Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin
Peter S. Topham, … , Fujio Shimizu, David J. Salant
Peter S. Topham, … , Fujio Shimizu, David J. Salant
Published December 1, 1999
Citation Information: J Clin Invest. 1999;104(11):1559-1566. https://doi.org/10.1172/JCI7728.
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Article

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin

Abstract

mAb 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.

Authors

Peter S. Topham, Hiroshi Kawachi, Samir A. Haydar, Sumant Chugh, Theresa A. Addona, Kathryn B. Charron, Lawrence B. Holzman, Michael Shia, Fujio Shimizu, David J. Salant

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Figure 1

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Representative Western blot analysis for mouse IgG in cross-linked immun...
Representative Western blot analysis for mouse IgG in cross-linked immunoprecipitates. Isolated Sprague-Dawley rat glomeruli were incubated with either mAb 5-1-6 or RVG-1. After washing, the antibody/antigen complex was cross-linked with DTSSP followed by detergent solubilization and immunoprecipitation with agarose bound goat anti-mouse IgG. The precipitates were resolved by nonreducing 5% SDS-PAGE, electroblotted to nitrocellulose, immunoblotted for mouse IgG, and developed using a chemiluminescent technique. A mouse IgG–containing band is visible at 150 kDa in both lanes, consistent with uncomplexed mouse mAb 5-1-6 or RVG-1. In addition, a band is specifically present in the mAb 5-1-6 IP lane at approximately 300 kDa, which represents the cross-linked complex of mAb 5-1-6 and its antigen.