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.
Abstract. Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Srcfamily kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was cotransfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin.
Proper organization of the actin cytoskeleton is essential for the normal structure and function of podocytes. RhoA modulates actin dynamics but its role in podocyte biology is controversial. Here, we generated transgenic mice that express a constitutively active form of RhoA in a podocyte-specific and doxycycline-inducible manner. Induction of activated RhoA with doxycycline resulted in significant albuminuria. Furthermore, both the degree of albuminuria and the histologic changes in the glomerulus positively correlated with the level of constitutively active RhoA expression: low levels of expression associated with segmental foot-process effacement without changes observable by light microscopy, whereas higher levels of expression associated with both extensive foot-process effacement and histologic features of focal segmental glomerulosclerosis (FSGS). In addition, induction of activated RhoA markedly upregulated glomerular mRNA expression of fibronectin and collagen IA1, and the degree of upregulation positively correlated with the level of albuminuria. Withdrawal of doxycycline led to a decline in albuminuria toward basal levels in most mice, but heavy albuminuria persisted in some mice. Taken together, these data suggest that activation of RhoA in podocytes leads to albuminuria accompanied by a range of histologic changes characteristic of minimal change disease and FSGS in humans. Although most changes are reversible, severe and prolonged activation of RhoA may cause irreversible glomerulosclerosis.
Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes. 20: 153320: -154320: , 200920: . doi: 10.1681 Podocytes are unique epithelial cells within the kidney glomerulus that comprise the outermost layer of the blood filtration barrier. 1 Upon differentiation, podocytes extend numerous actin-based foot processes from their cell bodies that interdigitate and surround the glomerular capillary wall. At the interface of adjacent foot processes, a specialized intercellular junction known as the slit diaphragm is formed. The slit diaphragm apparently contributes to the morphology of foot processes through physical connection to the underlying actin cytoskeleton, as mutations affecting numerous slit diaphragm-associated proteins lead to actin rearrangement and foot process effacement. 2 In addition to genetic alterations, injury to podocytes as a consequence of diseases such as diabetes and hypertension as well as inflammation may also result in impaired slit diaphragm filtration leading to loss of protein in the urine (proteinuria) and subsequent renal failure. 3 Nephrin, encoded by NPHS1, is a transmembrane protein of the Ig superfamily that forms a key structural component of the slit diaphragm. Nephrin also functions as an intracellular signaling scaffold to recruit proteins such as podocin and CD2AP J Am Soc Nephrol
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