Title: Sirolimus-Associated Heavy Proteinuria in a Renal Transplant Recipient: Evidence for a Tubular Mechanism
Abstract: American Journal of TransplantationVolume 6, Issue 2 p. 429-433 Free Access Sirolimus-Associated Heavy Proteinuria in a Renal Transplant Recipient: Evidence for a Tubular Mechanism L. Straathof-Galema, L. Straathof-Galema Department of NephrologySearch for more papers by this authorJ. F. M. Wetzels, J. F. M. Wetzels Department of NephrologySearch for more papers by this authorH. B. P. M. Dijkman, H. B. P. M. Dijkman Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, The NetherlandsSearch for more papers by this authorE. J. Steenbergen, E. J. Steenbergen Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, The NetherlandsSearch for more papers by this authorL. B. Hilbrands, Corresponding Author L. B. Hilbrands Department of Nephrology *Corresponding author: L.B. Hilbrands, [email protected] for more papers by this author L. Straathof-Galema, L. Straathof-Galema Department of NephrologySearch for more papers by this authorJ. F. M. Wetzels, J. F. M. Wetzels Department of NephrologySearch for more papers by this authorH. B. P. M. Dijkman, H. B. P. M. Dijkman Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, The NetherlandsSearch for more papers by this authorE. J. Steenbergen, E. J. Steenbergen Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, The NetherlandsSearch for more papers by this authorL. B. Hilbrands, Corresponding Author L. B. Hilbrands Department of Nephrology *Corresponding author: L.B. Hilbrands, [email protected] for more papers by this author First published: 19 January 2006 https://doi.org/10.1111/j.1600-6143.2005.01195.xCitations: 91AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Sirolimus is a new and potent immunosuppressive agent. Recently, increased proteinuria has been recognized as an important complication. However, the mechanism thereof has remained unclear. We describe a patient who received sirolimus as standard therapy after living donor kidney transplantation. Within 10 days the patient developed a severe proteinuria that disappeared completely after substituting tacrolimus for sirolimus. Renal biopsy disclosed normal glomeruli even without effacement of the podocytic foot processes. Using FITC labeled anti-albumin antibodies we noted complete absence of albumin in the proximal tubules, whereas an abundant albumin staining was observed in a control patient with a comparable level of proteinuria due to a recurrence of focal segmental glomerulosclerosis after transplantation. Our data suggest that sirolimus can induce severe proteinuria, and that reduced tubular protein reabsorption contributes to the protein loss. Introduction Sirolimus is a new and potent immunosuppressive agent that has been used successfully in renal transplant recipients (1). Sirolimus blocks an intracellular kinase (mTOR) that regulates growth and proliferation of lymphocytes. The introduction of sirolimus has fostered hope for the use of calcineurin-inhibitor free immunosuppressive regimens in renal transplant patients, thus limiting the risk of nephrotoxicity. However, sirolimus can cause other side effects such as hyperlipidemia (2), mouth ulcers (3), disturbed wound healing (4), impaired recovery of delayed graft function (5) and thrombotic microangiopathy (6). More recently, proteinuria has been reported as a consequence of sirolimus therapy, although the mechanism has remained unclear (7-9). We report a renal transplant recipient who developed heavy proteinuria during treatment with sirolimus. The renal biopsy findings were remarkable and suggested that impairment of tubular protein reabsorption was a major cause of the proteinuria. Case A 60-year-old Caucasian male received a renal allograft from his healthy son in February 2005. The pretransplant clinical course was characterized by slowly progressive renal insufficiency since 1991, without signs of a nephrotic syndrome. A renal biopsy was not performed. He had been on hemodialysis since November 2003. During the transplantation procedure, a venous thrombus at the site of the clamp on the renal vein was noted. Thrombectomy was performed immediately and the patient was treated with low-molecular-weight heparin for several weeks after the surgical procedure. Our patient participated in a clinical trial designed to compare sirolimus and tacrolimus, and he was allocated to immunosuppressive therapy consisting of sirolimus, prednisone, mycophenolate mofetil and daclizumab. The dose of sirolimus was 15 mg on day 