L, creatinine of 168 μmol/L, sodium of 133 mmol/L, and potassium of 5.2 mmol/L. Her hemoglobin was 7.7 g/dL, hematocrit 24 %, total peripheral white cell count of 17700 with a shift to the left, and a platelet count of 20 000 (Table 1). Arterial blood gases showed high anion gap metabolic acidosis. Initial ultrasound showed bilateral enlarged edematous kidneys. An abdominal computed tomography (CT) scan showed diffusely enlarged kidneys, with perinephric edema, extensive gas in the renal tissues and perinephric areas bilaterally (Figure 1). She was treated with intravenous fluids, ceftriaxone and amikacin.
An abdominal computed tomography scan showed diffusely enlarged kidneys, with perinephric edema, extensive gas in the renal tissues and perinephric areas bilaterally.
Laboratory Findings. Results of hematologic and blood chemical values.
Variable
On Admission
Second Day
Fifteenth Day
Hematocrit (%)
28
25
38
White-cell count (per mm3)
17,200
25,000
5800
Differential count (%)
Neutrophils
85
90
65
Lymphocytes
15
10
35
Platelet count (per mm3)
20, 000
15,000
110,000
Glucose (mmol/L)
31.5
22.2
7.5
Blood urea nitrogen (mmol/L)
32
39
15
Creatinine (μmmol/L)
168
247
124
Sodium (mmol/L)
133
130
148
Potassium (mmol/L)
5.2
5.7
4.8
Chloride (mmol/L)
102
92
107
Carbon dioxide (mmol/L)
13.2
9.2
23.5
Because of persistent high fever, hypotension, an increase in the total peripheral white cell count and worsening renal function (Table 1) the patient was transferred to the intensive care unit on day two and was treated with intravenous fluids, a tritrated insulin infusion, ceftriaxone, amikacin and inotropic support with dopamine. On review by the urologists, it was thought that conservative management should be attempted given the absence of obstruction of the urinary tract. Blood and urine cultures grew Escherichia Coli senstive to amikacin and ceftiraxone as well as ciprofloxacin.
The patient' s clinical condition improved remarkably over the fourth day of treatment, thus obviating the need for surgery.
A Follow-up CT scan obtained 7 days after the initial study showed global improvement with marked reduction of the gas within the kidneys and a decrease in perinephric edema.
Two weeks after admission to the hospital, during which time she made a steady clinical recovery, her antibiotic was switched to oral ciprofloxacin and fluconazole and was sent home 15 days after admission with a serum creatinine concentration of 124 μmol/L (Table 1). A CT scan on discharge showed complete resolution of the renal emphysema.
Upon further review she is clinically well and free of infection.
Conclusions
First described in 1898, emphysematous pyelonephritis (EPN) is an acute necrotizing parenchymal and perirenal infection caused by gas forming uropathogens [8-10]. Four factors appear to be involved in the pathogenesis of EPN: gas-forming bacteria, high tissue glucose, impaired tissue perfusion and a defective immune response [11]. Diabetics account for 70–90% of all cases [9,12].
The organisms most commonly associated with EPN are E. Coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Aerobacter aerogenes, Citrobacter and rarely yeast. Left untreated, EPN is uniformly fatal [13].
Estimates of mortality using current therapy range from 10% to 40% with patients treated medically having a higher mortality than those treated surgically, 70% versus 30%, respectively [8,9,12,14]. Thus, traditionally, it is thought that antibiotic therapy alone is usually ineffective, and prompt nephrectomy is necessary [12].
In a previous report of 48 cases of EPN patients were classified in four classes according to CT findings, from class 1(the mildest) to class 4 (the most severe form) [15]. Ninety-six per cent (96%) had diabetes mellitus with 22% also having urinary obstruction. The mortality rate in those receiving antibiotics alone was 40% (2 of 5). The success rate of those treated with percutaneous catheter drainage (PCD) plus antibiotics (27 of 41) was 66% and those who had nephrectomy was 90% (9 of the 10 patients). In EPN class 1 and 2, all the patients who were treated with PCD plus antibiotics survived. In extensive EPN (classes 3 and 4), 85% (17 of 20) were successfully treated with PCD and antibiotics. Eight of the 14 who had an unsuccessful treatment using a PCD underwent nephrectomy, seven of whom survived.
Hui reported a case of EPN treated with nephrectomy and stated that based on available data, surgical intervention appears to be the preferred treatment [16]. Chen et al described 10-year experience with 25 EPN patients [17]. Eighty per cent (80%) required antibiotics plus PCD only; 12% underwent nephrectomy and 8% died. Shokeir reviewed their 15-year experience of 20 patients with EPN in Egypt [18]. He emphasized that immediate nephrectomy, as soon as the patient is medically stable, should not be delayed.
Goldsmith [19], Kondo [20], Labussiere [21,22], Punnose [22], Jain [23], and Best [24] all have described individual cases of EPN treated successfully with antibiotics alone.
Angulo [25], Grozel [26], Shimizu [27] and Tahir [19] et.al. Each reported cases of bilateral EPN that were successfully treated with antibiotics alone, thus obviating the need for renal replacement therapy which would have been needed if they were treated with bilateral nephrectomies.
We believe that nephrectomy is not the preferred treatment anymore for all cases of emphysematous pyelonephritis. EPN shall be classified into grades of severity and treatment planned accordingly. Although difficult to perform because of the rarity of EPN, randomized controlled studies for management of EPN are greatly needed.
List of abbreviations
EPN:Emphysematous pyelonephritis
Competing interests
None declared.
Authors contributions
Author 1 GF, participated in medical treatment throughout the entire hospitalization and wrote and drafted manuscript. Author 2HN, participated in medical treament in the ICU and drafted the manuscript also. Authors 3 and 4, JC and FM are third and fourth year residents of internal medicine and participated in medical treatment and diagnosis. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent was obtained from the patient's relatives for publication of the study.
In the United States, transjugular renal biopsies using the Quickcore™ side cut needle system have previously been described primarily for transjugular renal biopsy in patients with concurrent liver and kidney disease.
Methods
We describe transjugular renal biopsy with the Quickcore™ system in 9 patients with nephrotic syndrome and contraindications to percutaneous renal biopsy, who underwent biopsy between 23 October 1996 and 12 April 2001. The most common contraindication was oral anticoagulation with coumadin (40%). Other contraindications included horseshoe kidney, severe renal failure, and spontaneous coagulopathy. A 62 cm straight catheter and 60 cm side-cut Quickcore™ biopsy needle were used to obtain cortical tissue. Packing of the biopsy tract with Gelfoam™ was used for venographically identified capsular perforation.
Results
Ten procedures were performed on 9 patients with one requiring re-biopsy (5% of all renal biopsies performed at our institution). There were 9 transjugular renal biopsy and one combined liver-kidney biopsy. A mean of 4 ± 2 passes were made, with a mean of 3 ± 1 cores obtained per procedure. Histologic diagnosis was made in 90% of biopsies and in 100% of patients. Two patients developed transient hydronephrosis associated with gross hematuria; both required transfusion. Capsular perforation occurred in 90%. One patient died of bacterial sepsis, unrelated to the biopsy, several days after the procedure.
Conclusions
Transjugular renal biopsy appears to be efficacious in high-risk patients, for whom the percutaneous approach is contraindicated, including patients on oral anticoagulation. The transfusion rate in the present study was similar to other American reports using this technique.
The number of patients who are not suitable candidates for percutaneous renal biopsy (PRB) may increase in the future because of increasing prevalence of bleeding diatheses, both spontaneous and due to wider use of anticoagulation for thrombotic disorders and dysrythmias. [1] The most common indication for renal biopsy in the United States is the nephrotic syndrome. [2] This condition has been associated with an increased risk of thromboembolism, particularly in patients with membranous nephropathy [3] or systemic lupus erythematosus, [4] and such patients may thus also require anticoagulation. Several alternative techniques have developed over the last decade for those who have contraindications to PRB. Of these, the most widely used are the various methods of endovascular biopsy. The current status of transjugular renal biopsy (TJRB) has been reviewed recently. [1] Most reports and the largest series, in a broad array of settings, have come from Europe. [5-7] In the United States, the only reports of experience with TJRB have been in patients with concurrent liver and kidney disease. [8]
Since the seminal report of Mal, et al [5], an even larger TJRB series has been reported by Cluzel, et al [6]. Four hundred patients undergoing TJRB were compared to 400 patients undergoing PRB, using the modified Colapinto™ aspiration needle system. Diagnostic tissue adequacy was 95.8%, with a major complication rate of 1%. Although excellent results were obtained, this technique appears to require a steep "learning curve," according to the authors. The endovascular automated side-cut core biopsy sets such as the Quick-Core™ (Cook, Bloomington, IN) may allow even higher yields of diagnostic tissue with possibly less operator-dependence. In addition, biopsy tract embolization to reduce bleeding risk post-biopsy is possible using this system. So far no American center has reported results on the use of the Quick-Core™ biopsy set in patients with contraindications to PRB (other than combined liver and kidney disease) referred for TJRB. We report our experience with this technique at our institution from October 1996–April 2001.
Materials and Methods
Data on 9 of 10 patients undergoing TJRB at Walter Reed Army Medical Center (WRAMC) from 23 October 1996 to 30 April 2001, including demographics, indications, technical details, and complications, were recorded and analyzed. The starting date reflects the time at which all TJRB began to be tracked by both Nephrology and Interventional Radiology at WRAMC. The protocol (WU # 1186) was submitted to the WRAMC institutional review board and approved in May 1997. Seven patients with biopsies performed between October 1996 and May 1997 were analyzed retrospectively. Data on 3 of 4 subsequent biopsies were obtained prospectively. (One patient declined consent, and thus no clinical data is available regarding the biopsy). Six different interventional radiologists performed the biopsies over the course of the study.
All biopsies were performed in the interventional radiology suite with biplane or single-plane anteroposterior and lateral fluoroscopic capabilities. Biopsy was performed only if patient blood pressure was <140/90 mm Hg. In most patients, a previous abdominal sonogram was available. Prothrombin time, partial thromboplastin time, INR (where indicated), platelet count, and serum creatinine level were obtained before each procedure. No patient had significant thrombocytopenia. Management of patients on anticoagulation was per the recommendations of Kearon, et al [9]. No patient was given fresh frozen plasma or cryoprecipitate, before or after the procedure. No bleeding times were performed. Patients were given pre-biopsy DDAVP or estrogen if the serum creatinine was ≥ 3 mg/dl or the creatinine clearance by the Cockcroft-Gault formula was ≤ 30 cc/min, as previously described (N = 5) [10]. No patients were dialysis-dependent, either acutely or chronically.
All biopsies were performed with the transjugular Quick-Core™ needle biopsy system (Cook, Bloomington, IN), which consists of a 7 F, 50.5-cm transjugular sheath with a 14 G inner-stiffening cannula; a 5 F, 80-cm multipurpose curved catheter; and a 60-cm biopsy needle with a 2-cm throw length. Biopsy was performed preferentially on the right kidney because the right renal vein is shorter than the left, and provides a better angle for access to the kidney. Biopsy specimens were obtained with the 60-cm biopsy needle oriented in a posterolateral direction to avoid inadvertent puncture of the colon.
The right neck was prepared and draped in sterile fashion. A guide wire was advanced through the distal lumen of 9F catheter into the inferior vena cava and the 9F catheter removed. This was replaced with a 14F short vascular sheath.
For the one patient who underwent a combined liver and kidney biopsy, a 5F angled multipurpose catheter was advanced through this sheath, over a wire, to the inferior vena cava and used to selectively cannulate the right hepatic vein. The catheter was passed distally into the hepatic vein. Hepatic wedge venography with carbon dioxide (because of the patient's renal insufficiency) was performed. An Amplatz™ (Cook, Bloomington, IN) wire was then placed and the catheter removed. Over the Amplatz™ wire the Cook Quick-Core™ biopsy sheath was advanced without difficulty into the proximal hepatic vein. The wire was removed and a 20-gauge Quick-Core™ biopsy needle used to obtain core biopsy specimens. The sheath was then removed over a wire.
For patients undergoing renal biopsy (either combined or separate), after cannulation of the right internal jugular vein, the multipurpose catheter was advanced and used to engage the right renal vein. Over a hydrophilic wire, the catheter was advanced until it wedged within the right lower pole of the kidney. This was verified by injection of 3 cc of contrast material. The guiding sheath was then angled posteriorly (to avoid inadvertent colonic puncture) and biopsy specimens obtained with the 18-gauge Quick Core™ needle as described for liver biopsy. Extravasation of contrast post venography through the sheath was taken as evidence of capsular perforation. If extravasation of contrast was seen, the tract was selected using a Terumo wire and the small catheter included in the liver access set. A Gelfoam™ (Pharmacia, Upjohn Inc., Peapack, New Jersey) pledget was then placed at the far aspect of this tract near the capsule until no further extravasation of contrast was demonstrated. The collecting system was assessed for free drainage of contrast or development of caliectasis. No further filling of the tract was demonstrated after the embolization. All biopsy devices were then removed. The catheter, its stiffeners, and sheath were all removed and hemostasis obtained with manual compression. For patients previously on oral coumadin, depending on the indication (as per Kearon, et al [9]), intravenous heparin was restarted in as soon as two hours (no bolus, maintenance infusion only). Oral coumadin was generally started the same night.
For the patient with the horseshoe kidney, access to the right internal jugular vein and inferior vena cava was as above. A wedged venogram with a very small amount of contrast material demonstrated some cortical staining. The Rosen guide wire was reintroduced and the catheter was exchanged for the 8-French vascular sheath with a stiffener. The guide wire was removed, and a small amount of contrast material again demonstrated intravascular placement of the sheath.
There was transgression of the collecting system with the first biopsy specimen; however, there was no filling of the collecting system following the introduction of a single pledget of GelfoamTM. After completion of the procedure, there was no evidence of intraluminal filling defect within the right renal collecting system (i.e., no significant thrombus within the collecting system). The extracapsular contrast collection was approximately 2 cm × 2 cm and was not observed to expand over the final 10 minutes of the procedure.
The length of core samples obtained was generally 10–14 mm, maximally 18–20 mm. Tissue for hematoxylin and eosin, periodic acid-Schiff and other special stains, as well as electron microscopy was fixed in 2% glutaraldehyde solution. Tissue for immunofluorescence was preserved in Michel's Fixative (Poly Scientific, Bay Shore, NY). Paraffin sections were used for light microscopy. Specimens were processed for immunofluorescence and electron microscopy in standard fashion. All specimens were examined by a single nephropathologist at the Armed Forces Institute of Pathology.
After biopsy, outpatients were admitted to a 23-hour recovery unit, and remained in bed for at least 12 hours, with frequent observation of vital signs. Hematocrit was assessed 4–6 hours post biopsy. Serial urines were collected to assess for hematuria. All but two of the patients in whom TJRB was performed as an outpatient procedure were discharged the next day. The patient found to have systemic amyloidosis was already an inpatient for clinical reasons.
Results
Patient characteristics, contraindications to conventional PRB, and outcomes are shown in Table 1 (see additional file 1). Ten biopsies were performed in 9 patients (1 patient had a successful repeat TJRB one month after the first attempt yielded inadequate tissue). No patient required open surgical biopsy. Mean age was 58 ± 21 years. Six were Caucasian; 2 African-American, and 1 Asian-American. There were 4 males and 5 females.
Four of 9 patients had substantial renal failure, i.e., serum creatinine ≥ 3 mg%, which constituted a relative contraindication. No patient was referred for morbid obesity. The most common contraindication to PRB was bleeding diathesis (5/9), either due to coumadin anticoagulation (2 with history of deep vein thrombosis, 1 with history of pulmonary embolus, and 1 with anti-cardiolipin antibody syndrome with associated deep vein thrombosis) or spontaneous coagulopathy. No patient had a platelet count < 100,000 cells/mm3. Mean INR (international normalized ratio) at the time of biopsy in these 5 patients was 1.4 ± 0.3. Two patients proved to have membranous glomerulonephritis, and were on coumadin prior to biopsy. Proteinuria had preceded thromboembolic events in these patients.
Tissue was adequate for diagnosis in 9/10 biopsies, and in all patients (due to successful re-biopsy). The mean number of passes was 4 ± 2, yielding 3 ± 1 biopsy cores, with a mean glomerular number of 9 ± 8. Capsular perforation occurred in 9/10 biopsies, but gross hematuria occurred in 6/10 biopsies-1 of which occurred in the only patient without capsular perforation. The biopsy that produced inadequate tissue was the only one in which capsular perforation did not occur. The hematocrit declined ≥ 4 % after biopsy in 3/10 biopsies. Transfusion was needed in only 2 patients; these two also developed hydronephrosis due to collecting system bleeding, which resolved without further intervention. One had horseshoe kidney, and the other was on coumadin for anti-cardiolipin antibody syndrome. She resumed intravenous heparin and coumadin the day of the biopsy due to high thrombosis risk. Only one other of the 4 patients on oral anticoagulants had intravenous heparin restarted the same day; the other two began oral coumadin that night. One patient died of bacterial sepsis (presumed due to line infection) several days after biopsy (an 88-year-old man with liver and renal amyloidosis). No patient developed significant renal failure after the procedure (defined as a serum creatinine level elevation ≥ 1.5 mg/dl or requirement for dialysis).
Discussion
Recent series have shown that tissue adequacy with TJRB is excellent (>95%) 5,7 and comparable to PRB [5]. The tissue adequacy of 90% for procedures and 100% for patients in our study is comparable to that in other reports. The mean number of glomeruli obtained by Sam, et al [8] was higher than in our series. This may be due to their routine use of pathology review during biopsy, which was not feasible at our institution.
Biopsy influenced management in all cases, except for Patient 9, who was diagnosed with amyloidosis, and who died from sepsis. Subsequent to biopsy, patients 1 and 2 were treated with chlorambucil and methylprednisolone [11]. Patient 7, who presented with heavy proteinuria and nephritic urinary sediment, proved to have mesangial lupus nephritis. Without renal biopsy, which excluded diffuse proliferative lupus nephritis, she might have been reasonably treated empirically with cyclophosphamide [12]. Patient 8 had minimal change nephropathy. While this 22-year-old patient might have been treated empirically with prednisone, his presentation was also consistent with focal segmental glomerulosclerosis, which would have been treated differently [13].
Although large series of TJRB using the modified Colapinto™ aspiration needle system have been reported [5-7], the largest series on the use of 18-g automated biopsy needle is that of Sam, et al, [8] which reported on 29 TJRB. In the series reported by Cluzel, et al [6], 8 of 400 TJRB were done with Quick Core™ automated systems. They commented that the stiffness of the Quick Core™ system virtually precludes a left jugular approach, but because of its thinness, allows deeper placement in the renal parenchyma. The Colapinto™ device appears to require more training to deploy properly, because of its flexibility and the requirement for manual aspiration of samples. In a randomized, unblinded study of liver biopsies, the time required for training, procedure time, and tissue adequacy were superior for automated biopsies vs. aspiration biopsies, with similar complication rates [14]. No such comparisons exist for TJRB.
During the same time period as this series, we performed 192 native PRB. There were no laparoscopic or open surgical biopsies. Thus, only 5% of biopsies at WRAMC were deemed "contraindicated" by the percutaneous approach, and all were able to be done using TJRB. Therefore, TJRB, at least in our practice setting, is unlikely to become a "high-volume" procedure, but despite this, is able to be effectively and safely done by interventional radiologists with transjugular liver biopsy experience and equipment, i.e., the Quick Core™ automated system. Transjugular liver biopsy is generally a more common procedure, and was performed 12–14 times a year at our institution during the time of the study.
In our series, 5/9 patients had a coagulopathy, either spontaneous or due to coumadin. Two (22%) had an INR > 1.5 at the time of biopsy, in comparison to 39% in the series of Sam, et al [8], whose patients had advanced chronic liver disease. They attempted to correct coagulopathy in all patients, although the details of correction were not specified. In contrast, because of the different etiology of coagulopathy in most of our patients, we performed TJRB through a heparin window as per Kearon, et al [9]. Theoretically, the same could be done for PRB, although bleeding risk would typically persist for six weeks, and the percutaneous tract would be associated with a greater bleeding risk than with TJRB, even given capsular perforation.
The issue of capsular perforation is also relevant to tissue adequacy. In animal models, better specimens with more glomeruli were obtained after unintentional capsular perforation, presumably due to more distal positioning of the biopsy needle [15]. The high rate of capsular perforation in our study was not associated with a high complication rate, given the high risk of the population. The transfusion rate of 20% is lower than the 29% rate of Sam, et al [8]. The use of a side-cut needle with a shorter (1 cm) throw and a blunt-tipped end to reduce the risk of capsular perforation and other organ damage 16 has been successful in animal models, but has not been reported in humans.
Cluzel, et al [6] reported low transfusion rates (about 1%) after TJRB. However, the population studied is not comparable to ours or that of Sam, et al [8]. Possibly the most common indication for TJRB in the Cluzel study was an elevated bleeding time, even with a normal creatinine or absent bleeding history. It was largely on this basis that 76% of the patients in the study were said to have had a "bleeding abnormality." In many centers, particularly in the United States, such patients are likely to be treated with DDAVP or conjugated estrogen before PRB [1]. In our study, only one patient with a "coagulopathy" required transfusion. The other transfusion was required by the patient with a horseshoe kidney, who had no other risk factors for bleeding, and is, to our knowledge, the first such patient to undergo TJRB.
The two cases of bleeding-associated hydronephrosis, one of which was in the patient with horseshoe kidney, were the result of a communication with the urinary tract. In the horseshoe kidney, this could have been due to aberrant location of the collecting system. In fact, contrast extravasation into the collecting system was observed during the procedure. In the other case, caliectasis was noted during the procedure, which was initially attributed to the pressure of injection. This patient had been on coumadin anticoagulation, although the INR at the time of biopsy was 1.2. She was restarted on heparin and coumadin the day of the biopsy because of high risk of thrombosis. The use of Gelfoam™ embolization did not prevent significant bleeding in these two patients, possibly because the tracts could not be exactly approximated, or the Gelfoam™ pledget became displaced. It is noteworthy that both transfusion and Gelfoam™ failure were associated with urinary tract perforation, because Gelfoam™ embolization is less likely to be successful in preventing bleeding in this setting. Gross hematuria, without the need for transfusion, has not been reported specifically as a complication after TJRB. Therefore, our gross hematuria rate of 60% cannot be compared with other studies. However, we speculate that in the process of obtaining a core specimen, adjacent vessels were disrupted and could have led to introduction of blood into the renal tubules, especially in patients with disturbed hemostasis.
The one death in our series was due to sepsis in a patient with systemic amyloidosis, unrelated to the biopsy. The patient's hematocrit declined only 1.4% post-procedure, despite a spontaneous coagulopathy.
The generalizability of our series is limited by its small size. Because none of the patients were obese, no conclusions about the safety of TJRB in obese patients can be drawn. As technology improves, the cost and complexity of TJRB is likely to decrease. In fact, the cost of TJRB at "experienced" institutions is less than twice that of PRB, and the technique is particularly advantageous in combined liver-kidney biopsy [5-8]. Given the "user-friendly" characteristics of the Quick Core™ transjugular biopsy set, interventional radiologists throughout the world with transjugular liver biopsy experience can reasonably apply this technique. The most common contraindications to PRB are likely to be maintenance anticoagulation or bleeding diathesis (due in some cases to acute or chronic renal failure), perhaps followed by congenital abnormalities. The present study shows that the transjugular technique can obtain diagnostic renal tissue in these circumstances. The safety of this procedure in high-risk populations is still uncertain due to the small numbers of patients studied with the equipment used in the present study. Due to the low volume of procedures, it is unusual to find institutions with experience in multiple methods of high-risk renal biopsy (ie, both TJRB and Laparoscopic renal biopsy). Because TJRB is considered only when conventional PRB is contraindicated, comparison with PRB is not possible. Future studies are suggested to determine the best method of embolizing the biopsy tract in TJRB, given the results of the present study.
Competing Interests
None declared.
Author's contributions
Dr. Abbott assumed the role of primary investigator for the research protocol after the departure of Dr. Musio, and was responsible for data collection, analysis, and manuscript preparation.
Dr. Musio originally submitted the research protocol, and as the first primary investigator, was responsible for submission of the initial abstract, which was presented a poster at the 1997 American Society of Nephrology meeting and published in the Journal of the American Society of Nephrology in 1997.
Dr. Chung was responsible for performance of the transjugular renal biopsy in the patient with the horseshoe kidney and was responsible for the diagnostic and therapeutic planning and description of the case, as part of the overall manuscript.
Dr. Lomis was responsible for the performance of the combined transjugular renal and hepatic biopsy, the first performed at Walter Reed Army Medical center, and was responsible for the diagnostic and therapeutic planning and description of the case, as part of the overall manuscript.
Dr. Lane was responsible for two of the transjugular renal biopsies and contributed to the development and description of the technique, as well as provided comparisons with other techniques of transjugular biopsies in the literature. Dr. Lane assisted in all aspects of manuscript preparation and data analysis.
Dr. Yuan was an associate investigator on the original protocol and assisted in all aspects of manuscript preparation and data analysis.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional File 1
Table1
TJRBPaperPaper dec02.doc
Acknowledgements
We would like to acknowledge Dr. Alan Meglin and Dr. Mark Lukens for their participation in this study and their contribution to previous work, without which this study would not have been possible.
The patient characteristics and mortality associated with autosomal dominant polycystic kidney disease (PKD) have not been characterized for a national sample of end stage renal disease (ESRD) patients on the renal transplant waiting list.
Methods
40,493 patients in the United States Renal Data System who were initiated on ESRD therapy between 1 April 1995 and 29 June 1999 and later enrolled on the renal transplant waiting list were analyzed in an historical cohort study of the relationship between hematocrit at the time of presentation to ESRD and survival (using Cox Regression) in patients with PKD as a cause of ESRD.
Results
Hematocrit levels at presentation to ESRD increased significantly over more recent years of the study. Hematocrit rose in parallel in patients with and without PKD, but patients with PKD had consistently higher hemoglobin. PKD was independently associated with higher hematocrit in multiple linear regression analysis (p < 0.0001). In logistic regression, higher hematocrit was independently associated with PKD. In Cox Regression analysis, PKD was associated with statistically significant improved survival both in comparison with diabetic (hazard ratio, 0.64, 95% CI 0.53–0.77, p < 0.001) and non-diabetic (HR 0.68, 95% CI 0.56–0.82, p = 0.001) ESRD patients, adjusted for all other factors.
Conclusions
Hematocrit at presentation to ESRD was significantly higher in patients with PKD compared with patients with other causes of ESRD. The survival advantage of PKD in ESRD persisted even adjusted for differences in hematocrit and in comparison with patients on the renal transplant waiting list.
Polycystic kidney disease (PKD) has recently been associated with decreased mortality compared with non-diabetic end stage renal disease (ESRD) patients. [1] Because Hematocrit levels have been reported to be higher in PKD patients than in other patients with ESRD, it has been suggested that the survival of these patients may be related to their elevated Hematocrit levels. [2] Another recent study adjusted for the level of Hematocrit at initiation of dialysis, and found the survival benefit of PKD persisted. [3] However, because PKD patients are much more likely to be placed on the renal transplant waiting list and receive renal transplantation than other ESRD patients [1,3], it is possible this survival advantage may also be due to their younger age and better general health. It would be useful to know if the differences in Hematocrit and survival persisted in analysis limited to patients enrolled on the renal transplant waiting list. Therefore, we analyzed data from the standard analysis files of the 2000 United States Renal Data System (USRDS) database. Our objectives were to analyze differences in Hematocrit and hemoglobin between patients with PKD and other causes of ESRD, whether these differences have changed over time, and whether this or other factors are associated with their relative survival advantage in ESRD.
Methods
We analyzed a national registry (the 2000 USRDS) in an historical cohort study of the association of Polycystic kidney disease with Hematocrit and patient survival among patients enrolled on the renal transplant waiting list. Information on comorbidity, as well as height and weight to calculate BMI, was obtained from the USRDS file SAF.MEDEVID. This file is derived from the Center for Medicare and Medicaid Studies (formerly HCFA) medical evidence form (2728) starting with a sample of ESRD patients prior to April 1995 and universal afterward, and has been validated for use in research. [4] We first selected patients who initiated ESRD therapy between 1 April 1995 to 29 June 1999 who had data sufficient to calculate hematocrit. From this cohort, we selected patients enrolled on the renal transplant waiting list, excluding any dates of listing prior to 1 April 1995. The date patients on the renal transplant waiting list first received a renal transplant was also extracted, also excluding any dates prior to 1 April 1995. Recipients of organs other than kidneys and of transplants without preceding dialysis were excluded. The variables included in the USRDS standard analysis files (SAF's), as well as data collection methods and validation studies, are listed at the USRDS website , under 'Researcher's Guide to the USRDS Database', Section E, 'Contents of all the SAF's' (Standard Analysis Files), and published in the USRDS. The demographics of the end stage renal disease population have been previously described (2001 USRDS report). Dialysis patients younger than age 65 are eligible for Medicare 90 days after starting dialysis, with a waiver granted to those choosing home therapies. Therefore, hospitalization and mortality data may be incomplete during the first 90 days after dialysis initiation for patients younger than 65, but start immediately after renal transplant.
The file SAF.PATIENTS was used as the primary data set, including cause of renal disease (PDIS) and cause and date of patient death. SAF.RXHIST was used to obtain follow-up dates. The file SAF.TXWAIT contains the date patients in the above cohort were first placed on the transplant waiting list. The file SAF.MEDEVID includes data from the Medical Evidence Form (2728). The file SAF.TXUNOS included information on transplant donor type, pre-transplant dialysis, previous transplant, and multiple organ transplants. Files were merged using unique patient identifiers. Details on anthropometric measurements or nutritional parameters other than serum albumin were unavailable. No information on patient medications was available (for the entire cohort of patients) except for the use of pre-dialysis erythropoietin. The USRDS researcher's agreement specifically prohibits patient contact or chart review.
All analyses were performed using SPSS 9.0 TM (SPSS, Inc., Chicago, IL). Files were merged and converted to SPSS files using DBMS/Copy (Conceptual Software, Houston, TX). Statistical significance was defined as p < 0.05. Univariate analysis was performed with Chi-square testing for categorical variables and Student's t-test for continuous variables. Variables with p < 0.10 in univariate analysis for a relationship with elevated BMI were entered into multivariable analysis as covariates. Continuous variables were examined for outliers, and values ≥ 3 SD from the mean were removed from analysis with the exception of height and weight, as above, as per prior reports.
Stepwise linear regression was used to calculate the independent association of Polycystic kidney disease, adjusted for other factors, with both Hematocrit and hemoglobin levels. Factors included BMI, race, age, gender, year of first dialysis session, cause of end stage renal disease (diabetes, PKD, or other causes), dialysis type (hemodialysis vs. peritoneal dialysis) and additional variables from the medical evidence form, including diabetes and hypertension as comorbidities distinct from causes of ESRD, and ESRD network, as the independent variables.
Stepwise logistic regression was used to model independent associations with Polycystic kidney disease, using the same variables as in linear regression.
Stepwise Cox Regression non-proportional hazards analysis modeled the association between polycystic kidney disease with all-cause mortality, controlling for the same variables as in linear regression above. Survival analysis compared all patients on the transplant waiting list, censoring patients for receipt of renal transplantation.
Survival time was calculated as the time from the date of the first listing for transplant until death, or latest available follow-up date or receipt of renal transplant. We did not remove patients from the category of listing for transplant if they were removed from this category at a later time, nor did we remove patients from the category of renal transplant recipient if they later experienced graft loss, in intent to treat fashion. Hierarchically well-formed models were used for assessment of interaction terms in all models.
Results
Of 348,615 patients who initiated ESRD therapy in the USRDS database from 1 April 1995 to 29 June 1999, 43,707 were subsequently entered on the renal transplant waiting list, of whom 40,493 were entered on the waiting list on or after 1 April 1995. Of these, 89% had sufficient information from the medical evidence form (2728) for hematocrit. The mean date of first ESRD service was 14 January 1997. The total study cohort had 12,127 recipients of cadaveric kidneys and 4200 recipients of living donor kidneys, in addition to wait-listed patients who did not receive transplants during the study period. The mean transplant date was 25 Dec 1997, and the most recent transplant date was 16 November 1999. The most recent follow-up date was 29 June 2001. Thus, the study had 51 months of accrual and 24 months of additional followup.
For patients with causes of ESRD other than PKD, median hematocrit in 1995 was 27.2 ± 5.3%, in 1996 27.4 ± 5.4%, in 1997 28.0 ± 5.4%, in 1998 28.8 ± 5.4%, and in 1999 30.5 ± 5.4%. There was significant increase in hematocrit by year in linear regression analysis (p < 0.01). For patients with ESRD due to PKD, median hematocrit in 1995 was 29.9 ± 5.7%, in 1996 29.6 ± 5.3%, in 1997 30.5 ± 5.4%, in 1998 30.9 ± 5.6%, and in 1999 31.3 ± 5.5%. There was also a significant increase in hematocrit by year for these patients in linear regression analysis (p < 0.01).
Table 1 shows descriptive statistics of the study population, which were comparable to previous USRDS reports. The far right column shows results of logistic regression analysis of factors independently associated with polycystic kidney disease.
Factors assessed in ESRD patients on the renal transplant waiting list who started dialysis on or after 1 April 1995–29 June 1999
Factor
Wait-Listed Patients
Univariate Odds Ratio for Polycystic Kidney Disease
Adjusted Odds Ratio for Polycystic Kidney Disease In Logistic Regression (95% CI)B
N
40,493
PKD
2949
Male
24,143 (59.6)
0.89 (0.83–0.97)
0.89 (0.79–0.99)
African American
11,193 (27.6)
0.27 (0.24–0.31)
0.25 (0.22–0.30)
Year of First Dialysis
1996.5 ± 1.1
1.02 (1.02–1.03)
Mean Age (Years)
45.6 ± 13.9
0.98 (0.97–0.99)
Mean Followup (Years)
1.4 ± 1.0
Renal Transplantation
16,327
1.48 (1.38–1.59)
1.23 (1.10–1.39)
Continuous Variables from Medical Evidence Form, 2728 (at initiation of dialysis):B
Recipient Weight (in kg)
75.7 ± 13.7
1.01 (1.01–1.02)
Body Mass Index (kg/m2)
25.8 ± 4.9
1.02 (1.01–1.03)
Hematocrit (%)
28.2 ± 5.5
1.04 (1.03–1.05)
Hemoglobin (gm/dl)
9.5 ± 1.9
Serum Albumin (gm/dl)
3.4 ± 0.6
2.67 (2.41–2.96)
Serum Creatinine (mg/dl)
10.0 ± 4.4
0.97 (0.95–0.98)
BUN (mg/dl)
94.4 ± 33.5
Categorical Variables from the Medical Evidence Form (2728), history of:
COPD
615 (1.5)
0.68 (0.47–0.97)
0.60 (0.37–0.96)
Congestive Heart Failure
4621 (11.4)
0.18 (0.15–0.23)
0.31 (0.23–0.41)
Ischemic Heart Disease
3179 (7.9)
0.58 (0.49–0.69)
0.69 (0.54–0.88)
Myocardial Infarction
1105 (2.7)
1.67 (1.17–2.38)
Peripheral vascular disease
1879 (4.6)
0.19 (0.13–0.27)
0.34 (0.22–0.54)
Diabetes (Primary)
8068 (22.1)
0.07 (0.05–0.09)
0.59 (0.44–0.79)
Hypertension
26,658 (73)
Cancer
451 (1.1)
0.41 (0.25–0.67)
Alcohol use
373 (0.9)
0.31 (0.16–0.61)
0.42 (0.20–0.88)
Pre dialysis EPO
11,108 (30.4)
1.10 (1.01–1.20)
Hemodialysis (vs. Peritoneal Dialysis)
28,003 (76.7)
0.88 (0.80–0.96)
In column one, data given as the number (% of total) or mean ± one standard deviation of patients with polycystic kidney disease who had the disease or factor. In column two, data are for all other ESRD patients. Odds ratios are the strength of the association of a disease or factor with polycystic kidney disease, with 1.0 being neutral. * = p < 0.01 vs. patients with all other causes of ESRD by Chi Square Test Ap < 0.01 vs. patients with all other causes of ESRD by Student's t-test Bcontrolled for all covariates listed in methods section. Only results with statistical significance (p < 0.01) are shown.
Table 2 shows results of linear regression analysis of factors associated with Hematocrit, while Table 3 shows results of linear regression analysis of factors associated with hemoglobin.
Linear regression analysis of factors associated with hematocrit level
Factor
Standardized Coefficient (Beta)
P Value
Polycystic Kidney Disease
0.062
<0.001
Pre-Dialysis EPO use
0.123
<0.001
Serum Albumin
0.157
<0.001
Male
0.113
<0.001
Age (per year)
0.034
<0.001
Year of First ESRD Service
0.06
<0.001
Smoking
0.015
0.007
Ischemic Heart Disease
0.013
0.027
Hemodialysis (vs. peritoneal dialysis)
-0.076
<0.001
Serum Creatinine (mg/dl)
-0.296
<0.001
Congestive Heart Failure
-0.029
<0.001
Dialysis modality was obtained from the Medical Evidence form (HCFA 2728), and was therefore only available for patients who started on ESRD therapy on or after 1995. Only variables that were significant in univariate analysis are shown.
Figure 1 shows unadjusted survival stratified by patients with ESRD due to Polycystic kidney disease, diabetes, and all other causes (excluding Polycystic kidney disease and diabetes). Polycystic kidney disease was associated with statistically significant reduced mortality (p < 0.001 by Log Rank Test) vs. either diabetics or non-diabetics. The hazard ratio for patients with Polycystic kidney disease was 0.64, 95% CI 0.53–0.77, p < 0.001 in Cox Regression. Figure 3 shows a survival plot of patients with Polycystic kidney disease vs. all other ESRD patients, excluding those with ESRD due to diabetes. Compared to non-diabetic ESRD patients, patients with Polycystic kidney disease had a hazard ratio for mortality of 0.68, 95% CI 0.56–0.82, p = 0.001.
Unadjusted survival, by cause of ESRD, patients on the renal transplant waiting list. U.S. Patients on the renal transplant waiting list, who started dialysis from 1 April 1995-29 June 1999, N = 40,493. 1 = Patients with Polycystic kidney disease, 2 = patients without Polycystic kidney disease or diabetes, 3 = patients with diabetes. All diseases listed are as causes of end stage renal disease.
Discussion
The present study confirms an association between elevated hematocrit, hemoglobin, and Polycystic kidney disease in a cohort of ESRD disease patients on the renal transplant waiting list. The association between increased hematocrit and polycystic kidney disease was initially noted in small observational studies, which noted higher erythropoietin levels and reticulocyte counts in patients with Polycystic kidney disease as well. [5,6] and later confirmed in registry studies of peritoneal dialysis patients. [7] Although other authors have noted an increase in hematocrit with increasing time on dialysis, [8] the present study was only able to measure hematocrit at the start of dialysis, prior to usual initiation of erythropoietin, although a substantial number of wait-listed patients received pre-dialysis erythropoietin. Regardless, the study again confirms that the survival advantage of Polycystic kidney disease was independent of hematocrit or hemoglobin at the start of dialysis, and in fact was independent of all measured factors. The present study is in agreement with previous reports of positive outcomes of peritoneal dialysis in patients with polycystic kidney. [1,9,10]
As we have previously reported, the reasons for the comparatively good survival experienced by patients with polycystic kidney disease are unclear, since these patients have known extrarenal manifestations that may impact longterm survival. While these extrarenal manifestations are emphasized in renal textbooks and training exams, they do not appear to be as significant as the extrarenal manifestations (ie, comorbidity) associated with other diseases causing ESRD, predominantly diabetes and hypertension, as shown in Table 1.
