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

Abstract 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.

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Publication Details

Journal
medRxiv
Published
2026-10-26
DOI
https://doi.org/10.64898/2026.10.24.26366391
Primary Topic
Renal Diseases and Glomerulopathies
Type
preprint

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preprint

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

Naoya Fujita, Yoshitsugu Kaku, Rika Fujimaru, Shoichi Maruyama et al.
medRxiv
Renal Diseases and Glomerulopathies
preprint

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

Naoya Fujita, Yoshitsugu Kaku, Rika Fujimaru, Shoichi Maruyama, Kandai Nozu, Taishi Nada, Riku Hamada, Hiroki Miyano, Yoshitaka Isaka, Tomoo Kise, Erika Hidawa, China Nagano, Amane Harada, Koichi Nakanishi, Tomohiko Yamamura, Hideki Ban, Kazumoto Iijima, Yuta Ichikawa, Yuta Inoki, Miwa Goto, Hiroshi Kaito, Kazumichi Fujioka, Yuko Shima, Yusuke Atarashi, Maria Kiriyama, Nana Sakakibara, Takaya Ozeki, Yusuke Okuda, Eri Yamamoto, Wataru Shimabukuro, Rie Kuroki, Bishnu Devi Maharjan, Masanori Miyanishi, Kenji Ishikura, Yohei Ikezumi, Tomoko Horinouchi, Koichi Kamei, Kazuma Kita, Aoi Tanaka, Jeeeun Kim, Natsumi Yamamura, Masaya Muto, Yumiko.T Sakai, Nanami Nagatsuka, Shin'ichi Akiyama, Keiko Miwa, Akshay Ganguly, Valentina Podolinnaia, Mami Onishi
preprint en

Abstract

Abstract 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.

medRxiv
Fujita Health University (JP), Wakayama Medical University (JP), Yokohama City University Medical Center (JP), University of the Ryukyus (JP), Osaka Women's and Children's Hospital (JP), Osaka City General Hospital (JP), Fukuoka Children's Hospital and Medical Center for Infectious Diseases (JP), Japanese Red Cross Kumamoto Hospital (JP), Children's Medical Center (IR), Japanese Red Cross Fukuoka Hospital (JP), Kobe Children's Hospital (JP), National Center For Child Health and Development (JP), Tokyo Metropolitan Children's Medical Center (JP), Juntendo University Nerima Hospital (JP), Sysmex (Japan) (JP), Nagoya University (JP), Kitasato University (JP), Kobe University (JP), University of Yamanashi (JP), Saitama Medical University (JP), Yokohama City University (JP), The University of Osaka (JP)
Japan Agency for Medical Research and Development
Renal Diseases and Glomerulopathies
DOIOpen Access PDFOpenAlex
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