0 (i.e. the day of surgery), 12 mg on day 1 and 6 mg daily thereafter, targeting blood levels between 10 and 15 ng/mL. After transplantation the serum creatinine level decreased slowly, compatible with acute tubular necrosis due to the complicated procedure (Figure 1). Proteinuria, measured in serial 24 h urine collections, was present and was initially attributed to hematuria and the result of cold perfusion injury. Over the next days, renal function improved and proteinuria decreased (Figure 1). However, from day 4 onward we observed a clear increase in proteinuria reaching values of more than 12 g/day despite improvement of renal function. There was no evidence of macroscopic hematuria, and the absence of blood was confirmed by measuring urinary hemoglobin (Table 1). Quantitative analysis of urinary proteins disclosed a nonselective proteinuria (Table 1). In addition, low-molecular-weight proteins were abundant. At the 8th postoperative day serum creatinine amounted 3.0 mg/dL, serum albumin 3.3 g/dL and proteinuria 11.8 g/day. The sirolimus blood level was 8.8 ng/mL (day 6). Since the primary kidney disease of our patient was unknown, we considered rejection or recurrence of focal segmental glomerulosclerosis (FSGS) as the most likely causes of the proteinuria. After a partial renal vein thrombosis was excluded by renal scintigraphy and Doppler ultrasound, a renal allograft biopsy was performed. Figure 1Open in figure viewerPowerPoint Time course of serum creatinine and proteinuria. Renal transplantation was performed at day 0. Serum creatinine levels are depicted by diamonds and proteinuria is shown as bars. Sirolimus was stopped after day 7 and replaced by tacrolimus. Table 1. Analysis of urinary proteins Normal values Day 8 post Tx Day 23 post Tx Day 41 post Tx Total protein (g/g creatinine) <0.100 9.2615 0.2359 0.158 Albumin (g/g creatinine) <0.017 6.3846 0.1769 0.024 IgG (g/g creatinine) <0.008 0.6019 0.0133 0.0028 Transferrin (g/g creatinine) <0.0017 0.3654 0.0103 0.0014 α1-Microglobulin (mg/g creatinine) <10 113.5 188.8 89.1 Free hemoglobin (μmol/L) 0 Selectivity index 0.55 Quantitative analysis of urinary proteins was done in random urine samples. Results were corrected for urinary creatinine concentration. Free hemoglobin was measured after freezing and thawing the sample to ensure lysis of the erythrocytes. The selectivity index was calculated by dividing the clearance of IgG by the clearance of albumin. The renal biopsy contained on average eight glomeruli per section and showed normal cortical architecture without significant interstitial edema, infiltrate or fibrosis (Figure 2A). The glomeruli did not show significant abnormalities. Notably, there was no hypercellularity which would indicate acute transplant glomerulopathy, nor were there any abnormalities of the glomerular basement membrane. There were no signs of acute rejection such as tubulitis or endovasculitis. There was moderate tubular epithelial cell damage with nuclei that appeared activated, and vacuolization, flattening and occasionally shedding of epithelial cells. Immunohistochemical staining was negative for C4d deposition in peritubular capillaries, which underscored the absence of acute humoral rejection. Additional immunofluorescence staining for immunoglobulins and complement components was not performed. Since glomeruli may appear normal in the initial stage of recurrent FSGS, electron microscopy was performed (Figure 2B). Notably, there was no significant foot-process effacement, thus excluding recurrent FSGS as cause of the proteinuria. We were even more puzzled by the normal appearance of the podocyte foot-processes since foot-process effacement is usually present in any glomerular disease associated with heavy proteinuria. Therefore, we have analyzed in more detail the process of tubular protein reabsorption. Frozen sections were incubated with FITC-labeled anti-albumin antibodies thus allowing visualization of reabsorbed albumin in the tubular cells. For comparison we have used biopsy sections of a transplant patient with recurrence of FSGS and a comparable level of proteinuria (10 g/day), who was not treated with sirolimus. The same batch of antibodies was used in both cases. The results are shown in Figure 2C and D. In our sirolimus treated transplant patient we could not detect albumin reabsorption droplets in the proximal tubular cells, whereas reabsorption droplets were abundantly present in the patient with recurrent FSGS. Essentially similar observations were made regarding the reabsorption of