A multi-institutional study found a possible role of anti-nephrin antibodies in post-transplant focal segmental glomerulosclerosis recurrence
Authors: Yoko Shirai, Kenichiro Miura, Kiyonobu Ishizuka, Taro Ando, Shoichiro Kanda, Junya Hashimoto, Yuko Hamasaki, Kiyohiko Hotta, Naoko Ito, Kazuho Honda, Kenji Tanabe, Tomoko Takano, Motoshi Hattori
Journal: Kidney International (2023)

Possible roles of anti-nephrin antibodies in post-transplant recurrent focal segmental glomerulosclerosis (FSGS) have been reported recently. To confirm these preliminary results, we performed a multi-institutional study of 22 Japanese pediatric kidney transplant recipients with FSGS including eight genetic FSGS and 14 non-genetic (presumed primary) FSGS. Eleven of the 14 non-genetic FSGS patients had post-transplant recurrent FSGS. Median (interquartile range) plasma levels of anti-nephrin antibodies in post-transplant recurrent FSGS measured using ELISA were markedly high at 899 (831, 1292) U/mL (cutoff 231 U/mL) before transplantation or during recurrence. Graft biopsies during recurrence showed punctate IgG deposition co-localized with nephrin that had altered localization with increased nephrin tyrosine phosphorylation and Src homology and collagen homology A expressions. Graft biopsies after remission showed no signals for IgG and a normal expression pattern of nephrin. Anti-nephrin antibody levels decreased to 155 (53, 367) U/mL in five patients with samples available after remission. In patients with genetic FSGS as in those with non-genetic FSGS without recurrence, anti-nephrin antibody levels were comparable to those of 30 control individuals, and graft biopsies had no signals for IgG and a normal expression pattern of nephrin. Thus, our results suggest that circulating anti-nephrin antibodies are a possible candidate for circulating factors involved in the pathogenesis of post-transplant recurrent FSGS and that this may be mediated by nephrin phosphorylation. Larger studies including other ethnicities are required to confirm this finding. Possible roles of anti-nephrin antibodies in post-transplant recurrent focal segmental glomerulosclerosis (FSGS) have been reported recently. To confirm these preliminary results, we performed a multi-institutional study of 22 Japanese pediatric kidney transplant recipients with FSGS including eight genetic FSGS and 14 non-genetic (presumed primary) FSGS. Eleven of the 14 non-genetic FSGS patients had post-transplant recurrent FSGS. Median (interquartile range) plasma levels of anti-nephrin antibodies in post-transplant recurrent FSGS measured using ELISA were markedly high at 899 (831, 1292) U/mL (cutoff 231 U/mL) before transplantation or during recurrence. Graft biopsies during recurrence showed punctate IgG deposition co-localized with nephrin that had altered localization with increased nephrin tyrosine phosphorylation and Src homology and collagen homology A expressions. Graft biopsies after remission showed no signals for IgG and a normal expression pattern of nephrin. Anti-nephrin antibody levels decreased to 155 (53, 367) U/mL in five patients with samples available after remission. In patients with genetic FSGS as in those with non-genetic FSGS without recurrence, anti-nephrin antibody levels were comparable to those of 30 control individuals, and graft biopsies had no signals for IgG and a normal expression pattern of nephrin. Thus, our results suggest that circulating anti-nephrin antibodies are a possible candidate for circulating factors involved in the pathogenesis of post-transplant recurrent FSGS and that this may be mediated by nephrin phosphorylation. Larger studies including other ethnicities are required to confirm this finding. Lay SummaryPossible roles of anti-nephrin antibodies in post-transplant recurrent focal segmental glomerulosclerosis (FSGS) have been reported recently. To confirm these preliminary results, we performed a multi-institutional study. Plasma anti-nephrin antibodies in 11 patients with post-transplant recurrent FSGS were high before transplantation or during recurrence. Graft biopsies during recurrence showed punctate IgG deposition colocalized with nephrin that had altered localization with