Establishment of an Anti-Nephrin Autoantibody Assay and the Predictive and Diagnostic Significance of Pre-treatment Antibody Titers in Pediatric Nephrotic Syndrome

Introduction: Anti-nephrin autoantibodies have been implicated in podocytopathies, including minimal change disease and focal segmental glomerulosclerosis. However, their prevalence, longitudinal dynamics, and clinical significance in first-onset nephrotic syndrome (NS) including steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) remain unclear. Methods: We established a highly sensitive anti-nephrin autoantibody assay using a modified recombinant nephrin antigen and the automated immunoassay platform, and applied it to a prospective Japanese pediatric, multicenter, cohort with first-onset NS and an independent validation cohort. Anti-nephrin autoantibody titers were evaluated before and after treatment and analyzed in relation to clinical phenotypes, genetic risk alleles, and tissue findings. The assay was validated using immunoprecipitation-Western blotting (IP-WB) in representative samples, assessing IgG-nephrin colocalization in evaluable kidney biopsy specimens, and antigen inhibition assays in all antibody-positive samples from the validation cohort. Results: In the pediatric discovery cohort (n = 103), all patients with SSNS (n = 83, 100%) and nine of 16 with SRNS (56.3%) were positive for anti-nephrin autoantibodies, whereas all patients with IgA nephropathy (IgAN) with NS onset were negative (n = 4, 0%). Standard-range positivity was associated with SSNS (77/77, 100%), high-titer positivity with SRNS (9/15, 60.0%), and antibody negativity was restricted to non-SSNS diseases, comprising SRNS (7/11, 63.6%) and IgAN (4/11, 36.4%) (P < 0.0001). Similarly, in the independent validation cohort (n = 42), high-titer positivity was associated with SRNS (1/1, 100%), standard-range positivity with SSNS (30/33, 90.9%), and antibody negativity identified IgAN and membranous nephropathy (5/8, 62.5%) or SRNS (3/8, 37.5%). Orthogonal validation by IP-WB, IgG-nephrin colocalization, and antigen inhibition assays was concordant. Conclusions: Anti-nephrin autoimmunity is a hallmark of NS and identifies distinct clinical phenotypes at disease onset. The automated immunoassay for anti-nephrin autoantibodies enables rapid, highly sensitive, and automated antibody measurement and may serve as a diagnostic triage tool for early etiologic stratification and clinical characterization of NS at disease onset.