Mechanisms of Ischemic Kidney Injury and Repair

描述（由申请人提供）：拟议研究的总体目标仍然是了解缺血性肾细胞损伤和修复的机制，长期目标是建立可用于预防和治疗人类急性肾损伤（阿基）的疗法。 在最后的支持期，我们已经确定存活的近端小管细胞（PTC）负责替换损伤诱导的损失的上皮细胞，没有证据表明专门的祖细胞/干细胞群替换了PTC。在适应不良的促纤维化修复中，近端小管不直接转化为间质成纤维细胞，而是呈现与细胞周期G2/M停滞相关的促炎分泌表型。此外，使用表达人白喉毒素受体的转基因动物对PT的原发性损伤导致炎症、纤维化、血管稀疏和肾小球硬化。我们还在大鼠中进行了确定性研究，导致FDA、EMA和日本监管机构将Kim-1（我们发现的）用于肾毒性的临床前评价。目前的建议将急性肾损伤与慢性肾脏疾病联系起来，因为它专注于急性损伤后通过G2/M调节运动并导致适应不良修复导致纤维化的决定因素。当通过细胞周期的进展被阻止时，特别是在G2/M转换的G2期，所产生的衰老表型导致促纤维化和炎性因子的分泌，其赋予对重复损伤和最终慢性纤维化的增强的敏感性。在第一个具体目标中，我们将定义ATM和ATR在异常修复中的作用， 急性肾小管损伤后细胞周期停滞、持续去分化以及炎症和纤维化增强。我们将确定ATM/ATR激活及其下游效应物（包括细胞周期蛋白D1、ELF 4/MEF、CHK 1、CHK 2、p53和gH 2AX）的时间特征，并将这些发现与PTC的分化状态相关联。我们将评估 肾小管中ATM或ATR的靶向基因缺失对细胞周期停滞和严重缺血/再灌注损伤、马兜铃酸肾毒性和白喉毒素诱导的局灶性近端小管细胞坏死的纤维化后果的影响。在第二个具体目标中，我们将定义sirtuin T2（SIRT 2）在损伤反应中的作用，并将SIRT 2表达和活性与G2/M转换控制和促纤维化分泌表型相关。SirT 2调节从G2到M的过渡，我们提出，我们在损伤中观察到的上调导致在G2中的长期驻留，较少的凋亡和促纤维化分泌表型。在第三个具体目标中，我们将确定增加的自噬和TOR-自噬空间偶联隔室（TASCC）形成是否是与肾损伤后G2/M期阻滞相关的促纤维化分泌表型的重要特征。我们将探索自噬，SIRT 2和细胞周期停滞相关分泌之间的潜在协同作用。这项工作将有希望确定新的目标，以加强适应性修复和抑制促纤维化修复。

DESCRIPTION (provided by applicant): The overall goal of the proposed studies remains to understand the mechanisms of ischemic kidney cell injury and repair with a long range goal to establish therapies that will be useful to prevent and treat acute kidney injury (AKI) in man. Over the last support period we have established that surviving proximal tubule cells (PTCs) are responsible for replacement of injury-induced lost epithelial cells with no evidence that a specialized progenitor/stem cell population replaces the PTCs. In maladaptive pro-fibrotic repair the proximal tubule does not directly convert to interstitial fibroblasts but rather assumes a pro-inflammatory secretory phenotype associated with cell cycle G2/M arrest. Furthermore primary damage to the PT using transgenic animals expressing the human diphtheria toxin receptor leads to inflammation, fibrosis, vascular rarefaction and glomerulosclerosis. We have also conducted definitive studies in the rat that led the FDA, EMA and Japanese regulatory agency to qualify Kim-1 (which we discovered) for preclinical evaluation of nephrotoxicity. The current proposal links acute kidney injury to chronic kidney disease by it focus on the determinants that regulate movement through G2/M after acute injury and result in maladaptive repair leading to fibrosis. When progression through the cell cycle is arrested, particularly in the G2 phase of the G2/M transition, the resulting senescent phenotype leads to secretion of profibrotic and inflammatory factors that confer enhanced sensitivity to repeated injury and ultimately chronic fibrosis. In the first Specific Aim we will define the roles of ATM and ATR in abnormal repair with cell cycle arrest, persistence of dedifferentiation and potentiation of inflammation and fibrosis after acute tubular injury. We will determine the temporal characteristics of ATM/ATR activation and their downstream effectors (including cyclin D1, ELF4/MEF, CHK1, CHK2, p53 and gH2AX) and relate these findings to the differentiation status of the PTC. We will evaluate the effects of targeted genetic deletion of ATM or ATR in the kidney tubule on cell cycle arrest and the fibrotic consequences of severe ischemia/reperfusion injury, aristolochic acid nephrotoxicity and focal proximal tubule cell necrosis induced by diphtheria toxin. In the second Specific Aim we will define the role of sirtuin T2 (SIRT2) in the response to injury and to relate SIRT2 expression and activity to G2/M transition control and the profibrotic secretory phenotype. SirT2 regulates the transition from G2 to M and we propose that upregulation, which we have observed with injury, leads to prolonged residence in G2, less apoptosis and a profibrotic secretory phenotype. In the third Specific Aim we will determine whether increased autophagy and TOR-autophagy spatial coupling compartment (TASCC) formation are important features of the profibrotic secretory phenotype that is associated with G2/M arrest after kidney injury. We will explore a potential synergy between autophagy, SIRT2, and cell cycle arrest-associated secretion. This work will hopefully identify new targets for enhancing adaptive repair and inhibiting pro-fibrotic repair.