In contrast to our previous study, the present study found that hematocrit was significant in multivariate as well as univariate analysis of factors associated with Polycystic kidney disease. In contrast, the present study did not find significant negative associations between Polycystic kidney disease and either cerebrovascular accident, use of pre-dialysis erythropoietin, or hemodialysis, as was noted in our previous analysis. This is most likely due to changes in population characteristics (the previous study included all patients with end stage renal disease) and the much more complete proportion of patients with evidence from the CMS Form 2728 in the present study (100%). However, it is notable that hemodialysis was still not more commonly used in Polycystic kidney disease, in contrast to general recommendations, [11] although this recommendation has not been universal. [12] Peritoneal dialysis has been a more frequent modality in patients with polycystic kidney disease than in patients with other causes of ESRD in the last several USRDS reports. However, this association has not previously been demonstrated corrected for age and other factors.
The limitations of the current study are similar to those of other retrospective studies. Variables not available in the USRDS were as stated in the methods section. Residual confounding may have persisted beyond the ability of statistical adjustment to correct. We were unable to follow laboratory values, particularly hematocrit, over time. Radiographic findings could not be confirmed. The USRDS did not distinguish between PKD1 and PKD2 or other genetic presentations of autosomal dominant polycystic kidney disease. The limitations of the CMS Form 2728 have been reviewed, [4] but the accuracy of this form is greatest for cardiovascular disease, which is the leading causes of death in the dialysis population. The population-based nature of this study minimized issues of selection and center bias. Our comparison of Polycystic kidney disease patients with other patients on the renal transplant waiting list, in addition to information on comorbidity and laboratory data, minimized issues of selection bias as much as a registry study is likely to be able. However, this very selection introduces problems with generalizability, and the results of the present study apply to patients on the renal transplant waiting list, not all patients with end stage renal disease.
In summary, the mean hematocrit of patients with polycystic kidney disease was significantly higher at presentation to ESRD than for patients with other causes of ESRD on the renal transplant waiting list, despite a significant overall increase in such levels in more recent years of the study. The survival advantage of patients with polycystic kidney disease compared to patients with other causes of ESRD was independent of other factors, including renal transplantation.
Competing Interests
None declared.
Author's Contributions
KA conceived the research plan, conducted primary analysis of the USRDS datafiles, and was responsible for primary preparation of the manuscript. LA, as an expert on the USRDS, supervised the research plan, analysis, and approved the final version of the manuscript.
All authors read and approved the final manuscript.
Authors's Note
The opinions are solely those of the authors and do not represent an endorsement by the Department of Defense or the National Institutes of Health. This is a U.S. Government work. There are no restrictions on its use.
Pre-publication history
The pre-publication history for this paper can be accessed here:
The greater susceptibility of children to renal injury in post-diarrheal hemolytic-uremic syndrome (HUS) may be related, at least in part, to heightened renal cell sensitivity to the cytotoxic effect of Shiga toxin (Stx), the putative mediator of kidney damage in HUS. We hypothesized that sexual maturation, which coincides with a falling incidence of HUS, may induce a relatively Stx-resistant state in the renal cells.
Methods
Cultured human glomerular endothelial (HGEN), human glomerular visceral epithelial (HGEC) and human proximal tubule (HPT) cells were exposed to Stx-1 after pre-incubation with progesterone, β-estradiol or testosterone followed by determination of cytotoxicity.
Results
Under basal conditions, Stx-1 potently and dose-dependently killed HPT and HGEC, but had relatively little effect on HGEN. Pre-incubation for 1, 2 or 7 days with physiologic or pharmacologic concentrations of progesterone, β-estradiol or testosterone had no effect on Stx-1 cytotoxicity dose-response on any cell type. In addition, no steroid altered Gb3 expression (Stx receptor) by any cell type at any time point.
Conclusion
These data do not support the notion that hormonal changes associated with puberty induce an Stx-resistant state within kidney cells.
Background
The factors responsible for the age-related incidence of post-diarrheal hemolytic uremic syndrome (HUS) are unknown. Clinical data indicate that the peak incidence of HUS occurs around two years of age, but the disease clearly occurs in significant numbers of older children [1]. Population-based studies suggest that there is a decline in the incidence of HUS from a peak at age 1–2 years until a nadir at about age 11 years [1,2]. In addition, a case-control study of the Washington State outbreak of HUS revealed that the mean age of patients with HUS was 8 years, while the mean age of patients without HUS was 15 years [3]. These studies raise the possibility, therefore, that the appearance or disappearance of a factor or factors in adolescents and adults reduces susceptibility to end-organ injury in HUS.
HUS is thought to be due, at least in large part, to Shiga toxin (Stx)-mediated cell toxicity [4]. Since the kidney is a primary target of Stx in patients with HUS, it has been hypothesized that the preferential renal injury occurring in children with HUS is related to heightened renal susceptibility to Stx in this age group. Such increased renal sensitivity to Stx in children, if it does indeed exist, could be due to a wide variety of factors. One immediately apparent factor that has been examined is age-related alterations in renal Stx binding and Stx receptor (galactose-α-1,4, galactose-β-1,4, glucose-ceramide (Gb3)) expression. An autopsy study determined that total kidney Gb3 levels were lower in infants than in adults (although only two individuals under 19 years of age were examined) [5]. Another study using immunostaining of biopsy samples found that glomeruli from infants (under two years of age), but not from older children or adults, bound Stx [6]. This provocative finding, albeit involving a relatively small sample size and patients who often had glomerulopathies, provided a potential explanation for augmented glomerular Stx sensitivity in young children. Confirmation of these observations is needed, however they do suggest that Gb3 levels, and in particular the pattern of renal Gb3 expression, may be a factor in the age-related incidence of renal injury in HUS.
The decreased incidence of HUS renal disease in adolescents and adults raises the possibility that factors associated with puberty may be involved. There have been no studies, to our knowledge, that have examined the effects of puberty-related hormonal alterations on renal cell sensitivity to Stx. Indeed, there is no information whatsoever on how any sex steroid affects Stx responsiveness of any cell type. It seemed reasonable, therefore, to examine whether sex steroids (estrogen, progesterone and testosterone) could modify the cytotoxic effect of Stx on renal cells. Further, since Gb3 levels may be altered with aging, the effect of sex steroids on renal cell Gb3 expression was examined.
Several renal cell types have been demonstrated to respond to Stx. Based on histologic evidence, glomerular endothelial cells have been invoked as being primarily involved in HUS renal injury [7]. In vitro studies have demonstrated that human glomerular endothelial cells (HGEN) bind Stx-1 and are modestly sensitive to the toxin's cytotoxic effect [8]. Recent work indicates that other renal cell types may be Stx targets in HUS: cultured human proximal tubule (HPT) [9] and human visceral glomerular epithelial (HGEC) [10] cells express abundant amounts of Gb3 and are highly sensitive to the cytotoxic effect of Stx-1. Consequently, the current study examined the effect of sex steroids on Stx-1 cytotoxicity on, and Gb3 expression by, HGEN, HGEC and HPT.
MethodsCell culture
Primary cultures of HPT were obtained from Clonetics (San Diego, CA) and studied at the third passage. The identity and purity of these cultures was established by immunofluorescent staining as previously described [9] (in addition to the extensive characterization by Clonetics, we have determined that these cells lack von Willebrand factor and platelet endothelial cell adhesion molecules (PECAM), but are positive for cytokeratin, immunofluorescence and alkaline phosphatase activity). HPT were maintained in 1:1 Dulbecco's Modified Eagle Media:Ham's F12 containing 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 250 μg/L amphotericin B, 1 μg/ml hydrocortisone, 10 μg/ml insulin, 5.5 mg/ml transferrin, 6.7 μg/ml selenium, 0.2 gm/L ethanolamine, 6.5 ng/ml L-thyroxine, 10 ng/ml epidermal growth factor, and 10% fetal bovine serum (FBS). Hydrocortisone was present in the media since HPT do not grow well in the absence of corticosteroids (personal observation).
Primary cultures of HGEN were obtained from Cell Systems (Kirkland, WA) and studied at the third passage. These cells have been extensively characterized by Cell Systems; in addition, we determined that these cells have von Willebrand factor and PECAM, but lack cytokeratin, immunofluorescence. HGEN were maintained in EGM2-MV media (Clonetics) which contains epidermal growth factor, hydrocortisone, vascular endothelial cell growth factor, basic fibroblast growth factor, insulin-like growth factor-1, ascorbic acid, heparin and FBS.
Primary cultures of HGEC were obtained from nephrectomies as previously described [10] and studied at the third passage. Briefly, glomeruli were obtained by sieving renal cortex, collagenase digested, and cultured in GEC media (Media 199 + 20% FBS + 100 μg/ml Endothelial Growth Supplement (PerImmune, Rockville, MD) + 100 U/ml penicillin/streptomycin). Primary cultures were passaged when 40% confluent and maintained in GEC media thereafter. Cell identity and purity was determined as previously described [10]: all cells stained (by immunofluorescence) negative for von Willebrand factor, PECAM and anti-myosin, but were positive for cytokeratin.
Stx-1 cytotoxicity and Gb3 expression
Confluent cells in 96 well plates were placed in their maintenance media without serum for 24 hr and exposed to either high or low concentrations (see Results) of progesterone, β-estradiol or testosterone (all from Sigma Chemical Co., St. Louis, MO) for 1, 2 or 7 days in serum-free media. Subsequently, Stx-1 (purified in our laboratory [9]) was added for 72 hr at concentrations of 10-5 – 10-11 grams/liter. The neutral red cytotoxicity assay was performed [9]. Briefly, 50 μg/ml neutral red in Media 199 containing 5% serum was added for 2–3 hours at 37°C. Cells were washed with 1% CaCl2 + 1% formaldehyde and solubilized in 1% acetic acid in 50% ethanol. Absorption was read at 450 nm.
For determination of Gb3 content, cells grown in 6-well plates were placed in serum-free media for 24 hr followed by addition of the steroids for 1, 2 or 7 days. Gb3 was quantitated as previously described [9]. Briefly, cells were scraped off the plates, centrifuged, the pellet extracted in chloroform:methanol:water and separated on high performance thin layer chromatography-silica plates (Mallinckrodt Baker Inc., Paris, KY) by ascending chromatography. The plates were dried, immersed in 0.5% polyisobutylmethacrylate in acetone, and sequentially incubated with Stx-1, anti-Stx-1 monoclonal antibody (purified from a hybridoma cell line, 13C4 (ATCC, Rockville, MD), as previously described [9]), and 125I-goat anti-mouse IgG (DuPont NEN, Boston, MA). Gb3 concentrations were calculated by densitometry (Eagle Eye II, Stratagene, LaJolla, CA) and standardized to total protein. Prior to centrifugation, a cell aliquot was solubilized in 0.1 N NaOH, mixed with Bradford reagent (Bio-Rad, Richmond, CA) and protein concentration determined by measuring absorbance at 590 nm.
Statistics
All data were analyzed by one way analysis of variance. Individual conditions were compared using Student's t-test after the Bonferroni correction. P values < 0.05 were taken as significant.
Results
Stx-1 (72 hr exposure to toxin) dose-dependently killed HGEN, HGEC and HPT (Figure 1). In HGEN, Stx-1 had an LD50 of about 10-5 g/L (140 pM), while in HGEC the LD50 for Stx- was about 10-8 g/L (140 fM) and in HPT the LD50 for Stx was about 10-9.5 g/L (4.4 fM).
Effect of Stx-1 (72 hr exposure) on cell survival in cultured human glomerular endothelial cells (HGEN), human glomerular epithelial cells (HGEC) or human proximal tubule cells (HPT). N = 9 for each data point. % control refers to control cells not exposed to toxin.
Exposure for 1, 2 or 7 days to high (1 μM) or physiologic concentrations (reference range of physiologic steroid concentrations based on Barnes Hospital Laboratory Manual [11]) of progesterone (30 nM), β-estradiol (100 pM), or testosterone (30 nM) had no effect on HGEN (Table 1), HGEC (Table 2) or HPT (Table 3) responsiveness to Stx-1. For the sake of brevity, the tables illustrate the effect of 10-5, 10-8, and 10-11 g/L Stx-1, however a full dose-response from 10-5 – 10-11 g/L Stx-1, in ten-fold concentration increments, was done for each condition: there was also no significant effect of these other concentrations of sex steroids on the cytotoxic effects of Stx-1 on any of the renal cell types. In addition, physiologic or pharmacologic concentrations of progesterone, estradiol or testosterone had no effect on baseline (without exposure to Stx-1) HGEN, HGEC or HPT survival.
Effect of sex steroids on Stx-1 cytotoxicity in human glomerular endothelial cells (HGEN). Cells were exposed to high and low concentrations of steroids for 1, 2 or 7 days followed by addition of varying concentration of Stx-1 for 72 hr. Cell survival was determined at the end of the experiment. % survival refers to percent of cells surviving after exposure to Stx-1 ± steroids. N = 9 each data point. Results are expressed as mean ± SEM.
HGEN
Dose
[Stx-1] gm/L
Stx alone (% survival)
Day 1 (% survival)
Day 2 (% survival)
Day 7 (% survival)
Estradiol
1 μM
-5
54 ± 4
49 ± 3
49 ± 6
48 ± 6
1 μM
-8
90 ± 7
86 ± 6
85 ± 8
95 ± 6
1 μM
-11
102 ± 8
102 ± 10
105 ± 4
91 ± 8
100 pM
-5
54 ± 4
51 ± 3
55 ± 3
58 ± 4
100 pM
-8
90 ± 7
87 ± 6
96 ± 9
85 ± 9
100 pM
-11
102 ± 8
96 ± 6
103 ± 11
115 ± 11
Progesterone
1 μM
-5
54 ± 4
51 ± 4
50 ± 4
45 ± 6
1 μM
-8
90 ± 7
84 ± 9
85 ± 8
95 ± 7
1 μM
-11
102 ± 8
100 ± 4
103 ± 6
95 ± 7
30 nM
-5
54 ± 4
53 ± 3
53 ± 4
52 ± 5
30 nM
-8
90 ± 7
91 ± 9
92 ± 7
94 ± 9
30 nM
-11
102 ± 8
102 ± 7
104 ± 7
100 ± 7
Testosterone
1 μM
-5
54 ± 4
58 ± 5
55 ± 2
56 ± 3
1 μM
-8
90 ± 7
91 ± 8
92 ± 9
94 ± 10
1 μM
-11
102 ± 8
103 ± 12
95 ± 8
102 ± 7
30 nM
-5
54 ± 4
52 ± 4
54 ± 4
55 ± 3
30 nM
-8
90 ± 7
86 ± 6
90 ± 6
94 ± 8
30 nM
-11
102 ± 8
96 ± 6
100 ± 5
104 ± 8
Effect of sex steroids on Stx-1 cytotoxicity in human glomerular epithelial cells (HGEC). Cells were exposed to high and low concentrations of steroids for 1, 2 or 7 days followed by addition of varying concentration of Stx-1 for 72 hr. Cell survival was determined at the end of the experiment. % survival refers to percent of cells surviving after exposure to Stx-1 ± steroids. N = 9 each data point. Results are expressed as mean ± SEM.
HGEC
Dose
[Stx-1] gm/L
Stx alone (% survival)
Day 1 (% survival)
Day 2 (% survival)
Day 7 (% survival)
Estradiol
1 μM
-5
54 ± 4
51 ± 4
51 ± 3
48 ± 5
1 μM
-8
90 ± 7
79 ± 10
94 ± 8
86 ± 5
1 μM
-11
102 ± 8
96 ± 10
107 ± 5
95 ± 8
100 pM
-5
54 ± 4
53 ± 4
52 ± 3
50 ± 4
100 pM
-8
90 ± 7
88 ± 8
89 ± 6
83 ± 7
100 pM
-11
102 ± 8
106 ± 7
96 ± 9
98 ± 9
Progesterone
1 μM
-5
54 ± 4
54 ± 49
56 ± 5
54 ± 2
1 μM
-8
90 ± 7
83 ± 9
90 ± 9
90 ± 5
1 μM
-11
102 ± 8
101 ± 3
101 ± 6
103 ± 7
30 nM
-5
54 ± 4
50 ± 6
58 ± 4
50 ± 5
30 nM
-8
90 ± 7
86 ± 4
92 ± 6
78 ± 9
30 nM
-11
102 ± 8
97 ± 7
105 ± 7
93 ± 7
Testosterone
1 μM
-5
54 ± 4
53 ± 4
55 ± 4
50 ± 4
1 μM
-8
90 ± 7
90 ± 11
90 ± 7
84 ± 6
1 μM
-11
102 ± 8
103 ± 9
100 ± 7
105 ± 5
30 nM
-5
54 ± 4
59 ± 6
59 ± 4
54 ± 9
30 nM
-8
90 ± 7
84 ± 8
89 ± 5
82 ± 7
30 nM
-11
102 ± 8
98 ± 6
106 ± 8
97 ± 6
Effect of sex steroids on Stx-1 cytotoxicity in human proximal tubule cells (HPT). Cells were exposed to high and low concentrations of steroids for 1, 2 or 7 days followed by addition of varying concentration of Stx-1 for 72 hr. Cell survival was determined at the end of the experiment. % survival refers to percent of cells surviving after exposure to Stx-1 ± steroids. N = 9 each data point. Results are expressed as mean ± SEM.
HPT
Dose
[Stx-1] gm/L
Stx alone (% survival)
Day 1 (% survival)
Day 2 (% survival)
Day 7 (% survival)
Estradiol
1 μM
-5
54 ± 4
49 ± 4
50 ± 4
46 ± 5
1 μM
-8
90 ± 7
81 ± 7
83 ± 7
84 ± 9
1 μM
-11
102 ± 8
97 ± 5
99 ± 7
107 ± 7
100 pM
-5
54 ± 4
49 ± 4
60 ± 4
49 ± 6
100 pM
-8
90 ± 7
95 ± 8
90 ± 7
87 ± 8
100 pM
-11
102 ± 8
97 ± 6
103 ± 5
92 ± 9
Progesterone
1 μM
-5
54 ± 4
56 ± 4
52 ± 3
60 ± 5
1 μM
-8
90 ± 7
99 ± 9
81 ± 7
91 ± 9
1 μM
-11
102 ± 8
107 ± 8
101 ± 5
106 ± 8
30 nM
-5
54 ± 4
50 ± 6
52 ± 3
54 ± 4
30 nM
-8
90 ± 7
86 ± 4
83 ± 5
90 ± 5
30 nM
-11
102 ± 8
97 ± 7
105 ± 5
92 ± 7
Testosterone
1 μM
-5
54 ± 4
57 ± 3
49 ± 4
58 ± 5
1 μM
-8
90 ± 7
92 ± 7
89 ± 5
91 ± 8
1 μM
-11
102 ± 8
94 ± 5
97 ± 6
100 ± 6
30 nM
-5
54 ± 4
51 ± 4
64 ± 7
57 ± 3
30 nM
-8
90 ± 7
98 ± 8
97 ± 7
85 ± 5
30 nM
-11
102 ± 8
109 ± 10
102 ± 6
103 ± 7
The effect of 1, 2 or 7 days of exposure to physiologic or pharmacologic concentrations of progesterone, estradiol or testosterone on Gb3 expression by HGEN, HGEC or HPT was also examined. A representative blot of Gb3 content is shown in Figure 2 (figure illustrates 2 day steroid exposure in HPT) to demonstrate that Gb3 isolated from cells migrated on the gel at the same position as purified Gb3. As for Stx-cytotoxicity, none of the steroids, at any dose or exposure time, modified Gb3 content in any cell type (n = 3 each condition, data not shown).
Representative blot of Gb3 expression by human proximal tubule cells after 48 hr exposure to media alone (control), 1 μM β-estradiol, 1 μM progesterone or 1 μM testosterone. A Gb3 standard is shown for reference. Identical results were obtained at 1 and 7 days of steroid exposure.
Immunoreactive steroid concentration was determined in media samples at the start and conclusion of the 7 day experiment (analyzed in clinical laboratory at Associated Regional University Pathologists, Salt Lake City, UT) in order to determine the stability of the added steroids. Estradiol and testosterone concentrations did not vary over 1, 2 or 7 days in culture media. Progesterone levels also did not fall after 1–2 days in culture media, however after 7 days in media the progesterone concentration significantly dropped (80% decrease). Consequently, fresh progesterone was added to the 7 day experiments every two days.
The failure of steroids to affect Stx cytotoxicity was not due to inactivity of the steroid preparations. Similar preparations and concentrations of steroids from the same supplier (Sigma) were tested by Dr. Wayne Meikle in the Division of Endocrinology at the University of Utah using an in-house estrogen and progesterone binding assay and a testosterone-dependent stimulation of prostate cancer cell apoptosis assay [12]; all the steroids retained biologic activity (data not shown).
Discussion and Conclusion
The reasons why HUS primarily affects children are unknown. It is evident that the peak incidence of HUS occurs in very young children (1–2 years of age). The factors responsible for this apparent heightened sensitivity to infection with Stx-expressing bacteria have not been extensively investigated and were not examined in the current study. Indeed, it is conceivable that not all of the factors involved in HUS occurrence in young children are identical to those involved in the diminished HUS incidence in adolescents and adults. Numerous factors could be involved in age-related Stx sensitivity, including differences in the degree of enteric invasion by Stx-producing E. coli; in Stx transport across the gut, in circulating red or white blood cell toxin binding and delivery to the kidney, in the immune response to Stx, in renal Stx binding, and/or in intrinsic or cofactor-regulated (e.g. inflammatory cytokines) renal Stx responsiveness. The current study evaluated one of these possibilities, namely that renal cell Stx-1 responsiveness decreases with age due, at least partially, to hormonal changes associated with puberty. As stated earlier, there is no precedent in the literature for examination of the effect of sex steroids on Stx responsiveness, however, the coincidental fall in clinically apparent HUS in adolescents and adults [1] raises the possibility that hormonal changes associated with puberty induce a relatively Stx-resistant state in the kidney.
HGEN, HGEC and HPT were chosen for examination in this study because these cell types, at least in vitro, have been shown to be injured by Stx-1 [6-8]. Mesangial cells were not examined since Stx-1 has not been demonstrated to affect their viability [13]. Previous studies have separately examined the cytotoxic effect of Stx-1 on HGEN [8], HGEC [10], and HPT [9], however this is the first study to directly compare Stx-1 sensitivity of these renal cell types. These data indicate that, as suggested from the previous studies, within the limits of the cell culture system, HPT are the most sensitive to Stx-1 cytotoxicity of the three cell types, HGEC are slightly less sensitive, and HGEN are at least three orders of magnitude less sensitive than either HGEC or HPT. Such differential sensitivity is quite interesting, particularly considering that glomerular endothelial cell damage is a prominent histologic feature in biopsies obtained from patients with clinically well established HUS [7]. The significance of these observations remains, therefore, to be determined. Although speculative, it is tempting to propose that HPT and HGEC may be early targets of Stx-1 and that this could play a role in the pathophysiologic processes that characterize renal injury in HUS.
Estrogen, progesterone or testosterone did not affect Stx-1 mediated renal cell cytotoxicity or Gb3 expression. Cells were exposed to the steroids for up to one week in order to allow time for steroid-regulated gene transcription and protein expression. In addition, both physiologic and pharmacologic concentrations of steroids were utilized. Despite these efforts to be insure that the system was optimized in order to detect a steroid-mediated alteration of Stx responsiveness, the concern still exists that this system may not really reflect the influences of sex steroids in vivo. Indeed, one might argue that an animal model, in which the testes or ovaries are removed and the animals are given Stx in the presence and absence of steroid replacement, would be better. Such studies would, however, be problematic. Traditional animal models, such as mice, rats, rabbits or pigs do not develop typical renal disease when given either Stx or E. coli 0157:H7 [14]. Further, animal models that most closely reflect human HUS are only in the early stages of development [14]. Hence, the cell culture system remains the most reasonable means to assess an effect of sex steroids on renal cell Stx-1 responsiveness; such systems have clearly been demonstrated to be responsive to sex steroids (e.g. testosterone-regulated KAP promoter activity in proximal tubule cells [15] as well as estrogen-regulated endothelial cell function [16]). Given these limitations, our data suggest that sex steroids do not alter renal cell sensitivity to the cytotoxic effects of this Stx-1. Consequently, the reasons why clinically apparent HUS primarily occurs in children remains to be determined.
Author 1 performed the analysis of cytotoxicity, maintained cell culture and was involved in experimental design. Author 2 performed Gb3 analysis and assisted with cell culture. Author 3 designed, interpreted and supervised the experiments.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was funded by National Institutes of Health grants RO1 HL56857 and RO1 DK52043 (both to D.E.K.).
There is little information on how target lipid levels can be achieved in end stage renal disease (ESRD) patients in a systematic, multidisciplinary fashion.
Methods
We retrospectively reviewed a pharmacist-directed hyperlipidemia management program for chronic hemodialysis (HD) patients. All 26 adult patients on chronic HD at a tertiary care medical facility were entered into the program. A clinical pharmacist was responsible for laboratory monitoring, patient counseling, and the initiation and dosage adjustment of an appropriate 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (statin) using a dosing algorithm and monitoring guidelines. The low-density lipoprotein (LDL) cholesterol goal was ≤ 100 mg/dl. A renal dietitian provided nutrition counseling and the nephrologist was notified of potential or existing drug interactions or adverse drug reactions (ADRs). Patients received a flyer containing lipid panel results to encourage compliance. Data was collected at program initiation and for 6 months thereafter.
Results
At the start of the program, 58% of patients were at target LDL cholesterol. At 6 months, 88% had achieved target LDL (p = 0.015). Mean LDL cholesterol decreased from 96 ± 5 to 80 ± 3 mg/dl (p < 0.01), and mean total cholesterol decreased from 170 ± 7 to 151 ± 4 mg/dl (p < 0.01). Fifteen adjustments in drug therapy were made. Eight adverse drug reactions were identified; 2 required drug discontinuation or an alternative agent. Physicians were alerted to 8 potential drug-drug interactions, and appropriate monitoring was performed.
Conclusions
Our findings demonstrate both feasibility and efficacy of a multidisciplinary approach in management of hyperlipidemia in HD patients.
Background
Patients on dialysis have more coronary artery disease (CAD) and CAD-related mortality than the general population [1]. Elevated low-density lipoprotein (LDL) cholesterol level is an independent risk factor for patients with end stage renal disease (ESRD) [2]. The 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been associated with decreased all-cause mortality in dialysis patients in a registry-based study [3] and clinical trials are underway to confirm this benefit [4,5].
Mortality benefits of statins has been attributed to lower lipid levels, but recent reports indicate that statins may reduce cardiovascular risk by other mechanisms, [6,7] and may also reduce cancer-related mortality [8]. Consensus is growing for statin use in all ESRD patients with atherosclerotic disease or diabetes [9]. If used this way, over 60% of all dialysis patients might be eligible for statins [10]. Despite an acceptable safety profile [6,11], fewer than 10% of dialysis patients were prescribed statins according to USRDS and Canadian studies, [3,12] even in known coronary heart disease [13].
Despite differences in the pattern of dyslipidemia and cardiovascular disease in ESRD patients compared with the general population, current use of statins focus on treating elevated LDL cholesterol levels. National Cholesterol Education Program (NCEP) guidelines recommend a target LDL cholesterol of 100 mg/dl in high-risk groups [14]. Pharmacists are becoming more involved in managing a variety of chronic diseases [15-19]. For lipid lowering, a team approach is more effective in the ambulatory care setting than management by a physician [20,21], but there are no published reports on using this approach in a dialysis population. Here, we describe the effectiveness and feasibility of our multidisciplinary lipid management program using the specific skills of pharmacist, dietitian and nephrologist in hemodialysis patients.
Walter Reed Army Medical Center (WRAMC) is a 235-bed, tertiary-care military hospital with 25–30 chronic dialysis patients. Prior to implementation of our program, two formulary statins (simvastatin and cerivastatin) were available. A hospital-wide switch to these statins was mandated by the Department of Defense Pharmacoeconomic Center to reduce cost and provide uniformity. A Statin Formulary Conversion Clinic switched all patients to cerivastatin (preferred agent) or simvastatin between January-April 2000 [22]. HD patients were given simvastatin during the conversion due to dose adjustment recommendations for cerivastatin in patients with renal insufficiency and lack of experience with this drug in ESRD. Consequently, there were several dialysis patients taking non-formulary statins when our HD lipid management program began, because they had failed to reach goals on formulary statins.
Our program was designed as an ongoing lipid management program, directed by the clinical pharmacist assigned to the Nephrology Service. The clinical pharmacist received approval from the hospital credentials committee, the pharmacy & therapeutics committee, and the director of dialysis to prescribe and make dosage adjustments and order laboratory tests as per a lipid management guideline. The WRAMC Human Use Committee approved a 6-month retrospective review of the program in April 2001.
MethodsGuideline development
A guideline for management of hyperlipidemia and conversion to formulary statin in HD patients was developed jointly between the nephrologists, clinical pharmacist and renal dietitian before program implementation (Table 1). Patients were converted at the start of the program, or whenever they initiated dialysis. Only simvastatin and atorvastatin were included in the guideline. Atorvastatin was prescribed for patients who failed simvastatin.
Guideline for lipid management & conversion to formulary statin
a. Order lipid panel and P3* every 4 months if patient is currently on cholesterol lowering medication.
b. If patient is at goal without drug therapy, draw lipid panel and P3 yearly.
c. Modify/Initiate patient's therapy per the following guideline:
If patient is on:
Action:
Cyclosporine
Refer to nephrologist
Gemfibrozil
Refer to nephrologist
If triglycerides >200
Refer to nephrologist
If patient's current cholesterol medication is:
None, and LDL>100, no contraindications
Begin Simvastatin 20 mg (1/2 of 40 mg tablet)
Provide pharmacy counseling, drug literature, dietary consult if needed.
Convert to Simvastatin 20 mg qd (1/2 of 40 mg tablet)
Atorvastatin 20 mg qd
Convert to Simvastatin 40 mg qd
Atorvastatin 40 mg qd
Convert to Simvastatin 80 mg qd
Atorvastatin 80 mg qd
Continue Atorvastatin 80 mg qd
d. Draw P3 and lipid panel 8 weeks after changing or initiating therapy.
If liver enzymes are >3 times upper limit of normal
Refer to nephrologist.
Hold Statin.
If patient complains of muscle aches or weakness
Refer to nephrologist.
Draw CPK.
e. If LDL>100 after 8 weeks, adjust dosing according to the following guideline:
If current dose is Simvastatin 20 mg qd
Increase to Simvastatin 40 mg qd
If current dose is Simvastatin 40 mg qd
Increase to Simvastatin 80 mg qd
If current dose is Simvastatin 80 mg qd
Change to Atorvastatin 80 mg qd
f. Repeat steps d. and e. until patient reaches LDL goal or is taking Atorvastatin 80 mg
g. Draw P3 and lipid panel 8 weeks after initiating Atorvastatin 80 mg. If patient still not at LDL goal, refer to nephrologist.
*P3 includes alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin
Inclusion/exclusion criteria
All WRAMC chronic hemodialysis patients were eligible. Exclusion criteria included age < 18 years, acute renal failure, pregnancy or nursing, use of cyclosporine, tacrolimus, or gemfibrozil, elevated liver associated enzymes (LAEs), and allergy to statins. All 25 hemodialysis patients met criteria for inclusion in September 2000 when the program was implemented. An additional patient who initiated dialysis during the study period was included, for a total of 26 patients.
Laboratory monitoring
Blood tests were routinely drawn every 4 weeks for monitoring of anemia, calcium and phosphate control, LAEs, nutritional indices, and dialysis adequacy. The pharmacist ordered a non-fasting lipid profile for each patient in accordance with the guideline (e.g. every 8 weeks, every 4 months, or yearly depending on monitoring requirement). The lipid profile included total cholesterol (TC), LDL cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol. Blood was analyzed in the WRAMC clinical chemistry laboratory using the Roche COBAS Integra 700 System® (Roche Diagnostic System, Somerville, NJ). Roche Precinorm® and Precipath® reagents were used for quality control for HDL and LDL cholesterol results. Bio-rad Liquicheck® Level 1 and Level 2, (Bio-rad Diagnostic Group, Irvine, CA), were used for quality control of TC and triglyceride measurements. Calibration was done daily. A non-fasting lipid profile was acceptable for LDL cholesterol and TC measurement because the assay measured parameters directly. If the non-fasting triglyceride level was ≥ 200 mg/dL, the physician was notified, and a fasting triglyceride level was ordered at his/her discretion.
Patient counseling and referral
After review of each lipid profile, the pharmacist provided verbal instruction, statin information handouts (at drug initiation or change), and a "Personal Cholesterol Management Report" to each patient. This reported the patient's own lipid profile result and goal values, as well as the new statin dose (or dose to be continued). This report also explained the importance of cholesterol control and listed common side effects of statins. At the time of each report, the pharmacist explained the lipid profile results, asked about side effects, and reviewed the patient's drug regimen for medication compliance and new or potential drug interactions. The pharmacist also gave the patient a new prescription with refills to last until the next scheduled lipid panel. Patients needing dietary reinforcement were referred to the renal dietitian. Adverse reactions or potential drug interactions were brought to the nephrologist's attention.
Documentation and data collection
The clinical pharmacist documented all interventions and referrals using an Excel® spreadsheet (Microsoft, Redmond, WA). Individual reports were generated that gave lipid profile and LAE results, any adverse drug reaction or drug interaction, dosage adjustments made, and the date of the next scheduled lipid profile. This report was placed in the patient's HD chart after each lipid profile. A copy was given to each nephrologist.
Lipid data was tabulated monthly by the pharmacist and submitted to the nephrology service quality improvement coordinator, the medical director of dialysis, and the chief of nephrology for ongoing program evaluation. Data submitted included the number of patients followed, number of patients at LDL cholesterol goal, average LDL cholesterol and total cholesterol, number of adverse drug reactions and potential drug interactions identified. HDL data was not analyzed or included as part of the guideline.
Statistical analysis
Data was tabulated in Microsoft Excel® and analyzed using SigmaStat® (SPSS, Chicago, IL). Fisher exact test was used for categorical variables and paired Student's t-test was used for continuous parametric variables. Wilcoxon signed-rank test was used for continuous variables that were not normally distributed. A p-value of < 0.05 was considered statistically significant.
Results
Patient demographics are shown in Table 2. After 6 months, 23 of 26 patients (88%) had reached target LDL cholesterol (Figure 1), compared with 15 patients (58%) at the start of the program (p = 0.015). Mean LDL cholesterol decreased 16.7%, from 96 ± 5 to 80 ± 3 mg/dl (p < 0.001), and mean total cholesterol decreased 11.1%, from 170 ± 7 to 151 ± 4 mg/dl (p = 0.004).
Change in LDL cholesterol 6 months after program initiation.
Patient Demographics (n = 26)
Mean age (years)
55.7 + 11
Gender (M/F)
17 / 9
Race (n, %)
Black
21 (81)
White
3 (11)
Asian
2 (8)
Etiology of chronic renal failure* (n, %)
Diabetes mellitus
11 (42.3)
Glomerulonephritis
9 (34.6)
Unknown or other
5 (19.2)
Hypertension
3 (11.5)
Median time on dialysis (months)
14.5 (range 0–153)
*totals >100% due to 2 patients with combined etiology of diabetes mellitus and hypertension
Eight adverse drug reactions were identified in 7 patients. Four patients (15.4%) experienced musculoskeletal symptoms, 2 of which had muscle aches and weakness (hand, arm and back muscles). Creatine phosphokinase (CPK) levels were normal in both cases. Drug therapy was not interrupted in either case, and other etiologies for the symptoms were pursued. The other 2 patients reported shoulder soreness, thought related to exercise, and leg cramping, thought to be due to the previous day's dialysis. CPK levels were not obtained and the symptoms spontaneously resolved after several days.
Two patients (7.6%) experienced an increase in LAEs. A 3-fold increase in serum transaminases occurred in one patient who was initiated on simvastatin 20 mg qd during the program. The drug was discontinued. One patient had a mild transient increase in alkaline phosphatase, from 115 IU/L to 131 IU/L, when starting statin therapy. The drug was not discontinued and the level returned to baseline within 2 months.
Diarrhea or gastrointestinal upset was reported in 2 patients (7.6%). Relationship to the statin was unclear. One patient was switched from simvastatin to atorvastatin with slight improvement. In the second patient, symptoms resolved when acetaminophen with codeine was discontinued.
Eight potential drug interactions were identified. Physicians were alerted to monitor for myopathy and changes in international normalized ratio (INR) in 3 patients taking warfarin because of potential for cytochrome P450 3A4 competition. Other drugs with cytochrome P450 interaction potential were identified, including verapamil (2 patients), and diltiazem (1 patient). Other drug interactions of a different/unknown mechanism included digoxin and levothyroxine (1 patient each). No drug interaction resulted in an adverse event or discontinuation of therapy.
Fourteen patients (54%) were on statins at the start of the program, 4 of whom were receiving non-formulary statins. (1 cerivastatin: 3 pravastatin.) Four patients were on simvastatin 20 mg qd; 6 patients were on simvastatin 40 mg qd. At 6 months, 15 (58%) were on statins. Of these, 7 patients were on simvastatin 20 mg qd, 3 patients were on simvastatin 40 mg qd, and 2 patients were taking simvastatin 80 mg qd. One patient each was taking atorvastatin 40 mg qd and 80 mg qd. Two patients were initiated on statins during the period studied, one of which was discontinued at 6 months due to increased LAEs. During the 6-month period, 1 patient died from cardiovascular disease. Her LDL cholesterol and TC were not elevated (55 mg/dl and 146 mg/dl, respectively). Three patients received renal transplants.
Table 3 summarizes the interventions made by the pharmacist. All patients received medication teaching and compliance assessment at each encounter. Most patient encounters resulted in continuation of current therapy (including those who were at goal LDL cholesterol with non-pharmacological therapy). Statins were stopped in 1 patient because of low LDL cholesterol level (37 mg/dl). The renal dietitian was consulted 3 times for specific dietary counseling relating to cholesterol control and compliance.
Type of intervention by pharmacist
Type of intervention
number
Continue current therapy
59
Change of therapy
15
Increase in dose (same drug)
6
Change drug
5
Initiation of therapy
2
Drug discontinued due to ADR
1
Drug discontinued (other)
1
Request dietary consult
3
Overall nutritional status remained stable. Median albumin at study entry was 3.73 g/dL (range 2.3 – 4.5 g/dL), vs. 3.86 g/dL (range 2.9 – 4.7 g/dL) at 6 months (p = 0.345).
Discussion
This study shows that improved LDL cholesterol control can be achieved in chronic HD patients using a multidisciplinary pharmacist-directed lipid program. Hemodialysis populations may be ideal for this program because of convenient blood sampling and frequent provider contact. Program costs included the minimal cost of patient pamphlets, and the cost of additional pharmacist time. We estimated that 8–12 additional hours/month were required for pharmacist interventions, documentation and compiling reports. The program did not incur additional laboratory costs, since lipid-monitoring guidelines were those of the NCEP. Overall, the program was well accepted by nurses, nephrologists and patients.
