UROLITHIASIS--ROLE OF NEPHRON DYSFUNCTION/INJURY

我们的工作假设是，草酸盐诱导的肾上皮细胞损伤在草酸钙结石病的发展中起着核心作用，草酸盐诱导的损伤促进了肾脏内草酸钙晶体的成核、生长和保留。我们实验室最近的研究支持这一假设，并表明草酸诱导的肾小管细胞损伤可能继发于自由基产生增加。这一工作假设是本建议及其3个相互关联的目标的重点。目的1将确定草酸毒性的机制，定义a)草酸晶体和/或草酸阴离子在草酸损伤中的作用，b)可以介导草酸作用的细胞过程c)细胞内自由基产生的相关位点，d)自由基作用的目标（质膜，线粒体等）e)先前“致敏”刺激对草酸毒性的影响。目的2将描述草酸盐诱导的肾上皮细胞基因表达变化，定义a)草酸盐诱导的基因表达变化模式，b)导致观察到的基因表达变化的细胞事件，以及c)增加自由基“缓冲”或促进草酸盐降解的基因操作的治疗益处。目的3将确定实验动物的高草酸尿是否会引起肾细胞功能的变化，这与在培养的肾上皮细胞中观察到的高草酸尿反应相似。拟议的体外研究将利用LLC-PK1细胞进行大部分研究，确认远端小管细胞（MDCK细胞）和人肾上皮细胞（RPTEC细胞）的关键发现。这些研究将确定草酸盐在体外的作用，并评估各种药理学和遗传操作的有效性，以限制草酸盐的毒性。这些体外研究的结果将为体内研究的设计提供信息和指导，以评估实验动物高血氧症的后果，并确定氧化应激在结石疾病发生/进展中的作用。这种体外和体内研究的结合应该为草酸钙结石疾病的过程提供重要的见解，并为这种疾病的管理提供可能的治疗策略。

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.