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Investigation of the Role of Nephrin Phosphorylation and Associated Signaling Pathways in Kidney Podocytes

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Title: Investigation of the Role of Nephrin Phosphorylation and Associated Signaling Pathways in Kidney Podocytes
Author: New, Laura Alexandra
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Jones, Nina
Abstract: The integrity of the glomerulus plays a key role in prevention of kidney damage. The specialized function of the glomerular filter has led to the evolution of a unique cell type—the podocyte—whose shape and function depends on its network of actin-based ‘foot processes’. Damage to the filter—the slit diaphragm junction—formed between foot processes leads to proteinuria and can result in kidney failure. The slit diaphragm supports podocyte architecture through the IgG protein nephrin, whose cytoplasmic tail contains several conserved tyrosine residues within YDxV motifs which are phosphorylated by Fyn kinase. The significance of these tyrosines has remained elusive, but they have recently been shown to recruit the essential podocyte protein Nck. We hypothesize that phosphorylated nephrin signaling via Nck is important in the podocyte and that removal of the YDxV motifs from nephrin in vivo will result in a loss of Nck signaling and the collapse of the podocyte actin cytoskeleton. To study nephrin phosphorylation on specific tyrosines, we generated phospho-nephrin antibodies against individual YDxV motifs. These antibodies allowed us to validate that these sites are indeed phosphorylated in vivo and to demonstrate that phosphorylation on these tyrosines is altered in a podocyte injury model. This work was complemented by further study of the regulation of nephrin phosphorylation, which outlined a mechanism whereby nephrin phosphorylation is positively regulated by Nck. Recruitment of Nck to phosphorylated nephrin via its SH2 domain enables its SH3 domains to bind and activate Fyn kinase, thus increasing nephrin tyrosine phosphorylation and promoting continued engagement of nephrin signaling pathways. Finally, we investigated the importance of nephrin tyrosine phosphorylation in vivo through the nephrinY3F mouse model, which contains mutations preventing nephrin phosphorylation on the YDxV tyrosine motifs. The inability to phosphorylate these tyrosines in vivo does not prevent slit diaphragm formation, but results in podocyte foot process effacement and proteinuria with age. These results highlight the importance of phospho-nephrin signaling pathways in the podocyte, and—as altered nephrin phosphorylation is observed in human kidney disease—may contribute to a better understanding of how these pathways can be therapeutically manipulated to restore damaged podocytes.
Date: 2014-04
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