ROLE OF CALCIUM IN ENDOTOXIN-INDUCED TUBULE CELL INJURY

缺血性和肾毒性急性肾功能衰竭的显著特征是 肾小管损伤。急性肾功能衰竭是一种常见而严重的 内毒素血症的并发症及体外实验中，内毒素对肾脏的细胞毒作用 小管。然而，最终导致 内毒素对肾小管细胞的细胞毒作用尚未见报道 之前已检查过。我的假设是内毒素诱导的细胞毒性 肾小管通过钙稳态和钙平衡紊乱来调节 可以通过随之而来的拟议调解人的释放来进行调整。这 假设的基础如下：a.最近有令人信服的 有证据表明钙稳态失调可能会导致 对亚致死到致死细胞损伤的进展；b.体内的几个 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(TXA2)、血小板- 激活因子(PAF)和肿瘤坏死因子(TNF)；c.体外 研究表明，内毒素可导致体内这些介质的释放。 其他类型的细胞，以及d.这些介体中的一些已被证明能引起 [Ca~(2+)]的升高I.此外，我还获得了初步数据 证明内毒素、脂质-A、TXA2和PAF诱导血管内皮细胞 LLC-PK1肾小管上皮细胞内钙离子浓度的变化这个 具体目的是：1.检测内毒素对大鼠的细胞毒性作用 近端小管和LLC-PK1细胞。钙在细胞毒性中的作用 内毒素的作用将通过抑制细胞内释放来评估 以及细胞外的内流。内毒素对细胞内钙离子的直接影响 使用荧光探针Fura-2进行监测，评估 内质网钙离子摄取和释放潜力的研究 线粒体膜，并通过监测血浆活性的变化 膜钙-三磷酸腺苷酶。钙离子变化之间的时间关系 并将检测其细胞毒性。3.研究TXA2、PAF的作用 和肿瘤坏死因子在内毒素诱导的细胞内钙稳态和细胞外基质代谢紊乱中的作用 细胞毒性。鉴于TXA2、PAF和TNF被认为是许多 内毒素在体内的作用及其对钙的直接影响 动态平衡以及它们在调节血管紧张素转换酶 将进行内毒素检查。4.进一步描述推定的 近端小管和LLC-PK1细胞的内毒素、TXA2和PAF受体 使用放射性配基结合分析。

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.