Epithelial cell structure and function depend on an intact cytoskeleton. It is known that alterations in the actin cytoskeleton during periods of ischemic injury and recovery have important consequences for cell and organ function. This is especially true of the renal proximal tubule epithelia which is the predominant site of injury in human ischemic acute renal failure. One of the dramatic consequences of these alterations is the loss of cell adhesion to the underlying tissue matrix. Myosin II is felt to play a key role in organizing the actin cyctyskeleton in areas of the cell critical to cell-matrix adhesion and thus cell and organ function. The regulatory processes involved in these alterations have not been well characterized; however, the Rho family GTPases have been shown to play a role in regulating distinct aspects of cytoskeletal rearrangement involved in normal cellular function. The overall hypothesis of this study is that myosin II activation is an important distal effector of the Rho regulatory cascade and plays a critical role in controlling the actin cytoskeleton during ischemia and recovery. We propose to evaluate the level of myosin activation during ATP depletion and recovery in a reversible cell culture model of ATP depletion that recapitulates the changes observed in ischemic acute renal failure. We plan to relate changes in myosin activation during ATP depletion and recovery with the number of actin stress fibers which are involved in focal adhesion complexes and cell matrix adhesion. We also propose to use recombinant forms of Rho, Rho- kinase, and MLCK which are dominantly active and negative to begin to determine the regulatory mechanisms involved in control of myosin II in epithelial cells during ATP depletion and recovery. Taken together these studies should provide new and important information about the mechanisms regulating pathologic rearrangements of the cytoskeleton in epithelial cells during ischemia.
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- National Institutes of Health