IgG. Figure 2Open in figure viewerPowerPoint Renal biopsy findings. Panel A: Light microscopy, original magnification 400×, methenamine silver staining. Shown is a representative part of the renal biopsy. Glomeruli are unremarkable. Notably, there are no abnormalities of the glomerular basement membrane or endocapillary hypercellularity. There is no significant interstitial infiltrate or tubulitis. There is moderate tubular epithelial cell damage as indicated by the presence of nuclei with prominent nucleoli and occasional vacuolization of the cytoplasm. Panel B: Electron microscopy, original magnification 4000×, Podocyte cell bodies are slightly swollen and vacuolated and there is minimal microvillous transformation. However, there is no significant foot process effacement. The glomerular basement membrane is normal and the endothelium is unremarkable. Panel C and D show frozen sections stained with FITC-labeled anti-albumin antibodies (polyclonal goat anti-human-albumin antibodies, Bethyl Laboratories Inc., Montgomery, Texas, USA). Original magnification 150×, inset 400×. Panel C shows a section from our transplant patient. Note that albumin-containing reabsorption droplets are not present in tubular epithelial cells. Panel D shows a section from a patient with recurrent FSGS who was not treated with sirolimus. Here, many albumin-containing reabsorption droplets are seen in tubular epithelial cells. The subsequent course in our patient is depicted in Figure 1. Sirolimus was withdrawn from the immunosuppressive regimen on the 8th postoperative day and tacrolimus was started. Urinary protein excretion dropped and proteinuria was no longer detectable after 2 weeks. Graft function recovered to a creatinine clearance of 90 mL/min. Discussion We describe a patient who developed heavy proteinuria within 1 week after renal transplantation. There are several reasons to consider the use of sirolimus to be related to the massive protein excretion. First, examination of the renal allograft biopsy showed no evidence of rejection, glomerulonephritis or recurrent FSGS. Second, the time course of the proteinuria argues against ischemia-related proteinuria (10, 11). Finally, replacement of sirolimus by tacrolimus led to complete disappearance of the proteinuria. Several studies have implicated that replacement of a calcineurin inhibitor (CNI) by sirolimus may induce or aggravate proteinuria (7-9, 12, 13). The interpretation of these studies is hampered by the fact that the majority of patients that were included had chronic allograft nephropathy (CAN) with already some degree of proteinuria at the time sirolimus was started. Since control groups were lacking in these studies, it cannot be excluded that the natural course of CAN was responsible for the increase in proteinuria in a considerable number of cases. Moreover, after switching from a CNI to sirolimus it is hard to determine whether the increase in proteinuria reflects the loss of the reducing effect on proteinuria of the CNI, or results from sirolimus itself. Few studies have provided detailed morphological analysis, including electron microscopy, in patients with increased proteinuria after initiation of sirolimus. Dittrich et al. described four patients with various glomerular diseases after conversion from a CNI to sirolimus (7). However, it cannot be completely excluded that a recurrence of the native kidney disease or glomerular abnormalities that are often associated with chronic transplant glomerulopathy were involved in these cases. Our patient developed heavy proteinuria during de novo treatment with sirolimus, while the time after transplantation excludes a contribution of CAN. Normally, only low-molecular-weight proteins such as β2-microglobulin and α1-microglobulin readily pass across the glomerular capillary wall. These proteins are normally reabsorbed by the proximal tubule epithelial cells by endocytosis, which involves binding to the brush border protein megalin (14). The normal filtration of albumin is most likely in the order of 1 g/day (15). The filtered albumin is reabsorbed in the proximal tubules after binding to megalin and cubulin, and subsequent endocytosis (14). In case of tubulo-interstitial injury the reabsorption of proteins is disturbed which results in tubular proteinuria. When the glomerular barrier is intact, this tubular proteinuria does not exceed values of 1–2 g/day. The initial proteinuria in our patient could have been the consequence of ischemia-reperfusion damage, which has been shown to be associated with glomerular