Anti-nephrin antibodies are not enriched in patients with primary and posttransplant recurrent podocytopathies
Francesco Pecoraro, Luca Perico, Federica Casiraghi, Paola Rizzo, Matias Trillini, Andrea Angeletti, Manuel Alfredo Podestà, Xhuliana Kajana, Agnese Spennacchio, Marta Todeschini, Marilena Mister, Giuseppe Castellano, Ariela Benigni, Giuseppe Remuzzi
Francesco Pecoraro, Luca Perico, Federica Casiraghi, Paola Rizzo, Matias Trillini, Andrea Angeletti, Manuel Alfredo Podestà, Xhuliana Kajana, Agnese Spennacchio, Marta Todeschini, Marilena Mister, Giuseppe Castellano, Ariela Benigni, Giuseppe Remuzzi
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Clinical Research and Public Health Autoimmunity Nephrology

Anti-nephrin antibodies are not enriched in patients with primary and posttransplant recurrent podocytopathies

Abstract

BACKGROUND Anti-nephrin autoantibodies have emerged as a putative pathogenic driver in a subset of patients with podocytopathies, including those with posttransplant disease recurrence.METHODS We measured anti-nephrin autoantibodies in a cohort of 65 patients with podocytopathy associated with steroid-sensitive nephrotic syndrome (n = 39) and steroid-resistant nephrotic syndrome (n = 26) and in 34 patients with posttransplant podocytopathy recurrence. Fourteen patients with membranous nephropathy and 20 healthy volunteers served as controls. ELISA and immunoprecipitation assays were performed to detect anti-nephrin IgG using 2 different recombinant human nephrin proteins. Immunofluorescence analysis was performed to assess gG deposition and its colocalization with nephrin in renal biopsies.RESULTS When using an ELISA based on murine cell-derived human antigen, the highest positivity was found in healthy volunteers (55%), correlating with levels of circulating natural anti–α-galactose-α-1,3-galactose antibodies. This cross-reactivity was abrogated with recombinant human nephrin expressed in human cells. In this setting, very low prevalence (<5%) of anti-nephrin antibody-positive patients was found in steroid-sensitive and -resistant nephrotic syndrome cohorts and in patients with posttransplant disease recurrence. These frequencies were comparable to healthy volunteers. Using confocal and super-resolution microscopy, only trace amounts of IgM, but no IgG, were found in the glomeruli of analyzed biopsies, which did not colocalize with nephrin.CONCLUSION With the methodology presented here, anti-nephrin reactivity was extremely rare and occurred at comparably low frequencies in healthy controls, native-kidney podocytopathies, and posttransplant disease recurrence. This suggests that these autoantibodies are not inherently disease specific and may not serve as a broad biomarker across podocytopathies.TRIAL REGISTRATION ClinicalTrials.gov NCT06334692.FUNDING The Medici di Marignano family.

Authors

Francesco Pecoraro, Luca Perico, Federica Casiraghi, Paola Rizzo, Matias Trillini, Andrea Angeletti, Manuel Alfredo Podestà, Xhuliana Kajana, Agnese Spennacchio, Marta Todeschini, Marilena Mister, Giuseppe Castellano, Ariela Benigni, Giuseppe Remuzzi

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Figure 2

Anti-nephrin antibody detection by ELISA and anti–α-Gal antibody cross-reactivity using human nephrin produced in murine cells.

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Anti-nephrin antibody detection by ELISA and anti–α-Gal antibody cross-r...
(A) Antibodies against the extracellular domain of recombinant nephrin produced in mouse cells were measured by ELISA in patients with steroid-sensitive nephrotic syndrome (SSNS), steroid-resistant nephrotic syndrome (SRNS), and posttransplant podocytopathy recurrence (Post-tx R). HVs and patients with MN served as controls. The dashed line indicates the threshold (180 AU/mL). Positive samples are highlighted in red. (B and C) Representative Western blot (upper panels) and Ponceau red staining (lower panels) of nephrin produced in murine cells (mouse), α1-3Galβ1-4Glc-BSA (α-Gal–BSA), nephrin produced in human cells (human), and unconjugated BSA under nonreducing (NR) and reducing (R) conditions. Membranes were probed with mouse anti–α-Gal antibody (green) and rabbit anti-nephrin antibody (red) to assess α-Gal modification (n = 3) (B) or with HV sera (n = 4) (C) to evaluate the reactivity of circulating antibodies against the different proteins. For each panel, MWs are expressed in kilodaltons. (D) Circulating anti–α-Gal antibodies quantified by ELISA in HV sera classified as positive or negative by ELISA with nephrin produced in mouse cells. The dashed line indicates the threshold (42.9 μg/mL). (E) Correlation between anti–α-Gal and anti-nephrin absorbance values at OD450 in HV sera by ELISA. Linear regression analysis with 95% CI is shown (r = 0.6618 and P = 0.0028). (F) Sera from HV identified as anti-nephrin positive were preabsorbed with unconjugated 10 μg BSA, 5 μg α-Gal–BSA, or 10 μg α-Gal–BSA prior to ELISA with nephrin produced in mouse cells. Data are presented as mean ± SEM and were analyzed using 1-way ANOVA with Tukey’s multiple-comparison test, unpaired 2-tailed t test, or paired t test, as appropriate. *P < 0.05, **P < 0.01, and ***P < 0.001.