Grant: Travel Awards - Institute Community Support

The transmembrane protein nephrin is a key component of the slit diaphragm (SD), a molecular complex that lies laterally between podocyte foot processes. It has recently been postulated that nephrin phosphorylation influences its turnover and this plays a key role in maintaining a healthy SD. Tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of several signaling proteins that regulate podocyte shape and thereby SD permselectivity. Previous work from our lab has identified and mapped the interaction between nephrin and the ShcA adaptor protein and demonstrated an early requirement for ShcA in maintenance of the SD in podocyte-specific ShcA knockout mice. Given ShcA's established role in endocytosis, we now hypothesize that ShcA may regulate nephrin endocytosis to ultimately maintain filtration barrier integrity. Puromycin aminonucleoside nephrosis (PAN) injury was induced in Sprague-Dawley rats and nephrin endocytosis was monitored via biotinylation and immunofluorescence experiments at multiple timepoints. We demonstrate that ShcA expression is increased in podocytes in response to PAN-induced injury, which is accompanied by activation of the stress-activated p38 and JNK MAP kinases. Interestingly, we found that transactivation of AP-1, which occurs downstream of nephrin as well as p38 and JNK, is attenuated by ShcA, and this effect is reversed by disruption of nephrin-ShcA interactions. Further investigation revealed that ShcA promotes nephrin endocytosis in a phospho-dependent manner by enhancing Src-mediated nephrin phosphorylation, its own binding to nephrin and endocytosis into EEA1-positive endosomes both in vitro and in PAN rats. Together these findings suggest a protective effect of ShcA in podocytes by influencing phospho-dependent nephrin endocytosis and possibly attenuating nephrin-mediated p38/JNK signaling in response to injury.