increased nephrin tyrosine phosphorylation and Src homology and collagen homology A expression. Plasma anti-nephrin antibody levels decreased after remission, and graft biopsies after remission showed no signals for IgG and a normal expression pattern of nephrin. In patients with genetic FSGS and nongenetic FSGS without recurrence, anti-nephrin antibody levels were comparable with those in controls consisting of healthy individuals and disease controls. These results suggest that circulating anti-nephrin antibodies are a possible candidate for circulating factors involved in the pathogenesis of post-transplant recurrent FSGS. Possible roles of anti-nephrin antibodies in post-transplant recurrent focal segmental glomerulosclerosis (FSGS) have been reported recently. To confirm these preliminary results, we performed a multi-institutional study. Plasma anti-nephrin antibodies in 11 patients with post-transplant recurrent FSGS were high before transplantation or during recurrence. Graft biopsies during recurrence showed punctate IgG deposition colocalized with nephrin that had altered localization with increased nephrin tyrosine phosphorylation and Src homology and collagen homology A expression. Plasma anti-nephrin antibody levels decreased after remission, and graft biopsies after remission showed no signals for IgG and a normal expression pattern of nephrin. In patients with genetic FSGS and nongenetic FSGS without recurrence, anti-nephrin antibody levels were comparable with those in controls consisting of healthy individuals and disease controls. These results suggest that circulating anti-nephrin antibodies are a possible candidate for circulating factors involved in the pathogenesis of post-transplant recurrent FSGS. Post-transplant recurrence of primary focal segmental glomerulosclerosis (FSGS) is a major challenge in kidney transplantation. Kidney transplant recipients with primary FSGS face a high risk of post-transplant recurrence in the form of nephrotic range proteinuria (>2.0 g/g creatinine), which leads to allograft damage.1Cravedi P. Kopp J.B. Remuzzi G. Recent progress in the pathophysiology and treatment of FSGS recurrence.Am J Transplant. 2013; 13: 266-274Google Scholar The role of circulating factors has long been presumed in the pathogenesis of primary FSGS, but the definitive identity of such factors remains unknown. Previous studies reported that an i.v. injection of monoclonal anti-nephrin antibodies in rats induced massive proteinuria in <24 hours2Orikasa M. Matsui K. Oite T. et al.Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody.J Immunol. 1988; 141: 807-814Google Scholar,3Topham P.S. Kawachi H. Haydar S.A. et al.Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin.J Clin Invest. 1999; 104: 1559-1566Google Scholar and led to a decrease in nephrin expression in 1 hour.4Kawachi H. Koike H. Kurihara H. et al.Cloning of rat nephrin: expression in developing glomeruli and in proteinuric states.Kidney Int. 2000; 57: 1949-1961Google Scholar In addition, anti-nephrin antibodies, when injected in rodents, changed the nephrin staining pattern from linear to coarse granular and induced nephrin tyrosine phosphorylation, likely via Src family kinase activation.5Takeuchi K. Naito S. Kawashima N. et al.New anti-nephrin antibody mediated podocyte injury model using a C57BL/6 mouse strain.Nephron. 2018; 138: 71-87Google Scholar,6Lahdenperä J. Kilpeläinen P. Liu X.L. et al.Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases.Kidney Int. 2003; 64: 404-413Google Scholar Furthermore, nephrin mRNA was decreased in the nephropathy induced by anti-nephrin antibodies.4Kawachi H. Koike H. Kurihara H. et al.Cloning of rat nephrin: expression in developing glomeruli and in proteinuric states.Kidney Int. 2000; 57: 1949-1961Google Scholar Recently, a patient who presented with early post-transplant massive proteinuria recurrence was reported to have pretransplant anti-nephrin antibodies.7Watts A.J.B. Keller K.H. Lerner G.L. et al.Discovery of autoantibodies targeting nephrin in minimal change disease support a novel autoimmune etiology.J Am Soc Nephrol. 