A new monoclonal antibody (MAb) was raised against human glomerular components to investigate the possible existence of glomerular antigens reflecting cellular adaptation in glomerulonephritis. The ...MAb recognized a podocyte antigen as well as those expressed on renal arterial endothelial cells and smooth muscle cells by indirect immunofluorescence. An additional, but weaker immunoreaction was found in epithelial cells of the Bowman's capsule. This MAb recognized a 30-kD protein on western blotting of glomerular extracts under non-reducing and reducing conditions. Immunoperoxidase electron microscopy revealed that this antigen is present within the cytoplasm, but not on the cell membrane of the podocytes. Moreover, the antigen was found to be reduced in the glomeruli of patients with minimal change glomerulonephritis. These results suggest that the 30-kD protein is a novel protein, which we hypothesize is involved in maintenance of the structural and functional integrity of the podocytes. In addition, reduced expression of the 30-kD protein in the podocytes may be related to the increasing proteinuria in minimal change glomerulonephritis.
The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin‐driven cell adhesions and processes is connected to podocyte dysfunction, ...proteinuria, and kidney diseases. FilGAP, a GTPase‐activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin‐based protein complex closely associated with stress fibers, that mediates cell‐extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process‐like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP‐depleted podocyte cells was rescued by inhibition of Rac1 or P21‐activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell‐ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.
The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. FilGAP, a GTPase‐activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis (FSGS). In this study, we addressed the function of FilGAP in useful in vitro podocyte models and found that the down‐regulation of Rac1/PAK1 signaling by FilGAP is responsible for cell‐extracellular matrix (ECM) adhesion and process formation of podocytes.
