Abstract P322: Interaction of Shroom3 With Fyn Impacts Phosphorylation of Nephrin Causing Proteinuria With Foot Process Effacement

Abstract

In the translational GoCAR study, we identified that a CKD-associated SHROOM3-SNP, and tubular Shroom3 expression correlated with the development of fibrosis in renal allografts. We showed that SHROOM3 facilitated TGF-B signaling suggesting its potential role as a therapeutic target. However, recent data suggest a protective role for SHROOM3 in proteinuria and glomerular development. To study the role of SHROOM3 in adult glomeruli, we used doxycycline-inducible (DOX), shRNA-mediated SHROOM3 knockdown, Podocin- and tubular-specific (PAX8)-RTTA mice, comparing these to non-transgenic DOX-fed littermates. After 2wks of DOX, adult Podo-RTTA mice developed significant albuminuria compared to littermates. Albuminuria was reversible on DOX-withdrawal, and reappeared on re-initiation. No podocyte loss [WT1 stain] was seen in these mice(8-wks DOX). EM revealed >50% foot process effacement. PAX8-RTTA mice did not show proteinuria. Glomerular RNA- seq identifed intracellular signaling/Small GTPAse signaling/integrin signaling/ actin-cytoskeleton among downregulated Gene-ontology terms in knockdown mice vs Controls. To identify protein interacting partners of SHROOM3, we performed mass spectrometry on protein lysates of 293-T cells overexpressing SHROOM3 immunoprecipitated (IP) with either anti-V5, -SHROOM3 or IgG. Among 491 unique interactions, we confirmed SHROOM3 as the top ranking protein. Interestingly, FYN - a src-kinase - was a top ranking candidate. Podocyte FYN is crucial for NPHS1-phosphorylation, and FYN-deficient mice show identical proteinuria phenotype. In human podocytes, we confirmed the interaction of endogenous SHROOM3 and FYN by IP. Glomerular protein extracts of Shroom3-knockdown mice showed decreased phosphorylation of FYN, and NPHS1. In human allografts from GoCAR, we identifed a corresponding reduced albuminuria (>1year post-transplant) associated with homozygosity of the risk allele in the donor. In summary, Podocyte-specific SHROOM3 knockdown causes a reversible proteinuria phenotype in adult mice, by interacting with FYN, a mechanism distinct from its effect on renal fibrosis in allografts.