2022; 33: 238-252Google Scholar We also reported a case of post-transplant recurrent FSGS (rFSGS) where circulating anti-nephrin antibodies were detected in the plasma before transplantation.8Hattori M. Shirai Y. Kanda S. et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant. 2022; 22: 2478-2480Google Scholar In the 1-hour post-implantation biopsy of this patient, IgG deposition, nephrin tyrosine phosphorylation, upregulation of Src homology and collagen homology A (ShcA), and altered nephrin distribution were observed together with foot process effacement (FPE).8Hattori M. Shirai Y. Kanda S. et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant. 2022; 22: 2478-2480Google Scholar ShcA is involved in nephrin endocytosis and signaling cascades that contribute to actin polymerization, cell survival, and barrier turnover.9Martin C.E. Jones N. ShcA expression in podocytes is dispensable for glomerular development but its upregulation is associated with kidney disease.Am J Transl Res. 2021; 13: 9874-9882Google Scholar To confirm these findings from a single patient, we conducted a multi-institutional study examining patients with FSGS who underwent kidney transplantation between 1986 and 2022, including those without post-transplant recurrence. We confirmed that anti-nephrin antibodies and associated changes in graft biopsy (IgG deposition, nephrin tyrosine phosphorylation, ShcA upregulation, altered nephrin distribution, and FPE) were observed only in rFSGS but not in non-rFSGS and genetic FSGS. In addition, in patients with rFSGS who achieved remission, anti-nephrin antibody levels were significantly decreased and graft biopsies showed the disappearance of IgG, nephrin tyrosine phosphorylation, and ShcA staining and recovered a normal expression pattern of nephrin and no FPE. Our results suggest that anti-nephrin antibodies may be causally linked to the post-transplant recurrence of FSGS. The clinical and research activities reported here are consistent with the Principles of the Declaration of Istanbul as outlined in the "Declaration of Istanbul on Organ Trafficking and Transplant Tourism," the ethical principles in the 1964 Declaration of Helsinki (2013 update), and the ethical guidelines for epidemiological studies issued by the Ministry of Health, Labour and Welfare, Japan. This study was approved by the Research Ethics Board of Tokyo Women's Medical University (approval number 2021-0184). All participants and/or their guardians provided informed consent to be enrolled in the study. Overall, 109 Japanese patients with childhood-onset FSGS underwent their first kidney transplantation from 1986 to 2022 at the Department of Pediatric Nephrology, Tokyo Women's Medical University; Department of Nephrology, Toho University Faculty of Medicine; and Department of Urology, Hokkaido University, Graduate School of Medicine. Patients with congenital nephrotic syndrome (onset within 3 months of birth) and repeat kidney transplantation were not included in this study. Of 109 recipients, 8 patients with secondary FSGS and 56 recipients with FSGS who did not undergo genetic testing were excluded (Figure 1). Of these, 45 patients underwent whole exome sequencing, which identified disease-causing variants in 65 genes associated with FSGS/steroid-resistant nephrotic syndrome in 28 patients (genetic FSGS) (Supplementary Tables S1 and S2).10Miura K. Kaneko N. Hashimoto T. et al.Precise clinicopathologic findings for application of genetic testing in pediatric kidney transplant recipients with focal segmental glomerulosclerosis/steroid-resistant nephrotic syndrome.Pediatr Nephrol. 2023; 38: 417-429Google Scholar The remaining 17 patients had no pathogenic variants in genes associated with FSGS/steroid-resistant nephrotic syndrome (nongenetic, presumed primary FSGS). Of 17 patients with nongenetic (presumed primary) FSGS, 12 had rFSGS and 5 did not (non-rFSGS).11Ohta T. Kawaguchi H. Hattori M. et al.Effect of pre- and postoperative plasmapheresis on posttransplant recurrence of focal segmental glomerulosclerosis in children.Transplantation. 2001; 71: 628-633Google Scholar, 12Hattori M. Akioka Y. Chikamoto H. et al.Increase of integrin-linked kinase activity in cultured podocytes upon stimulation with plasma from patients with recurrent FSGS.Am J Transplant. 