Overall mean proteinuria decreased significantly from 5.1±3.8 g/g during pretreatment to 2.1±2.8 g/g during posttreatment (P=0.003); this decrease was observed primarily among subjects who received rituximab in addition to plasmapheresis (Table 2). Among those who attained complete or partial remission (Table 3), mean pretreatment to posttreatment proteinuria declined significantly from 5.3±3.9 to 1.5±1.7 g/g (P=0.0005; Fig. 1B). This improvement persisted on the last available proteinuria measurements (mean peak to last available, 8.8±7.0 to 1.7±2.6 g/g; P=0.0002; Table 3). In contrast, individuals who did not respond to treatment (n=6) had persistent proteinuria, with a posttreatment mean proteinuria of 15.1±18.7 g/g.
The overall suPAR levels decreased from a median of 6.781 to 4.129 pg/mL (P=0.02) with treatment (Table 2; Fig. 1).
Improvements in these clinical parameters correlated with remarkable improvement in foot process effacement, which decreased from 55%±34% to 22%±23% (P=0.0009; Fig. 1C) in subjects who achieved complete or partial remission. Furthermore, the improvement in podocyte foot process effacements involved even patients who failed to respond to therapy in respect to proteinuria (Fig. 2).
C-Reactive Protein Levels in Individuals With Posttransplantation FSGS
To evaluate the association between suPAR and inflammation in posttransplantation FSGS subjects, C-reactive protein (CRP) levels were measured in 15 individuals with posttransplantation FSGS at time of diagnosis. Mean CRP levels were low (9.8±4.3; reference range, 0–20) in these patients (Table 1).
Our study confirmed that the first pathologic finding of recurrent renal allografts is podocyte foot process effacement. We demonstrated that circulating suPAR levels at the time of diagnosis correlate with the severity of effacement. Furthermore, a complete or even partial response to therapy results in significant resolution of foot process effacement and improvement in proteinuria. In the seven patients with suPAR measured before and after therapy, we noted a significant decrease in the levels after therapy.
Early reports implicated the importance of podocytes in the recurrence of FSGS after transplantation. Harrison et al. reported a case of a 62-year-old woman who suffered recurrent FSGS (14). Biopsies of native kidney and at the time of posttransplantation recurrence showed similar alterations in the podocyte junctions. Hoyer et al. reported in 1972 that minimal change disease preceded FSGS recurrence in two cases (15). In a report of a patient with immediate recurrent FSGS, the allograft biopsy at 1 hr after transplantation showed minor glomerular abnormalities with partial foot process effacement on electron microscopy (16). We also recently reported on 19 pairs of prereperfusion and postreperfusion biopsy results obtained from patients with FSGS undergoing renal transplantation (10). The mean number of effaced foot processes on the postreperfusion biopsy was higher among the seven subjects who developed recurrent FSGS within 30 days compared with those who did not. Cheong et al. also reported on six children with early FSGS recurrence, five of them had only foot process effacement initially. However, three of them later developed segmental sclerosis on subsequent renal biopsies (17). Our study, which represents the largest sample to date, confirms that the earliest detectable ultrastructural change in recurrent FSGS after renal transplantation is podocyte foot process effacement.
Whether these podocyte changes resolve with treatment, however, has not been well delineated in the current literature. Artero et al. reported on their 10-year experience of recurrent FSGS in adults and children (12). They noted one individual whose foot process effacement resolved with plasmapheresis accompanied by decline in proteinuria and three who achieved remission with treatment had only podocyte changes, whereas two who did not respond to therapy already had sclerosis before plasmapheresis. The recent case report by Gallon et al. in which an allograft from a recipient with early FSGS recurrence was subsequently retransplanted to a different patient without primary FSGS supports the reversibility of foot process effacement in FSGS. These changes resolved on postoperative day 14 (18). In our study, we found that our therapy resulted in significant improvement in podocyte effacement decreasing from a mean of 55% to 22%. The decrease in podocyte effacement involved not only responders but also patients who did not respond to therapy by way of a significant decrease in proteinuria. These improvements may be due to the early recognition of FSGS recurrence and institution of therapy.
