Endothelial Injury in Endotoxin-Induced Acute Renal Failure

描述(申请人提供)：急性肾功能衰竭影响约5%的住院患者，导致极高的发病率和死亡率，通常由脓毒症引起。初步研究表明，内毒素诱导的ARF是感染性肾功能障碍的一种模型，依赖于肿瘤坏死因子对其受体TNFR1的作用，并与体内和体外的肾内皮细胞凋亡、炎症和血管渗漏有关。具体目标1将使用原代培养的小鼠肾内皮细胞，通过使用药物抑制剂和siRNA方法来确定肿瘤坏死因子诱导的内皮屏障功能障碍的机制，并将研究对肿瘤坏死因子引起的细胞骨架破坏的影响。特定目的2将利用过度表达和缺乏肌球蛋白轻链激酶(MLCK)内皮异构体的转基因小鼠，确定这种内皮屏障功能障碍在脂多糖诱导的急性肾功能衰竭(ARF)过程中的作用。维持内皮屏障完整性的鞘氨醇-1-磷酸类似物也将被用来确定对内毒素诱导的ARF的影响。肾交叉移植将被用作一种工具来确定肾脏的贡献，而不是全身内皮损伤，长期植入的血压和肾脏血流探头设备将允许实时测量肾内血流动力学，以响应内毒素血症的上述操作。具体目标3将测试假设，即肿瘤坏死因子诱导的caspase激活放大肾内皮细胞炎症，并将确定这种串扰发生的机制，这可能涉及MLCK和其他细胞骨架事件。特异性目标4将利用缺失caspase-8的内皮特异性基因敲除小鼠和含有非功能性核因子-βB的内皮特异性转基因小鼠，来区分内皮细胞凋亡和炎症在脂多糖诱导的ARF转归中的各自作用。该项目将共同开发工具，以便更好地了解内皮损伤的重要性和机制，以及它在感染性ARF中的作用。

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.