Nck Proteins Maintain the Adult Glomerular Filtration Barrier

Abstract

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

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

“…Glomeruli were isolated from WT mice exposed to protamine sulfate or LPS challenge and immunoblotted with phosphospecific nephrin antibodies recognizing Y1176/Y1193 and Y1217. 16 Within the protamine sulfate model, we observed a significant increase in nephrin phosphorylation on Y1176/ Y1193 after protamine sulfate perfusion, which decreased after heparin sulfate recovery ( Figure 7, A and B). Relative to protamine sulfate-treated mice, Y1176/Y1193 was weakly phosphorylated in glomeruli perfused with buffer alone.…”

“…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.…”

“…Comparable levels of total nephrin relative to GAPDH were seen in all genotypes. Moreover, using our phosphospecific nephrin antibodies developed to recognize Y1176, Y1193, or Y1217, 16 we detected strong phosphorylation of nephrin in WT animals, which was severely decreased in nephrin WT/Y3F animals and completely absent from Y3F animals ( Figure 2B). …”

“…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.…”

“…Nephrin is tyrosine phosphorylated during glomerulogenesis and throughout life, 8,16,17 and decreases in nephrin phosphorylation on Y1217 are seen coincident with foot process effacement in human kidney diseases, including minimal change disease (MCD) 18 and membranous nephropathy. 19 Similarly, reduced phosphorylation of the downstream survival factor Akt on serine (S) 473 can be observed in MCD, 20,21 and inactivation of the actin binding protein cofilin is detected in patients with MCD, membranous nephropathy, and FSGS.…”