Rho GTPases and Actin Cytoskeletal Function in Renal Ischemia

缺血性急性肾功能衰竭的持续存在是住院患者发病率和死亡率的主要原因，而这些患者的预后相对缺乏改善，这对我们更好地理解这种损伤背后的细胞机制提出了挑战。通常支持近端小管上皮细胞结构和功能的肌动蛋白细胞骨架组织的破坏被认为是损伤的早期和关键方面，对肾功能和细胞存活有直接影响。在该奖项的前几年，我们已经证明Rho gtpase在介导缺血或使用ATP耗尽的细胞培养模型中观察到的细胞骨架改变中起关键作用。此外，我们还发现RhoA、Rac1和Cdc42的活性对ATP的耗尽很敏感，但每种蛋白受ATP耗尽的影响是不同的。我们提出，Rho GTPase活性的改变和随之而来的细胞骨架破坏是通过AMPK-TSC1/2通路信号传导导致II型（雷帕霉素不敏感）mTOR复合物2失活的结果，该复合物最近被证明通过Rho GTPase调节细胞骨架。我们建议通过细胞培养和肾缺血动物模型的补充研究来测试和阐明我们提出的机制。我们提出了四个具体目标来解决我们的中心假设：1)研究体外ATP消耗和体内缺血对AMPK的激活；2)在体外和体内研究TSC1/2和Rheb GTPase信号通路对Rho GTPase活性和细胞骨架组织的影响；3)确定AMPK活化对II型mTOR复合物的影响；4)确定AMPK和mTOR信号改变对缺血性肾损伤动物模型中肌动蛋白细胞骨架组织和近端小管细胞功能的影响。这些研究将确定将能量消耗与细胞骨架改变联系起来的关键机制，并为确定新疗法的靶点提供机会

The persistence of ischemic acute renal failure as a major cause of morbidity and mortality in hospitalized patients and the relative lack of improvement in outcomes for these patients challenges us to better understand the cellular mechanisms underlying this injury. Disruption of the actin cytoskeletal organization that normally underpins the structure and function of proximal tubule epithelial cells is well-established as an early and critical aspect of injury with direct consequences for renal function and cell survival. In the previous years of this award we have shown that Rho GTPases play critical roles that may mediate the cytoskeletal alterations observed with ischemia or in a cell culture model that uses ATP depletion. Moreover, we showed that activity of RhoA, Rac1 and Cdc42 is sensitive to ATP depletion, but that each protein is differentially affected by depletion. We propose that alterations in Rho GTPase activity and consequent cytoskeletal disruption are the result of signaling through the AMPK-TSC1/2 pathway resulting in inactivation of the type II (rapamycin insensitive) mTOR complex 2, which has recently been shown to regulate the cytoskeleton through Rho GTPases. We propose studies to test and elucidate our proposed mechanism using complementary studies in cell culture and in animal models of renal ischemia. We propose four specific aims to address our central hypothesis: 1) Investigate the activation of AMPK by ATP depletion in vitro and ischemia in vivo; 2) Investigate the role of TSC1/2 and Rheb GTPase signaling on Rho GTPAse activity and cytoskeletal organization in vitro and in vivo; 3) Determine the effect of AMPK activation on the type II mTOR complex; 4) Determine the effect of altered AMPK and mTOR signaling on actin cytoskeletal organization and proximal tubule cell function in animal models of ischemic renal injury. These studies will determine the key mechanism linking energy depletion to cytoskeletal alterations, and affords an opportunity to identify targets for novel therapies Ischemia (loss of blood flow) is a major cause of acute kidney failure. Currently there is a poor understanding of the way that kidney cells respond to ischemia, which makes it difficult to design therapies or drugs. The studies proposed will test a possible mechanism leading to cell injury that could be a target for drug therapy.