Side effects were minimal during the period, and several potential drug interactions were avoided. Saltissi, et al [11] recently reported the tolerability and effectiveness of simvastatin in doses of 5–20 mg in lowering LDL and non-HDL cholesterol in dialysis patients. Although the majority of our patients required simvastatin 20 mg to reach LDL goal, simvastatin in higher doses and atorvastatin were used and well tolerated, with dose changes made in a timely fashion, undoubtedly contributing to program effectiveness. It is unknown whether a longer treatment period with these doses would have produced more side effects. One complicating factor of statin therapy in HD patients is the high prevalence of hepatitis B and C, making it difficult to differentiate hepatitis-induced increases in LAEs from those due to the statin. This was not an issue in our study, although several patients were positive for hepatitis. Although CPK levels were not measured at baseline, in patients with musculoskeletal symptoms, this value was found to be normal. Of note, the 3 patients who did not reach LDL cholesterol goal were not those who experienced side effects requiring discontinuation of therapy. LDL levels in these patients were near goal and ranged from 102–105 mg/dl.
Limitations of the study include the small number of patients and lack of a control group. Since patients began the program after "physician only" lipid management, one presumes by the improvement in lipid levels and number of patients at LDL cholesterol goal that the team approach was more favorable. That 54% of patients were on statins and 58% were at goal LDL cholesterol at the beginning of the program can be attributed to the close monitoring by nephrologists in a fellowship program and the pharmacy-run statin conversion clinic. Even so, these results improved after implementing the multidisciplinary approach.
Although compliance was not documented by pill count, the pharmacist, with access to computer databases, assessed drug adherence by refill frequency, patient interviews, and lab results. In addition to increasing compliance, including patients in therapeutic decision-making may be cost-effective [23]. The patient handouts with lipid results were well received and may have improved patient's compliance and interest in lipid therapy, but we did not formally assess patient knowledge of hyperlipidemia goals, treatment, and side effects, nor document refill frequency.
Some studies suggest that dialysis patients with low LDL cholesterol levels have increased risk of mortality vs. those with higher LDL cholesterol levels [24]. LDL cholesterol and conventional cardiac risk factors are insensitive predictors of CAD in this population [25], and may reflect the high frequency of malnutrition [26] and established cardiovascular disease [27]. In our study, albumin remained stable as LDL cholesterol levels declined.
Although the guidelines for lipid therapy in HD patients are extrapolated from the general population, current recommendations are to treat high-risk patients to a target LDL cholesterol of ≤ 100 mg/dl. Most dialysis patients in the United States do not meet these targets. Tonelli et al [16] measured statin use in dialysis patients, but not whether target LDL cholesterol was achieved. Admittedly, lipid lowering and its benefits are less well defined in renal failure explaining why nephrologists may not be as aggressive in statin prescribing [28]. However, the anticipated shortages of nephrology manpower (nephrologists, nurses, physicians' assistants, dietitians) may make it more difficult in the future to achieve therapeutic goals [29]. Methods to help promote appropriate use of statins and other drug therapy would be beneficial. Pharmacists are not routinely assigned to dialysis units, however, Manley, et al [30] showed that every dollar spent on pharmaceutical care in ESRD results in a savings of $3.98. A recent American College of Physicians-American Society of Internal Medicine position paper speculates that pharmacist participation in patient care beyond patient education and hospital rounds will be time consuming for physicians [31]. In reality, pharmacist participation in the collaborative management of conditions such as hyperlipidemia through programs like ours, may free nephrologists' time for other activities and improve patient outcomes while still maintaining physician control of therapy. Yet, maintaining good communication, documentation, and quality assurance is essential.
The treatment of hyperlipidemia is readily suited to the algorithmic, multidisciplinary approach used in the present study. Moreover, similar programs could be employed for management of other ESRD complications, such as hyperphosphatemia and anemia, further improving outcomes in dialysis patients with the least possible impact on staffing and resources.
Despite editorial speculation that nephrologists are "late to the party" in adoption of cardiovascular risk reduction measures [9], underutilization of proven therapies is not unique to nephrology. The National Academy of Science's Institute of Medicine reported that our health care delivery system continues to suffer from many deficiencies, such as use of beta-blockers in only 50% of patients with myocardial infarction [32]. The percentage in dialysis patients is similar [2]. Setting targets and achieving them are quite different things. Undoubtedly, new methods for assuring the implementation of proven or therapies are needed. The present study adds new information on the process of improving statin use and other therapies that could improve outcomes in dialysis patients.
Conclusions
Text for this section.
Competing interests
None declared
Authors' contributions
RV participated in the development & implementation of the guideline described in the study, collected data, and wrote & edited the manuscript.
KA supervised development of the guideline and implementation of the program, participation in literature search and writing and editing the manuscript.
PW supervised development of the guideline and implementation of the program, and participated in the writing and editing of the manuscript.
RM participated in the development and implementation of the guideline, and editing of the manuscript.
AS participated in the development of the guideline and editing of the manuscript.
CY participated in the development and implementation of the guideline, statistical analysis of the data, and writing and editing of the manuscript.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Nephropathic cystinosis is an autosomal recessive disease resulting from intracellular accumulation of cystine leading to multiple organ failure.
Case report
We describe the clinical course of a patient managed from the age of six until his death at the age of 33 years. He underwent multiple surgery, including two renal transplants, developed transplant renal artery stenosis that was managed medically, and progressive heart failure at the age of 33 years. His death from a ruptured pseudoaneurysm associated with a restrictive cardiomyopathy is noteworthy. A limited cardiac autopsy revealed the presence of cystine crystals in interstitial cardiac histiocytes and one myocardial cell, along with 1000-fold higher tissue cystine content of the left ventricular myocardium compared to patients without cystinosis, suggesting the possibility of direct cystine mediated metabolic injury.
Nephropathic cystinosisRenal transplantCardiomyopathyPseudoaneurysmLong term survivorBackground
Nephropathic cystinosis is an autosomal recessive disorder characterized clinically by generalized proximal renal tubular dysfunction, progressive renal damage after intracellular accumulation of cystine [1,2]. The result is the buildup of cystine in various organs and eventual multiple organ failure. In the kidney, it leads to progressive renal damage, resulting in end-stage renal disease (ESRD) by 10 years of age on average [1,2]. The drug cysteamine reduces intracellular concentrations of cystine and can retard progression of renal damage if started early [1,2]. Renal transplantation is a viable option and the graft survival is excellent [3]. The gene for cystinosis which has been cloned and identified as CTNS, maps to chromosome 17p13 [4]. In the European population, mutations of CTNS include a 65-kb deletion-involving marker D17S829 and 11 other small mutations [4]. Other CTNS mutations have been confirmed in American-based populations of cystinosis patients [5]. We describe the clinical course of a nephropathic form of cystinosis diagnosed at the age of five years and managed at Albert Einstein College of Medicine from the age of six for the next 27 years. His survival despite multiple complications, and his death at 33 years of age due to a ruptured pseudoaneurysm associated with cardiomyopathy, is noteworthy.
Case report
A one-year old white male child was found to have rickets and was treated with oral vitamin D. He was noted to be growth retarded, and to have albuminuria and aminoaciduria. He received 50,000 units of vitamin D daily, off and on until the age of five. He was then seen at another institution where he underwent renal and bone marrow biopsies. The biopsies revealed cystine deposits; opthalmological examination confirmed the diagnosis of cystinosis. He was then referred to Albert Einstein College of Medicine for further management. At the age of six years, the patient had growth retardation, renal osteodystrophy, albuminuria, glucosuria and chronic renal insufficiency. A creatinine clearance measured later revealed 20% GFR. He was managed conservatively until he reached ESRD, at the age of 11 years. Four months after initiation of hemodialysis, he developed a persistent fever with fatigue and orthopnea. Physical exam revealed a blood pressure (BP) of 137/101 mm of Hg, an enlarged liver, distended neck veins and pericardial rub. The EKG revealed right atrial and left ventricular enlargement. A clinical diagnosis of uremic pericarditis was made and aggressive dialysis was initiated. The patient responded to aggressive dialysis and hydralazine, guanethidione and methyldopa, for BP control. Subsequently, he developed other complications of cystinosis, namely hypothyroidism, hypersplenism and corneal deposition of cystine with diffuse pigmented mottling of the entire eye and fundus. A splenectomy was performed at the age of 11 years. The spleen weighed 310 grams and was laden with cystine deposits. At that time his medications included antihypertensives, digoxin, bicarbonate, testosterone, L-thyroxine, calcium and vitamin D.
At the age of 12 years, he received a cadaveric renal transplant. He had mild reversible rejection in the first year but maintained normal renal function on azathioprine and prednisone. Four years later, he developed chronic rejection eventually needing dialysis two years later. An arterio-venous (AV) fistula was created on his right arm. His BP remained persistently above 170/110 mm of Hg. Nephrectomies of transplant and native kidneys along with propanolol, minoxidil, hydralazine, and methyldopa were needed to control his BP. At the age of 18 years he received his second cadaveric renal transplant. Four months later, due to persistent uncontrolled hypertension and elevated serum renin (>500 ng/dl/hr), he underwent a transplant arteriogram. It revealed a significant of narrowing of the transplant renal artery, one inch distal to the anastomosis, and a slight post-stenotic dilatation. It was decided to medically control hypertension with hydrochlorthiazide, minoxidil, and propanolol. At that time he had a complete slip of both femoral epiphyses and his bones showed signs of chronic renal osteodystrophy. With rehabilitation using crutches and surgical correction of his genu valgum he was able to ambulate.
For the ensuing 12 years he had a relatively unremarkable course. He had stable renal function with a serum creatinine of 0.8 mg/dl and satisfactory BP control. Annual echocardiograms revealed mild atrial enlargements, mild pulmonic insufficiency and left ventricular hypertrophy with normal systolic function with an ejection fraction (EF) of 68%. His medications included hydrochlorthiazide, propranolol, minoxidil, L-thyroxine, azathioprine and prednisone. His vision refracted to 20/20 in each eye with a correction for mixed astigmatism. He used artificial tears when needed. At the age of 30 he began reporting increasing fatigue, exercise intolerance, and generalized edema. Physical examination confirmed heart failure and furosemide and digitalis were needed for a short period to stabilize his condition. Repair of a large AV fistula did not alter his symptoms. He was readmitted with crampy, persistent abdominal pain. An ultrasound revealed a pseudoaneurysm at the site of the transplant renal artery anastomosis. Gallstones were also noted. After extensive consultations, he was considered a high operative risk and a decision was made to manage him conservatively.
At the age of 33 years he again presented with extreme fatigue and edema. A chest x-ray demonstrated cardiomegaly and bilateral pleural effusions. An echocardiogram showed biventricular hypertrophy with normal left ventricular EF (72%), bilateral atrial enlargement and a dilated aortic root. He was treated with diuretic therapy and digitalis but with limited response. He was subsequently admitted to the hospital in a dehydrated state following a short viral illness. He developed sudden hypotension and worsening of abdominal pain. A CT scan of the abdomen revealed a ruptured abdominal aneurysm. Surgical intervention was attempted, but the patient died on the operating table due to massive blood loss.
Post-mortem examination
A restricted post-mortem examination, limited by the family to cardiac biopsy, was performed within 10 hours of death. At the biopsy site 4 cm above the apex, the anterior left ventricular wall measured 2.5 cm in thickness. The left ventricular myocardium showed prominent myocyte hypertrophy with interstitial and perivascular fibrosis. Electron microscopy revealed rectangular intralysosomal crystals characteristic of cystine within interstitial histiocytes lying adjacent to myocytes (fig. 1). A single myocyte containing crystalline inclusions suggestive of cystine was identified (fig 2). Tissue cystine content was measured using a cystine binding protein assay [6]. The cystine content of the left ventricular myocardium was 18.5 nmol half cystine per mg of wet tissue as compared to myocardium from five control autopsy patients without cystinosis in whom it was 0.09–0.081 nmol half cystine per mg of wet tissue. Thus, the tissue cystine level was 1000-fold higher than normal.
Electron microscopy picture of the myocardium with the interstitium showing cystine rectangular crystal (× 15,000).
Myocardium with the interstitial histiocytes with rectangular cystine crystal.
Discussion
Cystinosis is an autosomal recessive disorder characterized by excessive storage of cystine in several organs, including kidney, spleen, liver, lymph node, cornea and thyroid gland [1,2]. Individuals typically present in the first year of the life with symptoms of severe fluid and electrolyte disturbance, a renal Fanconi syndrome, growth failure, and photophobia. Without specific treatment, they progress to end-stage renal failure (ESRD) by end of the first decade. A rare 'late-onset' form of cystinosis presents in older children with renal impairment but not necessarily a Fanconi syndrome. Adults with 'benign' cystinosis have asymptotic corneal cystine deposition but do not have progressive renal damage [1,2,7,8]. The diagnosis of nephropathic cystinosis can be confirmed by slit lamp examination of the cornea which reveals needle shaped, tinsel-like refractive opacities. The levels of cystine can be measured in bone-marrow cells, leukocytes, and cells of the rectal mucosa [1,2,7,8]. Renal involvement is characterized by cystine depositions in the interstitium as well as glomerular and tubular epithelium. With disease progression there is tubular atrophy, interstitial fibrosis, glomerulosclerosis and eventual obsolescence of the glomeruli. Though hypertension is relatively uncommon in these patients. When patients reach ESRD, dialysis or transplantation are reasonable options. Graft survival in these patients is similar to that observed in ESRD of other etiologies [3]. However, cystine continues to accumulate in other organs, leading to multisystem disease. Oral cysteamine can retard progression of renal damage if started early in the course of the disease and if given for prolonged periods [2,9]. Oral cysteamine must be continued even after transplant to prevent ongoing accumulation of cystine [2,9]. By the time oral cysteamine became routinely available the patient had significant progression and therefore was not treated.
Development of aneurysm or pseudoanerysm of a major vessel in patients with nephropathic cystinosis is quite rare. In fact, there is only one other report of a dissecting aortic aneurysm in a 7-year old boy in whom cystine crystals were absent from the aortic wall [10]. As the histopathological examination of pseudoanerysm was not carried out the exact etiology remains unknown. Cardiomyopathy associated with cystinosis was also not reported prior to the patient described here. In the literature there are two other reports of myocardial cystine deposition in patients with cystinosis [11,12]. The first report was of a 22-year old male with cystinosis and myopathy [11] who had a myocardial cystine level of 8.7 nmol half cystine per mg of wet tissue (as compared to 18.5 nmol half cystine per mg of wet tissue patient described here). The other patient was a 43 year old woman with a late-onset cystinosis in whom cystine crystals were observed in the myocardium at autopsy [12]. Neither of the two patients had cardiomyopathies. The myocardial cystine load in those two patients may not have been as heavy as in patient described here. It is possible that if the patient described by us had received cysteamine from a very early age, it could have reduced the cystine content in the myocardium. In another series of 13 post-renal-transplant cystinosis patients with distal vacuolar myopathy the cardiac ejection fractions and the wall motions were within normal limits and none of the patients had evidence of cardiac dysfunction [13]. The skeletal muscle cystine levels measured in those patients were similar to myocardial cystine level in the patient described here [13].
The pericarditis at the age of 11 years was attributed to insufficient dialysis and the response to more aggressive dialysis was considered as a satisfactory confirmation of the diagnosis. As the patient was ambulatory and had no further symptoms of heart failure for years after that episode, its role in the development of a restrictive cardiomyopathy twenty years later is unlikely. Annual echocardiograms and periodic cardiac evaluation allowed a fair evaluation of the cardiac status. The severe concentric left ventricular hypertrophy was out of proportion to the degree of hypertension of a long standing duration. Evidence of diastolic dysfunction manifested by pulmonary congestion and bilateral pleural effusions, with a normal left ventricular ejection fraction, point towards a restrictive cardiomyopathy. This could have been the consequence of long standing hypertension, uremia, a large AV fistula, and altered calcium-phosphorous metabolism due to renal osteodystrophy. However, the presence of cystine crystals in interstitial cardiac histiocytes and one myocardial cell, along with 1000-fold higher tissue cystine content of the left ventricular myocardium point towards the possibility of direct cystine mediated metabolic injury. Notably, his symptoms did not resolve after aggressive medical control of BP, surgical closure of the fistula and digitalis/diuretic therapy. The stress of profuse internal bleeding from the rupture of the pseudoaneurysm in conjunction with a weakened cardiac status, resulted in his death. As the patient described here never received cysteamine, the role of this drug in prevention of cardiomyopathy associated with cystinosis remains to be evaluated.
In summary, we describe the clinical course of a nephropathic cystinosis patient managed from the age of six until his death at the age of 33 years. His long-term survival, associated cardiomyopathy and death due to ruptured pseudoaneurysm are noteworthy.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We are grateful to Dr. W. A. Gahl and staff, including Isa Bernardini at the National Institute of Health for performing cystine tissue determinations.
The national incidence of and risk factors for hospitalized poisonings in renal transplant recipients has not been reported.
Methods
Historical cohort study of 39,628 renal transplant recipients in the United States Renal Data System between 1 July 1994 and 30 June 1998. Associations with time to hospitalizations for a primary diagnosis of poisonings (ICD-9 codes 960.x-989.x) within three years after renal transplant were assessed by Cox Regression.
Results
The incidence of hospitalized poisonings was 2.3 patients per 1000 person years. The most frequent causes of poisonings were immunosuppressive agents (25.3%), analgesics/antipyretics (14.1%), psychotropic agents (10.0%), and insulin/antidiabetic agents (7.1%). In Cox Regression analysis, low body mass index (BMI, <21.6 vs. >28.3 kg/m2, adjusted hazard ratio (AHR), 3.02, 95% CI, 1.45–6.28, and allograft rejection, AHR 1.83, 95% CI, 1.15–2.89, were the only factors independently associated with hospitalized poisonings. Hospitalized poisonings were independently associated with increased mortality (AHR, 1.54, 95% CI 1.22–1.92, p = 0.002).
Conclusions
Hospitalized poisonings were associated with increased mortality after renal transplantation. However, almost all reported poisonings in renal transplant recipients were due to the use of prescribed medications. Allograft rejection and low BMI were the only independent risk factors for poisonings identified in this population.
poisoningsdrug overdosemedication errorbody mass indexrejectiondiabetescomplicationsUSRDSpharmacistBackground
Poisonings are among the most frequent indications for hospitalization in the United States [1]. However, the frequency of hospitalizations for poisonings (drug overdose or toxicity) in renal transplant recipients has been infrequently reported, [2,3] in contrast to its occurrence in kidney donors [4,5]. Because many medications used by transplant recipients can be toxic, it might be expected that renal transplant recipients would have a higher risk of poisonings than the general population, especially in the first post-transplant year when dosages of immunosuppressive medications are usually at their highest. Analysis of the incidence and causes of poisonings represents an opportunity to explore the frequency and risk factors for medical errors, since many such complications may be avoidable. The impact of hospitalized poisonings on subsequent survival after renal transplantation has also not been assessed. Therefore, we analyzed national diata from the USRDS report. Our objectives were to determine the risk factors for and mortality associated with hospitalized poisonings after renal transplantation.
MethodsPatient population
This study used data from the United States Renal Data System (USRDS), using standard analysis files (SAF's) as of May 2000. The variables included in the USRDS standard analysis files (SAF's), as well as data collection methods and validation studies, are listed at the USRDS website, under 'Researcher's Guide to the USRDS Database', Section E, 'Contents of all the SAF's', and published in the USRDS. The demographics of the renal transplant population have been previously described (2001 USRDS report). SAF.TXUNOS was used as the primary dataset, and merged with variables from SAF.HOSP for hospitalization data, and SAF.PATIENTS for dates and causes of death as well as causes of renal disease, as previously reported [6-8]. Patient characteristics and treatment factors were those at the date of transplant. Recipients of organs other than kidneys were excluded.
Outcome definition
We conducted an historical cohort study of the incidence, risk factors and associated patient survival for hospitalized cases of poisonings (based on International Classification of Diseases-9th Modification Diagnosis Codes (ICD9) 960.x-989.x) as a primary discharge diagnosis in renal transplant recipients. Only the primary discharge diagnosis was used to ensure these were active diagnoses, ie, to exclude diagnoses with "history of poisonings." These diagnoses include potential overdoses for heroin, but not for cocaine or other illicit drugs. These diagnoses also include most known causes of environmental exposures, including minerals, pesticides, vaccinations, miscellaneous chemicals and certain foodstuffs. The first hospitalization for poisonings after the first renal transplant for a given individual occurring on or after 1 July 1994 and before 1 July 1998 (which could include a repeat transplant), with followup time truncated at three years was counted in analysis. Hospitalizations were chosen because they were more accessible in the database and less subject to interpretation than outpatient cases of poisonings, especially since the USRDS database has no information on confirmatory studies. Hospitalization data for transplant recipients may be unreliable after the patient has survived ≥ 3 years post transplant, when hospitalization reporting to Medicare for patients 65 years or younger is no longer required. However, Medicare reporting starts immediately after transplant, regardless of preceding dialysis status. All hospitalizations with a primary discharge diagnosis for poisonings were extracted from SAF.HOSP, merged with the transplant file, and hospitalizations outside the range of the study period were excluded. Hospitalizations for poisonings occurring at any time after renal transplant, including after graft failure (censored for patient death), were counted in analysis.
Variables used in analysis
The independent associations between patient factors and hospitalizations for poisonings were examined using multivariate analysis with stepwise Cox Regression (likelihood ratio method) including recipient and donor age, recipient race, gender, weight, pretransplant dialysis (yes/no), year of transplant, duration of dialysis prior to transplantation, total follow-up time, repeat transplant, donor cytomegalovirus serology, dialysis in the first week after transplant (delayed graft function, yes/no), rejection (diagnosis or treatment) occurring during the first six months after transplant, induction antibody therapy, maintenance immunosuppressive medications at time of discharge after transplant surgery, graft loss (analyzed as a time-dependent covariate as previously described), [9] and cause of ESRD (diabetes, systemic lupus erythematosus (because of the potential effect of corticosteroid therapy prior to renal transplantation), hypertension and glomerulonephritis). Episodes of rejection were not restricted to those occurring in the first year, in contrast to studies of allograft function, since there is no evidence that late (vs. early) rejection has a different impact on poisonings. The total cumulative dose of prednisone was not available in the USRDS. Both the USRDS and UNOS track the numbers of days of prednisone administered prior to initial hospital discharge, however values were missing for >90% of patients in both databases and could not be used as a covariate in the above analyses. Maintenance immunosuppressive medication use at the time of discharge after transplantation was also analyzed as a preexisting covariate. Information on use of medications (other than immunosuppressive medications), alcohol, tobacco, or radiologic procedures was not available.
Survival times
We defined patient survival times as the time from when patients had their first renal transplant during the study period to the time of death or most recent follow-up date (which was considered May 2000). For time to poisonings, survival time was defined as the time from first renal transplant until hospitalization for poisonings, with patients censored at death, loss to followup, or end of the study (which was considered 31 December 1999, since this was the most recent hospitalization date). The patient survival probabilities were estimated by using the Kaplan Meier method.
Statistical analysis
All analyses were performed using SPSS 9.0 TM (SPSS, Inc., Chicago, IL). Files were merged and converted to SPSS files using DBMS/Copy (Conceptual Software, Houston, TX). Univariate analysis was performed with Chi-square testing for categorical variables and Student's two-sided t-test for continuous variables. Variables with p < 0.05 in univariate analysis for a relationship with development of hospitalization for poisonings were entered into multivariate analysis as covariates. Kaplan-Meier analysis was used to construct survival plots of time to hospitalized poisonings after renal transplantation. Log-log plots were inspected to assess for proportionality of hazards over time. Stepwise Cox proportional hazards regression (likelihood ratio method) was used to model the association of patient factors with hospitalized poisonings. In order to control for misspecification errors, a random sample of 25% of patients was excluded from analysis and coefficients estimated for this sample. The sample was frozen, and coefficients estimated from the 25% sample. Because hospitalizations for poisonings were uncommon, Poisson Regression was used for validation, using the GENLOG approximation in SPSS. In order to estimate the association of hospitalizations with all-cause mortality, Cox-nonproportional hazards Regression was utilized. Times after hospitalization for poisonings were entered as 1, all other times were entered as 0, as previously described [10,11]. Patients with missing information for variables were excluded from the multivariate models, resulting in models smaller than the total population. Hierarchically well-formed models were used for the assessment of interaction terms [11].
Results
There were 42,096 solitary renal transplant recipients in the United States Renal Data System transplanted from 1 July 1994 to 30 June 1998, of whom 39,628 had complete enough information to calculate survival times. Mean followup was 1.89 ± 1.15 years (median, 1.91 years). Of the study population, 170 recipients were hospitalized with a primary discharge diagnosis of poisonings during the study period, with 188 total hospitalizations. The cumulative incidence of hospitalizations for poisonings during the study period was 2.3/1000 person years. Primary diagnoses for hospitalized poisonings are shown in Table 1. Suicide was not listed as an eventual primary cause of death among patients hospitalized for poisonings, nor was attempted suicide (E956, "Suicide and self-inflicted injuries by cutting and piercing instrument," or E958.9 "unspecified cause of suicide") listed as a secondary or tertiary discharge diagnosis among these patients. The mean length of stay for patients hospitalized for poisonings was 4.05 ± 4.15 days (range, 1–41 days). Among renal transplant recipients with diabetes, the most common cause of hospitalized poisonings was insulin/antidiabetic agents, while in patients who experienced allograft rejection, the most common cause of hospitalized poisonings was immunosuppressive agents.
Most Frequent Diagnoses for Hospitalized poisonings after Renal Transplantation
Diagnosis
ICD9 Code
N (%)
Anti-neoplastic/immunosuppression
963.1
53 (25.3)
Analgesics/Antipyretics
965.x
24 (14.1)
Opiates
965.90
14 (8.2)
Aromatic AnalgesicsA
965.4
6 (3.5)
Analgesics/Antipyretics
965.8
4 (2.4)
Salicylates
965.1
1 (0.6)
Psychotropic Agents
969.x
17 (10.0)
Benzodiazepine/Tranquilizer
969.4
10 (5.9)
Antidepressant
969.0
6 (3.5)
Psychostimulants
969.7
1 (0.6)
Insulin/Antidiabetic
962.3
12 (7.1)
Antihypertensives
972.6
4 (2.4)
Miscellaneous
977.8
15 (8.8)
Hydantoin
966.1
5 (2.9)
Corticosteroids
962.0
2 (1.2)
Antiallergic/antiemetic
963.0
2 (1.2)
A Not elsewhere classified, including Acetanilid, Paracetamol [acetaminophen] and Phenacetin [acetophenetidin]
The time to hospitalization for poisonings is shown in Figure 1. As shown, the risk of hospitalized poisonings were highest in the first few months after renal transplantation, with a consistent risk afterward. Figures 2 and 3 show the time to hospitalization for poisoning stratified by allograft rejection and body mass index, respectively. In Figure 2, analysis was limited to patients who survived at least 6 months after transplant. In figure 3, the risk of poisonings for all transplant recipients was similar for the first six months by both survival plots and log-log plots. Therefore, since this violated the proportional hazards assumption, multivariate analysis was also performed limited to patients who survived at least six months after transplant.
Time to hospitalizations for poisonings after renal transplantation in the United States, 1 July 1994–30 June 1998, N = 39,628. The risk of hospitalized poisonings was highest in the first 1–2 months after renal transplantation, followed by a consistent risk afterward.
Time to Hospitalization for Poisonings after Renal Transplantation, stratified by allograft rejection occurring within 6 months after transplant. Patients with a history of allograft rejection (REJ) had a significantly higher risk of hospitalized poisoning than those who did not (p = 0.01 by Log Rank Test).
Time to Hospitalization for Poisonings after Renal Transplantation, stratified by quartiles of BMI (1 =< 21.6 kg/m2, 2 = 21.6–24.9 kg/m2, 3 = 25.0–28.3 kg/m2, 4 => 28.3 kg/m2). Recipients with BMI < 21.6 kg/m2 had a significantly higher risk of hospitalized poisonings compared to recipients with BMI > 28.3 kg/m2, p = 0.01 by Log Rank Test.
Characteristics of the study population, including results of both univariate and multivariate analysis, are shown in table 2 (see additional file 1). In univariate analysis, factors associated with an increased risk of hospitalized poisonings were low body mass index, graft loss, cadaveric donor, rejection within 6 months after transplant, and diabetes, while male gender was associated with reduced risk of poisoning. In Cox Regression analysis, however, the only two factors independently associated with hospitalized poisoning were low body mass index and allograft rejection. There were no significant interactions between covariates, specifically no interactions between maintenance or induction medications, or between age and body mass index. In analysis limited to recipients age 18 and over, low body mass index (as assessed in Table 3, AHR, 2.74, 95% CI, 1.29–5.82, p = 0.009) and rejection (AHR, 1.88, 95% CI 1.16–3.05, p = 0.01) were again the only factors significantly associated with hospitalized poisonings.
Cox Regression Analysis of Factors Associated with Hospitalized Poisonings
N
HRA (95% CI)
P value
BMI <21.6 vs. >28.3 kg/m2
3.02 (1.45–6.28) B
0.003
Rejection within 6 months after transplant
1.83 (1.15–2.89) B
0.011
N in Final Model
21,575
AAdjusted hazard ratio by Cox Regression analysis of time to hospitalization for poisonings, 95%CI = 95% confidence interval Analysis using these variables was limited to patients who survived at least 6 months after transplant CAnalyzed as a time-dependent covarariate, all times after graft loss = 1, all times before graft loss = 0
Mortality after hospitalizations for poisoning, which was constant over time, is shown in Figure 4. Hospitalizations for poisonings were independently associated with increased mortality in Cox Regression (AHR, 1.54, 95% CI 1.22–1.92, p = 0.002).
Mortality after Hospitalization for Poisonings. The risk of mortality after poisonings was constant over time. Hospitalizations for poisonings were independently associated with increased mortality in Cox Regression (AHR, 1.54, 95% CI 1.22–1.92, p = 0.002).
Discussion
The present study demonstrated that almost all hospitalizations for poisonings after renal transplantation were due to the use of prescription medications, even though ICD9 codes 960–989.x also include poisonings from environmental toxins, vaccines, and anesthetic agents. Because many immunosuppressive medications are toxic, this was not a surprising finding. However, reports from the medical literature have been infrequent, as indicated previously. In fact, a Medline search for the terms "medication error transplant" yielded only 14 papers, [12,13] and none was returned for the search "medication error renal transplant." Therefore, review of the medical literature might give a misleading indication of the frequency of this complication. Although reported hospitalizations for poisonings as a primary diagnosis were not common, hospitalizations for poisonings were independently associated with increased mortality in renal transplant recipients, and also represented potentially avoidable morbidity with a mean length of stay of over 4 days.
Anti-neoplastic/immunosuppressive agents were the most common cause of poisonings in this population, as would be expected. The current study almost certainly underestimates the incidence of serious immunosuppressive drug toxicity in this population. In addition, renal transplant recipients have many potential indications for the use of analgesics/antipyretics, post-operative pain among them [14]. The frequent occurrence of poisonings due to analgesics after renal transplantation may therefore seem understandable. However, although the risk of poisonings was highest in the first few months after transplantation, the increase in relative risk in this time was not visually remarkable (Figure 1), and the risk of poisoning continued in a constant fashion after transplant. There was certainly no early peak followed by a dramatic stabilization in rates of poisonings, as has been demonstrated for hospitalizations for cytomegalovirus disease, [15] contrary to what might have been expected. This suggests the possibility that either the dosing or use of these medications may not be adjusted optimally after renal transplantation.
Benzodiazepines/tranquilizers were the third most common cause of poisonings in this study. It is noteworthy that calcineurin inhibitor toxicity may mimic anxiety or nervousness, [16] which are common indications for the use of benzodiazepines. In addition, many antidepressive agents may raise levels of calcineurin inhibitors [17]. Results of the present study raise the possibility that indications for treatment with benzodiazepines may actually represent toxicities of calcineurin antagonists, which might be best managed by adjusting the dose of the calcineurin inhibitors. Until then, we would suggest that the possibility of other drug toxicities, including drug interactions, be thoroughly excluded before prescribing benzodiazepines in this population. Often overlooked is the effect of erratic compliance with azole antifungals, anticonvulsants or other medications that inhibit or induce calcineurin inhibitors.
The high frequency of poisonings due to insulin/antidiabetic agents is also not surprising given the high frequency of diabetes among renal transplant recipients, although diabetes was not an independent risk factor for poisonings in the present analysis. This suggests that diabetic transplant recipients should be monitored with particularly close attention, perhaps in concert with regular visits with a pharmacist [18] or as part of a disease management program to reduce medical errors [19].
Considering that almost all causes of hospitalized poisonings after renal transplantation were attributed to prescription medications, the risk factors for hospitalized poisonings in the present study, namely allograft rejection and low body mass index, are all the more remarkable. The significance of a low body mass index as an independent risk factor for poisonings in this analysis as a risk factor for hospitalized poisonings, independent of age, suggests the possibility of suboptimal dose adjustment of medications. This particularly applies to calcineurin inhibitors, which are lipid soluble and have a large volume of distribution. Most calcineurin inhibitors are initiated adjusted for patient weight. However, in a small percentage of patients currently recommended doses may still result in toxicity. Area-under-the-curve monitoring gives an estimate of total drug exposure and may help minimize toxicity, particularly in patients with unusual clinical responses to standard dosing [20]. It would be useful to test this hypothesis in future studies.
The association of poisonings with allograft rejection in this category of patients may represent toxicity due to bolus intravenous or high dose oral corticosteroid therapy, or significant drug-drug interactions in this setting, which the database could not determine. However, the findings of the present analysis suggest a need for providers nationally to reevaluate the use of immunosuppressive agents in a systematic fashion after transplantation. The management of renal transplant patients is certainly not uniform; in some centers, the primary transplant surgeon may continue management of the patient up to a year or more after transplant, while in other centers a nephrologist or even primary care physician may assume primary management of the patient after the first transplant month. It is possible that more standardized guidance on dosing and the possibility of drug interactions would be beneficial. Currently, there are no clinical practice guidelines on the dosing of transplant related medications, although certain institutional guidelines do exist [21].
The present study is limited by use of primary hospitalization diagnoses for outcomes. The true frequency of poisonings and medication errors in the renal transplant population is most likely underestimated. Because of the study's retrospective nature, many other factors could not be validated. Patients with multiple potential causes for admission may not have been included among patients with hospitalized poisonings. However, the study is population based, and because accurate coding for hospitalization discharge is required, is likely to be less subject to reporting bias than case reports of poisonings in the medical literature.
The present study could not determine the reason for poisonings. In short, the possibilities of patient error vs. provider error could not be distinguished. However, consultation with pharmacists may reduce both [22]. Given the looming shortage of nephrologists, [23] nurses, and other medical providers, [24] multiple safeguards need to be established to minimize the possibility of error as much as possible in these complex and challenging patients. This is particularly true given the significant associations of both body mass index and allograft rejection with hospitalized poisonings in this analysis, which may indicate that more complex and individualized methods of medication dosing may be necessary after renal transplantation than are currently used. The present study identifies specific targets for investigation and improvement.
Competing interests
None declared.
Authors' contributions
Kevin C. Abbott conceived the study, conducted all analysis and wrote most of the manuscript. Rebecca Viola advised on toxic effects of drugs, and wrote substantial amounts of the discussion. Lawrence Agodoa is Project Director of the NIDDK and supervised all aspects of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
The incidence and risk factors for hospitalized atrial fibrillation have not been previously assessed in a national population of dialysis patients.
Methods
We analyzed the United States Renal Data System (USRDS) Dialysis Morbidity and Mortality Study (DMMS) Wave II in a historical cohort study of hospitalized atrial fibrillation. Data from 3374 patients who started dialysis in 1996 with valid follow-up times were available for analysis, censored at the time of renal transplantation and followed until November 2000. Cox Regression analysis was used to model factors associated with time to first hospitalization for atrial fibrillation (ICD9 code 427.31x) adjusted for comorbidities, demographic factors, baseline laboratory values, blood pressures, dialysis modality, and cardioprotective medications.
Results
The incidence density of atrial fibrillation was 12.5/1000 person years. Factors associated with atrial fibrillation were older age (> = 71 years vs. <48 years), extremes (both high and low) of pre-dialysis systolic blood pressure, dialysis modality (hemodialysis vs. peritoneal dialysis), and digoxin use. Baseline use of coumadin was associated with reduced mortality in patients later hospitalized for atrial fibrillation.
Conclusions
Dialysis patients had a high incidence of atrial fibrillation. This risk was largely segregated among those with established risk factors for atrial fibrillation, and hemodialysis patients. Use of coumadin was associated with improved survival among patients later hospitalized for atrial fibrillation.
Atrial fibrillation is thought to be more common in chronic dialysis patients than in the general population, although population based comparisons are not available. [1] Atrial fibrillation is worthy of separate study from other dysrythmias due to unique aspects of its natural history and management. [1] Risk factors for atrial fibrillation may be more common in dialysis, and include age, cardiac enlargement and an abnormal calcium-phosphorous metabolism. [2-5] However, the incidence, risk factors and associated mortality for hospitalized atrial fibrillation have not been reported for a national population of chronic dialysis patients. Previous studies have also not assessed the effects of cardioprotective medications on atrial fibrillation in dialysis patients. The management of chronic dialysis patients with atrial fibrillation is controversial, since studies of atrial fibrillation have generally excluded patients with chronic renal failure. [6,7] We therefore performed an historical cohort study of the United States Renal Data System (USRDS) Dialysis Morbidity and Mortality Study (DMMS) Wave 2, which includes information on blood pressure, lipid levels, medications, and other important clinical data. Our objectives were to determine the incidence, and risk factors for hospitalized atrial fibrillation (primary hospitalization discharge ICD9 code 427.31.x), occurring after the initiation of dialysis but prior to receipt of renal transplantation, as well as determine risk factors for mortality after hospitalized atrial fibrillation.
Methods
A historical cohort study of the USRDS DMMS Wave 2 was performed. Details on the inception, limitations, validity, variables and questionnaires used in the study are available online at the USRDS researcher's guide website, . This database has been used in many previous cross-sectional [8-11] and longitudinal studies, [12-15] including one by our own institution. [16] Briefly, DMMS 2 was a prospective cohort study of a random sample of 20% of all U.S. hemodialysis patients and virtually all peritoneal dialysis patients starting treatment in 1996 and early 1997. However, because outcomes such as hospitalization and mortality had to be merged with this study from other USRDS files, we did not consider it a true prospective cohort study for the purposes of this analysis. Characteristics of hemodialysis and peritoneal dialysis patients (abstracted from prospective surveys conducted specifically for DMMS 2) were matched and weighted to allow more appropriate comparisons between modalities. Baseline and follow-up data used in the study are shown in Table 1. In addition, a maximum of 15 medications prescribed to each patient at the study start date (day 60 of dialysis) were recorded. From this list, the use of beta-blockers (both cardioselective and non-selective), anti-arrhythmics, coumadin, digoxin, levo-thyroxine, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers (subcategorized as dihyropyridine and non-dihydropyridine), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), and aspirin was determined. For purposes of analysis, cardioselective beta-blockers were atenolol, betaxolol, bisoprolol, metropolol, and acebutolol. Carvedilol was approved for use by the FDA in February 1997, and was therefore not assessed. Blood pressure levels, systolic and diastolic, were obtained as the mean of three readings before and after dialysis, respectively. Pulse pressure, as the difference between systolic and diastolic blood pressure, was also assessed as a covariate in analysis.
