The role of the Rap guanine nucleotide exchange factor 1 (C3G) in the podocyte and in glomerular disease

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In most forms of glomerular diseases, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions. The cell adhesion protein Nephrin is necessary for establishing regular foot process architecture and junction composition in development by transducing signals that regulate actin cytoskeletal dynamics. Patients with mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and suffer from severe proteinuria. Nephrin transmits signals from the slit-diaphragm in podocytes via the cytoskeletal adapter proteins Crk1/2 and CrkL to induce lamellipodia formation in culture (George et al, J Clin Invest, 2012). In vivo in mice, Crk1/2 or CrkL deficiency protected from podocyte injury while Crk1/2 and CrkL deficiency resulted in abnormal podocyte process formation and proteinuria (George et al, Kidney International, 2014). As Nephrin activation induces lamellipodia in podocyte culture, we hypothesized that Nephrin may signal to Integrin at focal adhesions. Indeed, in podocyte culture Nephrin activation induces Integrin β activation. Preliminary data showed that this is dependent on the Rap guanine nucleotide exchange factor 1 (C3G) which is an activator of the small GTPase Rap1. Employing the Drosophila nephrocyte model, we showed that knockdown of the Nephrin ortholog Sticks-and stones (Sns) or gain-of-function results in mistargeting of Integrin (Dlugos, Picciotto et al, J Am Soc Nephrol, 2019). In the sequential grant period, we will dissect the mechanisms of Nephrin signaling to Integrin β and characterize the role of Rap1 and its activator C3G in podocyte culture and mice. We will test whether Nephrin-induced Integrin activation modulates podocyte adhesion. We already established a nephron-specific C3G knockout in mice (early podocyte knockout). Mice exhibit proteinuria and foot process effacement. We will now analyze the role of C3G in podocyte maintenance and reaction to injury. For this, we will cross available C3G flox mice with mice that express Cre recombinase driven by the Podocin promoter to generate and phenotype podocyte-specific C3G knockout mice. Additionally, we will test with podocyte-specific C3G knockout mice whether C3G plays a role during podocyte injury. RNA sequencing of prepared podocytes will uncover mechanisms of C3G signaling in vivo. Changes in Integrin activation may result in defective podocyte attachment to the glomerular basement membrane (GBM) and consecutive loss of podocytes into the urine. Podocyte loss is an important pathomechanism that results in chronic glomerular disease. Thus, molecular characterization of the Nephrin-Integrin pathway may expose potential therapeutic targets for glomerular disease.