Abstract
Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA, via its SH2 domain, was associated with several phosphorylated tyrosine residues on nephrin. Overexpression of ShcA promoted nephrin tyrosine phosphorylation and reduced nephrin signaling and cell surface expression in vitro. In a rat model of reversible podocyte injury and proteinuria, phosphorylated nephrin temporally colocalized with endocytic structures coincident with upregulation of ShcA expression. In vivo biotinylation assays confirmed that nephrin expression decreased at the cell surface and correspondingly increased in the cytosol during the injury time course. Finally, immunostaining in kidney biopsy specimens demonstrated overexpression of ShcA in several human proteinuric kidney diseases compared with normal conditions. Our results suggest that increases in ShcA perturb nephrin phosphosignaling dynamics, leading to aberrant nephrin turnover and slit diaphragm disassembly.
Original language | English (US) |
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Pages (from-to) | 92-103 |
Number of pages | 12 |
Journal | Journal of the American Society of Nephrology |
Volume | 29 |
Issue number | 1 |
DOIs | |
State | Published - Jan 2018 |
Funding
Education and National Training Program. C.E.M. is supported by an Natural Sciences and Engineering Research Council of Canada Alexander Graham Bell Canada Graduate Doctoral Scholarship and by an Ontario Graduate Scholarship. K.A.P. was supported by a CIHR Canada Graduate Masters Scholarship. M.T. is supported by a University of Guelph College of Biological Sciences Studentship. We gratefully acknowledge Dr. Laura New, Dr. Melanie Wills, Steve Hawley, Lauren Hampton, and Peihua Lu (all University of Guelph) for providing reagents, advice, and technical support. This work was supported by grants from Canadian Institutes of Health Research (CIHR) and the Kidney Foundation of Canada (to N.J. and T.T.), and CIHR/Sick Kids Foundation (to N.J.). N.J. holds a Tier 2 Canada Research Chair in Eukaryotic Cellular Signalling and was the recipient of a New Investigator Award from the Kidney Research Scientist Core
ASJC Scopus subject areas
- General Medicine