2008; 8: 1550-1556Google Scholar, 13Harita Y. Ishizuka K. Tanego A. et al.Decreased glomerular filtration as the primary factor of elevated circulating suPAR levels in focal segmental glomerulosclerosis.Pediatr Nephrol. 2014; 29: 1553-1560Google Scholar Furthermore, 10 of 12 patients with rFSGS achieved remission (responders). The remaining 2 did not achieve remission and had a progression to graft failure (non-responders). Patients without stored frozen plasma samples and graft biopsy specimens were excluded. Consequently, 8 patients with genetic FSGS and 14 patients with nongenetic (presumed primary) FSGS including 11 with rFSGS and 3 with non-rFSGS were analyzed in this study (Figure 1). Circulating anti-nephrin antibodies were quantified by enzyme-linked immunosorbent assay as previously described,7Watts A.J.B. Keller K.H. Lerner G.L. et al.Discovery of autoantibodies targeting nephrin in minimal change disease support a novel autoimmune etiology.J Am Soc Nephrol. 2022; 33: 238-252Google Scholar except that the recombinant extracellular domain of human nephrin was obtained commercially (R&D Systems, Inc). Briefly, Nunc MaxiSorp enzyme-linked immunosorbent assay plates (Thermo Fisher Scientific) were coated with 100 ng/well of the recombinant extracellular domain of human nephrin (R&D Systems, Inc.) and incubated overnight at 4 °C. Uncoated control wells were used to determine nonspecific binding (in the absence of antigen) for each patient sample, and this allowed for background subtraction. After washing, plates were blocked with 300 μl of SuperBlock (Thermo Fisher Scientific) for 1 hour at room temperature and then incubated overnight with patient samples diluted at 1:400. Plates were incubated with biotin-conjugated goat anti-human IgG Fc, highly cross-absorbed antibody (Thermo Fisher Scientific), followed by incubation with horseradish peroxidase–conjugated avidin (BioLegend). Then, 3,3ʹ,5,5ʹ-tetramethylbenzidine substrate was added, followed by stop solution 10 minutes later, and the absorbance at 450 nm was measured. Anti-nephrin antibody titers were determined using a standard curve derived from serial 2-fold dilutions of the index patient's sample (Supplementary Table S2, patient rFSGS1), whose titer was arbitrarily defined as 1000 U/ml. The cutoff for positivity was defined as the maximum antibody titer in controls consisting of healthy individuals (n = 13) and disease controls including membranous nephropathy (n = 13) and lupus nephritis (n = 4). Serum samples were obtained from patients with membranous nephropathy and lupus nephritis when they presented with proteinuria, with a median urinary protein excretion of 0.9 (interquartile range [IQR] 0.4–1.8) and 2.9 (IQR 2.3–5.1) g/g creatinine, respectively. Anti-nephrin antibodies were also measured using serum samples from 13 patients with minimal change disease (MCD), who had serial serum samples at relapse and remission. Of 13 patients at relapse, 8 had nephrotic range proteinuria (>2.0 g/g creatinine) and 5 showed proteinuria less than 2 g/g creatinine at the time of serum sampling during relapse. Dual immunofluorescence (IF) staining for IgG and nephrin was performed using frozen specimens of graft and native kidney biopsies as in our previous report.8Hattori M. Shirai Y. Kanda S. et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant. 2022; 22: 2478-2480Google Scholar Frozen specimens contained 3 to 5 glomeruli, and all glomeruli in the specimens were evaluated. IgG depositions colocalized with nephrin in 3 glomeruli per biopsy sample were evaluated by 2 experienced pathologists blinded to the groups with scores ranging from 0 to 3, with 0 representing "no IgG deposition colocalized with nephrin," 1 "IgG deposition colocalized with a small portion (<10%) of nephrin," 2 "IgG deposition colocalized with 10%–50% of nephrin," and 3 "IgG deposition colocalized with >50% of nephrin." The representative images of each score of IgG deposition are shown in Supplementary Figure S1. Dual IF staining for nephrin and IgG subclasses (IgG1–IgG4, 1:50, Sigma-Aldrich) and fluorescein isothiocyanate–conjugated polyclonal antibodies against IgM, C3, and C1q (Dako) with anti-nephrin was also performed using frozen specimens. Dual