Monitoring of Rho GTPase Activity in Podocytes.
The glomerulus is the filtration unit of the kidney, where the first step of urine formation occurs. Podocytes are characterized by their actin-based projections called foot processes. Podocyte foot processes play a critical role in the permselective filtration barrier, along with fenestrated endothelial cells and the glomerular basement membrane. The Rho family of small GTPases (Rho GTPases) is the master regulators of the actin cytoskeleton and functions as molecular switches. Recent studies have shown that disruption of Rho GTPase activity and subsequent changes in foot process structure cause proteinuria. Here, we describe an effector pull-down assay using GST-fusion proteins to monitor the activity of RhoA, Rac1, and Cdc42, which are prototypical Rho GTPases in podocytes.