Podocyte GIT1 and GIT2 ArfGAPs cooperatively sustain the ARHGEF7-Cdc42 axis and glomerular permselectivity

Podocytes are specialized glomerular epithelial cells characterized by actin-rich projections known as foot processes, which are essential for maintaining the filtration barrier. The Rho-family small GTPases Rac1 and Cdc42 are key regulators of actin cytoskeletal dynamics in podocytes, influencing cell adhesion, polarity and survival. However, the regulatory mechanisms of Rho GTPase activities in foot processes remain poorly understood. We previously identified GIT ArfGAP2 (GIT2) as a protective protein in podocytes that suppresses Rac1 activity at focal adhesions. However, the role of its paralog, GIT ArfGAP1 (GIT1), is unknown.

To study this, podocyte-specific GIT1 deficient and GIT1/GIT2 double deficient mice were generated and assessed for glomerular phenotypes. Cultured podocytes with GIT1/GIT2 single or double knockdown were established and characterized.

While podocyte-specific deletion of GIT1 in mice caused no overt phenotype and only mildly aggravated foot process effacement after injury, combined deletion of GIT1 and GIT2 resulted in severe foot process effacement, late-onset proteinuria, podocyte detachment, and focal segmental glomerulosclerosis in adulthood. In cultured podocytes, GIT2 knockdown or GIT1/GIT2 double knockdown, but not GIT1 knockdown alone, led to increased motility and accelerated focal adhesion turnover. Notably, motility of double knockdown podocytes lacked directionality and was accompanied by a significant reduction of Cdc42 activity and impaired nuclear translocation of Yes-associated protein (YAP). Mechanistically, the loss of Cdc42 activity was linked to enhanced degradation of the Cdc42-activating protein ARHGEF7, which was rescued by knockdown of the E3 ubiquitin ligase Cbl proto-oncogene B (CBL-b). Furthermore, in double knockdown podocytes, ARHGEF7 was not properly localized in focal adhesions, which was rescued by re-expressing either GIT1 or GIT2.

Our findings demonstrate that GIT1 and GIT2 cooperatively maintain the ARHGEF7-Cdc42 signaling axis, highlighting a previously unrecognized mechanism regulating Rho GTPase activity and cytoskeletal organization in podocytes.