IF staining for IgG and nephrin tyrosine phosphorylation in all specimens was performed using a rabbit polyclonal anti–Phospho-Nephrin (Tyr1176) antibody (PA5-105709, Thermo Fisher Scientific) at a dilution of 1:50,8 a rabbit polyclonal anti–Phospho-Nephrin (Tyr1193) antibody (PA5-72369, Thermo Fisher Scientific) at a dilution of 1:50, and a rabbit monoclonal anti–Phospho-Nephrin (Tyr1217) antibody (ab80298, Abcam) at a dilution of 1:50. Dual IF staining for nephrin and ShcA was performed as in our previous report.8Hattori M. Shirai Y. Kanda S. et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant. 2022; 22: 2478-2480Google Scholar All IF images were obtained by the 2-dimensional structured illumination microscopy mode with a Nikon microscope (N-SIM, Nikon), and image reconstruction was carried out using NIS-Elements software (Nikon) on the basis of a previous report with kind support from Dr. Noriko Tokai of the Imaging Core Laboratory (Institute of Medical Science, The University of Tokyo).8Hattori M. Shirai Y. Kanda S. et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant. 2022; 22: 2478-2480Google Scholar,14Gustafsson M.G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.J Microsc. 2000; 198: 82-87Google Scholar The average foot process width (FPW) in electron microscope images was calculated using the Deegens method,15Deegens J.K. Dijkman H.B. Borm G.F. et al.Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis.Kidney Int. 2008; 74: 1568-1576Google Scholar as previously reported.16Ishizuka K. Miura K. Hashimoto T. et al.Degree of foot process effacement in patients with genetic focal segmental glomerulosclerosis: a single-center analysis and review of the literature.Sci Rep. 2021 8; 1112008Google Scholar Post-transplant FSGS recurrence was defined as (i) the occurrence of nephrotic-range proteinuria (urine protein-creatinine ratio > 2 g/g creatinine) after kidney transplantation and/or (ii) diffuse FPE in graft biopsy on electron microscopy.10Miura K. Kaneko N. Hashimoto T. et al.Precise clinicopathologic findings for application of genetic testing in pediatric kidney transplant recipients with focal segmental glomerulosclerosis/steroid-resistant nephrotic syndrome.Pediatr Nephrol. 2023; 38: 417-429Google Scholar Remission was defined as urine protein excretion < 0.3 g/d.17Ban H. Miura K. Kaneko N. et al.Amount and selectivity of proteinuria may predict the treatment response in post-transplant recurrence of focal segmental glomerulosclerosis: a single-center retrospective study.Pediatr Nephrol. 2021; 36: 2433-2442Google Scholar Continuous variables of the patients' clinical characteristics are presented as median (IQR). Anti-nephrin antibody levels were compared using the Wilcoxon rank-sum test. Statistical evaluations were performed using JMP Pro 14.0.0 (SAS Institute). Patients' clinical characteristics are listed in Table 1 and Supplementary Table S2. The median (IQR) age of all 22 patients at kidney transplantation was 9.5 (8.1–15.2) years. Of 11 patients with rFSGS, prophylactic plasma exchange (PE) was performed in 7 patients, of whom 3 received rituximab before transplantation (Supplementary Table S2).17Ban H. Miura K. Kaneko N. et al.Amount and selectivity of proteinuria may predict the treatment response in post-transplant recurrence of focal segmental glomerulosclerosis: a single-center retrospective study.Pediatr Nephrol. 2021; 36: 2433-2442Google Scholar All patients with rFSGS were anuric-treated with dialysis therapy before transplantation. Post-transplant FSGS recurrence occurred immediately after transplantation in all 11 patients with rFSGS, with the median (IQR) onset of proteinuria at postoperative day 1 (1–1) (Table 1; Supplementary Table S2). The median (IQR) maximum proteinuria after transplantation was 17.9 (7.6–102.3) g/g creatinine in patients with rFSGS (Table 1; Supplementary Table S2). Of 11 patients with rFSGS, 9 achieved remission with and without treatment for recurrence including PE and rituximab (responders) whereas 2 had no response to PE and rituximab and had a progression to graft failure (non-responders) (Table 1; Supplementary Table S2). No patients with genetic FSGS had post-transplant FSGS