The research project proposed herein is designed to determine the mechanisms of cellular reactions to injury of renal proximal tubular epithelial cells (PTE). Understanding such reactions is fundamental to the elucidation of mechanisms of renal disease and of events following renal transplantation including ex vivo perfusion. In addition, the proximal tubule, as a highly polarized transporting epithelium, can also serve as a relevant model for cell injury, cell recovery, cell regeneration, and cell repair in other organs, tissues, and cells. In previous grant periods, this project has characterized many of the subcellular responses to injury of the PTE, investigated the morphologic and functional changes that are produced, and studied the mechanisms involved. Progress on this project has been facilitated by recent technologic developments that permit the detailed characterization of intracellular ion concentrations, including [Ca2+]i, and [H+]i, as well as the correlation of these analytes with other structural and functional changes in Ca binding proteins, gene expression, cytoskeletal structure and function, and cell viability. From its inception, this project has utilized in vitro models of PTE injury to enable precise in vitro studies to be correlated with in vivo observations in animals and humans. This theme will be continued and expanded in this proposal, using new technologies enabling simultaneous measurements of morphology, ion content, gene expression and cellular function to all be performed in the same living cell. For these studies, two highly relevant in vitro injury models for the PTE will be characterized: (1) ischemia (KCN/anoxia + IAA); and (2) oxidant stress to simulate in vivo ischemia by generation of superoxide using the xanthine-xanthine oxidase reaction in vitro). Both models will be characterized and modified by a series of interventions designed to elucidate the critical pathways of both injury and recovery. The use of sensitive computer-assisted microscopy, the capability of micro-injecting cells with one or more of a large number of unique fluorescent probes and caged compounds, and in situ methods for studying gene expression are permitting extraordinary advances in characterizing the relationship between Ca, pH, and a variety of cellular signalling processes including cytoskeletal changes in living cells, altered gene expression involved in both cell death and cell recovery, and direct manipulation of these events in living cells through the use of photohydrolysable """"""""caged"""""""" compounds. In summary, when these studies are completed, they will enable progress toward increased understanding, prevention, and treatment of renal disease in humans.
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