Nephrin Signaling Results in Integrin β1 Activation

Significance Statement The slit diaphragm protein Nephrin, which is essential for an intact glomerular filter, signals from the podocyte slit diaphragm to the Actin cytoskeleton and induces lamellipodia formation. The authors present evidence that Nephrin activation results in activation of Integrin β 1 in a cultured human podocyte model, and that C3G, a guanine nucleotide exchange factor of the small GTPase Rap1, is involved in Nephrin signaling to Integrin β 1. In vivo , in Drosophila nephrocytes, the Nephrin ortholog Sticks and stones is necessary for correct targeting of Integrin β 1. These findings indicate that Nephrin can mediate a signaling pathway that results in activation of Integrin β 1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix. Background Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. Methods To investigate Nephrin’s role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. Results Drosophila nephrocytes form a slit diaphragm–like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo , nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin β 1 in cultured podocytes. Conclusions Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin β 1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.