title: 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.affiliation: Renal Section, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: P S
authors.initials: PS
authors.lastname: Topham
authors.forename: H
authors.initials: H
authors.lastname: Kawachi
authors.forename: S A
authors.initials: SA
authors.lastname: Haydar
authors.forename: S
authors.initials: S
authors.lastname: Chugh
authors.forename: T A
authors.initials: TA
authors.lastname: Addona
authors.forename: K B
authors.initials: KB
authors.lastname: Charron
authors.forename: L B
authors.initials: LB
authors.lastname: Holzman
authors.forename: M
authors.initials: M
authors.lastname: Shia
authors.forename: F
authors.initials: F
authors.lastname: Shimizu
authors.forename: D J
authors.initials: DJ
authors.lastname: Salant
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000009:adverse effects / Q000737:chemistry / Q000276:immunology*; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D006801:Humans; D007678:Kidney Glomerulus / Q000276:immunology* / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009404:Nephrotic Syndrome / Q000276:immunology*; D011233:Precipitin Tests; D011506:Proteins / Q000737:chemistry / Q000276:immunology*; D011507:Proteinuria / Q000209:etiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D013388:Succinimides
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D011506:Proteins; D013388:Succinimides; C113267:nephrin; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)