Factors assessed in ESRD patients, DMMS Wave 2, 1996 only
Factor
N (%) or mean ± SD
Missing (%)
Univariate association with AFIB
Demographic Factors
N
3374
Female
1572 (46.6)
0
African American
933 (27.7)
0
0.41 (0.24–0.69)
Mean Age (Years)
58.9 ± 15.9
15 (0.4)
Quartiles of Age (vs. <48)
781
48–60
837
3.54 (1.17–10.76)
61–71
867
6.01 (2.06–17.52)
>71
874
15.26 (5.46–42.66)
Cardiovascular Factors
History of Coronary Heart Disease
1091 (34.9)
250 (7.4)
2.35 (1.60–3.44)
History of Congestive Heart Failure
1150 (36.2)
199 (5.9)
1.80 (1.24–2.61)
Chronic Obstructive Lung Disease
271 (8.6)
229 (6.8)
1.93 (1.13–3.40)
Stroke
336 (10.5)
164 (4.9)
1.69 (1.03–2.81)
Previous CABG
325 (9.6)
118 (3.5)
1.87 (1.14–3.06)
Previous PTCA
174 (5.2)
203 (6.0)
2.43 (1.36–4.35)
Previous coronary angiography
352 (10.4)
302 (9.0)
2.40 (1.51–3.81)
2nd quartile of pre-dialysis SBP (130–146 mm Hg) vs. 1st (<130 mm Hg)
0.49 (0.25–0.99)
CKD specific factors
Ever Transplanted (Yes/No)
460 (13.6)
NA
0.15 (0.05–0.48)
Peritoneal Dialysis
1662 (49.8)
37 (1.1)
0.59 (0.40–0.86)
Medications
Aspirin (Yes)
628 (18.6)
NA
Coumadin
198 (5.9)
NA
Beta-blockers
NA
Cardioselective
486 (14.4)
NA
Non-cardioselective
177 (5.2)
NA
Digoxin
142 (4.2)
NA
3.72 (2.17–6.38)
Amiodarone
19 (0.6)
NA
Calcium Channel Blockers
NA
Non-dihyrdopyridine
439 (13.0)
NA
Dihydropyridine
1371 (40.6)
NA
Statins
310 (9.2)
NA
ACE Inhibitors
772 (22.9)
NA
Levothyroxine
330 (9.8)
NA
In column one, data given as the number (% of total) or mean ± one standard deviation of patients. Univariate odds ratios (with significance testing by Chi Square) are the strength of the association of a disease or factor with time to hospitalization for atrial fibrillation (AFIB, ICD9 discharge code 427.31.x), with 1.0 being neutral.
SBP = systolic blood pressure, DBP = diastolic blood pressure, both in mm/Hg, obtained as the mean of three successive blood pressures. CABG = coronary artery bypass graft, PTca = percutaneous transluminal coronary angioplasty LVH = left ventricular hypertrophy, ECHO = echocardiography, EKG = electrocardiogram. Statin = HMG-CoA reductase inhibitor, NA = not available since variable was constructed from multiple variables (15 in the case of medications). Medication use was assessed at day 60 after initiation of dialysis. Comorbidities such as coronary heart disease and congestive heart failure refer to a history of these conditions within ten years prior to the study period. Selective and non-selective beta-blocker use does not total since some patients were on both agents Values for continuous variables more than 3 SD above and below the median were excluded.
Survival status was linked to the DMMS Wave 2 data from the 2000 USRDS Patients Standard Analysis File (SAF.PATIENTS) via unique patient identifiers assigned by the USRDS. The date and cause of death listed in a patient's SAF was obtained from a form submitted to the USRDS by the patient's nephrologist (form HCFA 2746). Patient survival status was complete through 6 November 2000. Hospitalization data was complete through 31 December 1999. Hospitalization data for atrial fibrillation (ICD9 codes 427.31x) were also linked to the DMMS Wave 2 data as above from SAF.HOSP, using only the first hospitalization for a given patient during the study period.
Time to hospitalization for atrial fibrillation was defined as the time from 90 days after the date of the first dialysis session until the first hospitalization for atrial fibrillation, censored for death, receipt of renal transplant, loss to followup or the end of the study period (which was considered 31 Dec 1999 since this was the most recent potential date of hospitalization available in the data files). Survival time was defined as the time from 90 days after the date of the first dialysis session until the date of death, censored for receipt of renal transplant, loss to followup or the end of the study period (which was considered 6 November 2000 since this was the most recent date of death specified in the data files).
Statistical analysis
Univariate analysis was performed with Chi-Square testing for categorical variables (Fisher exact test used for violations of Cochran's assumptions) and student's t-test for continuous variables (Mann Whitney test was used for variables without a Gaussian distribution). Variables with borderline significance (p value < 0.1 in univariate analysis) and those thought likely to have a clinical relationship with atrial fibrillation, such as cardioprotective medications and blood pressure, were entered into the multivariate models. In addition, variables thought to have a likely clinical relationship with atrial fibrillation were also entered into the multivariate model, in accordance with established epidemiological principles. [17] Stepwise (forward likelihood ratio method) Cox proportional hazards analysis was used to assess the association between baseline factors and hospitalized atrial fibrillation, independent of other factors. Log-log plots were inspected to verify the existence of proportional hazards. Estimated hazard ratios (HR) along with corresponding 95% confidence intervals (CI) and p values are reported for all regression covariates. Stepwise logistic regression was used to test the independent association of patient factors with preexisting coumadin and cardioselective beta-blocker use, using the same covariates as Cox Regression above. The association of atrial fibrillation with mortality as a time-dependent variable was tested by Cox non-proportional hazards regression analysis as previously described. [18] Hierarchically well-formed models were used in the assessment of interaction terms. [19] Collinearity diagnostics were evaluated both from the output of the statistical procedures and by manual generation of models including only one of several variables with close relationships (for example, congestive heart failure and left ventricular hypertrophy, which was assessed by chest X-ray, electrocardiogram, and echocardiography). Propensity scores [20,21] have been used to adjust for confounding in baseline medication use. Since this method is primarily undertaken to assess uniformity of risk among strata, model fitting was also tested by fitting models to a 70% random sample of the study population, and then running the same model for the remaining 30% of the population to test for agreement.
Results
A total of 4065 patients were included in the DMMS Wave 2 cohort. Of these, 3621 patients had valid dates for starting dialysis in 1996. From this cohort, 3374 had sufficient information to calculate follow-up times. During the time of the study period, 123 patients were hospitalized for a primary discharge diagnosis of atrial fibrillation, with an incidence density of 12.5/1000 person years.
Use of digoxin and particularly levothyroxine was common in dialysis patients. Among other anti-arrhythmic agents, only amiodarone was used by more than 10 patients at baseline, and so is the only other agent reported. Among the study population, cardioselective beta-blockers were used by 14.6% of patients hospitalized for atrial fibrillation vs. 14.4% of all other patients; digoxin was used by 12% of patients hospitalized for atrial fibrillation vs. 3.8% of all other patients; and coumadin was used by 8.1% of patients hospitalized for atrial fibrillation vs. 5.6% of all other patients. Characteristics of the study population, including univariate associations with hospitalized atrial fibrillation, are summarized in Table 1. Table 2 shows the percentage of patients who were hospitalized for atrial fibrillation by risk group. As shown, there were marked disparities by age, blood pressure, dialysis modality, and history of left ventricular hypertrophy.
Unadjusted rates of Atrial Fibrillation by high-risk groups
Patients in each risk group Hospitalized for atrial fibrillation
Age (Quartiles)
> = 71
76 (8.7)
61–70
29 (3.3)
48–60
14 (1.7)
< = 48
4 (0.5)
Pre-Dialysis SBP, mm Hg (Quartiles)
<130
40 (4.4)
130–146
25 (3.3)
147–162
15 (1.9)
>162
43 (5.0)
Hemodialysis
77 (4.6)
Peritoneal Dialysis
46 (2.8)
LVH by ECHO
Yes
20 (5.5)
No
66 (3.6)
Numbers given in the right column are the N(%) of patients in each risk group who were hospitalized for atrial fibrillation during the study period. SBP = systolic blood pressure, LVH = left ventricular hypertrophy on echocardiography as specified in the DMMS Wave II facility questionnaire, available at
Table 3 shows significant factors associated with hospitalized atrial fibrillation in multivariate analysis by Cox Regression. Factors positively associated with atrial fibrillation were advanced age (> = 71 years vs. age <48 years), hemodialysis (vs. peritoneal dialysis), and baseline digoxin use. Pre-dialysis systolic blood pressure had a "U" shaped association with atrial fibrillation, with highest risk associated with the low and high extremes of blood pressure (Figure 1). However, neither pre-dialysis diastolic blood pressure nor post-dialysis blood pressure were associated with the risk of atrial fibrillation, nor were serum albumin levels. No medications except digoxin were independently associated with atrial fibrillation. There were also no significant interactions between terms, specifically between medications, laboratory data or dialysis modality in patients with preexisting coronary heart disease.
Time to Hospitalization for Atrial Fibrillation by Quartile of pre-dialysis systolic blood pressure (mm Hg), (1 = <130, 2 = 131–146, 3 = 147–162, 4 = >162). As shown, patients at the extremes of predialysis systolic blood pressure had the greatest risk of subsequent hospitalizations for atrial fibrillation.
Cox Regression Analysis of Factors Associated with Hospitalized Atrial Fibrillation
There were 1961 total deaths in the cohort through 6 November 2000. Of the 123 patients hospitalized for atrial fibrillation, 90 died during the study period. Mean follow-up was 2.92 ± 1.14 years. Unadjusted survival after atrial fibrillation and for the entire cohort is also shown in Table 4. Survival was significantly worse after atrial fibrillation in Cox non-proportional hazards regression analysis, adjusted hazard ratio for mortality, 1.54, 95% confidence interval, 1.19–1.99, p = 0.001. The leading cause of death among patients hospitalized for atrial fibrillation was unknown (26%), followed by cardiac arrest of unknown cause (19%), cardiac arrthymia (8%), acute myocardial infarction (7%), atherosclerotic heart disease (6%), cardiomyopathy (3%) and stroke (3%). Of patients hospitalized for atrial fibrillation, the most common secondary diagnosis was hypertensive heart disease (15%), followed by congestive heart failure (11%).
Survival after Hospitalization for Atrial fibrillation
1-year
2-year
3-year
Survival after hospitalization for atrial fibrillationA
75
60
47
Survival for entire cohortB
84
68
55
P valueC
<0.01
<0.01
<0.01
Survival given in %. Atrial fibrillation = primary discharge diagnosis ICD9 427.31, N = 123. A Time to hospitalized atrial fibrillation as defined in the methods section. B Survival time as defined in the methods section. CBy univariate Cox non-proportional hazards Regression Test, atrial fibrillation modeled as a time-dependent variable.
Table 5 shows Cox Regression analysis of factors significantly associated with mortality in patients with hospitalized atrial fibrillation. Only two factors, the baseline use of coumadin (Figure 2) and higher systolic blood pressure, were associated with a lower risk of mortality after hospitalization for atrial fibrillation. Of note, calcium channel blockers, specifically non-dihyropyridine calcium channel blockers, were not significantly associated with mortality in patients with atrial fibrillation. In fact, the hazard ratio for mortality of patients with atrial fibrillation using non-dihydropyridine calcium channel blockers was 1.85, although the p value was 0.21 and 95% confidence intervals were 0.69–4.94. Dihydropyridine calcium blockers, as well as ACE inhibitors and aspirin, were also not significantly associated with mortality in patients with atrial fibrillation. Because use of coumadin might be biased by association with factors favorably associated with mortality, logistic regression analysis was performed to assess factors independently associated with use of coumadin. However, only three factors were independently associated with the use of coumadin: left ventricular hypertrophy on echocardiography (adjusted odds ratio, 1.88, 95% CI, 1.10–3.20, p = 0.02); chronic obstructive lung disease (adjusted odds ratio, 1.98, 95% CI, 1.05–3.74); and aspirin use (adjusted odds ratio, 0.31, 95% CI, 0.14–0.67, p = 0.002). When Cox regression analysis was performed limited to patients who had left ventricular hypertrophy, chronic obstructive lung disease, or did not use aspirin (N hospitalized for atrial fibrillation = 87), coumadin use was still significantly associated with improved survival, adjusted hazard ratio, 0.36, 95% CI, 0.16–0.82, p = 0.014. Models were robust to testing on random samples of the study population (70% vs. the remaining 30%).
Mortality after hospitalization for Atrial Fibrillation, limited to patients hospitalized for atrial fibrillation (M = 123), stratified by patients with baseline coumadin use (COUM) vs. no baseline coumadin use (NO COUM), P < 0.001 by Log Rank Test.
Cox Regression Analysis of Factors Associated with Mortality in Patients with Hospitalized Atrial Fibrillation
P Value
Hazard Ratio
95% CI
Coumadin use Quartiles of Predialysis SBP, mm Hg (vs. <130)
0.014
0.39
0.18–0.83
Quartiles of Predialysis SBP, mm Hg (vs. <130)
130–146
0.012
0.44
0.23–0.84
>162
0.024
0.53
0.31–0.83
Discussion
In agreement with some previous studies in the general population [1,4] and preliminary reports from the USRDS, [22] atrial fibrillation was associated with increased mortality among chronic dialysis patients. In the general population, cardiovascular events, and not thromboembolic events, are the most common cause of death after atrial fibrillation, [23] in agreement with the findings of the present analysis. However, it is also possible that thromboembolic events could have been underdiagnosed due to a low index of suspicion for thromboembolism in chronic dialysis patients, [24-26] because of the high number of deaths with an unknown cause, the low rate of autopsies in this population, or a combination of these factors. Vazquez et al [5] provide further evidence that causes of death may underestimate the incidence of embolic events in chronic dialysis patients with atrial fibrillation. They reported that 35% of chronic dialysis patients with atrial fibrillation suffered thromboembolic events at one year of followup.
Risk factors for atrial fibrillation in chronic dialysis patients were consistent with established risk factors [4,5,27], except for the association with hemodialysis (compared with those on peritoneal dialysis). In addition to age, left ventricular hypertrophy is also a well-established risk factor for atrial fibrillation in the general population. Many markers for cardiovascular comorbidity were associated with atrial fibrillation in univariate analysis (Table 1), but not in multivariate analysis (Table 3). While the efficacy of cardioversion in successful conversion of atrial dysrythmias may be helpful in some settings, it is less useful than the duration of the dysrythmia in patients with atrial fibrillation. [28] However, it has never been established whether the same guidelines apply to dialysis patients. The present study could not assess whether cardiac enlargement affected the success rate of cardioversion in dialysis patients hospitalized for atrial fibrillation.
Pre-dialysis systolic blood pressure exhibited a "U" shaped relationship with the risk of atrial fibrillation, which is similar to the "U" shaped relationship of blood pressure with mortality and other cardiovascular events in chronic dialysis patients. Recent reports have indicated that pulse pressure is a more predictive indicator of cardiovascular events in dialysis patients than either systolic or diastolic blood pressure separately. [29] However, pulse pressure was not independently associated with atrial fibrillation in our analysis.
Hemodialysis was associated with a significantly higher risk of atrial fibrillation than peritoneal dialysis in the present study. It is possible this association represents bias, since hemodialysis patients present for medical care much more frequently than peritoneal dialysis patients, and asymptomatic atrial fibrillation is more readily detected. However, there are legitimate reasons why atrial fibrillation might occur more frequently on hemodialysis than on peritoneal dialysis. First, Foley et al demonstrated that progressive cardiac enlargement was more frequent in hemodialysis patients than peritoneal dialysis patients, [2] although these findings were not replicated in a Japanese study. [30] Other investigators have confirmed that LVH progresses despite adequate blood pressure control in hemodialysis patients. [31] Also, hospitalized congestive heart failure is more common in hemodialysis than peritoneal dialysis patients, especially for those on the renal transplant waiting list. [32] The typical regimen for hemodialysis is likely far less physiologic than peritoneal dialysis. Residual renal function may also be preserved longer in patients on peritoneal dialysis, although this is controversial. [33,34] Some or all of these factors may therefore mediate the higher risk of atrial fibrillation associated with hemodialysis.
The effectiveness of coumadin for atrial fibrillation in chronic dialysis patients has not been assessed previously. In fact, previous reports considered chronic renal failure a contraindication to the use of anticoagulation in atrial fibrillation. [35] Wiesholzer et al concluded that non-rheumatic atrial fibrillation was not an independent risk factor for stroke in dialysis patients. [36] Therefore, Figure 2 suggests several possibilities. First, although the clinical indications for coumadin use in this cohort were unknown, coumadin is often given for a limited period of time [31], after which it is often discontinued, in the absence of known predisposing factors. Therefore, patients who were using coumadin at the time of the study assessment and later discontinued it might not have been taking coumadin at the time they were hospitalized for atrial fibrillation, negating a possible beneficial association of coumadin with mortality after hospitalization for atrial fibrillation. Another possibility is that many patients not taking coumadin at baseline may have later been treated with coumadin after hospitalization for atrial fibrillation. Given the growing evidence that dialysis patients in the modern era may actually be hypercoagulable, primarily due to defects of the fibrinolytic system, [25,37,38] clinical trials of the use of anticoagulation in atrial fibrillation and thromboembolic disorders are warranted. Of note, baseline coumadin use, in contrast to digoxin use, was not associated with hospitalization for atrial fibrillation and therefore presumably was not a marker for patients with pre-existing atrial fibrillation.
The decision to use coumadin in patients with atrial fibrillation in whom NSR cannot be established is based on an assessment of both their risk of serious bleeding and their risk of stroke. Coumadin therapy has been associated with improved survival in patients with reduced LV systolic dysfunction in the absence of dysrythmias as well, primarily through reduction in cardiovascular events rather than reduction of thromboembolic events. [39] This may be one explanation for the association of coumadin use with reduced mortality in chronic dialysis patients hospitalized for atrial fibrillation in the present study.
Study limitations
The present study was observational and not randomized, and therefore associations should not be construed as causative. The study is a random sample of all hemodialysis patients, not the total population, although the peritoneal dialysis population was almost fully captured. We did not have access to information on treatment or conversion to NSR in patients who were hospitalized for atrial fibrillation, nor did we have information on the type of atrial fibrillation for which patients presented and how it was classified within ACC/AHA/ESC guidelines. [40] We did not have access to information on which patients had self-limited episodes of atrial fibrillation vs. those who had persistent atrial fibrillation, nor the duration or presence of atrial fibrillation prior to hospitalization or the study start. We did not have information on left atrial size other than the presence of LVH on echocardiography. We also did not have information on thyroid functions (other than baseline use of thyroxine), alcohol use, or pre-existing atrial dysrythmias. Information bias could have arisen due to mistakes in coding or spelling, especially for medications. We were unable to follow changes in variables over time. Therefore, we could not follow changes in blood pressure, laboratory values, dialysis adequacy, or medications. We could not assess other factors that have shown to be associated with atrial fibrillation, such as hypokalemia or calcified aortic/mitral valves, [41] although there are many similarities between the risk factors for valvular heart disease and atrial fibrillation in chronic dialysis patients. [42] Our use of atrial fibrillation as an outcome variable required survival to reach hospitalization, and thus sudden death was not considered as an outcome. However, causes of death, especially if not verified by autopsy, are also of questionable validity in comparison to the rates of total mortality. [43]
In summary, the present observational, non-randomized study confirms the high incidence and mortality of atrial fibrillation in chronic dialysis patients. Traditional risk factors for atrial fibrillation such, as older age, were confirmed. Hemodialysis, possibly due to a greater frequency of progressive cardiac enlargement or more frequent observation, was also identified as a risk factor for atrial fibrillation. Of the greatest potential importance, the use of coumadin at the start of the study was associated with lower mortality in patients later hospitalized for atrial fibrillation. These findings should be confirmed in clinical trials.
Author' contributions
KCA conceived the original concept, performed the analyses, and wrote most of the manuscript
FCT collaborated in the manuscript and was instrumental in the initial preparation of DMMS 2 files for analysis, especially medications
AJT collaborated in the manuscript providing background on atrial fibrillation and its role in chronic renal failure.
LYA, as Project Director for the NIDDK, is a world expert on the USRDS and was instrumental in the interpretation of USRDS files.
Competing interest
None declared
Pre-publication history
The pre-publication history for this paper can be accessed here:
Atherosclerotic ischemic renal disease is a frequent cause of end-stage renal failure leading to dialysis among the elderly; Its prevalence is inferred from autopsy or retrospective arteriographic studies. This study has been conducted on 269 subjects over 50 with hypertension and/or CRF, unrelated to other known causes of renal disease.
Methods
All 269 patients were studied either by color-flow duplex sonography (n = 238) or by renal scintigraphy (n = 224), and 199 of the 269 patients were evaluated using both of these techniques. 40 patients, found to have renal artery stenosis (RAS), were subjected to 3D-contrast enhancement Magnetic Resonance Angiography (MRA) and/or Selective Angiography (SA). An additional 23 cases, negative both to scintigraphy and to ultrasound study, underwent renal angiography (MRA and/or SA).
Results
Color-duplex sonography, carried out in 238 patients, revealed 49 cases of RAS. MR or SA was carried out in 35 of these 49 patients, and confirmed the diagnosis in 33. Color-duplex sonography showed a PPV value of 94.3% and NPV of 87.0% while renal scintigraphy, carried out in 224 patients, had a PPV of 72.2% and a NPV of 29.4%. Patients with RAS showed a higher degree of renal insufficiency compared to non stenotic patients while there were no differences in proteinuria. RAS, based on color-duplex sonography studies, was present in 11% of patients in the age group 50–59, 18% in the 60–69 and 23% at age 70 and above.
Conclusions
A relatively large percentage of the elderly population with renal insufficiency and/or hypertension is affected by RAS and is at risk of developing end-stage renal failure. Color-duplex ultrasonography is a valid routine method of investigation of population at risk for renal artery stenosis.
Ischemic renal disease is a renal insufficiency associated with main renal artery stenosis or occlusion [1,2]. Chronic ischemia causes progressive renal damage with atrophy and permanent loss of structural integrity. The cause-and-effect relationship between anatomic renal artery disease and renal function is often difficult to establish [3]. Deterioration of renal function derives not only from reduction in blood flow, but also from long term hypertension and concomitant risk factors like diabetes, hyperlipidemia, atheroemboli and aging [4].
Ischemic nephropathy has been recognized in the last decade as a distinct cause of renal insufficiency, especially in patients with advanced age [5]. A substantial percentage of patients with endstage renal failure which requires entering a dialysis program, is primarily affected by ischemic renal disease [6,7]. Estimates of the prevalence of renovascular disease derive mainly from autopsy and angiographic studies of patients with renovascular hypertension or atherosclerotic disease involving other districts of the arterial tree [8-11]. Large population studies based on non invasive diagnostic methods are lacking.
The aim of this study is to evaluate the prevalence of renal artery stenosis in a relatively large population aged 50 and above, with arterial hypertension started over 50 years of age, or with chronic renal failure of late onset not explained by other diagnoses. The study is also aimed at comparing different diagnostic techniques for the detection of renal artery stenosis as well as clinical and biochemical indicators of renovascular disease.
Methods
This study has been carried out on 269 patients, consecutively referred to the nephrologic outpatient clinic for arterial hypertension and/or chronic renal failure. Patients were selected according to the following criteria: age above 50 years, arterial hypertension starting after this age and/or chronic renal failure of late onset, with or without hypertension and unexplained by other known renal pathologies such as chronic glomerulonephritis, chronic tubulointerstitial disease, polycystic kidneys and obstructive renal disease. Diabetes mellitus was not an exclusion criterion and cases of suspected diabetic nephropathy were included when extrarenal atherosclerotic arterial occlusive disease coexisted. None of the patients was on dialysis treatment or had received a kidney transplantation.
269 patients, 150 males and 119 females, were characterized for clinical and standard biochemical parameters, as reported in Table 1, and subjected to non invasive diagnostic manoeuvres, such as renal radionuclide scintigraphy and color-duplex ultrasonography of renal arteries. In particular, all 269 patients were studied either by color-flow duplex sonography (n= 238) or by renal scintigraphy (n= 224), and 199 of the 269 patients were evaluated using both of these techniques. 40 of 58 patients who were positive for renal artery stenosis with one or two techniques, underwent renal angiography either with three-dimensional contrast-enhanced Magnetic Resonance Angiography (3D-CE-MRA), and/or with Selective Angiography (SA). 7 of the remaining 18 patients could not be subjected to Magnetic Resonance Angiography because of technical problems (metallic prosthesis or pacemaker); 7 refused the procedure due to claustrophobia. In the remaining 4 cases, renal artery stenosis was on a small kidney. In particular, of 49 patients with a positive ultrasonography, 35 accepted to be submitted to renal angiography, while of 24 patients with a positive scintigraphy, 18 accepted to undergo renal angiography. In addition, 17 patients with negative results for renal artery stenosis with the non invasive manoeuvres, accepted to be submitted to Magnetic Resonance Angiography.
Clinical and laboratory data of the patient population (M ± SD;percent)
normal values
Patient, n°
269
Sex, (M/F)
150/119
Age, years
65,8 ± 11,9
Weight, Kg
70,8 ± 11,9
Creatinine, mg/dl
1,7 ± 1
(0,6–1,2)
Cr. Clearance, ml/min
54,8 ± 30,2
(90–120)
Urea, mg/dl
61,2 ± 33,2
(10–45)
Uric acid, mg/dl
6,5 ± 1,8
(3–7)
Sodium, mEq/L
142 ± 4,3
(136–145)
Potassium, mEq/L
4,6 ± 0,5
(3,6–5)
Cholesterol, mg/dl
243 ± 53
(150–220)
HDL cholesterol, mg/dl
49 ± 13,6
(>45)
Triglycerides, mg/dl
162 ± 83
(50–200)
Urinary protein, mg/24 h
654 ± 1103
(<150)
Systolic BP, mmHg
170 ± 25
(<140)
Diastolic BP, mmHg
96 ± 14
(<90)
Duration of hypertension, years
7,5 ± 6
n°
%
Arterial hypertension
238
88,5
Familiar hypertension
113
42,0
Smoking
89
33,0
Diabetes mellitus
42
15,6
Hypertensive retinopathy (stage III and IV) (163/269)
4
2,5
Interventricular septum (154/269)
121
78,5
Stenosis of epiaortic arteries (111/269)
60
54,0
Stenosis of iliofemoral arteries (52/269)
33
63,5
Renal dimensions asymmetry (>11 mm)
135
50,2
Doppler ultrasonography was carried out after fasting, following a three day of a low fibre diet and without smoking for a minimum of six hours before the procedure. Patients were studied with an Acuson 120 XP/4 (Acuson Corp., Mountain View, CA), equipped with a 3.5 MHz transducer, with longitudinal anterior, lateral and oblique approach, with at least threefold sampling of parameters along the artery. Subjects were asked to lie in the supine position for the study of the abdominal aorta and the origin of the renal arteries. Subsequently patients were turned on either side for the study of the renal arteries at the hilum and of parenchymal perfusion.
Standard criteria for the diagnosis of significant renal artery stenosis (≥ 60%) are [12]: 1. Systolic peak velocity above 180 cm/sec (sensitivity 94%, or 98% when peak velocity >200 cm/sec). 2. Renal aortic ratio, defined as the ratio between systolic peak velocity in the renal artery and systolic peak velocity in the abdominal aorta in the suprarenal tract, with a normal value of <3.5.
Renal radionuclide scintigraphy was performed with a gamma camera (Starcam 4000, General Electric, USA) with 99mTc-DTPA (diethylenetriaminepentaacetic acid) in 125 patients and with 99mTc-MAG3 (mercaptoacetyltriglycine) in 99 patients, with a total of 224 patients. MAG3 was chosen in patients with creatinine clearance <25 ml/min. Diuretics and/or ACE inhibitors were discontinued at least three days before, if treatment was underway. Criteria of positivity for renal artery stenosis were: Parenchymal transit time >4 minutes; a difference in split renal function >30%; Tmax >5 min, with a difference between kidneys >1 min. A captopril test was carried out in 161 patients, by administering captopril 50 mg p.o. one hour before the administration of the tracer. Following captopril administration, criteria of suspected stenosis were: with 99mTc-DTPA, a fall in glomerular filtration rate of the affected side >5%; with 99mTc-MAG3, an increase of at least 0.15 of the 20 minutes/peak count ratio (ratio between the activity after 20 minutes and peak radioactivity); or a lengthening of >2 min of Tmax value, or a delay of tracer elimination in the pelvis of >2 minutes [13,14].
Magnetic Resonance Angiography was performed with a 1.5 T magnet (Vision Plus; Siemens, Erlangen, Germany) with 25 mT/m gradient strengtth and 600 microseconds gradient rise time. In all cases a Flash 3D T1-weighted sequence (TR = 4.6; TE = 1.8, FA = 30°, NEX = 1, Slab thickness = 120, FOV = 500, matrix = 200 × 512, n° partitions = 34) was used. The acquisition time of the sequence was 23 s. A phased array body coil was used. Before the acquisition a test bolus was performed (TR = 5.8, TE = 2.4, FA = 10, Thickness = 10, matrix-128 × 256, FOV = 400, NEX = 40), injecting 2 ml of Gd-DTPA at 2 ml/s; in order to establish the correct delay time. The examination was performed administering 0.2 mmol/kg of Gd-DTPA with a power injector (Spectris, Medrad) at a flow rate of 2 ml/s. Images were reconstructed using a standard maximum intensity projection (MIP) algorithm and were reviewed by two radiologists obtaining a final report by consensus. This is a more advanced technique than TOF-MRA (Time of Fly) and PC-MRA (Phase-Contrast), and is especially useful for detection of renal artery stenosis. It is considered to have specificity and sensitivity values approaching selective angiography [15,16].
MR angiography was carried out in a total of 39 patients, 22 with positivity of at least one of the non invasive manoeuvers, and 17 cases with negative results. Selective angiography was carried out in 32 patients among those with at least one positivity for renal artery stenosis, diagnosed with non invasive techniques. Stenosis was considered significant both with MRA and SA when at inspection the reduction in arterial lumen diameter was ≥ 60%. In 14 patients, both MRA and SA were performed. The presence of renal artery stenosis, identified by MRA, was confirmed in all cases by SA. The total number of angiographic studies was 57.
Statistical analysis
Following a preliminary descriptive analysis, since the available data did not allow to evaluate sensitivity and specificity, contingency tables were created only for evaluation of positive and negative predictive values for the diagnostic techniques,. For each index the confidence interval at 95% level was calculated. Mann-Withney non parametric and Pearson χ2 tests were used. Significance threshold level was considered to be p < 0.05.
Results
The number of patients, sex distribution, average age, blood pressure values and several biochemical parameters for the entire population together with other clinical parameters of the population are reported in Table 1.
The population was found to have, on average, a moderate renal insufficiency, as shown by the mean creatinine clearance of 54.8 ± 30.2 ml/min. 88.5% of the patients had arterial hypertension. Hypertensive organ damage was evidenced by a thickening of interventricular septum in 78.5% of cases, revealed by echocardiography. In addition, on average the patients had increased cholesterol levels. Carotid and lower extremities artery stenosis were found in 54% and 63% respectively, of the patients studied. Diabetes mellitus was present in 15.6 %.
A synopsis of the results of the non-invasive techniques is reported in Table 2. Renal color-duplex sonography, performed in 238 patients, showed that 49 (20.6%) were affected by renal artery stenosis. MRA and/or SA were performed in 35 of these 49 patients. The diagnosis was confirmed in 33 cases, while 2 cases were false positive. 23 of 189 patients with a negative result of color-duplex sonography, accepted to undergo a renal angiography. In 20 cases the angiography confirmed the negativity, while 3 cases were found to be false negative. On the basis of these data the positive predictive value of color-duplex sonography was 94.3% (80.8–99.3% Confidence Interval) and the negative predictive value was 87.0% (66.4–97.2% C.I.).
Synopsis of results of non-invasive techniques
DDS
RS
Pos
Neg
Not Tested
All
Pos
22
0
2
24
Neg
27
150
23
200
No tested
0
39
6
45
All
49
189
31
269
Renal scintigraphy was carried out in 224 patients. 24 patients had a positive test for renal artery stenosis. 18 of 24 patients underwent an angiography. Positivity for renal artery stenosis was confirmed in 13, while 5 cases were false positive. Of 200 cases with negative results, 17 were further examined with angiography; 12 of these cases were false negative. The positive predictive value was 72.2% (46.5–90.3% C.I.), while the negative predictive value was 29.4% (10.3–56.0% C.I.).
The comparison between patients with and without renal artery stenosis (Table 3), based on the results of color-duplex sonography, has shown significant difference in the extent of renal failure. The levels of proteinuria did not reach statistical difference probably due to the groups sizes. Significant difference was also found in the prevalence of renal asymmetry and in percent of patients with diabetes mellitus (Table 3). 59% of the patients had a unilateral stenosis while it was bilateral in 41%. The comparison of renal damage between patients with unilateral and bilateral renal artery stenosis in reported in Table 4.
Humoral parameters (M ± SD) and percentage of clinical findings in patients with and without renal arterial stenosis
with
without
P
Patient, n°
49
189
Age, years
68,1 ± 8
66,3 ± 8,9
0,18
Creatinine, mg/dl
2,5 ± 1,5
1,6 ± 0,06
<,00001
Cr. Clearance, ml/min
34, 7 ± 19,2
58,2 ± 30,8
<,00001
Urea, mg/dl
84 ± 38
57 ± 29
<,00001
Uric acid, mg/dl
6,9 ± 1,8
6,4 ± 1,8
0,07
Sodium, mEq/L
142 ± 4,9
142,2 ± 4
0,6
Potassium, mEq/L
4,6 ± 0,5
4,5 ± 0,4
0,52
Cholesterol, mg/dl
236 ± 58
229 ± 54
0,15
HDL cholesterol, mg/dl
45,6 ± 12
49,6 ± 12
0,06
Triglycerides, mg/dl
176 ± 74
156 ± 76
0,08
Urinary protein, mg/24 h
659 ± 672
595 ± 980
0,14
Systolic BP, mmHg
175 ± 29
166 ± 21
0,07
Diastolic BP, mmHg
93 ± 15
94 ± 13
0,44
%
%
Smoking
28,4
31,8
ns
Diabetes mellitus
29,8
13,6
<,01
Hypertensive retinopathy (stage III and IV)
6,1
6,1
ns
Interventricular septum
88,0
74,5
ns
Stenosis of epiaortic arteries
30,0
15,2
ns
Stenosis of iliofemoral arteries
57,1
50,0
ns
Renal dimensions asymmetry (>11 mm)
75,6
43,9
<,001
Comparison of renal damage between patients with mono-and bilateral renal arterial stenosis.
monolateral
bilateral
P
Creatinine, mg/dl
2,4 ± 1,5
3.0 ± 1,8
0,22
Cr. Clearance, ml/min
41,2 ± 21,9
29,6 ± 16,5
0,15
Urea, mg/dl
76.0 ± 46,4
97,6 ± 36,5
0,08
Urinary protein, mg/24 h
575 ± 627
469 ± 329
0,7
Renal artery stenosis was found in 11.1% of patients in the 50–59 age group, in 18% in the decade 60–69 and 23% of patients above 70 years of age.
Discussion
The primary aim of this study is to evaluate the diagnostic role of the non invasive approach to renal artery stenosis and ischemic nephropathy in the elderly, to compare the clinical and biochemical data of patients with and without ischemic nephropathy and to establish the prevalence of this disease among the selected population in the different decades of life.
Based on a comparison between non invasive techniques such as renal scintigraphy and color-duplex sonography, and the diagnostic gold-standard of arteriography, that is both Magnetic Resonance and/or Selective Angiography, we attempted to assign predictive values to these non invasive techniques. There is sufficient agreement between MRA and SA to combine the outcomes for use as a goldstandard. The good agreement was probably due at least in part to the particular sensitivity and specificity of the MR technique utilized in this study, which is considered superimposable to selective angiography [15-17], at least for the stenosis at the origin or at main renal artery. All our cases tested with both arteriographic techniques had comparable results as far as the presence of renal artery stenosis is concerned. The identity of results has been reported by De Cobelli et al. [18] and by Ghantous et al [15].
There are some limitations in the use of Magnetic Resonance, such as claustrophobia and the presence of pacemakers or metallic prosthesis. Since Magnetic Resonance is generally not employed on a routine basis for screening purposes in large populations due to cost and availability, the less expensive non-invasive imaging techniques should come first in the preselection of affected patients.
In the comparison between color-doppler sonography and radionuclide scintigraphy, unfortunately based on a limited number of patients, the former was apparently clearly superior in terms of positive and negative predictive values, in agreement with data of the literature [19]. However this technique requires an experienced operator in order to reach high levels of reliability. On the other hand, in screening for ischemic nephropathy, among patients with renal insufficiency, radionuclide scintigraphy was clearly less reliable, either using DTPA or MAG3. This technique may give better results in terms of sensitivity and specificity in a population screened for renovascular hypertension, in general with normal renal function. Scoble et al [19] have underlined that radionuclide scintigraphy may give better results in the screening of renal artery fibrous dysplasia, where hyperreninism is more frequent, while it is of lower diagnostic value in the diagnosis of renal ischemia.
Comparing patients with and without renal artery stenosis in an elderly population, a higher grade of renal insufficiency in patients with renal artery stenosis emerged clearly, whether the stenosis was unilateral (59%) or bilateral (41%). Renal insufficiency was found in both groups (Table 4). Therefore renal artery stenosis in the elderly is associated to atherosclerotic vascular involvement of both kidneys, with bilateral damage to renal function. It is misleadingly simplistic to consider the overall renal damage as deriving from reduced blood perfusion of the kidney, due to renal artery stenosis. The poor correlation existing between the extent of renal artery stenosis and degree of renal insufficiency has been reported previously [3,20]. As for arterial hypertension, no difference was found between stenotic and non stenotic patients (Table 3).
The observation concerning proteinuria is also of interest. The extent of proteinuria did not seem to differ between patients with and without renal artery stenosis, probably due to the given groups sizes. Therefore the exclusion of patients with proteinuria higher than 500 mg/day from further diagnostic procedures for renal artery stenosis, appears to be unjustified [21,22]. More recently the occurrence of marked proteinuria in patients with ischemic nephropathy has been reported [23,24]. Therefore our data support the hypothesis that atherosclerotic renal involvement may be accompanied by pathologic proteinuria. On the other hand heavy proteinuria is of frequent occurrence in nephroangiosclerosis, focal glomerulosclerosis and cholesterol emboli, conditions found in ischemic nephropathy [25-27] among the elderly.
