Endothelial Injury in Endotoxin-Induced Acute Renal Failure

描述（由申请人提供）：急性肾功能衰竭影响约5%的住院患者，导致高发病率和死亡率，通常由败血症引起。初步数据表明，LPS诱导的ARF，脓毒性肾功能不全的模型，依赖于TNF对其受体TNFR 1在肾脏中的作用，并与肾内皮细胞凋亡，炎症，血管渗漏在体内和体外。具体目标1将使用小鼠肾内皮细胞（EC）的原代培养物，通过使用药理学抑制剂和siRNA方法来确定TNF诱导的内皮屏障功能障碍的机制，并将研究对TNF引起的细胞骨架破坏的影响。具体目标2将确定这种内皮屏障功能障碍在LPS诱导的急性肾功能衰竭（ARF）过程中的作用，使用过表达和缺乏肌球蛋白轻链激酶（MLCK）的内皮亚型的转基因小鼠。还将给予维持内皮屏障完整性的鞘氨醇-1-磷酸类似物，以确定对LPS诱导的ARF的影响。肾交叉移植将被用作一种工具，以确定肾脏相对于全身性内皮损伤的贡献，长期植入的血压和肾血流探针装置将允许实时测量肾内血流动力学，以响应内毒素血症背景下的上述操作。具体目标3将检验TNF诱导的半胱天冬酶激活放大肾内皮炎症的假设，并将定义这种串扰发生的机制，这可能涉及MLCK和其他细胞骨架事件。具体目标4将使用内皮特异性敲除小鼠缺乏半胱天冬酶-8，和内皮特异性转基因小鼠与非功能性NF-？B，区分内皮细胞凋亡和炎症对LPS诱导的ARF结果的各自贡献。总之，该项目将开发工具，使人们能够更好地了解内皮损伤的重要性和机制及其在脓毒性ARF中的作用。 公共卫生相关性：急性肾功能衰竭（ARF）是住院患者常见的并发症，导致高发病率和死亡率，通常由压倒性感染引起。该项目测试了肾内皮细胞（血管内皮细胞）的损伤和炎症是ARF的重要原因的假设。在这个提议中，使用具有改变的内皮功能的菌株的动物实验和细胞培养实验将提供对ARF所涉及的机制的更多理解，这最终将导致针对该综合征的更合理和靶向的治疗。

DESCRIPTION (provided by applicant): Acute renal failure affects approximately 5% of all hospitalized patients, causing great morbidity and mortality, and commonly results from sepsis. Preliminary data indicate that LPS-induced ARF, a model of septic renal dysfunction, depends on the action of TNF on its receptor TNFR1 in the kidney, and is associated with renal endothelial apoptosis, inflammation, and vascular leak both in vivo and in vitro. Specific Aim 1 will use primary culture of mouse renal endothelial cells (ECs) to determine the mechanisms of TNF-induced endothelial barrier dysfunction, via use of pharmacologic inhibitors and an siRNA approach, and will study the effect on cytoskeletal disruption caused by TNF. Specific Aim 2 will determine the role of this endothelial barrier dysfunction in the course of LPS-induced acute renal failure (ARF) using transgenic mice overexpressing and deficient in the endothelial isoform of myosin light chain kinase (MLCK). Sphingosine-1-phosphate analogues, which maintain endothelial barrier integrity, will also be administered to determine the effect on LPS-induced ARF. Renal cross-transplantation will be used as a tool to define the contribution of renal as opposed to systemic endothelial injury, and chronically implanted blood pressure and renal flowprobe devices will allow real-time measurement of intrarenal hemodynamics in response to the above manipulations in the setting of endotoxemia. Specific Aim 3 will test the hypothesis that TNF-induced caspase activation amplifies renal endothelial inflammation, and will define mechanisms through which this cross-talk occurs, which may involve MLCK and other cytoskeletal events. Specific Aim 4 will use endothelial-specific knockout mice lacking caspase-8, and endothelial-specific transgenic mice with nonfunctional NF-?B, to distinguish the respective contributions of endothelial apoptosis and inflammation to the outcome of LPS-induced ARF. Together, this project will develop tools which should allow greater understanding of the importance and mechanism of endothelial injury and its role in septic ARF. PUBLIC HEALTH RELEVANCE: Acute kidney failure (ARF) is a common complication in hospitalized patients that leads to great morbidity and mortality, and commonly results from overwhelming infection. This project tests the hypothesis that injury and inflammation to kidney endothelial cells, which line the blood vessels, are an important cause of ARF. In this proposal, animal experiments using strains with altered endothelial functions and cell culture experiments will provide greater understanding of the mechanisms involved in ARF, which should eventually lead to more rational and targeted therapies for this syndrome.