Loss of tubular epithelium contributes to the development of ischemic acute renal failure. Our long term objective is to elucidate the molecular mechanisms that are involved in hypoxic cell injury and to develop protective strategies. We have shown that Bax, a pro-apoptotic member of the Bcl-2 family, plays a major role in cell injury caused by hypoxia/ATP depletion, and in the apoptosis that develops during reoxygenation/ATP repletion. During hypoxia, Bax translocates from cytosol to mitochondria and forms oligomers. Together with associated Bak oligomers. Bax permeabilizes mitochondria to release cytochrome c (Cyt.c). Cyt.c facilitates the formation of a protein complex, the apoptosome, required for caspase activation and subsequent apoptotic execution. Expression of anti-apoptotic Bcl-2 does not prevent Bax translocation, but abrogates Bax and Bak oligomerization, and thereby Cyt.c leakage. Experiments in vivo showed reciprocal regulation of Bax and Bcl-2 in the kidney during reperfusion after ischemia. Based on these findings, we propose to pursue four specific aims: Aim 1 hypothesizes that Bax dependent cytochrome c release is a two step process involving translocation followed by oligomerization induced by a BH3-only protein. Studies in this aim will use gene silencing, in vitro reconstitution, as well as EM-Immuno histochemistry and FRET techniques to examine the mechanisms of mitochondrial outer membrane permeabilization. Aim 2 hypothesizes that pore structures formed by Bax and Bak contain other mitochondrial outer membrane proteins. These experiments will use immunoprecipitation, 2D-PAGE and mass spectrometry based peptide sequence analysis. Aim 3 hypothesizes differential ATP requirements for sequential stages of the apoptotic pathway. Mechanisms of caspase activation and its inhibition will be studied by immunoblotting, in vitro reconstitution, gene expression and mutagenesis. Aim 4 hypothesizes that Bax expression regulates tubular injury in the kidney. Transcriptional regulation of Bax promoter during ischemia and reperfusion will be studied using mobility shift assays, and in vitro foot-printing. In vivo gene silencing of Bax will be attempted in efforts to block up regulation of Bax and thereby prevent cell injury in the ischemic kidney.