The data of the medical literature on the prevalence of ischemic renal disease in the elderly are scarce, derived mainly from autoptic findings or from arteriography studies performed for coronary artery disease [7-10]. Our results based on color-duplex ultrasonography findings indicate an elevated occurrence of this disease in this cohort, with an increasing prevalence from the sixth decade, and reaching above 20% after age 70. Therefore the presence of ischemic nephropathy should be considered in elderly patients affected by unexplained renal insufficiency and/or hypertension of late onset. The possible evolution towards further deterioration of renal function in patients with renal artery stenosis and the results of relatively non-invasive renal stenting procedures for prevention and treatment, are indication for an extensive screening of at risk subjects.
Competing interests
None declared
Authors' contributions
1. GC conceived and coordinated the study
2. SC clinical work
3. SL expert in DDS
4. EM clinical work and participation in design of the study
5. IN biostatistical work
6. GR carried out renal scintigraphy
7. MR magnetic resonance performance
8. GV collaboration in renal scintigraphy
9. DS clinical laboratory work
10. MF clinical work and assistance in coordination
11. AZ endovascular surgery and selective angiography
12. RC DDS expert
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The study was supported by funds of La Sapienza University. The skillful contribution of Ms. Antonina Scuderi is acknowledged
As EPO treatment of chronic anemia of advanced renal disease is now the standard of care we examined if such treatment may slow the progression of renal function decline.
Methods
Data of 18 pre-ESRD patients were analyzed retrospectively 12 months prior and prospectively 12 months after the initiation of EPO. Mean creatinine was 5.0 ± 1.8 mg/dL (Mean ± SEM) when starting EPO at a weekly dose of 5000 ± 500 units once the hematocrit was below 30 %. EPO dose was titrated monthly for a hematocrit between 33.0% and 37.0%. Metabolic complications and hypertension were controlled.
Results
At month_0 the average blood pressure was 148/76 ± 5/4 mmHg and at month_12 it was 145/73 ± 6/3 mmHg (p = 0.75 by 2 tailed paired Student's t test). 12/18 patients were on an ACE-i or ARB before month_0 and 14/18 were on it after (p = 0.71 by Fisher's 2 tailed exact test). The average hematocrit rose from 26.9% ± 0.6 to 33.1 % ± 0.1. When linear regression analysis was applied to pre- and post-EPO 1/creatinine data the mean rate of decline was -0.0140 ± 0.0119 (mean ± SD) and -0.0017 ± 0.0090 (non-parametric Wilcoxon matched pairs signed rank sum test: Z value: -2.91; P = 0.004) respectively. 5/18 patients did not require dialysis 12 months after starting EPO (month_0).
Conclusion
Treatment of the anemia of chronic renal failure with erythropoietin, when instituted together with vigorous metabolic control may slow the rate of renal function decline.
Background
Chronic renal failure is often a progressive rather than a stable process which most frequently leads to end stage renal disease (ESRD). There are very few maneuvers that can ameliorate the course of renal insufficiency such as the use of ACE-inhibitors, aggressive blood pressure control or vigorous glucose control. As the renal function progressively declines complications of renal failure such as acidosis, uremia or volume overload become more and more significant and eventually may be the principal reason for the initiation of renal replacement therapy. These complications of chronic renal failure, however, can be managed for awhile with medical therapy. We noted that the control of anemia of renal failure not only manages the complication of chronic renal failure but also slows the rate of renal function decline or in some cases it arrests the process. As the treatment of anemia of pre-ESRD patients with EPO is now the standard of care we investigeted whether such treatment may indeed slow the progression of renal function decline.
MethodsStudy design
This is a prospective, observational-clinical cohort study of patients treated for cohort anemia. Data were analyzed to compare the rate of progression of renal disease based on serum creatinine values before the initiation of human recombinant erythropoietin (EPO) [Procrit® by Ortho Biotech] and after.
Inclusion criteria
Adult patients who developed a renal disease who progressed to the stage requiring treatment of their anemia by subcutaneous EPO. Patients had to have at least three months' data prior to the EPO therapy followed by at least three months' data after therapy was started in our outpatient nephrology clinic at a tertiary referral center. Data of patients of any renal disease with a severe renal failure were examined including glomerulonephritides, lupus nephritis, diabetic nephropathy and unknown renal diseases.
Exclusion criteria
Transplant recipient status, acute renal failure or anemia identified as other than secondary to chronic renal failure were exclusion criteria.
Methods
Patients with severe renal failure were identified at the time of initiating EPO treatment in a clinical setting. Data of 35 such patients were examined and 18 qualified for data analysis. Data were analyzed in a retrospective manner 1 year prior and prospectively one year after the initiation of EPO. The mean age of patients was 67.3 ± 0.05 (Mean ± SEM) years at the start of the observation, 2 were African Americans and one was Hispanic, 6 patients were women and 7 had type II diabetes mellitus. The exact pathology for the renal failure was not known in the majority of patients, but one was known to have polycystic kidney disease. The patients' mean creatinine was 5.0 ± 1.8 mg/dL at the time of starting subcutaneous EPO at a starting weekly dose of 5000 ± 500 units depending on the degree of anemia when the patient's hematocrit was less than 30 mg%. EPO dose was adjusted monthly to keep the hematocrit between 33.0% and 37.0%. All patients were monitored with monthly physical examination as well as laboratory tests that included hemoglobin, hematocrit, iron saturation, Blood Urea Nitrogen, creatinine and electrolytes. Iron was supplemented by oral iron preparations described to be taken daily but when the iron saturation reached less than 20% then intravenous iron dextran was given in an outpatient setting in divided doses to reach 1 gm per course. Metabolic acidosis was controlled to keep the "CO2" (bicarbonate) on the electrolyte panel greater than 22 mmol/L by either oral sodium bicarbonate or sodium citrate. Potassium was primarily controlled by controlling the metabolic acidosis, diuretics or occasionally by oral sodium polystyrene. Blood pressure control was maximized with a preference for an angiotensin converting enzyme inhibitor (ACE-i) or angiotensin receptor blocking agent (ARB). A nephrologist saw all patients monthly.
End points
Patients were monitored and their data collected until they required dialysis or death (there was none) or lost to follow up (1/18) whose data were censored. Necessity for dialysis was decided when patients started to have uremic symptoms or metabolic complications that could no longer be managed medically.
Results
5 of the 18 patients did not require dialysis at the end of the 12 months period after starting EPO (month_0). At month 0 the average blood pressure was 148/76 ± 5/4 (mean ± SEM) mmHg and at month_12 the mean blood pressure was 145/73 ± 6/3 mmHg (p = 0.75 by 2 tailed paired Student's t test). 12/18 patients were on an ACE-i or ARB before month 0 and 14/18 were on it after (p = 0.71 by Fisher's 2 tailed exact test). After starting EPO the average hematocrit rose from 26.9 ± 0.6 to 33.1 ± 0.1 where they were maintained. When linear regression analysis was applied to pre- and post-EPO 1/creatinine data the mean rate of 1/creatinine decline was -0.0140 ± 0.0119 (mean ± SD) and -0.0017 ± 0.0090 (using the non-parametric Wilcoxon matched pairs signed rank sum test: Z statistical value of -2.91 which corresponds to a two tailed P = 0.004) respectively. The slope of the creatinine rise changed to a negative value in 6 of the 18 patients' case with this treatment indicating actual reversal.
Discussion
Although treatment the anemic complication of renal failure has been available since the late 1980's when EPO became available treatment of it was delayed perhaps because of initial concerns that EPO treatment may actually worsen the progression of renal disease. This suspicion was based on rat studies that indicated that perhaps anemia has a nephro-protective effect and the correction of it would accelerate renal injury [1,2]. However, in a double-blind placebo-controlled clinical trial there was no adverse renal effect demonstrated over a 12-week period [3] as expressed by 1/creatinine versus time. Similarly, in a 24-week prospective open-label study the rate of decline in kidney function was not altered by the use of EPO [4]. We also used 1/creatinine values as the index of renal function instead of creatinine because of the linear correlation of 1/creatinine with the glomerular filtration rate as opposed to the pure creatinine values [5,6].
Lim at al. [7] who also compared the slopes of linear regression lines of the renal function of 26 patients over 24 months found that neither EPO administration nor a normal hematocrit had any adverse effect on the renal function. Others [8] have also confirmed that normalization of the hematocrit with EPO does not accelerate the loss of the residual renal function.
Our findings indicate that the treatment of anemia of chronic renal failure may prolong the renal survival. This is consistent with others' findings. A pediatric study from Germany by Krmar et al.[9] as well as by Kuriyama [10] who compared treated and untreated patients had similar results. When compared, the treated patients had a lower rate of progression of the renal decline in both studies albeit the number of patients in the pediatric study was 11 thus no statistical significance could be shown.
Recently, Jungers [11] also found that the rate of progression of renal failure is slower in patients who receive EPO than those who do not with a delayed need for initiation of dialysis.
The possibility of explanations is rather wide. One could speculate better oxygen delivery to the ischemic tissues may be responsible for this beneficial effect. This process would lower the ischemic burden perhaps by beneficial effects of EPO on endothelial dysfunction as evidenced by thrombomodulin/ creatinine ratios when treated with EPO [12]. Other mechanism could involve the suppression of hormones such as angiotensin and aldosterone, both of which are known to have important roles in tissue damage and fibrosis. Since captopril was shown [13] to increase the effective renal plasma flow and renal blood flow in anemic pre-dialysis patients and this effect could no longer be observed when treated with EPO it is possible that the correction of anemia may involve these hormones that act of the efferent arterial resistance or other hemodynamic modulators. Other fibrosis promoting participant substances could also include TGF-beta yet their role and response to EPO remains to be shown. While this is only an observational study from the clinical practice with a relatively small number of patients it could point towards the necessity for a larger prospective study designed to answer the question raised here whether the administration of EPO in the pre-ESRD population can indeed reverse or halt a process of continuous renal decline.
Conclusions
Treatment of the anemia of chronic renal failure with subcutaneous erythropoietin, when instituted together with the vigorous control of the metabolic complications of chronic renal failure may slow the rate of renal function decline and in some cases may even reverse the process of renal function loss.
Competing interests
As part of our full disclosure statement we declare that we had no financial or any other interest nor did we have any external support for this study.
Authors' contributions
MT conceived of and collected the data and compiled wrote the study.
SK had a major share in evaluating and interpreting the data as well as writing the manuscript.
MPC is a mathematician and statistician who performed the appropriate calculations and statistical analysis.
1/creatinine values before and after EPO treatment. The rate of progression of renal function decline as reflected by the linear regression lines slowed from -0.0140 dL/mg·month ± 0.0119 (mean ± SD) to -0.0017 dL/mg·month ± 0.0090 (non-parametric Wilcoxon matched pairs signed rank sum test: Z statistical value of -2.91 which corresponds to a two tailed P = 0.004) after starting EPO at Month 0.
Pre-publication history
The pre-publication history for this paper can be accessed here:
The use and possible effects of factors known to improve outcomes in patients with human immunodeficiency virus associated nephropathy (HIVAN), namely of angiotensin converting enzyme inhibitors (ACE) and antiretroviral therapy, has not been reported for a national sample of dialysis patients.
Methods
We conducted a historical cohort study of the United States Renal Data System (USRDS) Dialysis Morbidity and Mortality Study (DMMS) Wave 2 to identify risk factors associated with increased mortality in these patients. Data were available for 3374 patients who started dialysis and were followed until March 2000. Cox Regression analysis was used to model adjusted hazard ratios (AHR) with HIVAN as a cause of end stage renal disease (ESRD) and its impact on mortality during the study period, adjusted for potential confounders.
Results
Of the 3374 patients who started dialysis, 36 (1.1%) had ESRD as a result of HIVAN. Only 22 (61%) of patients with HIVAN received antiretroviral agents, and only nine patients (25%) received combination antiretroviral therapy, and only 14% received ACE inhibitors. Neither the use of multiple antiretroviral drugs (AHR, 0.62, 95% CI, 0.10, 3.86, p = 0.60), or ACE inhibitors were associated with a survival advantage. Patients with HIVAN had an increased risk of mortality (adjusted hazard ratio, 4.74, 95% Confidence Interval, 3.12, 7.32, p < 0.01) compared to patients with other causes of ESRD.
Conclusions
Medications known to improve outcomes in HIV infected patients were underutilized in patients with HIVAN. Adjusted for other factors, a primary diagnosis of HIVAN was associated with increased mortality compared with other causes of ESRD.
Previously limited to case series, [1-4] information on the patient characteristics and course of human immunodeficiency virus or acquired immunodeficiency syndrome associated nephropathy (HIVAN) after the onset of end stage renal disease (ESRD) has been reported for the national population of US ESRD patients. [5,6] Substantial improvements in the survival of dialysis patients with HIV infection have been noted after 1995,[6] and have been attributed to treatment with highly active antiretroviral therapy (HAART). [7] Despite these encouraging reports, the morbidity and mortality of these patients remains high compared with age-matched patients with ESRD from other causes. [6] Despite the improving information on the pharmacokinetics of these drugs in dialysis patients, a recent report suggested that only 58% of the 62 HIV-infected patients with ESRD were on antiretroviral therapy. [8] Use of angiotensin converting enzyme inhibitors (ACE) in patients with HIVAN is associated with delayed progression to ESRD. [9,10] Some other reports have suggested that use of ACE in dialysis patients may improve survival. [11] It is possible the use of ACE might be particularly beneficial in HIV infected patients with ESRD particularly those with HIV associated nephropathy as heavy proteinuria often continues after the onset of dialysis [12]. Alternatively, risk of hyperkalemia from ACE in patients with HIVAN due to associated tubulointerstitial inflammation and renal tubular acidosis may increase mortality. [13] Although a recent single-center report found no benefit of ACE in dialysis patients with HIVAN [14], it is not clear whether this represents national experience.
Recent anecdotal reports of remission of HIVAN with use of HAART are encouraging. However, the prevalence of patients with HIVAN who develop ESRD may not decrease, because of improvement in survival of these patients, allowing them to live long enough to reach ESRD. [15-17] Such patients may even be more difficult to manage, since the onset of HIVAN could be delayed until these patients develop resistance to the therapy. Therefore, HIVAN is likely to continue as a major cause of ESRD in young African-Americans. Better understanding of factors associated with poor outcomes in patients with HIVAN and ESRD would help in improving outcomes in these patients. More detailed information on the metabolic, cardiovascular and chronic kidney disease specific characteristics of HIVAN patients on dialysis, as well as the use and possible benefit of certain medications, especially ACE and antiretroviral drugs in this population, would supplement previous reports. Further, use of a national database would allow the comparison of HIVAN patients in a large at-risk population and allow comparison with single center studies that may reflect practices that may not be representative of the national as a whole. Therefore, we analyzed data from the standard analysis files of the 2000 United States Renal Data System (USRDS) Dialysis Morbidity and Mortality (DMMS) Wave 2 database. The primary objective of the study was to examine whether patients with HIVAN who present to chronic dialysis had important differences in clinical and laboratory parameters compared to patients with other causes of ESRD and to assess factors associated with better survival such as laboratory parameters and medication use.
Methods
A historical cohort study of the USRDS DMMS Wave 2 was performed. Details on the inception, limitations, validity, variables and questionnaires used in the study are available online at the USRDS researcher's guide website, . This database has been used in many previous cross-sectional and longitudinal studies including one by our own institution. [18]. Briefly, DMMS WAVE 2 was a prospective cohort study of a random sample of 20% of all U.S. hemodialysis patients and virtually all peritoneal dialysis patients starting treatment in 1996 and early 1997. Because patients who started dialysis in 1997 comprised only a small proportion of the study population, we limited analysis to patients who started dialysis in 1996. Also, because outcomes such as dates of death and follow-up had to be merged with this study from other USRDS files, we did not consider it a true prospective cohort study for the purposes of this analysis.
Characteristics of hemodialysis and peritoneal dialysis patients (abstracted from prospective surveys conducted specifically for DMMS WAVE 2) were matched and weighted to allow more appropriate comparisons between modalities. Baseline and follow-up data used in the study are shown in Table 1 [see Additional file: 1]. All results are reported as N (%) or the mean ± the standard deviation. Patients with ESRD due to HIVAN were determined from the variable PDIS (primary cause of renal failure) from the file SAF.PATIENTS and merged with the DMMS WAVE 2 files using unique patient identifiers. Causes of renal failure selected as HIVAN were 0429A, 0429Z, 0439Z, or 0449Z (various codes for HIV or AIDS associated nephropathy), from the variable PDIS in the file SAF.PATIENTS, Core CD and were coded as 1 in statistical analysis, with all other patients coded as 0. AIDS associated nephropathy was listed as a diagnosis in the years studied and therefore that term is still used in the present study. In addition, a maximum of 15 medications prescribed to each patient at the study start date (day 60 of dialysis) were recorded. From this list, the use of beta-blockers (both cardioselective and non-selective), angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers (subcategorized as dihyropyridine and non-dihydropyridine), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), and aspirin was determined. Carvedilol, which has been independently associated with improved survival in certain populations, was approved for use by the FDA in February 1997, and was therefore not assessed. Blood pressure levels, systolic and diastolic, were obtained as the mean of three readings before and after dialysis, respectively. Of the study cohort, 2198 (65.1%) of patients with the variable HIV and 2164 (64.1%) with the variable AIDS, respectively, were either missing, unknown, or coded as "cannot disclose," and these variables were therefore not used in analysis.
Survival status was linked to the DMMS Wave 2 data from the 2000 USRDS Patients Standard Analysis File (SAF.PATIENTS) via unique patient identifiers assigned by the USRDS. The date and cause of death listed in a patient's SAF was obtained from a form submitted to the USRDS by the patient's nephrologist (form HCFA 2746). Descriptions of these files are available under the "USRDS Researcher's Guide" at . Patient survival status was complete through March 2000. Survival time was defined as the time from 90 days after the date of the first dialysis session until the date of death, censored for receipt of renal transplant, loss to followup or the end of the study period. For comparison, models that did not censor for the date of receipt of renal transplant were also used.
Statistical analysis
Continuous variables are presented as mean ± standard deviation unless otherwise specified. Univariate analysis was performed with Chi-Square testing for categorical variables (Fisher exact test used for violations of Cochran's assumptions, meaning fewer than 5 cases per cell) and student's t-test for continuous variables (Mann Whitney test or logarithmic transformation was used for variables without a Gaussian distribution, verified by visual inspection and goodness of fit tests (Chi Square for linear trend). Variables with borderline significance (p value < 0.1 in univariate analysis) and those thought likely to have a clinical relationship with HIVAN were entered into the multivariate models. Stepwise (forward step likelihood ratio method) Cox proportional hazards analysis was used to assess the association between baseline factors and HIVAN, independent of other factors. This method used the most computationally intensive method for calculating mean hazard ratios (approximations of relative risk) entering each variable into the model and removing those that were no longer significant after adjustment for all other variables. Log-log plots were inspected to verify the existence of proportional hazards. Estimated hazard ratios (HR) along with corresponding 95% confidence intervals (CI) and p values are reported for all regression covariates. The association of HIVAN with mortality was also tested by Cox regression analysis using adjusted hazard ratios (AHR). Hierarchically well-formed models were used in the assessment of interaction terms. Collinearity diagnostics were evaluated both from the output of the statistical procedures and by manual generation of models including only one of several variables with close relationships (for example, malnutrition, body mass index and serum albumin). To assess for possible confounding, logistic regression was also performed to assess for significant associations with a history of HIVAN using the same covariates as for Cox Regression as above. Multiple methods were used to assess goodness of fit with all variables, including Nagelkerke r2 (an attempt to quantify the proportion of explained "variation" in the logistic regression model, although values for r2 in logistic regression are much lower than for linear regression) and the c-statistic, which is the receiver operating curve for the cumulative predicted probability of the logistic regression model (0.5 being equivalent to chance and 1.0 equivalent to 100% agreement).
Results
A total of 4065 patients were included in the DMMS Wave 2 cohort. Of these, 3621 patients had valid dates for starting dialysis in 1996. From this cohort, 3374 had sufficient information to calculate follow-up times. Thirty-six of these patients had ESRD as a result of HIVAN. Mean follow-up was 2.19 ± 1.14 (standard deviation) years. Characteristics of all the patients and patients with HIVAN are summarized in Table 1 (see Additional file 1). As shown, in this file factors positively associated with HIV in univariate analysis included male gender, African American race, younger age, lower body mass index, decreased comorbidity in general, increased rates of malnutrition and smoking, lower blood pressure and cholesterol, higher serum creatinine, lower serum albumin bicarbonate, and decreased use of aspirin and calcium channel blockers. The distribution of serum PTH levels in the study population was severely skewed, with a skewness of 2.49 and a kurtosis of 9.08. We elected to use non-parametric tests of association (the Mann-Whitney test) and a t-test of logarithmically transformed values of PTH. Although the difference in serum PTH levels between patients with HIVAN and patients with other causes of renal disease appeared large, it was not significant in Mann-Whitney testing (p = 0.34) or t-tests of logarithmically transformed values of serum PTH (p = 0.64).
Specific antiretroviral agents are also shown in this file. The most common agents used in HIVAN patients were zidovudine and lamivudine. Only 22 (61%) of patients with HIVAN received antiretroviral agents, and only nine patients (25%) received combination antiretroviral therapy. Five patients received both zidovudine and lamivudine (a combination later called CombivirTM). Other combinations included one each for zidovudine-zalcitabine, stavudine-saquinavir, stavidine-lamivudine, and indinavir-lamivudine.
Table 2 shows factors independently associated with HIVAN in logistic regression. Factors included antiretroviral therapy, male gender, African American race, younger age, lower serum albumin, higher rates of malnutrition and smoking, and lower use of calcium channel blockers. There were no significant differences in ACE use by either HIV or HIVAN status, either unadjusted or adjusted. There was no significant interaction between ACE and antiretroviral therapy.
Logistic Regression of Factors Associated with HIVAN, entire cohort
P value
Adjusted odds ratio for HIVAN
95% CI
Age <48 (vs. > = 71)*
0.005
9.35
1.99, 43.48
Serum albumin (per higher quartile)
<0.001
0.31
0.15, 0.69
Male (vs. female)
0.001
6.58
2.39, 18.11
African American
<0.001
11.03
4.09, 27.78
CCB use (vs. other medications)
0.02
0.23
0.07, 0.75
Malnourished (vs. adequate nutrition)
0.001
4.09
1.56, 10.72
Smoking (vs. non active smoking)
0.02
2.89
1.16, 7.18
HIVAN=Human immunodeficiency virus associated nephropathy. *Other age categories were not statistically significant when compared to age > = 71 years Nagelkerke r2 was 0.50, c-statistic was 0.97 (95% CI, 0.96, 0.99)
Patients with HIVAN had significantly lower survival compared to patients with other causes of ESRD (Table 3, Figure 1). Plots of survival by ACE use are shown for HIVAN patients (Figure 2) and for the entire study cohort (Figure 3). In Cox Regression analysis adjusted for other factors known to be associated with mortality, HIVAN was independently associated with increased risk of mortality, adjusted hazard ratio, 4.74, 95% CI, 3.12–7.32, p < 0.0001. ACE use was not significantly associated with mortality in the entire cohort in Cox Regression, and it was not significant as an interaction term with HIVAN (p = 0.38, adjusted hazard ratio, 1.64, 95% CI, 0.55–4.87). In stratified analysis limited to patients with HIVAN, ACE use was not significantly associated with mortality (p = 0.48, AHR = 1.88, 95% CI, 0.33–10.83). Although the risk of death of patients with HIVAN and ESRD treated with single antiretroviral drug was higher than those not taking antiretroviral agents, patients on multiple antiretroviral drugs had a lower hazard ratio in adjusted analysis (0.62, 95% CI, 0.10–3.86 by Cox Regression), however this was not statistically significant (Figure 4). Among patients with HIVAN, the leading causes of death were AIDS (50%), missing/unknown (10.7%), and cardiac arrest of unknown cause (10.7%). Factors independently associated with survival in HIVAN patients are shown in Table 4. The only factor independently associate with improved survival was African American race, compared to all other races.
Survival, Dialysis Morbidity and Mortality Wave II Cohort (DMMS II), 1996 only, N = 3374, patients with Human Immunodeficiency Virus (HIV) associated nephropathy (HIVAN) vs. patients with other causes of end stage renal disease (ESRD), p < 0.01 by Log Rank Test.
Survival in HIVAN infected patients on chronic dialysis, 1996 DMMS II, by angiotensin converting enzyme inhibitor (ACE) use. P = 0.53 by Log Rank Test.
Survival in patients on chronic dialysis, 1996 DMMS II, by angiotensin converting enzyme inhibitor (ACE) use. P = 0.85 by Log Rank Test.
Survival, Dialysis Morbidity and Mortality Wave II Cohort (DMMS II), 1996 only, N = 3374, patients with Human Immunodeficiency Virus (HIV) associated nephropathy (HIVAN) who received combination retroviral therapy (COMBO) vs. all those who did not (NO COMBO), including those who were treated with single anti-retroviral agents, p = 0.77 by Log Rank Test.
Percent survival
1-year
2-year
3-year
HIVANA
53
31
28
Patients at risk
19
11
10
Dialysis Patients with other causes of renal failure
83
66
49
Patients at risk
2707
2180
1722
AUnadjusted cumulative percent survival, censored for receipt of renal transplant HIVAN=ESRD due to HIV/AIDS associated nephropathy AIDS=acquired immunodeficiency syndrome
Cox Regression of Factors Associated with Mortality, DMMS WAVE 2 patients with HIVAN only
One-Year Unadjusted Survival Factor Present
One-Year Unadjusted Survival Factor Absent
P Value
AHR
95% CI
African American Race* (vs. all other races)
54%
40%
0.05
0.20
0.04, 0.99
Two-Year Unadjusted Survival
Two-Year Unadjusted Survival
Antiretroviral therapy (yes/No)
15%
39%
0.21
1.92
0.69, 5.37
Multiple antiretroviral agents (yes/No)
22%
33%
0.60
0.62
0.10, 3.86
ACE inhibitors (vs. nonuse)
13%
29%
0.28
2.26
0.55, 9.31
CCB (vs. nonuse)
42%
25%
0.36
0.56
0.16, 1.96
Male (vs. female)
33%
17%
0.31
0.44
0.09, 2.16
Peritoneal Dialysis (vs. Hemodialysis)
38%
26%
0.47
0.58
0.13, 2.53
N = 36, N in final model = 30 *Because so few patients with HIVAN were not African American, only one-year survival could be compared Only factors significant in the final model are shown.
Discussion
The present study found that only 61% of a national sample of US dialysis patients with HIVAN received antiretroviral therapy, consistent with previous regional reports of 58% use of anti-retroviral agents, [8] suggesting that underutilization of antiretroviral in dialysis patients with HIVAN is a national problem. HAART use, which the present study could not directly measure, is also low in dialysis patients with HIVAN, at 33% according to a recent single-center study [14]. We found no beneficial effect of ACE on survival in HIVAN, also consistent with previous studies [14]. The survival of HIVAN patients on dialysis in the preset study is also consistent with previous reports [5,6]. One-year survival of patients with HIVAN was only 53% compared to 83% for patients with all other causes of ESRD. This survival is lower than reported recently by Ahuja et al. [6] in HIV infected patients starting dialysis in the United States after 1997, likely from the fact that patients included in the DMMS wave 2 were recruited in 1996. Although data on CD4 counts and viral loads were unavailable from the database (the importance of which is illustrated in two reviews.[19,20] HIVAN is generally a late manifestation of HIV infection. [21] Therefore, use of antiretroviral therapy would be expected in patients with HIVAN in the absence of definite contraindications, even disregarding the previously cited reports of a possible beneficial effect of HAART on the course of HIVAN itself. However, in the present study only 61% of patients with HIVAN were receiving antiretroviral therapy and 41% of these were only on single antiretroviral therapy. Although not statistically significant, there was a trend towards better survival of patients on two antiretroviral drugs. None of the patients was on more then two antiretroviral drugs. This underutilization of antiretroviral therapy could be due hesitancy on the part of the infectious disease specialists and nephrologists for using these drugs due to unavailability of data on the pharmacokinetics of these drugs in patients on dialysis. Providers may also be concerned about the nephrotoxicity of certain antiretroviral agents, notably indinavir-associated crystal induced renal failure, [22] and other antiretroviral agents that require dose adjustment in renal failure. [22] Urgent studies are therefore needed to understand factors responsible for this underutilization of HAART in these patients. Similar underutilization of medications known to be beneficial in patients with chronic kidney disease has also been reported for cardiovascular disease. [18,23], suggesting such practice may not be an isolated phenomenon. However, the long term tolerability and efficacy of HAART has not been studied in patients on dialysis.
Although a benefit of ACE use in HIV infected patients has been reported previously, [9] the benefits of ACE in retarding progression of renal failure in patients with HIVAN did not translate into improvement of survival of these patients on dialysis. This is in agreement with some recommendations that ACE use, to be effective, must start early in the course of HIVAN. As the number of patients with HIVAN who were treated with ACE was small in the present cohort, further prospective studies are required to validate our initial observation.
In comparison to patients with other causes of ESRD, the patients with HIVAN included in the DMMS wave 2 study were younger, the majority were African-Americans and had lower serum albumin and more severe acidosis. Some reports have suggested that HIV can infect parathyroid cells and these patients have low parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D3 levels. [24] However, this did not prevent development of secondary hyperparathyroidism in patients with HIVAN and ESRD. The mean PTH level of the patients with HIVAN was 225 ± 42.6 pcg/L, although lower was not statistically different from patients with causes of ESRD.
The present study has several limitations: it is observational and not randomized, therefore, confounding might persist despite adjustment. It is a random sample of all hemodialysis patients, not the total population, although the peritoneal dialysis population is almost fully captured. Information bias could have arisen due to mistakes in coding, especially for medications. We did not know the duration of use of medications prior to the study start. We were also unable to follow changes in variables over time. Therefore, we could not follow changes in blood pressure, laboratory values, or dialysis adequacy. This most especially applied to possible changes in medication use and changes in patient dry weight. These same limitations apply to other published studies using this database. In addition the information on CD4 count and plasma viral load in patients with HIVAN was not available and this could be an important uncorrected confounding factor. We could not assess factors associated with HIV seropositivity or AIDS due to the high percentage of patients with missing values for these variables.
In summary, we conclude that patients with HIVAN have a lower survival compared to patients with ESRD from other causes. Antiretroviral therapy is underutilized and the use of ACE does not improve survival of these patients. Future prospective studies are required to determine efficacy and tolerability of HAART and ACE inhibitors in patients with HIVAN and ESRD.
Competing interests
None declared.
Authors' contributions
KCA performed the primary data analysis and collaborated in the writing of the manuscript.
FCT derived the algorithms for the medication variables and collaborated in the writing of the manuscript.
LYA provided the USRDS CD data files and supervised the project, and collaborated in the writing of the manuscript.
TSA collaborated in the writing of the manuscript.
Note
The opinions are solely those of the authors and do not represent an endorsement by the Department of Defense or the National Institutes of Health. This is a U.S. Government work. There are no restrictions on its use
Pre-publication history
The pre-publication history for this paper can be accessed here:
Oxidative stress may play a critical role in the vascular disease of end stage renal failure and hemodialysis patients. Studies, analyzing either discrete analytes and antioxidant substances, or the integrated total antioxidant activity of human plasma during hemodialysis, give contradictory results.
Methods
Recently, we have introduced a new automated method for the determination of Total Antioxidant Capacity (TAC) of human plasma. We have serially measured TAC and corrected TAC (cTAC: after subtraction of the interactions due to endogenous uric acid, bilirubin and albumin) in 10 patients before the onset of the dialysis session, 10 min, 30 min, 1 h, 2 h and 3 h into the procedure and after completion of the session.
Results
Our results indicate that TAC decreases, reaching minimum levels at 2 h. However, corrected TAC increases with t1/2 of about 30 min. We then repeated the measurements in 65 patients undergoing dialysis with different filters (36 patients with ethylene vinyl alcohol copolymer resin filter -Eval-, 23 patients with two polysulfone filters -10 with F6 and 13 with PSN140-, and 6 patients with hemophan filters). Three specimens were collected (0, 30, 240 min). The results of this second group confirm our initial results, while no significant difference was observed using either filter.
Conclusions
Our results are discussed under the point of view of possible mechanisms of modification of endogenous antioxidants, and the interaction of lipid- and water-soluble antioxidants.
Background
Hemodialysis represents a chronic stress status for its recipients [1-3]. Although life salvaging, this procedure, by the application of a modified circulation and the forced passage of blood through a number of filters, activates endogenous inflammatory mechanisms and induces chronic release of molecules resulting in an increased production of reactive oxygen species [reviewed in [4,5]]. In addition, uric acid, an endogenous metabolite eliminated by hemodialysis, possesses significant antioxidant activity [6], while fluctuations in other endogenous antioxidant systems (plasma proteins, vitamins, etc) may lead to major variations of the internal redox state [1,3,6-8].
Circulation of oxidative molecules has been incriminated in protein, carbohydrate and lipoprotein oxidation and the generation of an increased arterial deposit, leading ultimately to atherosclerosis [9,10]. Indeed, accelerated development of atherogenesis and a number of vascular episodes characterize patients with chronic renal failure subjected to hemodialysis. In these patients oxidative stress relies on three major components: (1) The dialysis membrane, (2) the microbial contamination or pyrogen content of the dialysate, (3) the possible prooxidant effect of a number of metabolites, found at high concentrations in the patients' plasma, including uric acid [11].
Cross-sectional studies of dialysis patients reveal that, while traditional cardiovascular risk factors (hypertension, hypercholesterolemia) do not discriminate as well as in the general population, markers of inflammation and protein-calories malnutrition are highly correlated with cardiovascular mortality. Interesting hypotheses have been advanced, linked to the presence of oxidant stress and its sequelae as a unifying concept of cardiovascular disease in uremia [12]. A number of preventive strategies have been recently introduced, during and after hemodialysis, in order to counteract vascular disease. They include administration of antioxidant vitamins, the use of new biocompatible filters, presumably less immunogenic, and the addition of vitamin, hormone or trace metals in the patients' diet [1,2,13-23]. Nevertheless, although it is generally accepted that oxidative stress may result from dialysis therapy, no direct evidence exists confirming this hypothesis. A number of reports, either measuring specific analytes or enzymes [8,15,22,24-30], or estimating the total antioxidant activity of the plasma [1-3,6,7,9,26,29,31,32] give contradictory and non-conclusive results.
Recently, we have introduced a new automated method for the estimation of the plasma total antioxidant capacity [33]. This method is based on the inhibition of oxidation by plasma of an exogenously added marker (crocin) by an added pro-oxidant (ABAP). In this respect, it integrates the totality of circulating pro- and antioxidants, and gives a rough (although accurate) estimate of the antioxidant status of plasma at a given moment. In addition, we have also corrected these results for a number of analytes, directly affecting redox potential, thus introducing the concept of "corrected antioxidant capacity". We have used this assay in order to evaluate changes of the antioxidant capacity of patients during a cycle of hemodialysis. Our results indicate that, although the total antioxidant capacity of hemodialyzed patients shows a decrease during the procedure, the corrected antioxidant capacity increases, indicating that counterbalancing mechanisms might occur in human plasma, equilibrating the loss of uric acid and other antioxidant metabolites.
MethodsPatients and controls
Ten patients dialyzed with an ethylene vinyl alcohol copolymer resin (Eval) filter were analyzed. Seven samples were obtained from each patient, before the initiation of dialysis, 30 min, 1, 2 and 3 hours into the session and upon its completion. Sixty-five additional patients were examined (32 males [age range 15–91 years, mean 62.7, median 54.3 years] and 34 females [age range 26–88 years, mean 60.9, median 55.7 years]) and 56 volunteer blood donors on a normal diet (38 males and 18 females, age range 21–52 years, median 43.5 years). Polysulphone dialysis membranes were used on 23 patients (F6: 10 patients and PSN140: 13 patients), hemophan membranes on 6 (GFS 12 Plus) and ethylene vinyl alcohol copolymer resin filters (Eval 1.6 or 1.3) on 36. Dialysis age varied from 1 to 13.3 years (mean ± SE 6.0 ± 0.4 years, median 5.2 years). No significant changes among the different groups were found. Renal failure was due to diabetes (4 cases), polycystic kidney disease (8 cases), vasculitis (4 cases), tuberculosis (1 case), chronic pyelonephritis (4 cases), interstitial nephritis (2 cases), chronic glomerulonephritis (1 case), glomerulosclerosis (2 cases), hypertension (2 cases), while 37 patients were refered to the hospital with renal failure of unknown cause. All patients received non-steroid antiinflammatory, Vitamin B, and erythropoietin treatment. An informed consent was obtained from all participants in the study. Three samples (times 0, 30 and 240 min) were withdrawn from each patient, while a single sample was obtained from each blood donor on K3-EDTA. Plasma was immediately separated by centrifugation (2000 g, 4°C), aliquoted and stored at -80°C until assayed.
Determination of TAC
Plasma total antioxidant capacity (TAC) was measured on an Olympus AU-600 chemistry analyzer using the TAC kit, described previously [33] (Medicon SA, Gerakas, Greece). Briefly, antioxidants in the sample inhibit the oxidation (bleaching) of crocin from ABAP [2,2-Azobis-(2-amidinopropane) dihydrochloride] to a degree that is proportional to their concentration. The assay was performed at 37°C in the following steps: 2 μl of sample, calibrator or control were mixed with 250 μl of crocin reagent (R1) and incubated for 160 s. Subsequently, 250 μl of ABAP (R2) were added and the decrease in absorbance at 450 nm was measured 256 s later. Values of TAC were expressed as mmol/l of Trolox (a hydrophilic Vitamin E derivative) equivalents. During the initial validation of the TAC assay [33] we found that uric acid, bilirubin, and albumin accounted for 0.11, 0.11, and 0.01 mmol/mg of the antioxidant capacity, respectively. Subtraction of these interferences from the TAC value results in the calculation of corrected TAC (cTAC), an estimate of the redox state attributed, mainly to circulating exogenous antioxidants.
Routine clinical chemistry
Plasma uric acid, albumin, total and direct bilirubin, cholesterol, HDL-cholesterol and triglycerides were determined on an Olympus AU-600 chemistry analyzer using Olympus reagents provided by Medicon SA (Gerakas, Greece), as follows: uric acid OSR6136, albumin OSR6102, total bilirubin OSR6112, direct bilirubin OSR6111, cholesterol OSR6116, HDL-cholesterol OSR6187 and triglycerides OSR6133. LDL-cholesterol was estimated by the Friedewald equation [LDL-cholesterol = total cholesterol - (HDL-cholesterol + triglycerides/5)], when triglyceride values were <400 mg/dl. For the group of ten patients dialyzed with Eval membranes, a full blood count (white blood cells, polymorphonuclear cells, hemoglobin concentration, hematocrit value) accompanied all serial measurements.
Statistics
Statistical analysis of data was performed by the use of the SyStat v 10.0 program (SPSS Inc, Chicago, IL). Group differences were compared at each data point using ANOVA with the Bonferroni correction for small data sets. The residual variance was used as a common estimate of the Standard Error, and group means were therefore compared by the Student's t-test. The Origin v 5.0 program (MicroCal, Northampton, MA) was used for curve fitting.
ResultsMetabolite variation during dialysis
Kinetics of a number of analytes during dialysis is presented in Figure 1. Uric acid concentrations decrease during dialysis, following an exponential decay curve with t1/2 of 104 min. Albumin, on the other hand presents a gradual increase during dialysis, following an exponential growth curve, with t1/2 of 101 ± 12.8 min, due to hemoconcentration, as reflected by the hematocrit count. In contrast, minor changes of bilirubin were found during the dialysis cycle. Cholesterol on the other hand, as well as HDL and LDL cholesterol present minor changes during the dialysis cycle. A slight increase of triglycerides was observed, due probably to the feeding of subjects during hemodialysis.
