Nephrin mediates actin reorganization via phosphoinositide 3-kinase in podocytes

Abstract

Nephrin is a slit diaphragm protein critical for structural and functional integrity of visceral glomerular epithelial cells (podocytes) and is known to be tyrosine phosphorylated by Src family kinases. We studied the role of phosphoinositide 3-kinase (PI3K), activated via the phosphorylation of nephrin, in actin cytoskeletal reorganization of cultured rat podocytes. Phosphorylation of rat nephrin by the Fyn kinase markedly increased its interaction with a regulatory subunit of PI3K. Stable transfection of rat nephrin in the podocytes with podocin led to nephrin tyrosine phosphorylation, PI3K-dependent phosphorylation of Akt, increased Rac1 activity, and an altered actin cytoskeleton with decreased stress fibers and increased lamellipodia. These changes were reversed with an inhibitor of PI3K and not seen when the nephrin-mutant Y1152F replaced wild-type nephrin. Rac1 and Akt1 contributed to lamellipodia formation and decreased stress fibers, respectively. Finally, in the rat model of puromycin aminonucleoside nephrosis, nephrin tyrosine phosphorylation, nephrin-PI3K association, and glomerular Akt phosphorylation were all decreased. Our results suggest that PI3K is involved in nephrin-mediated actin reorganization in podocytes. Disturbed nephrin-PI3K interactions may contribute to abnormal podocyte morphology and proteinuria.

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture

“…20 Reductions in nephrin phosphorylation, Nck/p85 binding, and Akt activation have also been detected in the puromycin aminonucleoside model of reversible podocyte injury 12,14,16,18 in parallel with increased expression of the phosphatase PTP1B, which can dephosphorylate nephrin Y1217. 58 By contrast, phosphorylation of nephrin on Y1176/Y1193 is increased with protamine sulfate as noted previously.…”

“…Within this segment, there exists a number of highly conserved tyrosine (Y) residues ( Table 1) that, on phosphorylation by Fyn kinase, [7][8][9] serve as docking sites for intracellular signaling proteins ( Figure 1A). 10 Through recruitment of actin adaptors, such as p85/PI3K, 11,12 the Cas/Crk complex, 13 and Nck1/2, 8,9,14 nephrin phosphorylation is postulated to facilitate direct and dynamic connection to the podocyte cytoskeleton. Moreover, Nck enhances nephrin phosphorylation via activation of Fyn, 15 and loss of Nck within podocytes leads to reduced nephrin tyrosine phosphorylation and widespread foot process effacement, 15,16 inferring a reciprocal relationship between Nck and nephrin in the maintenance of podocyte structure.…”

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats

“…Treatment of diabetes mellitus through PI3K signaling pathway has recently been of great interest. PI3K simultaneously regulates Rac1 and Cdc42, which are associated with the actin cytoskeleton of kidney podocytes (Zhu et al, 2008). All PI3Ks are inhibited by the drugs wortmannin and LY294002.…”

Renal TRPathies

“…The recently proposed mechanosensitivity of TRPC6 channels 29 is not detectable at physiologically relevant pressures of Ͻ80 mmHg, 30 and three additional, independent studies failed to demonstrate that TRPC6 per se is mechanosensitive. [31][32][33] The second TRPC6-interacting protein, nephrin, is an essential constituent of the slit diaphragm contributing to actin reorganization in podocytes by engaging a phosphoinositide 3-kinase pathway 34 subsequent to phosphorylation by the Src family protein tyrosine kinase Fyn. 35 The functional relevance of TRPC6 phosphorylation by Fyn 36 was called into question on the basis of functional analyses of TRPC6 phosphorylation site mutants.…”