Furthermore, we found that suPAR levels correlated significantly with the degree of the ultrastructural changes in podocytes. In line with such a quick correction of podocyte ultrastructure are observations from a mother with FSGS who gave birth to a child with transient proteinuria (19). Both mother and child had high suPAR blood levels; the baby’s resolution of proteinuria is likely due to the decrease of suPAR serum levels and correction of podocyte injury.
In our study, posttransplantation CRP levels were normal, arguing against the contribution of inflammation to the high suPAR levels observed among our posttransplantation FSGS subjects. However, CRP represents only one marker of the activity of inflammatory pathways, and further analysis is needed.
Although clinical improvement among recipients with posttransplantation FSGS associated with treatment may have been mediated by plasmapheresis-related declines in suPAR, rituximab was found by Fornoni et al. to have direct stabilizing effects on podocyte function (3). Taken in the context of variable response to plasmapheresis with or without rituximab and the fact that suPAR binds and activate β3 integrin expressed on podocytes (6), prospective studies are needed to determine whether the gene expression that encodes for β3 integrin (ITGB3) in the recipient or allograft kidney (20) also has prognostic bearing on recurrent FSGS and response to treatment.
In spite of these significant findings of our study, it has a few limitations. Our main limitation was the retrospective design of our study, which may have led to missing some important cofounders that could have influenced our results. Although a prospective study in this field will be of great importance, our overall findings will have significant clinical application in kidney transplant recipients with posttransplantation FSGS. In addition, our study size was small, and findings reflect the clinical practice of a single center; therefore, our results may not be generalizable to other renal transplant centers. Furthermore, our study lacks control groups with nonrecurrent FSGS or non-FSGS patients. This was in most part due to the limited available sera obtained at the same time points (as per our study methods) for suPAR measurements, in addition to incomplete clinical data on these individuals. Finally, although median suPAR levels exceeded 3000 pg/mL, reported as the threshold to discriminate between FSGS cases and other nephrotic syndromes, these data were reported in a population composed primarily of individuals with primary FSGS (6, 7). Thus, this threshold may not be applicable for posttransplantation FSGS.
In summary, early posttransplantation recurrent or de novo FSGS manifests histologically by podocyte foot processes effacement. Response to therapy with plasmapheresis with or without rituximab may prevent light microscopic changes of FSGS from developing. suPAR may be one of the main circulating permeability factors that cause recurrent FSGS via binding to the podocytes. However, larger and multicenter prospective studies are needed to clarify the utility of suPAR measurements in predicting FSGS recurrence after transplantation and in monitoring response to therapy.
MATERIALS AND METHODS
The study was approved by the Johns Hopkins Medicine Institutional Review Board (Baltimore, MD). This is a retrospective case series study of all adult renal transplant recipients who underwent renal transplantation between January 1, 2003 to December 31, 2011 in our center and developed recurrent or de novo FSGS after kidney transplantation. Twenty-five individuals included in this study developed FSGS after transplantation.
All subjects received thymoglobulin and high-dose steroid for induction and a three-drug regimen consisting of mycophenolate mofetil, tacrolimus, and prednisone for maintenance immunosuppressions. Four patients with recurrent FSGS were switched from tacrolimus to cyclosporine during their treatment course for FSGS. None of the subjects received sirolimus.