Variation of uric acid, albumin, hematocrit, bilirubin and lipids during dialysis Data obtained from 10 dialysis patients. Mean ± SEM are depicted.
Total and corrected plasma antioxidant capacity during dialysis
Figure 2 presents the variation of TAC during dialysis, in the ten hemodialyzed patients. As expected, due to the presence of a number of endogenous metabolites dotted with antioxidant activity (for example uric acid) initial TAC values of hemodialyzed patients are high, as compared to those of control individuals. During dialysis however, these elevated values decrease, according to an exponential decay model, with t1/2 of 24.8 min. Thereafter, they remain constant during the whole time of dialysis.
Variations of TAC and corrected TAC during dialysis Up and down arrows depict values obtained in normal blood donors. Mean ± SEM of 10 patients and 56 blood donors
Corrected TAC is also depicted in Figure 2. As stated in our previous work [33], this calculated parameter represents the fraction of circulating antioxidants, after the elimination of interference of endogenous metabolites. Our previous work has shown that uric acid and bilirubin, and to a lesser degree albumin, are the major analytes interfering linearly with coefficients of 0.11, 0.11 and 0.01 mmol/L of TAC per mg/dL of each analyte respectively. We have therefore calculated the corrected TAC values in the same patients. As shown, corrected TAC increases during the dialysis procedure, following a sigmoidal curve, with t1/2 of 174 min. It is interesting to note that this value is slightly higher from the t1/2 of uric acid decay (174 ± 14.1 min, as compared to 101.2 ± 12.8 min respectively).
Comparing TAC and corrected TAC values with those obtained in normal blood donors (depicted in Figure 2 as up and down triangles respectively), it is observed that total TAC values are significantly decreased (t = 3.75, p < 0.001) in hemodialyzed patients as compared to controls. In contrast, while initial corrected TAC values are significantly lower than those of controls (t = 2.97, p < 0.01), they reach normal values at the end of dialysis.
Effect of different dialysis filters on metabolites and TAC values
Previous works have suggested that hemodialysis-related oxidative burden relies greatly on the dialysis membrane used. In this respect, as antioxidants might be consumed during a surge of oxidative molecules, TAC (as well as corrected TAC) might be decreased with the use of different membranes. We therefore attempted to investigate the effect of filters on TAC and corrected TAC values, in 65 patients (33 males and 32 females). Thirty-six patients were dialyzed using an ethylene vinyl alcohol copolymer resin (Eval) filter. In 10 and 13 patients, F6 and PSN140 polysulfone filters were used, respectively. Finally, 6 patients were dialyzed using a GFS12+ hemophan filter. The obtained results are presented in Figure 3. As shown, no significant differences were observed in any group (Kruskal-Wallis test statistics with p > 0.05 in any case), indicating that at least the filters used in the present study do not modify drastically the redox state of patients, during the dialysis procedure.
Effect of different filters used in dialysis on the concentration of different analytes and TAC levels Parameter variation in 65 patients under dialysis. 23 patients were dialyzed with polysulphone dialysis membranes (F6: 10 patients and PSN140: 13 patients), 6 patients with hemophan membranes (GFS 12 Plus) and in 36 patients ethylene vinyl alcohol copolymer resin filters (Eval 1.6 or 1.3) were used. Mean ± SEM of vaues.
Discussion
The primary defense against oxidative stress in extracellular fluids results from a number of low molecular weight antioxidant molecules being either water- (ex. ascorbic acid) or lipid-soluble (ex. Vitamin E). These antioxidants are either generated during normal metabolism (ex. uric acid, bilirubin, albumin, thiols) or introduced in the body by the consumption of dietary products rich in antioxidants (olive oil, fruits and vegetables, tea, wine, etc) [34]. The sum of endogenous plus exogenous (food-derived) antioxidants represents the total antioxidant capacity of extracellular fluids. Changes of these antioxidants reflect their consumption during acute oxidative stress states. It should be noted that cooperation between different antioxidant pathways provides greater protection against attack by reactive oxygen or nitrogen radicals, compared to any single compound. Thus, the overall antioxidant capacity may give more relevant biological information compared to that obtained by the measurement of individual biomarkers, as it considers the cumulative effect of all antioxidants present in plasma and body fluids [35]. A theory has recently been proposed, taking into account the redox potentials of exogenous and endogenous antioxidants. It postulates a cascade of reactions, in which following an oxidative stress, a lesser antioxidant is generated from a more potent one. Through this cascade, interactions among the lipid and the aqueous phases could be established [36].
A great variety of methods have been proposed for the assay of total antioxidant activity or capacity of serum or plasma [reviewed extensively and critically in [34,35]]. They stress the fine distinction between antioxidant activity and antioxidant capacity: Antioxidant activity corresponds to the rate constant of a single antioxidant against a given free radical; a ntioxidant capacity, on the other hand, is the number of moles of a given free radical scavenged by a test solution, independently of the capacity of any one antioxidant present in the mixture [35]. In the case of plasma, being a heterogeneous solution of diverse antioxidants, the antioxidant status is better reflected by antioxidant capacity rather than activity alone. This capacity is a combination of all redox chain antioxidants, including several analytes such as thiol bearing proteins, and uric acid. It thus appears that plasma antioxidant capacity is rather a concept than a simple analytical determination. Indeed, an increase of antioxidant capacity of plasma indicates absorption of antioxidants and improved in vivo antioxidant status [37], or the result of the activation of an adaptation mechanism to oxidative stress. It should be noted that, due to the contribution of diverse metabolites to the antioxidant capacity of human plasma, its increase may not necessarily be a desirable condition. Indeed, in some cases, such as renal failure (uric acid), icteric status (bilirubin), hepatic damage (hypoalbuminemia) the increase or decrease of several metabolites modifies plasma antioxidant capacity, a situation returning to physiological values after correction of the underlying disease [38]. In addition, high concentration of a number of metabolites, including uric acid, can lead to prooxidant effects, introducing a further decrease of the plasma antioxidant capacity [11]. Recently, we have introduced a new automated method for the assay of the plasma antioxidant capacity, based on the bleaching of crocin. This method (the TAC assay) gives an estimation of the integrated plasma antioxidant capacity. Furthermore, we also determined the interference of a number of endogenous analytes, such as uric acid, and bilirubin, which have been found to produce a major interference of TAC, while albumin results in a smaller interference [33]. The subtraction of these interferences in TAC assay resulted in the calculation of corrected TAC, cTAC representing the amount of antioxidant capacity due to the action of (mainly) exogenous antioxidants.
In the present work, we have assayed simultaneously TAC and the concentrations of these analytes during a single episode of hemodialysis. Although uremic plasma is almost unique in its concentration of numberless metabolites and toxins, identified or unknown, measurable or not, the concept of the TAC assay, measuring the inhibition of an exogenously added oxidant makes the assay suitable for the identification of the antioxidant capacity of uremic plasma. Concerning cTAC, we are aware that the subtraction of the interaction of albumin, bilirubin and uric acid only, may not take into account other (known or unknown) endogenous metabolites and toxins with a proper antioxidant or prooxidant activity. Therefore, the results concerning the cTAC measurements must be considered as indicative. In addition, a number of medications may influence the plasma antioxidant capacity. Nevertheless, an almost similar medication regime was followed by the parients' group. Therefore, the effect of medication (if any) on the results of the TAC assay, may be considered as constant.
Major modifications of a number of endogenous metabolites were observed during dialysis. Namely, uric acid is rapidly eliminated, with t1/2 of 101 min (Figure 1), while albumin plasma concentration is increased (t1/2 21 min) probably due to the hemoconcentration during dialysis. Bilirubin, on the other hand, follows a biphasic pattern with an initial decrease (possibly due to elimination) followed by an increase due to hemoconcentration. Lipids do not present major variations during the dialysis episode studied, with the exception of triglycerides due to the feeding of patients. As significant changes were not observed, the implication of lipids on the TAC assay was omitted. Total TAC measurements present equally major changes, following those of the above analytes. Indeed, results presented in Figure 2 show that the plasma antioxidant capacity of patients is higher before than during or after a session of dialysis. This can be due to the elimination of a number of metabolites, such as uric acid and bilirubin (Figure 1). In this respect our results are similar to those presented in previous reports, in which a comparable decrease of plasma antioxidant capacity was observed during renal dialysis [2,3,8-10,15,26,32,39,40]. Total TAC was found to increase later in hemodialysis, most probably due to either hemoconcentration [31], adaptation [41], or to a possible exchange of antioxidants between the lipid and aqueous phases [36]. Whether urate by itself and other analytes, at the concentrations encountered in dialysis patients and in the milieu of uremic plasma, exert a pro-oxidant [11] or antioxidant effect remains a matter of debate.
Calculation of the corrected TAC appears to provide a better estimate of the actual antioxidant activity of the organism, especially in cases such as renal dialysis, in which major fluctuations of endogenous metabolites and the elimination of a number of toxins occur. Indeed, as shown in Figure 2, the curve of corrected TAC is different from that of TAC. Specifically, a gradual increase of plasma antioxidant capacity is observed, with t1/2 of about 30 min. Various explanations for these results could be proposed: (1) Water elimination during dialysis causes increased concentration of endogenous antioxidant substances [31]. (2) Elimination of uric acid modifies the equilibrium between oxidized and reduced states of endogenous and exogenous antioxidants [41,42]. (3) It has been recently proposed that elimination of water-soluble metabolic antioxidants (bilirubin, uric acid) modifies the equilibrium of lipid- and water-soluble antioxidants [36]. (4) The presumed "antioxidant effect" of hemodialysis, detected here by cTAC, has also been attributed to the plasma glutathione increase by hemodialysis [25,43,44].
Redox state in uremic patients undergoing dialysis is rather confused. Several reports provide possible pathophysiological explanations of the observed changes in redox state and antioxidant status. It appears that patients with malnutrition and a low plasma albumin concentration have significantly reduced plasma antioxidant capacity due to the diminished availability of thiol groups [45]. Serologic evidence of an activated inflammatory response has been reported [46], as well as the contribution of phagocytes and cytokines to increased production of ROS [47-50]. Several lines of evidence indicate that further oxidative modification of retained solutes in the uremic milieu (ex. β2 microglobulin, homocysteine, cysteine) may potentiate their pathogenicity [51-53]. Dialytic therapy, which acts to reduce the concentration of oxidized substrates, improves the redox balance [12,54]. However, processes related to repetitive extracorporeal dialytic therapies (prolonged use of catheters for vascular access, use of bioincompatible dialysis membranes) can incite further inflammatory and oxidative stimuli (via complement and leukocyte activation), thus contributing to a pro-atherogenic state [55,56].
Our data, presented in Figure 3 indicate that no major changes in both analytes and TAC are observed with the use of modern filters. It has already been reported that hemodialysis decreases the oxidation levels of plasma protein-associated thiol groups [51]. It would be of interest to measure TAC and cTAC in patients dialyzed with vitamin E-modified membranes. It should be noted, however, that even with the use of vitamin E-modified filters, results on oxidative stress markers can be confounding.
In conclusion, our data suggest that although during hemodialysis several factors contribute to the generation of oxidative radicals, the organism is able to successfully resist the flood of oxidative substances. Oxidation and peroxidation reactions of renal failure patients must be reevaluated under this point of view, taking into account the auto-oxidation of excess antioxidants, as was recently reported for vitamin C and tocopherols [57-60], rather than a decrease of the plasma antioxidant capacity. Nevertheless, more extensive studies must be performed, taking into acoount the possible abrupt change of the plasma oxidative status at the end of dialysis, or the hours following it [12].
Pre-publication history
The pre-publication history for this paper can be accessed here:
Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by inflammatory serositis (fever, peritonitis, synovitis and pleuritis). The gene locus responsible for FMF was identified in 1992 and localized to the short arm of chromosome 16. In 1997, a specific FMF gene locus, MEFV, was discovered to encode for a protein, pyrin that mediates inflammation. To date, more than forty missense mutations are known to exist. The diversity of mutations identified has provided insight into the variability of clinical presentation and disease progression.
Case Report
We report an individual heterozygous for the M680I gene mutation with a clinical diagnosis of FMF using the Tel-Hashomer criteria. Subsequently, the patient developed nephrotic syndrome with biopsy-confirmed fibrillary glomerulonephritis (FGN). Further diagnostic studies were unremarkable with clinical workup negative for amyloidosis or other secondary causes of nephrotic syndrome.
Discussion
Individuals with FMF are at greater risk for developing nephrotic syndrome. The most serious etiology is amyloidosis (AA variant) with renal involvement, ultimately progressing to end-stage renal disease. Other known renal diseases in the FMF population include IgA nephropathy, IgM nephropathy, Henoch-Schönlein purpura as well as polyarteritis nodosa.
Conclusion
To our knowledge, this is the first association between FMF and the M680I mutation later complicated by nephrotic syndrome and fibrillary glomerulonephritis.
Background
Familial Mediterranean Fever (FMF) is an autosomal recessive disease primarily affecting individuals of Armenian, Turkish, Arab, and Jewish origin [1]. The disease is characterized by recurrent episodes of fever, pleuritis, peritonitis, synovitis, and pericarditis [2-4]. The most serious complication of FMF is the development of amyloidosis (AA variant), leading to nephrotic syndrome and end-stage renal disease [4]. Amyloidosis remains the leading cause of FMF-related mortality in affected individuals not receiving colchicine prophylaxis [5]. Additionally, other non-amyloid renal diseases are reported to occur in the FMF population with variable prevalence; for example, IgA and IgM nephropathy, and vasculitides including both polyarteritis nodosa (PAN) and Henoch-Schonlëin purpura [6]. Differentiation of the underlying renal disease in FMF patients who present with nephrotic syndrome is difficult, often requiring further diagnostic studies to identify the etiology. Moreover, the identification of the etiology of nephrotic syndrome is critical in determining an effective treatment regimen. For example, patients with amyloidosis (AA variant) usually respond to oral colchicine, which is known to prevent the insidious onset of end-stage renal disease [5]. In contrast, patients with PAN or Henoch-Schonlëin purpura usually respond very well to corticosteroids, with some experiencing complete resolution of proteinuria. [6]
In 1992, the gene responsible for Familial Mediterranean Fever (FMF), MEFV, was found to reside on the short arm of chromosome 16 [7]. Five-years later, the MEFV gene locus was discovered to encode for a protein, pyrin (marenostrin), a member of the RoRet gene family [8,9]. Pyrin is known to play a role in mitigating an inflammatory response [8,10]. To date, more than forty missense mutations are noted in association with FMF. One specific mutation, M694V, has been implicated as a risk factor for amyloidosis, especially in North African Jews homozygous for the M694V missense mutation [11]. However, individuals with mutations other than M694V are known to develop amyloidosis with renal involvement [11]. For the first time, we report an individual with symptomatic FMF, heterozygous for the M680I missense mutation, who developed progressive proteinuria, hematuria, and subsequent nephrotic syndrome secondary to fibrillary glomerulonephritis.
Case Report
A forty-nine year-old American man of Armenian ancestry was diagnosed clinically with Familial Mediterranean Fever (FMF) at age 39. At that time, he presented with recurrent, episodic fevers, peritonitis, synovitis, and pleuritis. Since the initial diagnosis, his treatment consisted of oral colchicine (1.2 mg/day), resulting in a decrease in the frequency, duration, and intensity of his inflammatory episodes. However, over the subsequent decade, his inflammatory episodes became more frequent, occurring every 7–10 days, with an average duration of approximately 3 days. His symptoms consisted of diffuse abdominal pain, sharp, non-radiating chest pain, and fevers as high as 103 degrees F. An extensive workup for other etiologies responsible for febrile illness was unremarkable (e.g. infection, malignancy, connective tissue disorders). Upon further examination, the previous diagnosis of FMF was confirmed using the Tel-Hashomer criteria [12].
Despite an increase in the oral colchicine dose to 1.8 mg/day, his symptoms persisted, prompting further diagnostic studies to correlate the clinical diagnosis with genotype. A serum sample was sent to the UCLA Medical Center for genetic analysis. Polymerase chain reaction, testing for the nine most common mutations [Table 1], was performed by methods previously described [13]. The results demonstrated the presence of an MEFV missense mutation (M680I) on one allele. Several months later, he presented with complaints of lower extremity edema without associated symptoms of cough, shortness of breath, dyspnea on exertion, orthopnea, paroxysmal nocturnal dyspnea, or chest pain. Upon further questioning, the patient admitted to gradual development of lower extremity edema over the past 2 to 3 months. He denied complaints of dysuria, polyuria, hematuria, or any discoloration of his urine. The remainder of the review of systems was unremarkable. A 2-dimensional echocardiogram revealed a normal size heart, normal valvular structure and function, and preserved left ventricular systolic function with a left ventricular ejection fraction of 55–60 percent. Relevant serological studies were conducted to evaluate the etiology of nephrosis. In addition to a urinalysis, the urine protein and creatinine were measured. Results from the urinalysis were positive for proteinuria (3+) and red blood cells (50–100 RBC's/HPF). The spot urine protein/creatinine ratio was 4.0. The patient's blood urea nitrogen (BUN) and serum creatinine were 17 mg/dl and 0.9 mg/dl, respectively. Erythrocyte sedimentation rate was 72 mm/hr with a normal complete blood count and differential. Serological tests for HIV (types I and II), HIV p24 antigen, Hepatitis A, B, and C titers were negative. Furthermore, tests for ANA, RPR, rheumatoid factor, c-ANCA, p-ANCA, cryoglobulin, lupus anticoagulant and anticardiolipin antibodies were negative. Both C3 and C4 complement levels were within normal limits. Additionally, serum protein electrophoresis was negative for a monoclonal gammopathy. The kappa/lambda ratio was 1.93 (NML range: 1.47–2.95). Urine protein electrophoresis was also unremarkable without evidence of Bence-Jones proteins.
MEFV missense mutations evaluted in our patient
Exon 2
Exon 10
E148Q
M680I
M694V
M694I
692delI
LS95R
A744S
V726A
* Analysis performed at the UCLA Medical Center using Polymerase Chain Reaction
As a result of the clinical and diagnostic findings consistent with nephrotic syndrome, a renal biopsy was performed. Biopsy results demonstrated sclerosis of approximately one-third of the glomeruli. The remaining glomeruli were hypercellular, exhibiting both mesangial and epithelial hyperplasia. Deposition of homogenous eosinophilic material was noted in capillary walls and mesangial areas, and stains for Congo red, thioflavin T and amyloid AA protein were all negative. Direct immunofluorescence showed a linear pattern of staining for IgG and C3, with equal staining for kappa and lambda light chains. Electron microscopy showed accumulation of fibrillary material in mesangial areas and capillary basement membranes [Figures 1, 2]. The individual fibrils averaged 15–20 nm in diameter. In light of the noncongophilic staining and fibril size, a diagnosis of fibrillary glomerulonephritis was rendered.
Renal biopsy (Light Microscopy). Light microscopy image of a glomerulus demonstrating negative staining for both Congo-red and thioflavin-T. Expansion of the mesangium with increased hyalinization of the basement membrane (arrow) and thickening capillary loops is present.
Fibrillary Glomerular Deposition (Electron Microscopy). Electron micrograph of renal biopsy showing randomly arranged microfibrils (asterisks) measuring 15–20 nm in diameter located in the basement membrane and mesangium.
Discussion
Once regarded simply as an inflammatory, autosomal recessive disease of unknown etiology, the spectrum of FMF has evolved as a complex genetic puzzle during the genomic era. More than forty missense mutations resulting in abnormal pyrin proteins have led to a new paradigm for FMF as an inherited state of dysregulated inflammation. The altered gene products – the so-called pyrins – are fundamental to understanding FMF. Pyrin-containing proteins play a pivotal role in the immune response by serving as key inflammatory mediators. Unsurprisingly, pyrin is found in large quantities in neutrophils and released in response to inflammatory stimuli. Additionally, a recently discovered pyrin-like domain was found to exist at the amino-terminal of several proteins involved in cell-signaling pathways inherent to inflammation. One pyrin protein, belonging to a larger class of the pyrin domain family, is involved in the Nuclear Factor-kappa B (NF-kB) cell-signaling pathway. Nuclear Factor-kappa B is an important transcription factor involved in inflammation via its induction of pro-inflammatory gene products. In individuals with the "wild-type" MEFV gene, pyrin serves a key role in regulating the intensity of the inflammatory response. In contrast, individuals with one or more missense mutations at the MEFV locus produce a pyrin protein with altered or absent function. Consequently, the response to inflammation is altered. The resultant dysregulated inflammatory response often times exceeds physiologic parameters and is disproportionate to the insult. [18-22]
The inflammation of FMF may not only be characterized as dysregulated, but is also chronic and recurrent. Its severity and duration have been associated with the secondary development of amyloidosis (AA variety). The amyloid fibrils that lead to nephrotic syndrome in FMF are derived from a serum precursor, serum amyloid A (SAA), itself the result of chronic inflammation. Amyloidosis, with renal involvement and nephrotic syndrome, has become the expected complication of long-term FMF. [14-17]
The novel association between FMF and fibrillary glomerulopathy in this patient raises two contingent questions. First, is the association itself a chance one, but if it is not, is fibrillary glomerulopathy like amyloidosis the result of material deposition that reflects chronic, dysregulated inflammatory activity? The present state of knowledge regarding fibrillary glomerular diseases strongly suggests that the presence of fibrils in the glomeruli of affected individuals represents fibrillar derivation from circulating immunologic molecules, fragments, or immune complexes [14]. Since their etiology, deposition and structure are analogous to that of amyloid in FMF, it is highly intriguing to postulate that this patient's renal involvement is more than a chance occurrence. Prior disease associations with the fibrillary glomerulopathies involve the presence of immune response-associated products [14,15]. Future reports of individuals or kindred with FMF, nephrotic syndrome and fibrillary glomerulopathy will be required to substantiate our theory.
Conclusions
Although the association between chronic inflammation and amyloidosis (AA) is well known, the present description of fibrillary glomerulopathy in FMF requires further elucidation. Nevertheless, the new association among FMF, nephrotic syndrome, fibrillary glomerulopathy, and an M680I (Figure 3) heterozygous genotype may represent a novel renal complication of FMF. Furthermore, it is suggested that symptomatic heterozygous individuals may actually be compound heterozygotes for a mutation not yet identified [22]. As the number of MEFV mutations discovered continues to increase, more data will become available revealing further relationships among the many different MEFV missense mutations and their phenotypic expression in various ethnic groups.
Competing interests
None declared.
Authors' contributions
1. Dr. Patrick Fisher served as primary author of this manuscript. Furthermore, he served as primary care physician for the patient described in this manuscript. Additionally, he was also responsible for the clinical evaluation leading to the diagnosis of nephrotic syndrome and subsequent fibrillary glomerulonephritis. Furthermore, Dr. Fisher was responsible for preparation and revision of the manuscript.
2. Dr. L. Tammy Ho served a consulting role as the clinical attending nephrologist. She served a significant role in the clinical evaluation of the patient's nephrotic syndrome, including the performance of the renal biopsy. Furthermore, she assisted in providing her clinical expertise in manuscript preparation.
3. Dr. Robert Goldschmidt served as the clinical pathologist who evaluated the renal biopsy specimens. In addition, he was responsible for the pathological diagnosis of fibrillary glomerulonephritis. Furthermore, he contributed to the preparation of the manuscript, focusing on the description of the clinical pathology.
4. Dr. Ronald Semerdjian served a significant role through his contribution as the patient's former primary care physician. During this time, he made the initial clinical diagnosis of Familial Mediterranean Fever in our patient. He also contributed to the preparation of the manuscript through his careful review and contributions.
5. Dr. Gregory Rutecki played a significant role as a consultant nephrologist and contributed significantly to the preparation and the revision of this manuscript.
M680I Missense Mutation. Illustration of the unspliced MEFV gene locus with exons 1–10 depicted (a), the spliced mRNA transcript revealing exon 2 and exon 10 and their respective codon number identifying where the nine most common mutations for FMF have been identified (b); a diagram illustrating the derivation of the M680I mutation from exon 10 with the amino acid methionine (wild-type) substituted with isoleucine at codon 680 (c).
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent was obtained from the patient for publication of the patient's details. I would like to acknowledge this patient for his perseverance, motivation, and unrelenting will to overcome the many obstacles he has and will encounter in the future.
I would also like to acknowledge the following physicians:
Dr. Gabriel Berlin†
Director, Outpatient Clinic for Adults and Children, Evanston Northwestern Healthcare, Evanston, IL
†For his clinical wisdom and dedication to the care of this patient.
‡Mark Haas, MD, PhD
Associate Professor of Pathology, Director, Electron Microscopy Laboratory The Johns Hopkins University School of Medicine, Baltimore, MD
‡For his expert analysis of the renal biopsy specimens and confirmation of the diagnosis of fibrillary glomerulonephritis.
Renal involvement in patients with chronic hepatitis C virus infection has been suggested to be due to a variety of immunological processes. However, the precise mechanism by which the kidneys are damaged in these patients is still unclear.
Case presentation
A 66 year old man presented with the sudden onset of autoimmune hemolytic anemia. Concomitant with a worsening of hemolysis, his initially mild proteinuria and hemoglobinuria progressed. On admission, laboratory tests revealed that he was positive for hepatitis C virus in his blood, though his liver function tests were all normal. The patient displayed cryoglobulinemia and hypocomplementemia with cold activation, and exhibited a biological false positive of syphilic test. Renal biopsy specimens showed signs of immune complex type nephropathy with hemosiderin deposition in the tubular epithelial cells.
Conclusions
The renal histological findings in this case are consistent with the deposition of immune complexes and hemolytic products, which might have occurred as a result of the patient's underlying autoimmune imbalance, autoimmune hemolytic anemia, and chronic hepatitis C virus infection.
Background
Extra-hepatic manifestations of hepatitis C virus (HCV) infection are diverse and appear during late middle age [1-3]. Among these, glomerulonephritis, arthritis, dermal vasculitis and sialadenitis are thought to develop as a result of the deposition of immune complexes (IC). It is generally accepted that B-cells infected with HCV clonally expand and produce autoantibodies [4,5]. When antigen-bound, these autoantibodies, along with anti-HCV antibodies that target viral epitopes on the surface of cells are known to circulate as IC. These IC may participate in the pathogenesis of HCV-associated glomerulonephritis, though it has been difficult to detect these IC clinically. While immunosuppressive agents have been used successfully to diminish the production of autoantibodies, such a strategy is contraindicated in HCV patients since it would lead to an increase in viral replication.
We herein report the case of a patient with extra-hepatic manifestations of chronic HCV infection that developed autoimmune hemolytic anemia (AIHA) prior to evident nephropathy.
Case presentation
A 66 year old man, with no prior history of blood transfusion, drug addiction, or the acquisition of tattoos, was diagnosed with hemolytic anemia in June of 2000. From the time he was 50, his yearly health check-ups have revealed faint hematuria (±) by urine dipstick testing, and he first exhibited proteinuria when he was 60 years old (see Table 1 for the patient's clinical history). At no time did the patient shows signs of liver dysfunction. Two of the patient's five brothers suffered from chronic liver disease (no further information was available) but none had a history of anemia.
Past laboratory findings before admission
date
1998/
1999/
2000/
2001/
3/27
3/4
3/24
7/21
9/11
12/18
1/30
2/7
normal range
RBC
(/μl)
525
528
334
272
236
213
226
225
(374–502)
Hb
(g/dl)
16.9
16.7
12.3
10.2
9.5
8.5
9.3
8.1
(11.1–15.3)
Htc
(%)
49.6
49.5
34.5
28.8
26.2
24.1
25.6
24.8
(33.2–45.3)
MCV
(fl)
94.5
93.8
103.3
105.9
111.0
113.1
113.3
110.2
(85.0–100.0)
MCH
(pg)
32.2
31.6
36.8
37.5
40.3
39.9
41.2
36.0
(30.0–35.0)
MCHC
(g/dl)
34.1
33.7
35.7
35.4
36.3
35.3
36.3
32.7
(32.0–36.0)
LDH
(U/l)
371
878
1032
1348
1194
1022
1082
(220–430)
TP
(g/dl)
8.1
7.6
7.1
7.4
6.2
6.3
6.3
(6.5–8.0)
Urine
Protein*
+
+
+
±
2+
3+
3+
3+
(mg/dl)
73
75
78
114
480
246.8
99.8
Occult blood reaction*
±
±
±
±
2+
3+
3+
3+
*: urine dip-stick test RBC: red blood cell, Hb: hemoglobin, Htc: hematocrit, MCV: mean corpuscular volome, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, LDH: lactate dehydogenase, TP: total protein
In March of 2000, the patient was diagnosed with sudden onset anemia characterized by an increase in both mean corpuscular volume and corpuscular hemoglobin. Result of the Coomb's, acidified serum, sucrose hemolysis, and cold agglutinin tests were negative. Because spherocytes predominated in his hemogram and the results of his erythroresistant test (Ribiere's test) were positive, he was provisionally diagnosed as having hemolytic anemia caused by spherocytosis. Since he was asymptomatic, he was followed without medication.
Starting in September of 2000, the patient's hematuria and proteinuria progressed and his hemolysis worsened. Laboratory data began to indicate an increase in urinary protein excretion and a lowered serum protein concentration. The patient began to experience mild pretibial edema in March of 2001, at which time he was admitted to our hospital and a renal biopsy performed. Hematological tests at that time revealed the hyperchromic anemia with a macrocytic pattern and an increased numbers of reticulocytes. A hemogram (Figure 1) revealed the presence of unusually-shaped red blood cells (RBC), and polychromatic cells and spherocytes. Ferokinetic parameters such as Fe, unsaturated iron binding capacity (UIBC), ferritin and transferrin levels were all within the normal range. However, low levels of haptoglobin (less than 10 mg/dl; normal range = 41–341 mg/dl) and extremely high levels of erythropoietin (114 mU/ml; normal range = 8–36 mU/ml) were detected. Unlike his earlier results, his direct Coombs test was now positive and binding of IgG to the RBC surface was observed using anti-human globulin monospecific antibodies. Screening for the target antigen was performed using a panel of RBCs (Resolve® Panel C, Lot no. RC246, Ortho-Clinical Diagnostics, Inc.) and revealed aggregation with all types of RBCs. Serum chemistry tests showed high levels of bilirubin and LDH, presumably caused by the excessive hemolysis (Table 2). Moderate levels of protein were found in the urine but serum total protein levels were slightly below the normal range. Urine dipstick testing gave a false positive result for occult blood detection. The slide precipitation test for syphilis also resulted in a false positive reading, though anti-cardiolipin β2GPI antibodies were absent. Hypocomplementemia with cold activation was demonstrated with the serum titer of CH50 being low though plasma titers were recoverable (his blood was collected in EDTA-containing tubes). Cryoprecipitation under cold storage (4°C, 72 hrs) followed by immunoelectrophoresis revealed the presence of a mixed type II cryogobulinemia. HCV viremia was confirmed using an HCV-RNA PCR test (genotype: 1B). Antibodies directed against HBV, HIV, EBV, and CMV were not found. An abdominal echogram revealed the absence of a splenomegaly and showed that his liver was of normal size and that it had a smooth surface.
Hemogram (May of 2001). Red blood cells showed anisocytosis, polychromatosis, and poikilocytosis. Note the presence of polychromatic cells (arrow) and spherocytes (double arrow) in the figure.
Laboratory findings on admission
CBC
normal range
WBC
14700
/μl
3600–9100
RBC
221
×104/μl
374–502
Hb
8.7
g/dl
11.1–15.3
Htc
24.7
%
33.2–45.3
MCV
112
fl
85.0–100.0
MCH
39.4
pg
30.0–35.0
MCHC
35.1
g/dl
32.0–36.0
Platelet
29.3
×104/μl
13.0–37.0
Reticulocyte
236
‰
8.0–20.0
Biochemistry
T. bilirubin
2.18
mg/dl
0.30–1.20
D. bilirubin
0.69
mg/dl
0.05–0.40
AST
41
U/l
8–38
ALT
16
U/l
4–44
LDH
1044
U/l
220–430
ALP
172
U/l
117–335
ChE
219
U/l
200–450
BUN
16.0
mg/dl
8.0–19.0
Cr
1.07
mg/dl
0.80–1.30
UA
9.1
mg/dl
3.9–7.8
Na
140
mmol/l
136–148
K
4.3
mmol/l
3.6–5.0
Cl
107
mmol/l
98–109
TP
6.3
g/dl
6.5–8.0
Albumin
3.69
g/dl
3.8–5.3
T. cholesterol
161
mg/dl
130–220
Triglyceride
137
mg/dl
55–150
Syphilis
STS slide precipitation test
1+
negative
TPHA
-
negative
Hepatitis virus
HBs Ag
-
negative
HCV
Ab
+
negative
108.5
S/CO
RNA
220
KIU/ml
genotype
1B
Immunology
IgG
1497
mg/dl
870–1700
IgA
246
mg/dl
110–410
IgM
152
mg/dl
35–220
ANA
40
dilution
<80
anti-ds DNA ab
<2.0
IU/ml
<6.0
anti-CL-β2GPI ab
1.8
U/ml
<3.5
RF
<12
IU/ml
<20
ASO
20
IU/ml
<200
CRP
<0.1
mg/dl
<0.20
C3
68
mg/dl
60–140
C4
16
mg/dl
10–40
CH50
21.5
U/ml
30.0–45.0
Immune complex
C1q-assay
<1.5
μg/ml
<3.0
C3d-assay
8.8
μg/ml
<3.0
Cryoglobulin
2+
negative
(monoclonal IgMk+polyclonal IgG)
Urinary examination
Protein
2.2
g/day
0.03–0.12
Creatine clearance
83.5
ml/min.
70.0–130.0
NAG
5.0
U/l
<5.0
β2-microglobulin
137
μ g/l
5–300
Urinalysis
pH
6.0
5.0–7.0
SG
1.010
1.005–1.020
Protein
2+
-
Occult blood
3+
-
Keton
-
-
Sediment
RBC
5–9/HF
WBC
10–19/HF
Epithel
1–4/HF
Tubular cell
1–4/HF
Cast
Hyalin
0–1/LF
Glanular
-
Oval fat body
+
Because the patient was symptom free, he was discharged after his renal biopsy and was followed without treatment. One year later, the patient had the same degree of hemolysis (hemoglobin = 7.4 g/dl, LDH = 756 U/l, total bilirubin = 2.7 g/dl) though his creatinine clearance had decreased (69.5 ml/min.). However, his urinary protein was stable (1.6 g/day) and his serum total protein recovered into the normal range (7.1 g/dl). His liver function was near normal (AST = 57 U/l, ALT = 19 U/l, albumin = 3.5 g/dl). And his level of HCV-RNA was slightly reduced (130 KIU/ml).
Renal histology (Figure 2 and 3): Five glomeruli were visualized in the patient's biopsy specimen. One of these glomeruli was intact while the other four, which were mildly lobulated, appeared to show a slight increase in mesangial cells and matrix. The glomerular basement membrane (GBM) was partially thickened and PAS staining revealed small spikes and bubble formation. No glomeruli had GBM cryoglobulinemic deposits. Small fibro-cellular crescents were found in two glomeruli. The proximal tubular epithelial cells contained numerous hemosiderin granules.
Renal tissue (Light microscopy, PAS stain, × 200). The patient's glomeruli showed partial thickening of the GBM and exhibited some mesangial proliferation. Hemosiderin deposits were seen in the cytosol of proximal tubular epithelial cells (arrowhead).
Renal tissue (Immunofluorescence, × 100). Extensive deposition of IgG was demonstrated in the GBM using an anti-human IgG monoclonal antibody.
Immunofluorescence histochemistry demonstrated the presence of IgG in the GBM that was distributed in a course granular pattern, as well as the presence of IgA in the GBM and mesangial area. Staining for IgM and C3c was faint in the mesangial area, while C3d, C4d, C1q and fibrinogen were undetectable. Electron microscopy revealed small subepithelial deposits and a few para-mesangial deposits in the glomerulus.
Conclusions
In our patient, IgG autoantibody, which binds to RBCs, was detected in our patient using the direct antiglobulin test (Coomb's test), leading us to conclude that AIHA was the primary pathogenic mechanism that resulted in his hemolysis. Since his serum reacted with the full panel of RBCs tested, his autoantibodies probably recognized a common RBC surface antigen. Recent studies which suggest that the possible target antigens in AIHA are band 3 or 4.1 peptides, Rh, or glycophorin A, all of which are expressed on RBCs [6-8]. Our patient had not been treated with any antiviral drugs, such as ribavirin®, which have been shown to induce hemolytic anemia by a mechanism involving the oxidation-induced aggregation of band 3, leading to the binding of autologous anti-band 3 antibodies (natural antibody); when activated by complement, these antibodies induce intra- or extra-capillary hemolysis [9]. As a matter of course, ribavirin® was contraindicated in our case. In any event, a similar hemolytic mechanism might have resulted in this patient's production of circulating immune complex and free iron which lodged into his kidney.
HCV-associated glomerulonephritis is characterized by two types of histological changes i.e., membranoproliferative glomerulonephritis and membranous nephropathy (MN) [10,11]. Since the deposition of immunoglobulin and complement is detected on the glomeruli in this condition, these histological changes are thought to be due to immune complex disease [10-12]. In our case, proliferative changes were slight, and immunoglobulins and complement breakdown products were found to be deposited primarily in the GBM. Therefore, the primary histological changes in our patient's kidney were classified as being of the MN type. We speculate that glomerular deposition of IC occurred during chronic progression of the disease when the ratio of antigen and antibody was optimized.
There were no direct evidence of a causal relationship between chronic HCV infection and the occurrence of AIHA in our patient, though such a relationship has been suggested in four published reports [13-16]. Two of four patients that received an orthotopic liver transplant (OLT) were reported to have developed AIHA [13,14]. Though the pathogenesis of AIHA was not discussed in those studies, it may have been related to the strong immunosuppressive therapy that the post-OLT patients received. Another reported case of AIHA was reported in a patient who suffered from B cell chronic lymphocytic leukemia who was being treated with intermittent chemotherapy [15], while still another case was reported in a patient who had a complete congenital deficiency of IgA [16]. Thus, the immune systems of the above four patients were compromised prior to their development of hemolysis. What is not known is how chronic HCV infection leads to AIHA. The following evidence suggested that this patient's chronic immunological imbalance was triggered by chronic HCV infection. 1) HCV viremia was confirmed. 2) Our patient was of late middle age and this age group was shown to have a high frequency of extra-hepatic manifestations of HCV infection [1,3]. 3) It is accepted that the presence of cryoglobulinemia, hypocomplementemia (cold activation), and a biological false positive on the syphilic test are characteristic abnormalities in patients with chronic HCV infection [1,12,17,18]. In just recently paper, Ramos et al. reviewed highly suspected 17 cases with HCV-related AIHA [19]. These authors suggested that a higher prevalence of immunologic markers including cryoglobulinemia and hypocomplementemia all support the hypothesis that HCV-related AIHA has an autoimmune pathogenesis caused by chronic HCV infection.
We concluded that renal involvement in our patient post-dated his development of AIHA, which occurred against the backdrop of a progressive autoimmune imbalance induced by chronic HCV infection.