recurrence (Table 1; Supplementary Table S2).Table 1Patients' clinical characteristicsCharacteristicGenetic FSGSNongenetic FSGSrFSGSNon-rFSGSNo. of patients8113Ethnicity: Japanese8 (100)11 (100)3 (100)Male4 (50)7 (64)3 (100)Age at the onset of NS, yr3.2 (2.8–4.4)2.1 (1.5–4.8)5.5 (4.8–6.4)Age at kidney failure, yr10.2 (5.2–14.1)5.2 (2.4–12.2)7.1 (6.0–8.2)Age at transplantation, yr12.3 (9.2–15.5)8.1 (6.9–15.0)12.4 (10.8–12.8)Donor type: living donor7 (88)7 (64)3 (100)Prophylactic maneuver3 (38)aThree of 8 recipients with genetic FSGS underwent a prophylactic maneuver because they did not undergo genetic analysis before transplantation.7 (64)bThree of 11 patients with rFSGS did not receive a prophylactic maneuver because they underwent deceased donor kidney transplantation. A patient with rFSGS had no pathogenic variants in genes associated with FSGS/SRNS, but his brother also presented with FSGS, so the patient was considered to have variants in undiscovered causative genes associated with FSGS/SRNS and did not undergo a pretransplant prophylactic maneuver.3 (100)Induction and immunosuppressant regimen3 (100)0 (0) Basiliximab, Tac, MMF, MP7 (88)11 (100) Basiliximab, CsA, MMF, MP1 (12)0 (0)1 h graft biopsy1 (12)6 (55)cGraft biopsies: we did not perform 1 h biopsy for a certain period.3 (100)Graft biopsies8 (100)9 (82)dGraft biopsies during recurrence were not performed in 2 patients with rFSGS, but 1 h biopsies were analyzed in these 2 patients.3 (100)Days from transplantation to recurrenceN/A1 (1–1)N/AMaximum proteinuria after transplantation, g/g creatinineN/A17.9 (7.6–102.3)N/ATreatment for recurrence Plasma exchangeN/A9 (82)eSpontaneous remission was achieved in 1 patient, and MP pulse monotherapy induced remission in 1 patient.N/A RituximabN/A3 (27)fPretransplant prophylactic rituximab infusion was not used before 2008.N/ADays from transplantation to graft biopsy during recurrenceN/A22 (20–55)gGraft biopsy during recurrence was performed in 9 cases.N/ATreatment response ResponderN/A9 (82)N/A Non-responderN/A2 (18)N/ACsA, cyclosporine A; FSGS, focal segmental glomerulosclerosis; MMF, mycophenolate mofetil; MP, methylprednisolone; N/A, not applicable; non-rFSGS, nonrecurrent focal segmental glomerulosclerosis; NS, nephrotic syndrome; rFSGS, recurrent focal segmental glomerulosclerosis; SRNS, steroid-resistant nephrotic syndrome; Tac, tacrolimus.Data are expressed as median (interquartile range) or n (%).a Three of 8 recipients with genetic FSGS underwent a prophylactic maneuver because they did not undergo genetic analysis before transplantation.b Three of 11 patients with rFSGS did not receive a prophylactic maneuver because they underwent deceased donor kidney transplantation. A patient with rFSGS had no pathogenic variants in genes associated with FSGS/SRNS, but his brother also presented with FSGS, so the patient was considered to have variants in undiscovered causative genes associated with FSGS/SRNS and did not undergo a pretransplant prophylactic maneuver.c Graft biopsies: we did not perform 1 h biopsy for a certain period.d Graft biopsies during recurrence were not performed in 2 patients with rFSGS, but 1 h biopsies were analyzed in these 2 patients.e Spontaneous remission was achieved in 1 patient, and MP pulse monotherapy induced remission in 1 patient.f Pretransplant prophylactic rituximab infusion was not used before 2008.g Graft biopsy during recurrence was performed in 9 cases. Open table in a new tab CsA, cyclosporine A; FSGS, focal segmental glomerulosclerosis; MMF, mycophenolate mofetil; MP, methylprednisolone; N/A, not applicable; non-rFSGS, nonrecurrent focal segmental glomerulosclerosis; NS, nephrotic syndrome; rFSGS, recurrent focal segmental glomerulosclerosis; SRNS, steroid-resistant nephrotic syndrome; Tac, tacrolimus. Data are expressed as median (interquartile range) or n (%). Anti-nephrin antibody levels in membranous nephropathy (n = 13) and lupus nephritis (n = 4) were comparable with those in healthy individuals (n = 13). Therefore, these 3 groups were collectively considered controls (n = 30) and the cutoff for anti-nephrin antibody positivity (231 U/ml) was defined as the maximum antibody level observed in controls. In 11 patients