Recurrent FSGS was defined by the presence of proteinuria as measured by urine protein-to-creatinine ratio and confirmed by 24-hr urine collection. A ratio of greater than 1 g/g in individuals who were anuric before renal transplantation (n=8) or a persistent increase in urine protein-to-creatinine ratio by greater than 1 g/g from baseline proteinuria among those who made urine before renal transplantation (n=16) was consistent with the diagnosis (data of urine before transplantation was not available on one patient). The diagnosis was confirmed by histologic findings of FSGS on biopsies obtained before or within 10 days of treatment commencement. On the contrary, de novo FSGS posttransplantation was defined by (a) new onset of more than 1 g/g proteinuria (or +2 on urinalysis in one patient with unavailable urine protein-to-creatinine ratio at time of diagnosis) in recipients whose primary cause of end-stage renal disease was not attributed to FSGS (n=2, IgA nephropathy and lupus nephritis) or was unknown (n=2, native kidney biopsy was not performed) in addition to (b) allograft biopsy with light microscopic changes consistent with FSGS, which is not explained by other pathologic findings.
Sociodemographic and clinical data were abstracted from patient medical records from the time of renal transplantation to 3 years after renal transplantation or the latest available clinical follow-up. Donor clinical characteristics included donor vital status, relatedness to the recipient, and ABO-compatibility with the recipient. Recipient clinical characteristics included age at FSGS diagnosis, number of prior renal transplantations, duration of dialysis, serum creatinine, and proteinuria estimated by urine protein-to-creatinine ratio. eGFR was measured using the CKD-Epi equation, which adjusts for the variation in serum creatinine associated with age, gender, and race (21).
Treatment with plasmapheresis was initiated at the time of diagnosis. A 100% albumin replacement fluid was routinely used. We used fresh-frozen plasma when patients were to undergo a renal allograft biopsy within 48 hr or in cases of daily plasmapheresis. One or two rituximab infusions (dose of 375 mg/m2 of body surface area) were administered to patients who failed to achieve complete or partial remission with plasmapheresis therapy alone. Complete remission was defined by a decrease in proteinuria to below 1 g/g upon completion of the treatment course. Partial remission was defined as a decline in proteinuria by 50% from the peak proteinuria level but remaining 1 g/g or greater at the end of therapy.
Renal histopathology was assessed by a renal pathologist using light microscopy, immunofluorescence, and electron microscopy. The degree of podocyte foot process effacement was based on biopsy reports as well as second assessments of the electron microscopy by another renal pathologist blinded to the original biopsy report and the recipient’s outcome. These two assessments were then averaged to obtain the mean podocyte foot process effacement, which was then categorized into mild (≤25%), moderate (26%–74%), and severe (≥75%). Baseline electron microscopy results were obtained from renal biopsies performed before or within 10 days of initiating therapy. Posttreatment foot process effacement was assessed from renal biopsies performed after the completion of the plasmapheresis sessions and rituximab infusion in refractory cases. A minimum of 10 capillaries was used to determine podocyte effacement.
Among recipients with stored sera available before and after treatment, suPAR levels and CRP levels were measured. suPAR levels were measured by using Quantikine Human uPAR immunoassay (R&D Systems, Minneapolis, MN), whereas CRP was measured by using Quantikine ELISA kit (R&D Systems). Individuals who underwent ABO-incompatible transplantation were excluded from the suPAR and CRP analyses.
We performed descriptive analyses to evaluate the distribution of the recipients’ baseline sociodemographic and clinical characteristics. The Kruskal–Wallis test was used to compare medians of nonnormal continuous variables across categories, whereas the Mann–Whitney U test was used to compare means of normally distributed variables across categories. Pretreatment and posttreatment comparisons were performed using paired t test and the Wilcoxon sign-rank test, as appropriate. Comparisons of pretreatment and posttreatment clinical parameters were conducted using the overall study population and then restricted only to recipients who attained complete or partial response to treatment. All statistical analyses were performed using Stata/MP version 11.2 (StataCorp, College Station, TX).
The authors thank Rachel Marino, NP, for her help in collecting some of the clinical data.
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Keywords:© 2013 by Lippincott Williams & Wilkins
Kidney transplant; Podocyte effacement; FSGS; suPAR; Rituximab