IO, YU, and YF were participated in the medical treatment of our patient. SH carried out the serological studies, and cryoprecipitate analysis. ME performed the renal biopsy with help from IO. TF, HO, and YY performed the pathological analyses.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent to publish the details of his medical record was obtained from our patient prior to preparation of this manuscript.
Calciphylaxis cutis is characterized by media calcification of arteries and, most prominently, of cutaneous and subcutaneous arterioles occurring in renal insufficiency patients.
Case Report
A 53-year-old woman with chronic cardiac and renal failure complained of painful crural, non-varicosis ulcers. She was hospitalized in an immobilized condition due to both the crural ulcerations and the existing heart-failure state (NYHA III-IV) having pleural and pericardial effusions, atrial fibrillation and weight loss of 30 kg over the past year. Despite normalization of calcium-phosphorus balance and improvement of renal function, the clinical course of crural ulcerations deteriorated during the following 3 months. After failure of surgical debridements, multiple courses of sterile-maggot therapy were introduced at a late stage to stabilize the wounds. The patient died of recurrent wound infections and sepsis paralleled by exacerbations of renal malfunction.
Conclusions
The role of renal disease in vascular complications is discussed. Sterile-maggot debridement may constitute a therapy for the ulcerated calciphylaxis at an earlier stage, i.e. when first ulcerations appear.
Background
Calciphylaxis cutis is characterized by media calcification of arteries and, most prominently, of cutaneous and subcutaneous arterioles [1], as well as by intimal proliferation and fibrosis [2]. The pathogenesis of Calciphylaxis remains unclear as it differs from intima-based common atherosclerosis and extravascular calcium-phosphate deposits in organs such as the skin in Calcinosis cutis are not involved. However, it appears to be associated with an elevated serum calcium or phosphate concentration elicited either by hyperparathyroidism or by vitamin-D intake. Female gender appears to be a risk factor. Although singular cases associated with non-renal diseases including inflammatory bowel disease, cancer, and primary hyperparathyroidism were reported [2], Calciphylaxis cutis mainly occurs in end-stage renal insufficiency, almost exclusively in patients undergoing kidney-replacement therapy [2-4]. The prognosis in Calciphylaxis patients is dramatically poor, with up to 6 months life expectancy after appearance of ulcerations [3]. Emphasizing its difference from intima-based atherosclerosis, it is also called calcific uremic arteriolopathy [1].
Case Report
A 53-year-old woman with a body mass index of 27.5 kg/m2 having chronic renal failure and chronic heart failure complained of painful crural, non-varicosis ulcers that started 8 months previously as livid palpable plaques or nodules within and underneath the skin, transforming into ulcers five weeks before admission and showing little sign of healing since. While a single, 2.5-cm ulcer closed, other ulcerations appeared at the right calf (Figure 1). Hence, the patient was admitted to a dermatologic department in an immobilized, weak condition having heart failure due to hypertensive and ischemic heart disease (left ventricular ejection fraction: 30 %, NYHA III-IV). Furthermore, she had a loss of appetite and lost 30 kg during the past year. Hypertension, recurrent ascendant urinary-tract infections and renal atherosclerosis were likely causes of renal failure. In addition, the patient had regularly taken analgesics. Systemic atherosclerosis was indicated by history of a myocardial infarction 5 years previously and by limitations of walking distance. In a coronary angiogram, a single-vessel coronary-artery disease was diagnosed necessitating an elective percutaneous coronary recanalization and stent deployment as well as three more percutaneous coronary interventions for repeated late in-stent restenosis during the previous 5 years. Clinical signs of the heart-failure state included both pleural and pericardial effusions, secondary mitral regurgitation due to cardiac dilation, and atrial fibrillation. The prevalent pericardial effusion was not deemed significant for cardiac filling as determined by echocardiography. Six months before admission, an Implantable Converter/Defibrillator (ICD) was inserted because of pre-syncope, non-sustained ventricular tachycardias in the Holter-ECG and a positive electrophysiological test result.
Calciphylaxis cutis on admission. After transfer to the dermatologic department, about 1 month after first incidence of a calciphylactic ulcer of the lower right leg.
Laboratory tests revealed a normal plasma concentration of parathyroid hormone (5.02 pmol/l, normal: 1.1–6.9), Vitamin (1,25-OH)-D3 deficiency (7.1 pg/ml, normal: 20.2–46.2), however, slightly elevated calcium (2.66 mmol/l, normal: 2.02–2.6) concentration. Potassium (4.3 mmol/l) and phosphate (1.26 mmol/l) concentrations were normal. Glomerular filtration rate estimated by MDRD equation [5] was 25.4 ml/min (creatinine: 207 μmol/l; urea: 30.2 mmol/l; albumin: 35.1 g/l) at admission indicative for renal failure. Further, low HDL cholesterol (0.86 mmol/l, normal: >0.9), high triglycerides (3.06 mmol/l, normal: <2.3), yet a normal LDL cholesterol (3.49 mmol/l, normal: <4.2) in absence of statins to which the patient claimed to be allergic, were found. Leukocyte count and C-reactive protein were found to be elevated at least during the 12 months prior to admission, ranging from 10–15 Gpt/l (normal:4–9) and 60–200 mg/l (normal: <5), respectively. Ferritin was in the normal range between 200 and 300 ng/ml (normal: 13–300), neither supporting an infection as an acute-phase reactant nor being the reason for the prevalent normocytic, normochromic anemia (hemoglobin 6 mmol/l, hematocrit 31%). However, an iron deficiency could have limited further rises of ferritin due to infection. The anemia was not due to erythropoietin deficiency as reticulocytes were 15 Gpt/l. Urine tests showed a microproteinuria (0.25 g/l) and leukocyturia (25/μl). After cessation of coumadin therapy for atrial fibrillation, the International Normalized Ratio leveled off at 1.42 (normal: 1) in presence of low-dose heparine. Liver enzymes including serum alcaline phosphatasis were between 16 and 19 μmol/l*s (normal: <4.2) and gamma-glutamyl transferase between 9 and 14 μmol/l*s (normal: <0.6), while transaminases were within normal range. Ultrasound examination revealed small kidneys (9 cm longitudinally) with loss of clarity of architecture and multiple cysts. The renal cortex was narrowed to 7 mm, ureters were not dilated. A whole-body bone scintigraphy was negative for bone metastases or osteomyelitis. Ultrasonographic examinations of the thyroid gland were unrevealing; parathyroid bodies were not identified. The radiologic search for nonvascular soft-tissue calcifications was negative. Systemic lupus erythematosus or other types of autoimmune diseases were ruled out by negative antibody screening tests. The diagnosis Calciphylaxis cutis was established by cutaneous biopsy.
Hypercalcaemia was readily normalized by diuretic therapy with torasemide in combination with standard heart-failure therapy including aldosterone antagonist, ACE inhibitor, beta-adrenergic blockade and digitalis over a one-month period. Possible reasons for the hypercalcaemia like hyperparathyroidism or high vitamin D serum levels were ruled out, parathyroid hormon-related peptide was not determined. Glomerular filtration rate improved to 33.1 ml/min under balanced fluid-substitution. Thus, renal function in this patient was mid-stage impaired with potential to recover under therapy. However, fluid overload had to be avoided because of the heart-failure condition. Once electrolytes, including plasma potassium fell, an incessant ventricular tachycardia triggered the ICD. Therefore, potassium was given to obtain a high-normal plasma concentration, and an amiodarone therapy was started.
Despite wound care on a daily basis both in hospital and in ambulatory care, the cutaneous ulcers worsened. 2 months after first admission, the patient was readmitted because of exacerbated ulcers on both lower legs (Figure 2). Hydrocolloid dressings failed because of the size and number of wounds. Instead, an active-charcoal absorband dressing combined with repeated surgical necrectomy was applied to stabilize the wound situation in conjunction with antibiotic therapy guided by wound-secretion-derived microbiological cultures. Parathyroid function remained normal. Following a 1-month consolidation therapy the patient was discharged with a stable wound condition to continue wound care at home. However, the large extent of those ulcers affecting both lower legs remained unchanged at the time of discharge compared to admission.
Calciphylaxis cutis, 2 months after admission. Large ulcerations at both lower legs with signs of superinfection.
About 4 months after first admission, the patient was readmitted with signs of sepsis necessitating an escalated antibiotic treatment and surgical wound care. Yet conventional necrectomy proved ineffective. Therefore, maggot debridement therapy using sterile maggots of the greenbottle fly Lucilia sericata was introduced. Following four courses, the wounds gradually improved, granulated and showed signs of healing, yet the large extent of those wounds remained, rendering the patient at high risk of reinfection (Figure 3). As an adjunct antibiotic therapy, a combination of piperacillin-combactam was given over 9 days. The subcutaneous nodule-like tumors continued to occur at new locations. The patient's overall condition remained poor. Six days after being transferred to a primary hospital, she was readmitted because of acute renal failure. In addition, she suffered from the consequences of a stroke, with right-side hemiparesis, as well as from worsened crural ulcerations and a new pre-sacral pressure-sore lesion. A urinary-tract infection with Pseudomonas aeruginosa was diagnosed. The patient died within 4 days of a systemic-inflammatory response syndrome primarily due to her superinfected, poorly controllable wounds.
Calciphylaxis cutis, 4 months after admission. Size enlargement of calciphylactic ulcerations. Maggot debridement therapy abolished superinfection to a large extent.
The diagnosis of Calciphylaxis cutis was confirmed by autopsy, revealing small- and medium-sized blood-vessel calcifications within the ulcerated skin of the lower legs as previously seen in the biopsy (Figure 4 and 5). Even capillaries were found to be surrounded by calcific deposits. The patient was shown to have severe calcified atherosclerotic lesions occasionally involving the whole circumference of the aorta and the coronary arteries with an in-stent restenosis within the right coronary artery. Furthermore, the patient presented intermediate-grade atherosclerosis in renal as well as in lower-leg peripheral arteries. Unusual locations of calcifications were the wall of the left atrium of the heart (Figure 6) and pulmonary arteries.
Calcifications of skin arteries, arterioles and capillaries. Biopsy and autopsy revealed calcifications of small arteries, arterioles and capillaries within the ulcerated skin.
Calcifications of skin arteries, arterioles and capillaries. Biopsy and autopsy revealed calcifications of small arteries, arterioles and capillaries within the ulcerated skin.
Calcifications of the left atrial wall of the heart. Autopsy revealed calcifications and endocardial fibrosis within the wall of the left atrium.
Discussion
Here we report a case of ulcerated Calciphylaxis cutis in the presence of a moderate renal insufficiency in contrast to most previous observations that suggest Calciphylaxis cutis to be associated with terminal renal insufficiency [3,4]. Moreover, the actual plasma levels of calcium and phosphorus seemed to play a minor role than assumed from previous evidence [3]. However, in spite of measures to preserve renal function, the clinical course of Calciphylaxis cutis was rapidly progressive and virtually undeterred by treatment in our patient. The skin ulcerations due to Calciphylaxis cutis constantly deteriorated over less than 6 months from first admission. Alternative therapeutic options including glucocorticoids were not deemed salutary in the presence of ulcerations [3]. Sterile maggot debridement therapy was more successful than surgical debridement in this patient. However, it was introduced late. In line with previous evidence [6], we strongly encourage its use in ulcerated Calciphylaxis cutis in combination with a broad-spectrum, Pseudomonas – effective antibiotic earlier on to avoid superinfections and to facilitate the granulation process. Overall, screening of renal-disease patients for Calciphylaxis cutis in the non-ulcerated stage seems to be crucial to timely address risk factors associated with the disease. Classic and putative risk factors for Calciphylaxis cutis derived from this case are summarized below.
Cardiovascular risk factors were low HDL cholesterol and high triglycerides, history of cigarette smoking during the previous 10 years (20-pack years) and arterial hypertension. The prevalent renal disease appears to be both a cardiovascular risk factor [7] and risk factor for Calciphylaxis [3]. Further, the patient may have had a genetic disposition to cardiovascular disease, as her mother died of myocardial infarction prematurely. Lipoprotein (a) and homocysteine levels were not determined. C-reactive protein elevation is likely due to the skin ulcers. However, it was high during the 12 months prior to death and was probably high even before that as her history of urinary-tract infections revealed, thus, making it eligible as a contributing factor for atherosclerosis [8]. A slight disturbance of calcium-phosphorus metabolism as well as a history of obesity, former coumadin use, and a possible underlying vitamin-K deficiency based on a steadily elevated international normalized ratio after coumadin discontinuation are prevalent, putative risk factors for Calciphylaxis [2]. Yet, the mechanism of these disseminated calcifications in small cutaneous arteries, and capillaries as well as in conductive arteries, pulmonary arteries and the left atrium is by far not explained. Future research will clarify whether matrix-protein dysfunction elicited by Vitamin-K deficient state is in play [9,10] which may affect both pyrophosphate and calcium metabolism [1,11] as well as wound healing [3].
Taken together, Calciphylaxis cutis is thought to arise from the arterial media layer rendering it different from intimal calcifications seen in common atherosclerosis and from extravascular organ calcifications such as Calcinosis cutis. The in-stent restenosis problem in our patient may reflect a propensity of intimal proliferation and fibrosis usually seen in Calciphylaxis [2]. Here, we speculate that the patient actually had both Calciphylaxis cutis represented by calcifications of small arteries, arterioles and capillaries of the skin as well as systemic atherosclerosis as primarily seen in conductive arteries.
List of abbreviations used
ACE = angiotensin converting enzyme
ECG = electrocardiogram
HDL = high-density lipoprotein
ICD = implantable cardioverter defibrillator
MDRD = Modification of Diet in Renal Disease (Study)
NYHA = New York Heart Association class
Competing interests
None declared.
Authors' contributions
RUP was the attending physician for this patient. He collected all results and drafted the paper. JS performed the autopsy, added his insights to all parts of the case report and helped in the process of revision. RP, head of the department, has overseen the drafting process and gave critical inputs to the manuscript. HA as the attending chief physician knowledgeably participated in the discussion and in the revision. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent for autopsy to further medical science and teaching was given by the patient before she died.
Although recovery of renal functions in dialysis dependent patients is estimated to be greater than 1%, there are no indicators that actually suggest such revival of renal function. Residual renal function in dialysis patients is unreliable and seldom followed. Therefore renal recovery (RR) in dialysis dependent patients may remain unnoticed. We present a group of dialysis dependent patients who regained their renal functions. The aim of this project is to determine any indicators that may identify the recovery of renal functions in dialysis dependent patients.
Methods
All the discharges from the chronic dialysis facilities were identified. Among these discharges deaths, transplants, voluntary withdrawals and transfers either to another modality or another dialysis facility were excluded in order to isolate the patients with RR. The dialysis flow sheets and medical records of these patients were subsequently reviewed.
Results
Eight patients with a mean age of 53.8 ± 6.7 years (± SEM) were found to have RR. Dialysis was initiated due to uremic symptoms in 6 patients and fluid overload in the remaining two. The patients remained dialysis dependent for 11.1 ± 4.2 months. All these patients had good urine output and 7 had symptoms related to dialysis. Their mean pre-initiation creatinine and BUN levels were 5.21 ± 0.6 mg/dl and 72.12 ± 11.12 mg/dl, respectively. Upon discontinuation, they remained dialysis free for 19.75 ± 5.97 months. The mean creatinine and BUN levels after cessation of dialysis were 2.85 ± 0.57 mg/dl and 29.62 ± 5.26 mg/dl, respectively, while the mean creatinine clearance calculated by 24-hour urine collection was 29.75 ± 4.78 ml/min. One patient died due to HIV complications. One patient resumed dialysis after nine months. Remaining continue to enjoy a dialysis free life.
Conclusion
RR must be considered in patients with good urine output and unresolved acute renal failure. Dialysis intolerance may be an indicator of RR among such patients.
Cessation of dialysisDialysis dependenceRecovery of renal functionsResidual renal functionBackground
Although guidelines for the initiation of hemodialysis in End Stage Renal Disease (ESRD) patients are clearly laid out, little is known about the indicators of Renal Recovery (RR) for the cessation of dialysis either on a temporary basis or indefinitely. Although there are studies citing recovery of renal function in dialysis dependent patient, but the indicators or pointers to such recovery is not elucidated. We identified eight patients who were declared dialysis dependent and were initiated on maintenance hemodialysis. Later, these patients were noted to have either complete or partial recovery of renal functions, and were taken off dialysis. The aim of this paper is to emphasize the need for watchfulness in assessing RR in patients who are declared to be dialysis dependent. We also attempt to identify the factors that may predict or alert a clinician to the possibility of RR that is sufficient enough for the cessation of maintenance dialysis.
Methods
We identified all patients who were discharged from our chronic dialysis facilities. Deaths, transplants, voluntary withdrawals and transfers to other modalities or dialysis centers were identified and excluded. The medical records of the remaining patients were reviewed, and the symptoms on dialysis, interdialytic weight gains, pre dialysis and post dialysis serum chemistries such as BUN, and creatinine were recorded. Creatinine clearance if determined prior to initiation of dialysis was noted. A note of the symptoms on dialysis such as hypotension, muscle cramps, lethargy and extreme weakness after dialysis was also made. The previously recorded follow-up chemistries and symptoms after the cessation of dialysis if any, was documented. Outcomes of death, return to dialysis, and duration of dialysis-free time was recorded. A total of 8 dialysis patients over the preceding three years who had recovered their renal function were identified (Table 1).
Table illustrating the characteristics, clinical course and outcome of the 8 patients presented in the study.
No
Age
Race/Gender
Renal Biopsy
Presumed Etiology
Serum Cr at initiation
Initial Serum BUN
Reason for Initiation of HD
Duration (months)
Symptoms on HD
Serum Cr after cessation
BUN after cessation of HD
Cr. Cl after cessation
Time off HD (months)
1
34
W/M
FSGS & AIN
HIV, AIN, HTN Nepscl
3.34
53
Nau/Vom
4
Leg cramps Fatigue
2.76
28
34
1 Died of HIV compl.
2
81
LA/F
FSGS
HTN Chol. Embolization
4.2
60
Fluid overload + rising cr
12
None
1.73
36
18
21
3
50
BM
FSGS
FSGS + ATN
8.61
114
Mental status changes
30
Washed out, cramps Low BP.
4.12
30
40
15
4
71
WM
Mem GN
Mem GN
6.35
68
Nau/vom Weight loss
29
Washed out, cramps Low BP.
5.36
48
9
9
5
60
BF
FSGS
Obesity related FSGS
6.88
69
Anasarca, ARF on CRF
6
Washed out, cramps Low BP.
1.91
48
36
26
6
31
BM
No biopsy
HIV Nephropathy
3.39
25
Acidosis + Increasing Cr
2
Nau/Vom Cramps
1.71
4
20
2
7
68
WM
Contrast Tox, no biopsy
ATN, Contrast. RCC with single kidney
4.34
121
Acidosis Hyperkalemia
2
Washed out, cramps Low BP.
2.4
17
30
33
8
36
AF
FSGS
FSGS
5.57
67
Nau/vom
4
Washed out, cramps Low BP.
1.85
26
51
51
Results
In the preceding 3 years we identified 8 patients in our two chronic dialysis facilities who had renal recovery following the discontinuation of dialysis (either on a temporary or an indefinite basis). Our chronic dialysis units have an incident patient population of approximately 25 patients per year, and a prevalent population of approximately 150 patients. The annual growth rate is approximately 7%. Among the 8 patients that recovered their renal function, 3 were Caucasians, 3 African Americans, 1 Hispanic and 1 Asian. Five were male and three female. The mean age of these patients was 53.87 ± (SEM) 6.7 with a range of 31 years to 81 years (Table 1). Four of these patients had an underlying glomerulopathy, and two had hypertensive glomerulosclerosis. Two patients had HIV disease, one patient had probable cholesterol embolization and one patient had ischemic nephropathy sustained during the removal of a kidney that had a cancerous growth. Six patients had symptoms attributed to uremia, 2 patients also had acidemia and hyperkalemia and 2 patients had fluid overload in the form of either resistant anasarca or pulmonary edema. Their mean BUN and creatinine was 72.12 ± 11.1 (range 25 – 121) mg/dl and 5.21 ± 0.6 (range 3.3 – 8.6) mg/dl respectively. These patients remained on dialysis for a mean of 11.13 ± 4.0 (range 2–30) months. It was unclear if the patients had good urine output at the time of dialysis initiation. However, all the patients vocalized an increment in urine output while on dialysis, and two patients had to frequently interrupt the dialysis session to pass urine. Seven of these patients were consistently symptomatic during the dialysis session. All seven patients had severe leg cramps, and 5 had a washed-out feeling after dialysis and felt better on the non-dialysis days. These seven patients requested a reduction in either their dialysis treatment time or the frequency of dialysis. Patients also had episodes of nausea, vomiting, and hypotension during dialysis. Their mean serum creatinine and BUN levels at the time of cessation of dialysis were noted to be 2.85 ± 0.5 (range 1.71 – 6.34) mg/dl and 30.86 ± 4.5 (range 14 – 48) mg/dl, respectively. Since these patients' creatinine levels were not in a steady state at the time of initiation, creatinine clearance was not determined by using 24-hour urinary creatinine excretion. After the cessation of hemodialysis, the patients were followed on a weekly basis in the nephrology clinics and their creatinine clearance was determined by 24-hour urinary creatinine excretion once the creatinine had stabilized. The mean creatinine clearance determined by 24-hour urinary creatinine excretion was 29.75 ± 4.8 (range 9 – 51) ml/min.
Of the 8 patients, three insisted to be taken off dialysis. These patients remained dialysis free for a mean duration of 19.75 ± 5.6 (range 1 – 51) months. One patient with HIV disease that was complicated by disseminated MAI infection and CMV retinitis infection died 1 month after discontinuation of dialysis due to infections. His creatinine clearance after the cessation of the dialysis was estimated to be 53 ml/min. One patient, whose creatinine clearance was 9 ml/min, and who remained without dialysis for nine months, had to be restarted on dialysis due to progressive loss of weight and appetite. His creatinine clearance remains at 9 ml/min. It is interesting to note that 6 of the 8 patients had a renal biopsy performed and 5 of these 6 patients with renal biopsy had focal segmental glomerulosclerosis (FSGS) as the primary glomerular disease. Although they all may have had acute renal failure in the setting of chronic kidney disease (CKD) they all were assumed to have renal failure secondary to their primary glomerulopathy.
Discussion
Although there are guidelines for the initiation of hemodialysis and for the care of patients on maintenance hemodialysis and peritoneal dialysis, there is very little in the literature regarding the recovery of renal function. This is mainly because renal failure patients who are labeled as dialysis dependent rarely regain their renal function. Even when they do, it may not be clinically evident, and such marginal renal recovery hardly justifies cessation of dialysis. There are case reports that cite delayed recovery of renal functions in dialysis dependent patients who have had ischemic nephropathy secondary to renal artery stenosis. When the renal artery occlusion to the solitary kidney was surgically repaired as late as six months after initiation of dialysis, the patient showed recovery of renal function [1]. Kato and his colleagues have reported recovery of renal function in their dialysis dependent patient who had cholesterol embolization to the kidneys causing loss of renal functions [2], while Morales and his colleagues reported spontaneous recovery of renal function in their patient who supposedly had renal failure due to FSGS associated with human immunodeficiency virus (HIV) infection [3]. Both of these conditions are widely believed to cause irreversible renal failure. Urologic conditions such as inadvertent ligation of the renal vein of a single functioning kidney and chronic obstructive uropathy due to chronic ureteric obstruction especially in a single functioning kidney, can also reveal evidence of delayed resolution of renal failure upon relieving the obstruction, as cited by Cancarini and Shokeir and their colleagues [4,5]. Alkhunaizi and his colleague reported a case of acute renal failure due probably to secondary oxalosis related to excessive ingestion of vitamin C. Their patient also had delayed renal recovery, which they attributed to slow removal of oxalates by aggressive hemodialysis [6]. Probably the most reported dialysis dependent cases that eventually regained renal functions are vasculitis related acute renal failure patients and those with accelerated hypertension, who were later noted to have an adequate amount of renal functions to evade dialysis therapy [7-9]. Sekkarie and colleagues reviewed 10-year data of 7404 patients from the Michigan Kidney Registry to evaluate the rate and associated factors for recovery of renal function. They observed that in patients with glomerulonephritis associated with a systemic illness, vasculopathies had threefold to fourfold higher recovery rates. White race, older age, and later year of ESRD were associated with significantly higher recovery rates. They also observed that in approximately 48% of those who regained renal function, recovery occurred within 6 months of initiating dialysis therapy, 74% within 1 year, and lasted at least 1 year in 75% of such cases [10]. However, they did not allude to the factors that forewarned the physicians about recovery of renal function. These authors cautioned the physicians to be vigilant before declaring them as having ESRD. Spanish medical literature, however, addresses the issue of recovery of renal function. Two different authors proposed periodic monitoring of residual renal functions at least once in two months [11,12].
In the United States, in contradistinction to the peritoneal dialysis patients, residual renal function in hemodialysis patients is usually not monitored. In our hemodialysis facilities, urine output is neither routinely measured nor residual renal function estimated. The only indication that alerted the physician was marginal decrement in the pre-dialysis serum creatinine in a patient who requested reducing the treatment time due to intolerance to dialysis procedure. All except one of our patients had symptoms on dialysis such as nausea, vomiting, hypotension and leg cramps. In our institution approximately 1% to 2% of the patients recover renal function. This may be an underestimation because only those patients who had severe symptoms on dialysis and requested a reduction in dialysis time, prompted us to re-evaluate their renal functions. Most of the symptoms are presumably secondary to excessive ultrafiltration on dialysis. There probably are more patients who are not subjected to significant fluid removal during dialysis, and therefore may not exhibit symptoms on dialysis. Such patients may continue to receive dialysis for undetermined period of time that they do not need. Our second patient may be a representation of such group.
Six of the 8, i.e. 75% percent of our patients who came off dialysis had a glomerulopathy listed as the cause of renal failure, which is supposedly an irreversible cause of renal failure. Clearly, these patients may have had an acute component of renal failure that either was ignored or was considered to be irreversible. One might argue that during multidisciplinary mandatory monthly patient care conferences (PCC), the decreasing values of BUN and creatinine should attract the attention of the caregivers to the possibility of RR. However, this does not seem to be the case as low BUN and creatinine can also be attributed to worsening of nutritional status. This may be perceived to be due to under-dialysis that may prompt even more aggressive dialysis that the patient does not tolerate. In some instances, such as our last patient, the creatinine may start to fall precipitously only after the cessation of dialysis. This possibly could not be evident until the dialysis was withheld. All these patients explicitly expressed a significant improvement in their quality of life after the cessation of dialysis. The most significant difference was observed in patient number four, who despite his GFR being less than 10 ml/min, could not tolerate hemodialysis session. He remained most symptomatic immediately following dialysis, experiencing better health the following day, but again cyclically worsening after dialysis. He remained off-dialysis for only 9 months but enjoyed his dialysis-free life during these 9 months. Although most of these patients had such symptoms for several months, their residual renal function was never estimated, and even if it were, that would not have made much difference because there are no guidelines for the interpretation of these results. The creatinine if not in a steady state, cannot be used for creatinine clearance utilizing the conventional formulae. The Cockroft and Gault formula, employing the predialysis serum creatinine, has its own limitations. In addition, progressive decline in serum creatinine is an independent indicator of poor nutritional status [13]. This, as mentioned earlier is often attributed to inadequate dialysis, and the patient therefore can be subjected to more aggressive dialysis, which is poorly tolerated and can make the patient more miserable.
Conclusion
Approximately 1% to 2% of the patients declared to have end stage renal disease has significant recovery of their renal function and is taken off dialysis schedule. More patients may have a potential for recovery of renal function that could be missed. There are no guidelines to monitor residual renal function. Additionally, many patients with various glomerulopathies with chronic kidney disease may develop acute renal failure and get erroneously labeled to have "reached ESRD" and become dialysis dependent. Once these patients even partially recover their renal function, they tend to develop symptoms such as muscle cramps, hypotension and tired or "washed out" feeling following their dialysis sessions. These patients then either tend to skip the dialysis sessions and/or reduce the dialysis time. We recommend that residual renal function be checked in all dialysis patients who are not anephric, and those who are symptomatic on dialysis with good urine out-put. These patients should be evaluated periodically for possible recovery of renal function.
Bar diagram depicting the level of serum creatinine in each patient at the time of initiation and cessation of maintenance hemodialysis. Series 1 represents the serum creatinine in mg/dl at initiation of dialysis and series 2 at discontinuation of hemodialysis.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Hemodialysis (HD) patients are at risk for medication-related problems (MRP). The MRP number, type, and appearance rate over time in ambulatory HD patients has not been investigated.
Methods
Randomly selected HD patients were enrolled to receive monthly pharmaceutical care visits. At each visit, MRP were identified through review of the patient chart, electronic medical record, patient interview, and communications with other healthcare disciplines. All MRP were categorized by type and medication class. MRP appearance rate was determined as the number of MRP identified per month/number of months in study. The number of MRP per patient-drug exposures were determined using: {[(number of patients) × (mean number of medications)]/(number of months of study)} /number of MRP identified. Results were expressed as mean ± standard deviation or percentages.
Results
Patients were 62.6 ± 15.9 years old, had 6.4 ± 2.0 comorbid conditions, were taking 12.5 ± 4.2 medications, and 15.7 ± 7.2 doses per day at baseline. Medication-dosing problems (33.5%), adverse drug reactions (20.7%), and an indication that was not currently being treated (13.5%) were the most common MRP. 5,373 medication orders were reviewed and a MRP was identified every 15.2 medication exposures. Overall MRP appearance rate was 0.68 ± 0.46 per patient per month.
Conclusion
MRP continue to occur at a high rate in ambulatory HD patients. Healthcare providers taking care of HD patients should be aware of this problem and efforts to avoid or resolve MRP should be undertaken at all HD clinics.
Background
The Institutes of Medicine report highlighting the burden of medication errors in the United States has brought the issue of patient safety to the forefront of medical concern. [1] In the United States healthcare system, medication-related problems (MRP) cause significant patient morbidity, mortality, and cost. [2-4] MRP are implicated in 16.1% of internal medicine ward hospital admissions.[4] Surprisingly, 58.9% of admissions could definitely or possibly be avoided. Once admitted to the internal medicine ward, greater than 18% of patient deaths can be attributed to one or more drugs.[5] Adverse drug events contribute to over 100,000 deaths annually and 25% of ambulatory patients report experiencing at least one adverse drug event.[3,6] The economic burden of MRP on the healthcare system is estimated to be in excess of $177 billion.[2]
Identification and resolution of MRP can occur through provision of pharmaceutical care. Pharmaceutical care is defined as a practice in which a pharmacist takes the responsibility for the patient's drug related needs, and is held accountable for this commitment.[7] The provision of pharmaceutical care has made substantial contributions to patient morbidity and mortality in critical care and congestive heart failure patients.[8,9]
Currently, there are approximately 350,000 end-stage renal disease (ESRD) patients in the United States.[1] In addition to renal failure, these patients have a mean 5 comorbid conditions that require complex medical regimens of a median 8 medications.[10,11]
Several studies have demonstrated that ambulatory hemodialysis (HD) patients are at risk for medication-related problems. [12-17] Medication-related problems can be classified into eight general categories: untreated indications; improper drug selection; sub-therapeutic dosage; overdose; adverse drug reactions; drug interactions; failure to receive drugs; and drug use without indication.[18] Factors associated with medication-related problems in these patients include: more than three concurrent disease states present; medication regimen changed four or more times during the past 12 months; five or more medications in present drug regimen; twelve or more medication doses per day; history of noncompliance; presence of drugs that require therapeutic monitoring, and presence of diabetes.[17] Nearly all HD patients are risk due to their multiple risk factors present.
Several single-center, short-term studies in ambulatory HD patients have shown that a mean 4 – 8 MRP exists per patient.[12-14,17] Although the reports identified numerous MRP of various type and significance, results presented were obtained with average patient follow up time of 2.6 ± 1.9 months. It is unknown if the number, type, or severity of MRP continues at the same rate after a few months time. This study was conducted to determine the number, type, severity, and appearance rate of MRP, as identified through pharmaceutical care activities, in ambulatory HD patients.
Patients and methods
Over a 10 month period (August 2001 through May 2002) randomly selected patients at our freestanding, non-profit outpatient dialysis unit (Dialysis Clinic, Inc., Kansas City, MO) were evaluated. Patients were eligible for inclusion if they were greater than 18 years of age, planned to be continuously enrolled in HD therapy at the same dialysis clinic throughout the duration of the study, and agreed to participate with monthly pharmacist visits. All patients were recruited within a 2 month period and followed for 10 months.
All patients were typically hemodialyzed for three to four hours per treatment, three days per week. Patients were under the care of a private group of nephrologists. Standard practice at the HD clinic included regular patient visits by a nephrologist, averaging two to three times per week. At these encounters, the nephrologist assessed the patient and made adjustments to the HD prescription or medication list as deemed appropriate. On a monthly basis, nephrologists rotate clinic responsibilities among the group.
At each monthly visit the pharmacist reviewed the patient chart, electronic medical record including laboratory measurements, conducted a patient interview with review of all medications, and communicated with other healthcare disciplines (medicine, nursing, dietary, social work) about the health status of the patient. With the data collected, the pharmacist then evaluated appropriateness of medical therapy, identified MRP, and communicated interventions to the nephrologist via pharmacist progress note and e-mail correspondence.
Data collected included: patient demographics (age, gender, race, reason for and duration of ESRD), documented comorbid conditions, medication type and number, and number of medication doses per day. Medications were classified similar to that previously reported[11,19] as follows: anemia (erythropoietin, iron), renal bone disease (calcium or aluminum salts, sevelamer, vitamin D analogs), cardiac (any agent used for hypertension, congestive heart failure, coronary artery disease, arrhythmia), cholesterol-lowering (niacin, fibric acid agent, HMG-CoA reductase inhibitor), endocrine (any agent used for diabetes, thyroid disorders, menopause), anti-infective (including antiviral), antithrombotic (agents that may affect platelet function or prolong coagulation), psychotropic (antidepressants, antipsychotics), gastrointestinal (histamine-2 receptor antagonist [H2RA], proton pump inhibitor [PPI], promotility agents, laxatives), vitamins, analgesics, antipruritics, and other (agents with a prevalence of less than 10%).
Identified MRP were categorized as: indication without drug therapy (IWD – patient has a medical problem that requires medication therapy (an indication for medication use) but is not receiving a medication for that indication); drug use without indication (DWI – patient is taking a medication for no medically valid indication); improper drug selection (IDS – patient has a medication indication but is taking the wrong drug); sub-therapeutic dosage (UD – patient has a medical problem that is being treated with too little of the correct medication); over-dosage (OD – patient has a medical problem that is being treated with too much of the correct medication); adverse drug reaction (ADR – patient has a medical problem that is the result of a ADR or adverse effect); drug interaction (DI – patient has a medical problem that is the result of a medication-medication, medication-laboratory, or medication-food interaction); failure to receive drug (FRD – patient has a medical problem that is the result of their not receiving a medication); and other (O – all those not able to be classified as above).
Statistics
Results are expressed as mean ± standard deviation or percentages (frequency data) as appropriate. MRP appearance rate was determined as follows: the number of MRP identified per month/number of months in study. The number of MRP per patient-drug exposures were determined using the following calculation: { [(number of patients) × (mean number of medications)]/(number of months of study)} /number of MRP identified.[17]
Results
Over the ten-month period 145 patients received HD (66 pharmaceutical care group; 79 usual care group). Data from twelve patients was excluded from analysis due to patient death (n = 9), transfer (n = 2), or transplant (n = 1) during the study period. Patient demographics are shown in table 1. Overall, patients were an average of 62.6 ± 15.9 years old (range 26–92) and received dialysis for 3.1 ± 2.5 years (range 0 – 13.3). The majority of patients were male (56%) and African American (78%). Overall, diabetes mellitus was the primary cause of ESRD (47.8%), followed by hypertension (22.4%). Patients receiving pharmaceutical care had 6.4 ± 2.0 (range 3–12) comorbid conditions, were taking 12.5 ± 4.2 (range 6–21) medications, and 15.7 ± 7.2 (range 4–34) doses per day at baseline.
Patient Demographics
Patient Characteristic
All (n = 133)
Pharmaceutical Care (n = 66)
Usual Care (n = 67)
p value
Age (years)
62.8 ± 15.0
62.0 ± 14.3
63.5 ± 15.8
0.64
Male gender (%)
55.6
45.5
65.7
0.22
Race (%)
Black
78.2
71.2
85.1
0.08
Caucasian
17.3
22.7
12.0
0.06
Other
4.5
6.1
3.0
0.66
ESRD Reason (%)
Diabetes mellitus
48.1
60.6
35.8
0.007
Hypertension
21.8
16.7
26.9
0.23
Glomerulonephritis
14.3
12.1
16.4
0.84
Other
15.8
10.6
20.9
0.10
ESRD Duration (years)
3.1 ± 2.5
3.0 ± 1.9
3.3 ± 2.9
0.43
# Medications
11.4 ± 4.2
12.5 ± 4.2
10.2 ± 3.8
0.007
# Comorbid Conditions
5.8 ± 1.9
6.4 ± 2.0
5.1 ± 1.5
0.00003
Over the 10-month period the pharmacist reviewed 5,373 medication orders and identified 354 MRP in 66 different patients. The MRP type and frequency can be seen in Figure 1. Most commonly, the pharmacist identified medication-dosing problems (33.5%) or adverse drug reactions (20.7%) of the time. An indication that was not currently being treated (IWD) was discovered 13.5% of the time. The "other" category included interventions aimed at education of the patient and/or staff, recommendations due to lack of supportive laboratory data, and recommendations due to inappropriate medication treatment duration. A MRP was identified for every 15.2 medications reviewed. Overall the MRP appearance rate was 0.68 ± 0.46 per patient per month. At the end of the study period, 0.45 MRP per patient per month were identified (Figure 2).
Frequency distribution of medication-related problems in ambulatory hemodialysis patients. ADR = adverse drug reactions; DI = drug interactions; DWI = drug use without indication; FRD = failure to receive drugs; IWD = indication without drug therapy; O = other; OD = over-dosage; UD = sub-therapeutic dosage; IDS = improper drug selection.
Number of medication-related problems per patient over time.
MRP were classified according to medication class involved (Table 2). Cardiovascular-related medications accounted for 29.7% of MRP: 13.3% cardiovascular medication; 8.2% cholesterol lowering medications; and 8.2% antithrombotic medications (e.g., aspirin). Endocrine medications accounted for 15.5% of identified MRP. Nephrology-specific medications (anemia and renal bone disease medications) accounted for 15% of MRP.
