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.