Ischemic renal tissue damage is a significant factor in the mortality and morbidity of patients suffering from numerous renal disorders. A primary consequence of ischemic injury is the loss of epithelial cell polarity through large-scale disruption of the actin cytoskeleton and cell-cell junctional complexes (adherens junctions and tight junctions). Junctional complexes are necessary for maintenance of polarized ion transport systems, receptors and enzyme distributions required for proper kidney function. ATP-depletion serves as an in vitro model for renal ischemia. Here we propose to analyze mechanisms that regulate tight junctions during ATP-depletion, and determine how these regulator mechanisms may protect cells from damage or accelerate recovery. Experiments will examine how adherens junctions (cadherin-mediated cell-cell adhesion) regulate tight junction assembly and function. We hypothesize that ATP- depletion causes tight junction disassembly as a consequence of adherens junction disassembly. Cadherin cell adhesion molecule junction during ischemia will be examined by manipulating cadherin junction (expressing mutant cadherins) in epithelial cells, and effects on tight junction assembly and on cell polarity will be assayed. Signaling processes leading through the adherens junction may also be disrupted during ATP-depletion. A major hypothesis to be tested in this project is that Rho-family GTPases functions in epithelial cells are inhibited during renal ischemia leading to disruption of junctional complexes. Rho-family GTPases (Rho, Rac and Cdc42) are members of the Ras gene superfamily, and have been shown to regulate actin cytoskeleton assembly. Preliminary evidence suggests that Rho-family GTPases control cell-cell junctional complex assembly in epithelial cells. Activation of these signaling systems may also protect cells from ischemic injury. Our studies will provide new and fundamental insight into critical regulatory mechanisms that are disrupted during ischemia.
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