UROLITHIASIS--ROLE OF NEPHRON DYSFUNCTION/INJURY

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Our working hypothesis is that oxalate-induced injury to renal epithelial cells plays a central role in the development of calcium oxalate stone disease, with oxalate-induced injury promoting the nucleation, growth and retention of calcium oxalate crystals within the kidney. Recent studies in our laboratory support this hypothesis and indicate that oxalate-induced injury to renal tubular cells may be secondary to increased free radical production. This working hypothesis serves as the focus of the present proposal and its 3 interrelated aims. Aim 1 will determine the mechanism(s) of oxalate toxicity, defining a) the role of oxalate crystals and/or the oxalate anion in oxalate injury, b) cellular processes that could mediate oxalate actions c) relevant sites of free radical production within the cells and d) targets of free radical actions (plasma membrane, mitochondria, etc.) e) the effects of prior "sensitizing" stimuli on oxalate toxicity. Aim 2 will characterize oxalate-induced changes in gene expression in renal epithelial cells, defining a) the pattern of oxalate induced changes in gene expression, b) the cellular events leading to the observed changes in gene expression, and c) the therapeutic benefits of genetic manipulations which increase free radical "buffering" or facilitate oxalate degradation. Aim 3 will determine whether hyperoxaluria in experimental animals will evoke changes in renal cell function which are comparable to those observed in response to hyperoxaluria in cultured renal epithelial cells. The proposed in vitro studies will utilize LLC-PK1 cells for the majority of the studies confirming key findings in distal tubular cells (MDCK cells) and in human renal epithelial cells (RPTEC cells). These studies will define oxalate actions in vitro and evaluate the effectiveness of various pharmacological and genetic manipulations to limit oxalate toxicity. Results from these in vitro studies will inform and guide the design of in vivo studies to assess the consequences of hyperoxaluria in experimental animals and to define the role of oxidant stress in the development/progression of stone disease. This combination of in vitro and in vivo studies should provide important insights as to processes involved in calcium oxalate stone disease and suggest possible therapeutic strategies for management of this disease.