damage, characterized by swelling of the podocytes and detachment from the glomerular basement membrane (16). Indeed, we have observed heavy proteinuria in the first hours after living donor kidney transplantation, while this proteinuria decreased to values below 1 g/day within 24 h (11). In contrast, 24 h after cadaveric donor transplantation proteinuria was found to range from 1.5 to 10 g/day, and values below 1 g/day were only reached at 3–5 days after transplantation (10). The course of the proteinuria in our patient more closely followed the pattern of recovery after cadaveric donor transplantation, which is most likely the consequence of the injury sustained during the surgical procedure. However, the increase in proteinuria observed from day 4 onward does not fit the time course of proteinuria caused by ischemia-reperfusion damage. Furthermore, we did not note detachment of podocytes, the typical histological pattern of cold perfusion injury (16). When we compared the albumin content of tubular epithelial cells between our patient and a patient with recurrent FSGS, a strong decrease of albumin reabsorption was noted in our patient. This suggests that altered tubular handling of protein contributed to the proteinuria at the time of the renal biopsy. However, the magnitude of the proteinuria as well as the presence of large amounts of high-molecular-weight proteins was not completely compatible with classical tubular proteinuria. An increased excretion rate of albumin and IgG and the high selectivity index suggested damage of the glomerular filtration barrier. Curiously in this respect, we did not observe any podocyte injury in the renal biopsy. In fact, the presence of normal foot processes argues against massive glomerular proteinuria. Taken together, there is a constellation of paradoxical findings for which alternative explanations should be considered. We think that there are two theoretical possibilities to account for the combination of a high urinary excretion rate of albumin, a decreased tubular reabsorption and normal podocytes. First, during the recovery phase of the ischemia-reperfusion damage there might have been a residual amount of glomerular protein leakage that was not accompanied by podocyte alterations. Increased tubular reabsorption would normally minimize the degree of proteinuria. However, interference of sirolimus with this tubular reabsorption might have resulted in the observed amount of proteinuria. The second possibility to explain our finding implies a disturbance of the recently proposed albumin retrieval pathway (17, 18). Experimental studies of Comper and colleagues have indicated that in the glomerulus larger amounts of protein are filtered than thus far assumed (17). These filtered proteins are recovered by a high-capacity retrieval pathway, which returns intact albumin to the renal vein. Sirolimus might also interfere with this pathway. Either explanation would implicate a toxic effect of sirolimus on tubular epithelial cells, which fits with observations of increased phosphate loss (19) and impaired recovery from delayed graft function (5) during treatment with sirolimus. In conclusion, we describe a renal transplant patient who developed heavy proteinuria during treatment with sirolimus. A decrease in tubular protein absorption appeared to contribute to the proteinuria. Although it is difficult to infer a cause-effect relationship from one single case, the course in our patient and several theoretical considerations suggest a causal role for sirolimus. Therefore, future studies on sirolimus-associated proteinuria should especially take into consideration the effect of sirolimus on tubular reabsorption and transport of proteins. References 1 Saunders RN, Metcalfe MS, Nicholson ML. Rapamycin in transplantation: A review of the evidence. Kidney Int 2001; 59: 3– 16. 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CrossrefCASPubMedWeb of Science®Google Scholar 19 Schwarz C, Bohmig GA, Steininger R, Mayer G, Oberbauer R. Impaired phosphate handling of renal allografts is aggravated under rapamycin-based immunosuppression. Nephrol Dial Transplant 2001; 16: 378– 382. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume6, Issue2February 2006Pages 429-433 AST and ASTS members - please log in via your Society website for full journal access.AST Members >> ASTS Members >> FiguresReferencesRelatedInformation
Publication Year: 2006
Publication Date: 2006-02-01
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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