CALCIUM AND ENDOTOXIN-INDUCED TUBULE CELL INJURY

钙和内毒素引起的肾小管细胞损伤

• 批准号: 2143986
• 负责人: Philip R. Mayeux
• 金额: $ 10.61万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143986
• 关键词: acute renal failure; adenosine triphosphate; adenylate kinase; animal tissue; calcium; calcium flux; cellular respiration; cytotoxicity; endoplasmic reticulum; endotoxins; fluorescent dye /probe; laboratory rat; membrane activity; mitochondrial membrane; platelet activating factor; renal toxin; renal tubule; thromboxanes; toxicant interaction; trypan blue; tumor necrosis factor alpha; tumor necrosis factor beta

The salient feature of both ischemic and nephrotoxic acute renal failure is renal tubular injury. Acute renal failure is a frequent and serious complication of endotoxemia and in vitro, endotoxin is cytotoxic to renal tubules. However, the biochemical events that lead ultimately to endotoxin-induced cytotoxicity to renal tubular cells have not been previously examined. My hypothesis is endotoxin-induced cytotoxicity to renal tubules is mediated through derangements in calcium homeostasis and can be modulated by the concomitant release of proposed mediators. This hypothesis is based on the following: a. There is compelling recent evidence which suggests that derangements in calcium homeostasis may lead to the progression of sublethal to lethal cell injury; b. Several in vivo studies have indicated that the effects of endotoxin may be mediated through the release of mediators including thromboxane A2 (TXA2), platelet- activating factor (PAF), and tumor necrosis factor (TNF); c. In in vitro studies endotoxin has been shown to cause release of these mediators in other cell types, and d. Some of these mediators have been shown to cause a rise in [Ca2+]i. In additions, I have obtained preliminary data demonstrating that endotoxin, lipid-A, TXA2 and PAF induce a rise in intracellular calcium [Ca2+]i in the LLC-PK1 renal tubular cell line. The specific aims are: 1. To examine the cytotoxic effects of endotoxin on rat proximal tubules and LLC-PK1 cells. The role of calcium in the cytotoxic effects of endotoxin will be evaluated by inhibiting intracellular release and extracellular influx. The direct effects of endotoxin on [Ca2+]i will be monitored using the fluorescent probe fura-2, evaluating changes in the Ca2+ uptake and release potential of the endoplasmic reticulum and mitochondrial membrane, and by monitoring changes in the activity of plasma membrane Ca2+-ATPase. The temporal relationship between changes in Ca2+ and cytotoxicity will be examined. 3. To examine the roles of TXA2, PAF and TNF in the endotoxin-induced derangement of [Ca2+]i homeostasis and cytotoxicity. Given that TXA2, PAF and TNF are proposed mediators of many of the effects of endotoxin in vivo, their direct effects on calcium homeostasis as well as their potential for mediating the effects of endotoxin will be examined. 4. To further characterize the putative receptors for endotoxin, TXA2, and PAF proximal tubules and LLC-PK1 cells using radioligand binding assays.

缺血性和肾毒性急性肾衰竭的显著特征是 肾小管损伤 急性肾衰竭是一种常见的严重的 在体外实验中，内毒素对肾脏具有细胞毒性， 小管 然而，最终导致 内毒素诱导的对肾小管细胞的细胞毒性尚未被 以前检查过。 我的假设是内毒素诱导的细胞毒性 肾小管通过钙稳态的紊乱介导， 可以通过同时释放提议的介质来调节。 这 假设基于以下内容：a.最近有令人信服的 有证据表明，钙稳态的紊乱可能导致 亚致死性至致死性细胞损伤的进展; B.几项体内 研究表明，内毒素的作用可能是通过 通过释放包括血栓素A2（TXA 2）、血小板- 活化因子（PAF）和肿瘤坏死因子（TNF）; c.体外 研究表明，内毒素可引起这些介质的释放， 其他细胞类型，和d.其中一些介质已被证明会导致 [Ca 2 +]i升高。 此外，我还获得了初步数据， 这表明内毒素、脂质-A、TXA 2和PAF诱导 LLC-PK 1肾小管细胞系中的细胞内钙[Ca 2 +]i。 的 具体目标是：1.观察内毒素对大鼠的细胞毒作用 近端小管和LLC-PK 1细胞。 钙离子在细胞毒性中的作用 通过抑制细胞内释放来评价内毒素的作用 和细胞外流入。 内毒素对[Ca ~（2+）]i的直接影响 使用荧光探针Fura-2进行监测，评估 内质网的Ca 2+摄取和释放潜力， 线粒体膜，并通过监测血浆中的活性变化， 膜Ca ~（2+）-ATPase。 Ca 2+变化之间的时间关系 并检查细胞毒性。 3.探讨血栓素A2、血小板活化因子在脑缺血中的作用 和TNF在内毒素诱导的[Ca 2 +]i稳态紊乱中的作用， 细胞毒 考虑到TXA 2、PAF和TNF被提议为许多肿瘤的介质， 内毒素在体内的影响，其对钙的直接影响 体内平衡以及它们介导 将检查内毒素。 4.为了进一步描述假定的 内毒素受体、TXA 2和PAF近端小管和LLC-PK 1细胞 使用放射性配体结合测定。