Background: We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. Methods: We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. Results: Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate. We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct fromits ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. Conclusions: We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.
|Original language||English (US)|
|Number of pages||17|
|Journal||Journal of the American Society of Nephrology : JASN|
|State||Published - Nov 2018|
Bibliographical noteFunding Information:
We thank the shared microscopy research facility and Dr. Richard Gordon of the electron microscopy core at Icahn School of medicine at Mount Sinai, as well as Electron Microscopy Facility at the University of Minnesota Imaging Center. Dr. Jenny Xiang at The Genomics Core Research Facility at Weill-Cornell School of Medicine, New York. We acknowledge critical feedback from Prof Detlef Schondorff and Prof. Peter Heeger during different stages of this work and manuscript. We acknowledge Dr. Lawrence Holzman for the gift of Nphs1 antibody. M.C.M. would like to acknowledge philanthropy from Nina and Homer Eaton, and Pablo Legorreta.
This work was supported by American Heart Association grant 15SDG25870018 (to M.C.M.) and National Institutes of Health grant RO1 DK102420 (to B.M.).
© 2018 by the American Society of Nephrology.