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Abstract: The kidneys regulate total body fluid volume, acid-base status, and electrolyte composition through the filtration of large amounts of plasma by glomerular capillaries, followed by selective reabsorption of solutes and water by renal tubules. To sustain filtration, the hydraulic pressure within glomerular capillaries is much higher than in other systemic capillaries. While the glomerular capillary wall is extremely water permeable, it is nearly impermeable to larger plasma proteins. This “permselectivity” is attributed to the highly specialized nature of the glomerular capillary wall, which consists of three layers: glomerular endothelial cells, basement membrane and podocytes. The podocytes are specialized epithelial cells that wrap primary and secondary actin-base projections around the exterior of the glomerular capillary loops. They counteract the high intracapillary pressure, and they contribute significantly to the size-selective retention of plasma proteins in the capillary lumen. This thesis explores the role of CLIC5A (chloride intracellular channel 5A) in regulating the structure and function of podocytes. Our laboratory previously reported that CLIC5A mRNA is nearly 800-fold more abundant in renal glomeruli than in other tissues. This level of enrichment suggested a specialized function of CLIC5A in glomeruli. CLIC5A belongs to a family of highly conserved proteins (CLICs) that associate reversibly with lipid bilayers. CLICs are often found in in the same location as ERM (ezrin, radixin, moesin) proteins, and deletion of some CLICs can functionally mimic the deletion of specific ERM proteins. ERM proteins connect the cytoplasmic domain of integral membrane proteins to cortical actin, and consequently shape and control the cell cortex. Even though a functional interaction between CLICs and ERM proteins had been postulated, a mechanism had not been described. Studies underlying this thesis revealed that ectopic expression of CLIC5A in COS-7 and HeLa cells, which do not express CLIC5A at baseline, significantly increased ezrin and moesin phosphorylation and their association with the actin cytoskeleton. CLIC5A expression also induced the formation of apical membrane projections, accompanied by increased actin polymerization. Since ERM protein phosphorylation indicates ERM activation, I was able to conclude that CLIC5A expression results in ERM protein activation and consequent cell-surface remodeling. ERM proteins can exist in an inactive, auto-inhibited form due to interactions between their N- and C-termini. PI(4,5)P2 docking, which is a required first step in ERM activation, produces a conformational change that allows binding of the ERM N-terminus to integral membrane proteins and the C-terminus to F-actin. PI(4,5)P2 docking also exposes a highly conserved Thr residue in the ERM C-terminus that is then phosphorylated, stabilizing the activated form. I observed that ectopic expression of CLIC5A increased PI(4,5)P2 production in clusters at the apical membrane and that silencing of endogenous PI4P5Kα inhibited CLIC5A-dependent ERM protein activation. HA-PI4P5Kα and -β as well as HA-PI5P4Kα and -β were pulled from cell lysates by GST-CLIC5A, indicating that CLIC5A and PI(4,5)P2 generating kinases can exist in the same complex. In podocytes, in vivo, CLIC5A was localized to the apical domain of podocyte foot processes, and it co-localized strongly with ezrin, podocalyxin and NHERF2 in glomeruli. Ezrin was previously shown to couple podocalyxin to cortical F-actin, in part through the intermediary NHERF2. In CLIC5 deficient mice, ezrin phosphorylation and ezrin abundance in podocytes were profoundly reduced, NHERF2 was uncoupled from the cytoskeleton, and podocalyxin abundance and electrophoretic mobility were altered. Furthermore, ultrastructural evaluation showed that podocyte foot processes were much broader and fewer in number in CLIC5 deficient mice when compared to wild-time mice. CLIC5 deficient mice also had microalbuminuria at baseline and they were more susceptible to Adriamycin-induced glomerular injury. Thus, CLIC5A serves to activate ezrin at the apical domain of glomerular podocyte foot processes. Studies in COS-7 cells indicate that this CLIC5A effect is due to the localized production of PI(4,5)P2 and involves activation of a PI4P5- or a PI5P4 kinase. In the absence of CLIC5A the podocyte Podocalyxin/NHERF2/Ezrin complex dissociates from cortical actin, associated with a reduction of the number of foot processes, microalbuminuria, and enhanced susceptibility to glomerular injury.