Ischemic acute renal failure is a major cause of morbidity and mortality. Cells of the kidney proximal tubule are particularly vulnerable to ischemic injury, and this injury is characterized by breakdown of normal cellular architecture with consequent loss of polarized distribution of surface proteins. This disruption of cell structure and function correlates with rapid and reversible redistribution of components of the actin cytoskeleton. We propose that this abnormal distribution of actin filaments results from inactivation of rho family GTPases. Rho proteins are the branch of the ras superfamily of small GTPases that regulate cell morphology and motility, and their role in controlling actin function has been extensively studied in fibroblasts, but is less well characterized in epithelial cells. We propose three specific aims to test the involvement of rho proteins and their effectors in actin reorganization in a cell culture model of ischemia, using ATP depletion induced by substrate depletion and antimycin A treatment. We will use microinjection and transfection to introduce dominant active and dominant negative mutants of Rho, Rac and Cdc42 into LLC-PK cells to test the effect of blocking inactivation or activation of pathways through these proteins on the actin cytoskeletal response to ATP depletion or recovery. We will measure the activation state of rho family GTPases under conditions of ATP depletion and recovery by measuring the ratio of GTP:GDP bound to the GTPase, and analysing cellular localization of the GTPase using GFP-tagged proteins. Cytoplasmic isoforms of myosin II play an important role in function of the actin cytoskeleton in polarized epithelia, and are an important target of Rho regulation. We will examine myosin localization and light chain phosphorylation in control cells and in response to ATP depletion, and ezrin regulation, and we will determine the effect of regulators of myosin activity, Rho-kinase and myosin light chain kinase (MLCK), using dominant negative and constitutively active mutants. The data derived from the proposed studies will provide a basis for understanding the cellular mechanisms that underlie the abnormal response of the cytoskeleton to ischemia, and a mechanism for the known beneficial effects of growth factors. This will provide a basis for the development of improved therapeutic approaches to the management of human ischemic acute renal failure.
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