Injury to the tubule compartment is a prominent feature of ischemic and related forms of acute renal failure. For functional recovery of the tissue, sufficient numbers of tubule cells must survive, resume adequate metabolism, repair structural damage, and, potentially, undergo proliferation to replace lost cells. The investigations supported by this grant during the past 12 years have provided insight into some of the major determinants of these events and have led us to hypothesize that: a) The barrier property of the plasma membrane that most fundamentally permits maintenance of cell viability during ATP depletion and related acute injury states is dependent on the presence of glycine; b) Protein dephosphorylation/rephosphorylation determines the extent and reversibility of sublethal structural alterations in glycine- protected cells and, as a consequence, their capacity for functional recovery. Focal adhesion disassembly/reassembly provides an approachable and highly relevant instance of this behavior; and c) Progressive impairment of energetic function in glycine- protected cells limits their ability to engage in ATP-dependent repair functions and to tolerate glycine withdrawal. This impairment is secondary to development of the mitochondrial permeability transition, occurs in an all or none fashion in individual cells, and can he improved by pharmacological and other approaches. We will test and further investigate these hypotheses in four Specific Aims: l) Optimize a novel approach using measurements of protein tyrosine phosphorylation to assess the energetic state of individual tubule cells and apply it to clarify the basis for the progressive energetic defect that develops in populations of glycine-protected, hypoxic, isolated tubules and the behavior of tubules during reperfusion after ischemia in vivo; 2) Directly visualize development of the mitochondrial permeability transition in the tubule cells, define its relationship to maintenance of the mitochondrial membrane potential, and assess promising new maneuvers to alleviate the energetic deficit; 3) Further define the nature and mechanisms of the prominent alterations of integrins and focal adhesion proteins that occur during hypoxia/reoxygenation of the isolated tubules and ischemia/early reperfusion in vivo and the relationships of these changes to the cellular energetic state; 4) Test the involvement of major factors potentially mediating the observed ATP level. dependent, disassembly/reassembly of focal adhesions in the glycine-protected, isolated tubules.
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