with rFSGS, the median (IQR) anti-nephrin antibody levels before prophylactic PE or during post-transplant recurrence were markedly high at 899 (831–1292) U/ml, with large variability among patients (Figure 2). The median (IQR) anti-nephrin antibody levels before transplantation in patients with non-rFSGS and those with genetic FSGS were 165 (153–244) and 113 (33–172) U/ml, respectively, which were comparable with those in controls (Figure 2). In MCD, anti-nephrin antibody levels in 5 of 13 patients at relapse (38%) were higher than the cutoff, with a significant reduction at remission (Supplementary Figure S2), which was consistent with a previous report.7Watts A.J.B. Keller K.H. Lerner G.L. et al.Discovery of autoantibodies targeting nephrin in minimal change disease support a novel autoimmune etiology.J Am Soc Nephrol. 2022; 33: 238-252Google Scholar Nephrin had a normal linear staining pattern along the glomerular capillary wall in pre-perfusion graft biopsies but had a granular staining pattern in 1-hour postperfusion biopsies obtained from 6 patients with rFSGS with available samples (Table 2 and Figure 38Hattori M. Shirai Y. Kanda S. et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant. 2022; 22: 2478-2480Google Scholar). All six 1-hour biopsies showed punctate deposition of IgG colocalized with nephrin (Table 2 and Figure 3; Supplementary Table S3 and Supplementary Figure S3). These findings were consistent with our previous report.8Hattori M. Shirai Y. Kanda S. et al.Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis.Am J Transplant. 2022; 22: 2478-2480Google Scholar Similar punctate IgG colocalized with nephrin was observed in the subsequent biopsies obtained from all recurrent patients during recurrence (Table 2 and Figure 3; Supplementary Table S3 and Supplementary Figure S3). IgG depositions were located at the slit diaphragm and podocyte intracellular areas in 1-hour biopsies, and podocyte intracellular depositions were more frequently observed during recurrence (Figure 3; Supplementary Table S3 and Supplementary Figure S3). IgG depositions that did not colocalize with nephrin were observed in 7 patients (Supplementary Table S3 and Supplementary Figure S4). Trace IgM depositions were observed in some biopsies obtained during post-transplant recurrence, but these IgM depositions did not colocalize with nephrin (Supplementary Figure S5). The graft biopsies obtained from patients with non-rFSGS and genetic FSGS did not show IgG deposition (Table 3; Supplementary Figure S6).Table 2Serial changes in circulating anti-nephrin antibody levels and pathological findings in responders and non-respondersVariableRespondersaRemission was defined as urine protein excretion < 0.3 g/d. (n = 9)Non-responders (n = 2)Anti-nephrin antibody levels (U/ml) Before KTx or during recurrence860 (829–1231)1000, 2516 After remission155 (53–367)bSerum samples were available in 5 of 9 patients after remission.N/AIgG deposition colocalizing with nephrin 1 h graft biopsy4/4 (100)c1 h graft biopsies were performed in 3 of 9 responders.2/2 (100) Graft biopsy during recurrence7/7 (100)dGraft biopsies during recurrence were performed in 7 of 9 responders.2/2 (100) Graft biopsy after remission0/8 (0)N/AIncreased expression of pY1176/pY1193/pY1217 nephrin 1 h graft biopsy3/3 (100)c1 h graft biopsies were performed in 3 of 9 responders.2/2 (100) Graft biopsy during recurrence7/7 (100)dGraft biopsies during recurrence were performed in 7 of 9 responders.2/2 (100) Graft biopsy after remission0/8 (0%)eGraft biopsies after remission were available in 8 of 9 responders.N/AIncreased ShcA expression 1 h graft biopsy3/3 (100)c1 h graft biopsies were performed in 3 of 9 responders.2/2 (100) Graft biopsy during recurrence7/7 (100)dGraft biopsies during recurrence were performed in 7 of 9 responders.0/2 (0)fNon-responders showed glomerulosclerosis and decreased expression of nephrin, which might affect the expression of ShcA. Graft biopsy after remission0/8 (0)eGraft biopsies after remission were available in 8 of 9 responders.N/AFPW 1 h graft biopsy, nm2184 (2050–2449)c1 h graft biopsies were performed in 3 of 9 responders.2272, 2204 Graft biopsy during recurrence, nm4190 (3571–4442)