Establishment of an Anti-Nephrin Autoantibody Assay and the Predictive and Diagnostic Significance of Pre-treatment Antibody Titers in Pediatric Nephrotic Syndrome
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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.
Lay Summary
Nephrotic syndrome is a kidney disease in which proteins leak into the urine because of a damaged kidney filtration barrier. Recent studies suggest that some patients produce autoantibodies against nephrin, a key protein required for normal kidney filtration. However, accurately measuring these antibodies has been difficult. In this study, we developed a highly sensitive automated blood test for anti-nephrin autoantibodies and used it in Japanese children with newly diagnosed nephrotic syndrome. We found that all children with steroid-sensitive nephrotic syndrome were positive for anti-nephrin autoantibodies at disease onset. In contrast, children with very high antibody levels or no detectable antibodies were more likely to have steroid-resistant disease or other kidney diseases. These findings were confirmed in an independent validation cohort. This blood test may help improve diagnosis, predict treatment response, and support more personalized management of nephrotic syndrome.