Medication Class Involvement
Medication Class
Number (% Occurrence)
Endocrine
55 (15.5)
Cardiac
47 (13.3)
Gastrointestinal
45 (12.7)
Renal bone disease
31 (8.8)
Cholesterol-lowering
29 (8.2)
Antithrombotic
29 (8.2)
Anemia
22 (6.2)
Psychotropic
19 (5.4)
Anti-infective
17 (4.8)
Analgesic
15 (4.2)
Other
15 (4.2)
Vitamins
6 (1.7)
Antipruritic
6 (1.7)
Discussion
We determined that MRP continue to occur at a relatively high rate month after month in ambulatory HD patients. At the end of the study period, 0.45 medication-related problems per patient per month were identified. Extrapolating these findings to the 246,121 United States HD population, nearly 111,000 MRP occur each month. If the same frequency of MRP type were to occur as we found at our clinic, then each month 35,409 dosing problems occur, 21,645 adverse drug events occur, and 14,430 medical problems are not treated with appropriate medication therapy. These numbers of MRP that could occur each month appears staggering. However, these patients have multiple disease states which require multiple medications and intense follow-up. HD patients take a median 8 medications for an average 5 comorbid conditions.[10,11] Additionally, others have demonstrated that patients with chronic kidney disease are not prescribed medications, despite having a medical necessity for pharmacologic treatment.[20,21] The impact of MRP in other ESRD populations, i.e., peritoneal dialysis and transplant patients, warrants further investigation.
Patients having cardiovascular- and endocrine-related MRP are not surprising given that over 42% of patients have diabetes and 75.2% of patients have a history of hypertension.1 Cardiovascular disease (CVD) is the leading cause of mortality in ESRD patients, accounting for approximately 50% of deaths.[1] There is much opportunity to increase use of medications with known cardioprotective benefit. Unfortunately, dialysis patients are infrequently prescribed known cardioprotective medications. [20-24] The 1998 United States Renal Data System Annual Data Report (USRDS ADR) stated that 44% of patients less than 65 years of age with diabetes as the cause of ESRD were not treated with a hypoglycemic agent. This number fells 41% for patients over 65 years of age. Data from the USRDS Dialysis Morbidity and Mortality (DMMS) Wave-2 study identified 9.7% of patients treated with an HMG-CoA reductase inhibitor as of day sixty following dialysis initiation.[11] The Dialysis Outcome Practice Patterns Study (DOPPS) study identified 6.9% of United States HD patients treated with HMG-CoA reductase inhibitors.[24] This included 13.8% of patients with coronary artery disease and 16.1% of patients with a previous myocardial infarction.
Analysis of USRDS and DOPPS data also show that relatively few patients are prescribed beta-blocker therapy despite having multiple indications for therapy.[11,22] Data from the DMMS Wave-2 study showed that 18.7% of incident dialysis patients with diagnoses of coronary artery disease, chronic heart failure, and previous myocardial infarction, were prescribed a beta-blocker. This includes 25% of patients with hypertension, 24% with coronary artery disease, and 24% with a previous myocardial infarction.[22] Approximately 19% of new HD patients are given aspirin therapy.[11] The percent of point prevalent patients prescribed aspirin was higher than incident patients, suggesting greater use of aspirin with time on dialysis.[11] Finally, we also performed a medication use audit at our HD unit.[23] Over 96% of patients had at least one cardiovascular risk factor present with an average of 2.4 ± 1.3 cardiovascular risk factor per patient. Of those patients with a diagnosis of chronic heart failure, 34.4% were prescribed an ACE inhibitor and 25.0% were prescribed a beta-blocker. Patients with a previous myocardial infarction were given a lipid-lowering agent, a nitrate, a beta-blocker, and aspirin 23.1%, 23.1%, 15.4%, and 69.2% of the time, respectively. These studies highlight that medication prescribing in the treatment of cardiovascular diseases is in need of improvement in order to maximize care of the ESRD population.
We have shown that MRP continue to exist at a high frequency in these medically complex patients. To address this problem, we advocate the inclusion of pharmacists as a member of the healthcare team taking care of ambulatory HD patients. A pharmacist is specially trained to identify MRP. Pharmacist provided pharmaceutical care services to resolve MRP has shown to improve medication compliance, provide drug information, raise awareness of inappropriate medication prescribing, and improve biochemical and therapeutic responses to medication. [12-17] This will increase the number of patients achieving standard of care and improve overall patient care.[12] A recent review estimated that for every $1 spent on pharmaceutical care activities in ESRD patients, approximately $4 to the healthcare system is saved.[25]
List of Abbreviations
ADR = adverse drug reactions
DI = drug interactions
DMMS = Dialysis Morbidity and Mortality
DOPPS = Dialysis Outcome Practice Patterns Study
DWI = drug use without indication
ESRD = end-stage renal disease
FRD = failure to receive drugs
HD = hemodialysis
IDS = improper drug selection
IWD = indication without drug therapy
MRP = medication-related problem
O = other
OD = over-dosage
UD = sub-therapeutic dosage
USRDS = United States Renal Data System
Competing Interests
All authors have no competing interests with the material presented in this manuscript.
Authors' contributions
HJM and DKD mutually conceived the study, participated in its design, collected data, and prepared the manuscript. RSM contributed to the manuscript preparation an editorial comment. All authors read and approved the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Intravenous iron is typically administered during the hemodialysis (HD) procedure. HD patients may be prescribed high-flux (HF) or high-efficiency (HE) dialysis membranes. The extent of iron sucrose and iron dextran removal by HD using HF or HE membranes and by ultrafiltration rate (UFR) is unknown.
Methods
Two in vitro HD systems were designed and constructed to determine the dialyzabiltiy of iron from a simulated blood system (SBS) containing 100 mg iron sucrose or iron dextran (system A) or 1000 mg iron sucrose (system B). Both in vitro systems utilized a 6-L closed-loop SBS system that was subject to 4 different HD conditions conducted over 4 hours: HE membrane + 0 ml/hr UFR; HE membrane + 500 ml/hr UFR; HF membrane + 0 ml/hr UFR; HF membrane + 500 ml/hr UFR. Blood flow and dialysate flow rates were 500 ml/min and 800 ml/min, respectively. The dialysate compartment was a 192-L open system for system A and a 6-L closed-loop system for system B. Samples from the SBS and dialysate compartments were taken at various time points and iron elimination rate and HD clearance was determined. Iron removal from the SBS > 15% was considered clinically significant.
Results
The greatest percentage removal from the SBS was 13.5% and -0.03% utilizing system A and B, respectively. Iron sucrose and iron dextran dialysate concentration was below the lower limits of assay (< 2 ppm) for system A. Dialysate recovery of iron was negligible: 0 – 5.4 mg system A and 5.47 – 23.59 mg for system B. Dialyzer type or UFR did not affect iron removal.
Conclusion
HF or HE dialysis membranes do not remove clinically significant amounts of iron sucrose or dextran formulations over a 4-hour HD session. This effect remained constant even controlling for UFR up to 500 ml/hour. Therefore, iron sucrose and iron dextran are not dialyzed by HE or HF dialysis membranes irrespective of UFR.
iron sucroseiron dextranin vitrohemodialysisclearanceBackground
Iron deficiency is a common problem in hemodialysis (HD) patients [1]. Reasons for iron deficiency include increased requirements due to erythropoetin-stimulated red blood cell production, blood loss through the HD procedure, and impaired absorption of oral iron [2]. As such, many patients require supplementation with intravenous (IV) iron to maintain transferrin saturations > 20% and ferritin concentrations > 100 ng/mL [1].
Iron dextran (InFed®, Watson Pharmaceuticals; DexFerrum®, American Regent, Inc.) was the first IV iron preparation used in HD patients. However, concerns over iron dextran side effects, specifically anaphylactic and anaphylactoid reactions [3], lead to development of other safer IV iron preparations. One such IV iron preparation available for use in HD patients is iron sucrose (Venofer®, American Regent Laboratories, Inc.).
It is recommended that iron sucrose be given by slow injection or infusion during the HD session to patients that require supplemental IV iron [4]. Previous reports have shown that very little iron dextran (InFed®, Watson Pharmaceuticals, molecular weight 90,000 daltons [5]) is removed via HD [6-8]. However it is unknown to what extent the HD procedure removes the other iron dextran product (DexFerrum®, American Regent, Inc., molecular weight 265,000 daltons [5]) or iron sucrose (Venofer®, American Regent, Inc., molecular weight 34,000 – 60,000 daltons [4]).
Iron sucrose, also known as iron saccharate, is a complex of polynuclear iron (III)-hydroxide in sucrose for intravenous use. Iron sucrose has a molecular weight of approximately 34,000 – 60,000 Daltons and a proposed structural formula: [Na2Fe5O8(OH)*3(H2O)]n*m(C12H22O11). The molecular weight of iron sucrose ranges 34,000 – 60,000 daltons as during the manufacturing process, the number of sucrose molecules bound to iron varies [4].
Given the differences between iron dextran products and iron sucrose regarding molecular weight and pharmacokinetic profile [9], one cannot assume that the HD clearances will be the same.
HD patients may be prescribed high-flux (HF) or high-efficiency (HE) dialysis membranes. HF and HE dialysis membranes have very different solute clearance capabilities. These membranes can remove molecular weight substances up to approximately 15,000 daltons[10]. Iron removal by HD may also be dependant upon convection (i.e., solute drag), which is controlled by the ultrafiltration rate (UFR) The extent of iron removal in HD patients using HF or HE membranes or varying UFR needs to be investigated.
Therefore, an in vitro HD study was conducted to determine whether HF or HE dialysis membranes, under various UFR, removed clinically important amounts of iron dextran or iron sucrose from a simulated blood system. The null hypothesis was that newer HF or HE dialysis membranes removed iron in clinically significant amounts and that the removal was dependent of UFR.
MethodsIn-vitro hemodialysis system A
A closed-loop fixed volume reservoir of 6-L normal saline solution (5750 ml reservoir + 250 ml tubing and dialysis membrane volume) was prepared for a simulated blood system (SBS). A 1000 ml graduated cylinder was used to measure out the normal saline. The normal saline had a pH = 5.0 and an osmolarity of 308 mOsmol/l. The SBS was maintained at 37°C and continuously mixed throughout the in vitro HD session. The blood flow rate (BFR) and dialysate flow rate (DFR) was maintained at 500 ml/min and 800 ml/min, respectively. To assess the contribution of convection to drug removal, UFR was set at 0 ml/hr for one-half of the HD sessions and at 500 ml/hr for the other half of the HD sessions. A volumetric HD machine (model 2008H, Fresenius USA, Walnut Creek, CA) controlled all BFR, DFR, and UFR. Two dialysis membranes were tested: CA210 (Baxter) a HE dialyzer and F80A (Fresenius) a HF dialyzer. HD was conducted for 4 hours with UFR a 0 ml/hr and for 4 hours at 500 ml/hr. The dialysate solution ran counter current to SBS within the dialysis membrane. The dialysate temperature was maintained at 37°C. Each HD procedure was conducted in duplicate.
In-vitro hemodialysis system B
Similar to system A, a closed-loop fixed volume reservoir of 6-L normal saline solution (5750 ml reservoir + 250 ml tubing and dialysis membrane volume) was prepared for a SBS. A 1000 ml graduated cylinder was used to measure out the normal saline. The normal saline had a pH = 5.0 and an osmolarity of 308 mOsmol/l. The SBS was maintained at 37°C and continuously mixed throughout the in vitro HD session.
The dialysate compartment was also a closed-loop 6-L compartment. A 1000 mL graduated cylinder was used to measure out the dialysate. The dialysate compartment consisted of a holding tank and a pressure chamber. A Shur-Flo® pump (model 4UN54) was used to pump dialysate from the holding tank to the pressure chamber. The pressure chamber was pressurized to at least 20 psi to provide sufficient fluid pressure for the dialysis machine. This dialysate pressure was necessary for the dialysis machine to operate and control the dialysate flow rate at 800 ml/min. Spent dialysate from the hemodialysis machine was collected in the dialysate holding tank. The holding tank dialysate compartment construction allowed for sampling to occur through the top of the holding tank.
The SBS and dialysate compartment was maintained at 37°C and continuously mixed throughout the in vitro HD session. The BFR and DFR was maintained at 500 ml/min and 800 ml/min, respectively.
Sample preparation and collection
For the in vitro system A study, iron sucrose 100 mg (5 ml of 20 mg/ml solution) or iron dextran 100 mg (2 ml of 50 mg/ml solution) was injected into the SBS. Samples (5 ml) were collected from SBS at 0, 15, 30, 60, 120,180, and 240 min. Collected samples were stored at 25°C until analysis.
Dialysate was sampled (5 ml) at 0, 60, 120, 180, and 240 min. All dialysate during the simulated HD session was collected pooled, volume measured, and then sampled (5 ml) for iron sucrose or iron dextran concentration. The dialysate sample was stored at 25°C until analysis.
For the in vitro system B study, iron sucrose 1000 mg (50 cc of 20 mg/ml solution) was injected into the SBS. Samples (15 ml) were collected from SBS and dialysate compartment at 0, 15, 30, 60, 120,180, and 240 min. Collected samples were stored at 25°C until analysis. All dialysate during the simulated HD session was collected pooled, volume measured, and then sampled (15 ml) for iron sucrose. The dialysate samples were stored at 25°C until analysis.
The iron sucrose sample preparation and data analysis occurred at a Dialysis Clinic, Inc dialysis unit in Kansas City, MO. The iron dextran sample preparation and data analysis occurred at a Renal Research Institute dialysis unit in Albany, NY. Both centers utilized bicarbonate based dialysate solutions.
Control for potential iron contamination
Disposable supplies (e.g., plastics) were utilized whenever possible and the use of glassware was not permitted to minimize iron loss. Additionally, to control for potential iron contamination within the HD procedure, an additional HD session was conducted without iron sucrose or iron dextran injected into the reservoir [SBS]. Dialysate and SBS samples were then collected at same time points as described previously. Any detectable iron found in the SBS or dialysate compartments (native iron) was then subtracted from measured iron concentration from the iron-containing HD sessions.
Samples analysis
The determination of iron concentration in study samples was performed using atomic absorption spectrophotometry. A Perkin-Elmer AAnalyst 300 was set up and operated as per previously described methods.[11] The instrument is equipped with a Perkin-Elmer Lumina™ Fe hollow cathode lamp, with an emission line of 248.3 nm and an air acetylene flame. A stock standard solution of ferrous ammonium sulfate in calcium chloride was used to run a 5-point standard calibration curve, ranging from 2.0 to 10.0 ppm of iron. This method has been validated for ruggedness, method and system precision, giving an overall testing variation of approximately 2%.
In vitro system A
Prior to analysis samples were prepared by pipetting 2.0 mL of sample into a 10.0 mL volumetric flask using a "to contain" pipette. Pipettes were then rinsed with a calcium chloride solution and 0.5 mL of HCl was added to the volumetric flask. Sufficient amount of calcium chloride solution as then added to yield theoretical concentration of 3.33 μg/mL (100 mg/6000 mL × 2.0 mL/10.0 mL × 1000 μg/mg).
In vitro system B
Prior to analysis samples from the SBS compartment were prepared by pipetting 2.0 mL of sample into a 50 mL volumetric flask using a "to contain" pipette. Pipettes were then rinsed with a calcium chloride solution and 2.0 mL of HCl added to the volumetric flask. Sufficient amount of calcium chloride solution (q.s. to 50 mL) was added to yield theoretical concentration of 6.664 μg/mL (1000 mg/6000 mL × 2.0 mL/50 mL × 1000 μg/mL).
Analysis of samples from the dialysate compartment was prepared by pipetting 3.0 mL of sample into a 10 mL volumetric flask using a "to deliver" pipette and adding 0.5 mL of HCl to the volumetric flask. Sufficient amount of calcium chloride solution (q.s. to 10 mL) was added to yield theoretical detection limit of no less than 1.666 μg/mL (i.e. 1% iron transfer)
SBS initial iron concentration (1000 mg/6000 mL = 166.6 μg/mL).
166.6 μg/mL × 1% / 100 = 1.666 μg/mL
1.666 μg/mL × 3 mL/10 mL = 0.5 μg/mL
In the event that a dialysate sample's determined concentration falls outside of the system's calibration curve of 10 ppm (i.e., greater than 20% iron transfer), the sample's preparation was adjusted so that the final determined concentration would fall within the system's calibration range of 0 to 10 ppm elemental iron.
All sample analysis occurred at a central laboratory (American Regent, Inc. Shirley, NY). Each system was visually inspected for color change in either SBS or dialysate compartment.
Data analysis
The mean ± SD iron concentrations were calculated for each sample taken at each time point for iron sucrose or iron dextran. The percent change in iron concentration from beginning to end of HD session and absolute amount of iron eliminated was calculated. A mean loss of greater than 15% from the SBS of the initial concentration was considered as a clinically important loss of iron.
Iron sucrose and iron dextran concentration results were modeled using PK-Analyst® (MicroMath, version 1.0, Salt Lake City, UT) pharmacokinetic data analysis software. A monoexponential model was assumed and pharmacokinetic parameters were calculated using the following formulae:
1. Elimination rate constant for HD period (Khd) was calculated:
A positive rate constant Khd suggests accumulation of substance in the tank; whereas a negative rate constant Khd suggests removal of substance from the tank.
2. The amount removed (A) by HD was calculated as the mean of:
A = (preHD conc - end of HD conc) * Volume of distribution (Vd) and
A = total iron sucrose or iron dextran collected in dialysate
Vd = SBS volume + tubing volume + dialyzer volume
3. Clearance HD (Clhd) = Slope of regression line × Vd.
Clearance values that are positive relate to accumulation of substance from the system; negative clearance values represent removal of substance from the system.
Statistics
Iron product pharmacokinetic parameters determined from samples obtained during HF and HE dialysis membranes were compared using two-sample t-test. Pharmacokinetic parameter values at 0 ml/hr and 500 ml/hr UFR were compared using a paired t-test. Finally pharmacokinetic parameters between iron sucrose and iron dextran compounds were compared using two-sample t-test. All t-tests were two-tailed and a p-value < 0.05 was considered significant.
ResultsIn vitro system A
Instillation of iron sucrose or iron dextran into the SBS resulted in a light brown colored solution. The dialysate compartment consisted of a clear solution. During the entire study there was no visual evidence of color change in the SBS or dialysate compartments.
The mean ± SD dialysate iron recovery values for iron sucrose from each HD session utilizing HF or HE dialyzers and 0 ml/min and 500 ml/min UFR test condition is reported in Table 1. At no time was any iron detected in the dialysate in the iron sucrose experiments, regardless of the dialysis membrane used. The amounts of iron measured in the dialysate compartment were below the limits of detection for the assay (< 2 ppm). The mean ± SD dialysate iron recovery for iron dextran from each HD session utilizing HF or HE dialyzers and 0 ml/min and 500 ml/min UFR test condition is reported in Table 2. In contrast to iron sucrose, some iron (5.44 ± 7.7 mg) from the iron dextran was found in the dialysate when using HE membranes at 0 UFR. However, under other dialysis conditions with HE or HF membranes, no iron was found.
Iron sucrose removal by high-flux or high-efficiency hemodialysis membranes over 240 minutes. (System A)
Iron Sucrose (Venofer®)
Study condition
SBS % iron concentration change(mean ± SD)
Khd (hr-1)(mean ± SD)
Dialysate Iron Recovery (mg)(mean ± SD)
Clearance† (ml/min)(mean ± SD)
F80: 0 ml/hr UFR
13.55 ± 1.56
0.0006 ± 0.0001
0.00 ± 0.00
3.62 ± 0.45
F80: 0 ml/hr UFR Re-run*
6.99 ± 8.55
0.0003 ± 0.0004
0.00 ± 0.00
1.85 ± 2.29
F80: 500 ml/hr UFR
-16.03 ± 8.57
-0.0006 ± 0.0003
0.00 ± 0.00
-2.46 ± 1.23
CA210: 0 ml/hr UFR
-6.20 ± 1.78
-0.0003 ± 0.0001
0.00 ± 0.00
-1.49 ± 0.42
CA210: 500 ml/hr UFR
-14.79 ± 31.51
-0.0003 ± 0.0019
0.00 ± 0.00
-6.87 ± 7.74
SBS = simulated blood solution; UFR = ultrafiltration rate; Khd = hemodialysis elimination rate; % = percent; * = The re-run study condition was an unique experiment that replicated all conditions present in the original F80: 0 ml/hr UFR experiment. † = Clearance values that are positive relate to accumulation of substance from the system; negative clearance values represent removal of substance from the system.
Iron dextran removal by high-flux or high-efficiency hemodialysis membranes over 240 minutes. (System A)
Iron Dextran (DexFerrum®)
Study condition
SBS % iron concentration change(mean ± SD)
Khd (hr-1)(mean ± SD)
Dialysate Iron Recovery (mg)(mean ± SD)
Clearance† (ml/min)(mean ± SD)
F80: 0 ml/hr UFR
-9.69 ± 3.31
-0.0004 ± 0.0001
0.00 ± 0.00
-2.29 ± 0.75
F80: 500 ml/hr UFR
-7.84 ± 0.73
-0.0003 ± 0.0000
0.00 ± 0.00
-1.26 ± 0.11
CA210: 0 ml/hr UFR
-8.77 ± 6.76
-0.0003 ± 0.0003
5.44 ± 7.70
-2.06 ± 1.54
CA210: 500 ml/hr UFR
-7.48 ± 3.54
-0.0003 ± 0.0001
0.00 ± 0.00
-1.20 ± 0.55
SBS = simulated blood solution; UFR = ultrafiltration rate; Khd = hemodialysis elimination rate; % = percent; † = Clearance values that are positive relate to accumulation of substance from the system; negative clearance values represent removal of substance
In general, no iron or insignificant levels in the case of iron dextran was lost from the SBS reservoir for either iron product. However in the experiment in which iron sucrose and HF membranes were run at 0 UFR, significant amount of iron (13.55% ± 1.56) were lost from the SBS reservoir at 240 minutes (Table 1.). This finding contributed to greater elimination rate (p = 0.003), clearance (p = 0.003), and greater amount of iron removed (p = 0.005) between the HF and the HE dialysis membranes. Since these results were very disparate to other HD conditions tested and since no other iron was recovered from the dialysate under these conditions, the entire experiment was re-run under the identical condition (HF membrane; 0 UFR; 500 mL/min BFR; 800 mL/min DFR; 4 hours). Results from the re-run test condition are provided in table 1. During the re-run experiment, some iron (6.99% ± 8.55) was again lost from the SBS reservoir, but again no comparable increase in iron was found in the dialysate. Statistical analysis of the data including data from the re-run yielded non-significant comparisons between elimination rate (p = 0.24), clearance (p = 0.24), and greater amount of iron removed (p = 0.22) between the HF and the HE dialysis membranes. In conclusion, no significant amount of iron was found in the dialysate regardless of dialysis membrane used or UFR. Furthermore, no significant loss of iron was observed in the SBS reservoir irrespective of iron product used or the test conditions.
In vitro system B
Instillation of 1000 mg iron sucrose into the SBS resulted in a dark brown (nearly black) colored solution. The dialysate compartment consisted of a clear solution. During the entire study there was no visual evidence of color change in the SBS compartment. The dialysate compartment became lightly yellowish in color by the end of 240 minutes.
The mean ± SD percentage recovery from each HD session utilizing HF or HE dialyzers and 0 ml/min and 500 ml/min UFR test condition are reported in Table 3. Iron sucrose SBS percent removal ranged from – 3.13 ± 0.06% to -0.30 ± 1.15%. Iron sucrose elimination rate from the SBS was less than 0.0001 hr-1 for all conditions. The dialysate recovery of iron sucrose ranged from 2.79 ± 1.89 mg to 23.59 ± 3.96 mg when 1000 mg was instilled in the SBS reservoir (i.e., 0.3 – 2.4% injected iron from SBS compartment). Within dialyzer type and between dialyzer types statistical analysis revealed non-significant comparisons between elimination rate, clearance, and percent removed by dialysis. These non-significant results remained while controlling for UFR.
Iron sucrose removal in a closed-loop system by high-flux or high-efficiency hemodialysis membranes over 240 minutes. (System B)
Iron Sucrose (Venofer®)
Study condition
SBS % iron concentration change(mean ± SD)
Khd (hr-1)(mean ± SD)
Dialysate Iron Recovery (mg)(mean ± SD)
Clearance† (ml/min)(mean ± SD)
F80: 0 ml/hr UFR
-3.13 ± 0.06
-0.0001 ± 0.0000
13.82 ± 13.34
-0.76 ± 0.01
F80: 500 ml/hr UFR
-0.03 ± 1.15
0.0000 ± 0.0000
23.59 ± 3.96
0.05 ± 0.19
CA210: 0 ml/hr UFR
-1.41 ± 3.77
-0.0001 ± 0.0002
5.47 ± 1.52
-0.34 ± 0.92
CA210: 500 ml/hr UFR
-1.58 ± 3.08
-0.0003 ± 0.0002
2.79 ± 1.89
-1.08 ± 0.93
SBS = simulated blood solution; UFR = ultrafiltration rate; Khd = hemodialysis elimination rate; % = percent; † = Clearance values that are positive relate to accumulation of substance from the system; negative clearance values represent removal of substance
Discussion
Intravenous iron is frequently given to HD patients to treat or prevent iron deficiency anemia[1]. Clinicians may have concern over the dialyzability of iron sucrose or iron dextran administered at anytime point during the HD session. This is the first study to investigate the in-vitro dialyzability of iron sucrose (Venofer, American Regent, Inc.) or iron dextran (DexFerrum, American Regent, Inc.).
The appearance of iron sucrose or iron dextran in the dialysate compartment is the best determinate of the respected agent's dialyzability. The results of our study demonstrate that no iron sucrose was ever detected in the dialysate and a negligible amount of iron from the iron dextran (maximum 5.44% of administered dose) was recovered from the dialysate compartment only with the HE membranes at 0 ml/hr UFR. In all other conditions, no iron was detected in the dialysate for iron dextran. These effects remained constant even controlling for UFR up to 500 ml/hr. The HF and HE dialysis membranes do not remove clinically significant amounts of these iron formulations from the SBS compartment over a 240-minute HD session.
Our results are consistent to previous in-vitro and in-vivo reports [6-8]. In a 30-minute HD in-vitro study iron dextran removal was determined for HF and HE dialyzers [7]. The greatest amount of iron removed (8%) was observed with a F8 (Fresenius) HF dialyzer [7]. Other investigators utilizing a cuprophane coil dialyzer in-vitro system determined a 0.5% iron dextran removal over 240 minutes [6]. The only in-vivo dialyzability study utilized iron dextran and HE membranes (Terumo 165 cuprammonium dialyzer) [8]. The investigators determined a maximal 2% iron dextran removal over 160 minutes.
At no time point over the sample collection period additional iron was injected into the SBS or dialysate compartments. However, negative regression analyses (Khd) and clearance results reported in the tables suggest that there are increasing amounts of iron in the system (i.e., appearance of additional iron). These findings may be the result of error amplification through multiplication of measured iron concentration and volume of dialysate or SBS reservoir at various time points. Sources of potential error include human (sample preparation), device (1000 mL graduated cylinder) and the atomic absorption spectrophotometry assay. Nonetheless, these negative value results suggest that no iron sucrose or iron dextran was removed from the SBS.
The positive iron sucrose regression analyses (Khd) and clearance results (Table 1) obtained from the HF membrane 0 UFR HD session, support the decreasing amounts of iron in the SBS system (6.99%). However, no iron sucrose was found in the dialysate compartment. A potential reason for the drop in iron sucrose in the SBS compartment is iron sucrose adsorption to the dialysis membrane or tubing. This effect was not seen with the HE membrane nor was it observed under dialysis conditions with a positive UFR.
In order to determine whether the decreasing amounts of iron were due to either adsorption to the dialysis membrane or actual loss of iron to the dialysate compartment, the closed-loop studies were conducted (in vitro system B). In those studies between 0.3 and 2.4% (23.59 mg of 1000 mg dose) of iron sucrose was recovered in the dialysate compartment when using 1000 mg of iron sucrose (10 times the current recommended dose) in the SBS reservoir. Therefore, the majority of loss seen in the earlier experiments with HF membranes is not due to transfer of iron to the dialysate compartment; rather the loss is probably due to adsorption to the dialysis membranes.
A potential limitation to our in-vitro model is that we utilized normal saline instead of blood in our SBS. Use of an in-vitro model raises concerns over the ability to extrapolate the data to humans. In regards to the various intravenous iron formulations, iron appears to dissociate from sucrose rapidly allowing for more immediate systemic iron utilization. This is illustrated in a study which compared iron sucrose, iron dextran and iron gluconate [12]. In that study, serum iron, ferritin and transferrin saturation increased more rapidly and were significantly higher in those patients who received iron sucrose compared to the other iron products. The pharmacokinetic profile of iron sucrose in normal healthy adults suggests that the dissociation of iron from the iron sucrose complex allow for immediate and substantial iron availability. However, it is unknown whether HD would remove the iron more rapidly than it is utilized. Even if the iron in our in-vitro study was 100% dissociated from the iron sucrose complex, we recovered virtually no iron in the dialysate and the percent change in iron concentration from baseline was minimally lower with HE dialyzers under 0 ml/hr UFR. No loss of iron was detected under other test conditions with HE or HF membranes. Therefore, HD would not remove significant amounts or iron sucrose prior to it being utilized in the body.
Conclusion
It is therefore concluded that iron sucrose (Venofer®, American Regent, Inc.) and iron dextran (DexFerrum®, American Regent, Inc.) are not significantly removed by either HF or HE dialysis membranes. Furthermore, under laboratory conditions in a closed-loop system, a maximum of 2.4% of iron sucrose is found in the dialysate, well below the clinically relevant level of 15%. These data show that neither iron dextran or iron sucrose are significantly dialyzed under conditions that mimic the clinical situation.
Competing interests
The authors received funding for this study by American Regent, Inc. The authors have no other competing interests.
Authors contributions
HJM and DWG mutually conceived the study, participated in its design, collected samples (HJM – iron sucrose; DWG – iron dextran), and prepared the manuscript.
HJM performed the statistical analysis.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors would like to express their sincerest gratitude to Mary Ann
McCracken , RN and Marcy L. McClaran, RN for their assistance in this project. This study was supported by an unrestricted research grant from American Regent, Inc. Data were presented at the 2003 American Society of Nephrology Meeting in San Diego, CA, USA.
Hyponatremia secondary to the syndrome of inappropriate secretion of antidiuretic hormone is an uncommon complication of treatment with the new class of antidepressant agents, the selective serotonin reuptake inhibitors. The risk of hyponatremia seems to be highest during the first weeks of treatment particularly, in elderly females and in patients with a lower body weight.
Case Presentation
A 61-year-old diabetic male was admitted to the hospital because of malaise, progressive confusion, and a tonic/clonic seizure two weeks after starting citalopram, 20 mg/day. On physical examination the patient was euvolemic and had no evidence of malignancy, cardiac, renal, hepatic, adrenal or thyroid disease. Laboratory tests results revealed hyponatremia, serum hypoosmolality, urine hyperosmolarity, and an elevated urine sodium concentration, leading to the diagnosis of inappropriate secretion of antidiuretic hormone. Citalopram was discontinued and fluid restriction was instituted. The patient was discharged after serum sodium increased from 124 mmol/L to 134 mmol/L. Two weeks after discharge the patient denied any new seizures, confusion or malaise. At that time his serum sodium was 135 mmol/L.
Conclusions
Because the use of serotonin reuptake inhibitors is becoming more popular among elderly depressed patients the present paper and other reported cases emphasize the need of greater awareness of the development of this serious complication and suggest that sodium serum levels should be monitored closely in elderly patients during treatment with citalopram.
Background
Hyponatremia secondary to the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) is an uncommon complication of treatment with the new class of antidepressant agents, the selective serotonin reuptake inhibitors (SSRIs) [1,2]. Estimations of the occurrence of hyponatremia during treatment with SSRIs range between 0.5% and 25%, and the risk of hyponatremia seems to be greatest during the first weeks of treatment with SSRI, in the elderly, in female patients and in patients with lower body weights [3,4]. However, severe consequences of hyponatremia caused SSRIs, such as tonic/clonic seizure, have not been reported. We describe the case of a 61-year-old male with tonic/clonic seizure caused by SSRI-induced hyponatremia
Case Presentation
We recently saw a 61-year-old male referred to us because of a 3-day history of malaise, progressive confusion, and a tonic/clonic seizure. Two weeks before, he had been started on a regimen of citalopram 20 mg at bedtime. The patient and his wife reported that he became progressively confused, lethargic and had difficulty performing simple tasks. He is a type 2 diabetic treated with metformin 500 mg twice daily and glyburide 2.5 mg once daily. Upon admission, the patient was afebrile with normal vital signs. He appeared euvolemic without evidence of congestion or dehydration. Neurologic examination was normal except for decreased strength on lower extremities. Significant laboratory findings included sodium of 124 mmol/L (136–145 mmol/L), potassium of 4.3 mmol/L (3.5–4.5 mmol/L), chloride 86 mg/dL (98–106 mmol/L), blood urea nitrogen of 3.2 mmol/L (3.6–7.1 mmol/L), creatinine 79.56 μmol/L (< 133 μmol/L), glucose of 10.49 mmol/L (4.2–6.4 mmol/L), a uric acid of 150 μmol/L (150–480 μmol/L) and a serum osmolarity of 263 mosm/L (285–295 mOsm/L). Urine sodium and urine osmolarity were elevated, 141 mEq/L, and 400 mosm/L, respectively. A CT of the head and an EEG were both normal. An AM cortisol level, thyroid-stimulating hormone (TSH) and free thyroxine levels were within normal limits. Results of a urine toxicology screen revealed no presence of ethanol or recreational drugs. A citalopram pill count confirmed compliance with the drug regimen without evidence of overdose.
A diagnosis of SIADH was made based on clinical euvolemia in the presence of hyponatremia with a urine osmolarity and sodium that were inappropriately high. Normal renal, thyroid and adrenal function with relative hipouricemia, all supported SIADH. Extensive investigations ruled out malignancy, pulmonary, hepatic cardiac or renal disease or any other known causes of SIADH.
On the day of admission, citalopram was discontinued and the patient was treated with 2 liters of intravenous 0.9% sodium chloride, phenytoin (5 mg/kg), and subcutaneous insulin. Approximately 24 hours after admission the patient's serum sodium increased to 129 mmol/L (136–145 mmol/L) and the chloride increased to 89 mmol/L (98–106 mmol/L), thereafter, fluids were restricted to 1200 ml/day. His mental status improved over the next 48 hours. Five days after admission serum sodium was 134 mEq/L (136–145 mmol/L) and serum chloride was 99 mmmol/L (98–106 mmol/L). Patient was fully alert, had no more seizures and was subsequently discharged. At this time phenytoin treatment was stopped. A follow up serum sodium three weeks after discharge was 135 mmol/L (136–146 mmol/L).
This patient's seizures appear to have been induced by hyponatremia that was secondary to SIADH, a diagnosis that is supported by the low serum sodium concentration, concentrated urine, and clinical evidence of euvolemia. The laboratory values and history were inconsistent with a diagnosis of psychogenic polydipsia. The finding of SIADH secondary to citalopram use may reflect dysregulation of serotonergic control of ADH release or metabolism. Experimental evidence in rodents has demonstrated the presence of serotonin's neurons in the hypothalamic supraoptic nucleus, which is where the ADH prohormone is synthesized[5]. Other studies suggest that serotonin may be involved in the regulation of ADH release[6]. The occurrence in this case of a seizure secondary to SIADH-associated hyponatremia suggests a possible mechanism for citalopram-induced convulsions and corroborates previous reports of citalopram-induced SIADH.
Conclusions
The present case and others previously reported, emphasize the need for greater awareness of the development of this serious and potentially fatal complication in association with citalopram therapy. Review of the present and previous cases has shown that the onset of citalopram-induced hyponatremia or SIADH ranges from 6 to 20 days after the therapy has been started [7-16]. Potential risk factors for SIADH due to citalopram included advanced age, female gender, concomitant use of medications known to cause SIADH or hyponatremia, and possibly, higher citalopram doses [7,8,17]. Therefore, a high level of suspicion, close and careful monitoring of serum sodium concentration particularly in elderly patients during the first month of therapy with citalopram may reduce the incidence of this serious and likely, not rare, adverse effect.
Although information is not conclusive, other SSRI's should also be avoided if treatment with an antidepressant had to be restarted in patients with past medical history of hyponatremia or SIADH induced by citalopram [17,18].
Competing interests
None declared.
Author's contributions
GF was the attending physician and wrote the paper; SP is a fourth year resident in internal medicine that participated in the care of the patient; CC-A is second year resident in internal medicine that participated in the care of the patient; JV is a third year resident in internal medicine that participated in the care of the patient. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent was obtained from the patient and his wife for publication of the study
SpigsetOHedenmalmKHyponatremia in relation to treatment with antidepressants: a survey of reports in the World Health Organization database for spontaneous reporting of adverse drug reactions1997173483529085327WooMHSmytheMAAssociation of SIADH with selective serotonin reuptake inhibitors199731348352BoumanWPPinnerGJohnsonHIncidence of selective serotonin reuptake inhibitor (SSRI) induced hyponatremia due to the syndrome of inappropriate antidiuretic hormone (SIADH) secretion in the elderly1998131215948957510.1002/(SICI)1099-1166(199801)13:1<12::AID-GPS718>3.3.CO;2-6WilkinsonTJBeggFJWinterACSainsburyRIncidence and risk factors for hyponatremia following treatment with fluoxetine or paroxetine in elderly people1999472112171019065710.1046/j.1365-2125.1999.00872.xVacherCMFretierPCreminonCCalasAHardin-PouzetHActivation by serotonin and noradrenaline of vasopressin and oxytocin expression in the mouse paraventricular and supraoptic nuclei2215132211880481JorgensenHRiisMKniggeUKjaerAWarbergJSerotonin receptors involved in vasopressin and oxytocin secretion2003152422491258851210.1046/j.1365-2826.2003.00978.xFisherADavisMCroft-BakerJPurcellPMcLeanACitalopram-induced severe hyponatraemia with coma and seizure. Case report with literature and spontaneous reports review20022117918712503252BarclayTSLeeAJCitalopram-associated SIADH200236155815631224360610.1345/aph.1C071BourgeoisJABabineSEBahadurNA case of SIADH and hyponatremia associated with citalopram2002432412421207504110.1176/appi.psy.43.3.241HullMKottlorsMBrauneSProlonged coma caused by low sodium and hypo-osmolarity during treatment with citalopram2002223373381200690810.1097/00004714-200206000-00018OdehMBenyAOlivenASevere symptomatic hyponatremia during citalopram therapy20013211591601121781910.1097/00000441-200102000-00009RiquelmeAMendezFOrtizAMMullerHCamposCRochaVValdiviesoASevere and recent hyponatremia and hypokalemia associated to the use of hydrochlorothiazide, enalapril and citalopram. Clinical case19991271223122810835739PradalierADevars du MayneJFBarzegarCVincentDHyponatremia and probable inappropriate secretion of antidiuretic hormone due to citalopram19985360060210070243SpigsetOAdielssonGCombined serotonin syndrome and hyponatraemia caused by a citalopram-buspirone interaction19971261639179637ChristensenOSorensenHAAlmdalTPAdverse effects of selective serotonin uptake inhibitors. Hyponatremia caused by Schwartz-Bartter syndrome1996158692069228984756VoegeliJBaumannPInappropriate secretion of antidiuretic hormone and SSRIs19961695245258894212ArinzonZHLehmanYAFidelmanZGKrasnyanskyIIDelayed recurrent SIADH associated with SSRIs200236117511571208655010.1345/aph.1A337KirbyDAmesDHyponatraemia and selective serotonin re-uptake inhibitors in elderly patients2001164844931137646410.1002/gps.367oai%3Apubmedcentral.nih.gov%3A411039!!!pmc!bmcneph