The chloride intracellular channel 5A (CLIC5A) protein, one of two isoforms produced by the CLIC5 gene, was isolated originally as part of a cytoskeletal protein complex containing ezrin from placental microvilli. Whether CLIC5A functions as a bona fide ion channel is controversial. We reported previously that a CLIC5 transcript is enriched approximately 800-fold in human renal glomeruli relative to most other tissues. Therefore, this study sought to explore CLIC5 expression and function in glomeruli. RT-PCR and Western blots show that CLIC5A is the predominant CLIC5 isoform expressed in glomeruli. Confocal immunofluorescence and immunogold electron microscopy reveal high levels of CLIC5A protein in glomerular endothelial cells and podocytes. In podocytes, CLIC5A localizes to the apical plasma membrane of foot processes, similar to the known distribution of podocalyxin and ezrin. Ezrin and podocalyxin colocalize with CLIC5A in glomeruli, and podocalyxin coimmunoprecipitates with CLIC5A from glomerular lysates. In glomeruli of jitterbug (jbg/jbg) mice, which lack the CLIC5A protein, ezrin and phospho-ERM levels in podocytes are markedly lower than in wild-type mice. Transmission electron microscopy reveals patchy broadening and effacement of podocyte foot processes as well as vacuolization of glomerular endothelial cells. These ultrastructural changes are associated with microalbuminuria at baseline and increased susceptibility to adriamycin-induced glomerular injury compared with wild-type mice. Together, the data suggest that CLIC5A is required for the development and/or maintenance of the proper glomerular endothelial cell and podocyte architecture. We postulate that the interaction between podocalyxin and subjacent filamentous actin, which requires ezrin, is compromised in podocytes of CLIC5A-deficient mice, leading to dysfunction under unfavorable genetic or environmental conditions.

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Chronic progressive renal fibrosis leads to end-stage renal failure many patients with chronic kidney disease (CKD). Loss of the rich peritubular capillary network is a prominent feature, and seems independent of the specific underlying disease. The mechanisms that contribute to peritubular capillary regression include the loss of glomerular perfusion, as flow-dependent shear forces are required to provide the survival signal for endothelial cells. Also, reduced endothelial cell survival signals from sclerotic glomeruli and atrophic or injured tubule epithelial cells contribute to peritubular capillary regression. In response to direct tubular epithelial cell injury, and the inflammatory reaction that ensues, capillary pericytes dissociate from their blood vessels, also reducing endothelial cell survival. In addition, direct inflammatory injury of capillary endothelial cells, for instance in chronic allograft nephropathy, also contributes to capillary dropout. Chronic tissue hypoxia, which ensues from the rarefaction of the peritubular capillary network, can generate both an angiogenic and a fibrogenic response. However, in CKD, the balance is strongly tipped toward fibrogenesis. Understanding the underlying mechanisms for failed angiogenesis in CKD and harnessing endothelial-specific survival and pro-angiogenic mechanisms for therapy should be our goal if we are to reduce the disease burden from CKD.

Obeidat M, Li L, Ballermann BJ. TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells. Am J Physiol Renal Physiol 307: F623-F633, 2014. First published July 9, 2014 doi:10.1152/ajprenal.00070.2014.-The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro angiogenesis. To deplete TIMAP, the EC were transfected with TIMAP-specific or nonspecific small interfering (si) RNA. The rate of electrical impedance development across subconfluent EC monolayers, a measure of the time-dependent increase in EC number, was 93 Ϯ 2% lower in TIMAP-depleted than in control EC. This effect on cell proliferation was associated with reduced DNA synthesis and increased apoptosis: TIMAP silencing reduced 5-ethynyl-2=-deoxyuridine incorporation by 38 Ϯ 2% during the exponential phase of EC proliferation, and cleaved caspase 3 as well as caspase 3 activity increased in TIMAPdepleted relative to control cells. Furthermore, TIMAP depletion inhibited the formation of angiogenic sprouts by glomerular EC in three-dimensional culture. TIMAP depletion strongly diminished growth factor-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation, suggesting a specific effect on the PI3K/ Akt/PTEN pathway. Endogenous TIMAP and PTEN colocalized in EC and coimmunoprecipitated from EC lysates. The inhibitory PTEN phosphorylation on S370 was significantly reduced in TIMAP-depleted compared with control EC, while phosphorylation of PTEN on the S380/T382/T383 cluster remained unchanged. Finally, the PTEN inhibitor bpV(phen) fully reversed the suppressive effect of TIMAP depletion on Akt phosphorylation. The data indicate that in growing EC, TIMAP is necessary for Akt-dependent EC proliferation, survival, and angiogenic sprout formation and that this effect of TIMAP is mediated by inhibition of the tumor suppressor PTEN. endothelial cells; PTEN; protein phosphatase 1-regulatory subunit; PPP1R16B; apoptosis; clectric cell-substrate impedance sensing MECHANISMS THAT REGULATE ANGIOGENESIS have received massive attention since Folkman (15) first advanced the theory that tumor growth depends on new blood vessel formation. Angiogenesis involves the sprouting and elongation of new blood vessels from preexisting vessels followed by stabilization and vessel specification (28). This process is crucial for normal blood vessel formation and patterning during embryonic development, for postembryonic blood vessel repair (5), and remodeling in response to hypoxia, in the female reproductive cycle, and for the establishment of the placental vasculature. Pathological retinal and tumor vascularization also depend on angiogenesis. While endothelial cells (EC) at the tip of the angiogenic sprout determine the direction and pattern of new blood vessel growth, elongation of the new vessel stalk requires EC proliferatio...

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