Characterization of signaling pathways regulating nephrin endocytosis in kidney podocytes: novel roles for Nck and ShcA adaptor proteins
Kidney podocytes maintain blood filtration selectivity through a network of actin-based projections termed foot processes. The slit diaphragm is a molecular barrier that lies laterally between foot processes and acts as a discerning filtration pore. Nephrin is a key component of this barrier and disrupted nephrin trafficking is suggested to be a source of filtration breakdown, although evidence of this within the complexity of the body remains incomplete. Further, although nephrin’s tyrosine phosphorylation is implicated in its trafficking, the binding partner(s) that facilitate these phospho-dependent mechanisms remain unidentified. Using several acute injury mouse models, we first demonstrate that nephrin tyrosine phosphorylation is commonly disrupted in the injured podocyte and that this disturbs connections between nephrin and Nck, a cytoskeletal adaptor. Targeted genetic disruption of nephrin/Nck interactions further exacerbates disease in mice, verifying a crucial role for nephrin/Nck signaling in withstanding insult. We next characterized a fundamental role for Nck in mediating recruitment of actin and the endocytic scission engine dynamin in late stages of nephrin endocytosis, which required nephrin’s tyrosine phosphorylation. Disruption of nephrin/Nck binding in podocytes in vivo and in cell models led to accumulation of nephrin in endocytic pits on the cell surface and this accompanied progressive barrier demise in mice. Interestingly, aberrant activation of this mechanism could also initiate disease, highlighting a requirement for tight regulation of this apparatus for barrier maintenance. Our final investigations revealed ShcA as a novel nephrin phosphotyrosine binding partner and modulator of nephrin trafficking. ShcA is normally expressed in low levels within podocytes and we identified a stark upregulation of ShcA in a rat model of kidney disease, concurrent with internalization of nephrin and barrier breakdown. In cell–based studies, we demonstrated that ShcA overexpression promotes phospho-mediated nephrin internalization. Upregulated ShcA gene and protein expression were also observed in several human kidney diseases, supporting the clinical relevance of this signaling pathway. Collectively, this work has identified Nck and ShcA as two novel mediators of phospho-dependent nephrin endocytosis. Integration of these pathways into the larger framework of nephrin trafficking reamins an important objective for future work.