This application addresses mechanisms underlying cell death in the kidney in response to hypoxia. Renal ischemia is a common cause for acute renal failure and such ischemia-induced injury is induced by hypoxia. Injury is observed both during the hypoxic phase and during the phase of reoxygenation, and one mechanism that may account for such injury is the generation of reactive oxygen species. The present application proposes to study a mechanism for such injury that is centered on the translocation of Bax, a death-promoting protein from the cytosol to the mitochondria which in turn induces the release of cytochrome c from the mitochondria into the cytosol. The principal investigator shows that the morphology of injury is apoptosis and does not require molecular oxygen for the damage to occur. The principal investigator hypothesizes that the morphology and the nature of cell death following the release of cytochrome c from the mitochondria into the cytosol is dependent upon a supply of ATP. If ATP is available, then the form of death that would occur is apoptosis. If ATP is not available, then necrosis would result. This hypothesis is based on the recognition that apoptosis is an ATP-dependent process. The loss of cytochrome c from the mitochondria impairs the ability of the cell to generate ATP through oxidative phosphorylation which necessitates integrity of the electron transport chain in the mitochondrion. Thus, if the cells do have a ready supply of ATP through preserved glycolysis, then apoptosis would be the form of cellular demise. Thus, the critical elements of this application are the study of translocation of Bax to the mitochondrion, the changes in mitochondrial permeability that are induced in the outer mitochondrial membrane induced by Bax, the release of cytochrome c into the cytosol and the cell injury that ensues. The principal investigator proposes four specific aims. The first three utilize a cell culture model of rat proximal tubular cells. The first specific aim will investigate the role of Bax in cytochrome c release from mitochondria during hypoxia as well as the mechanisms by which it induces the permeability change.
This specific aim will determine the requirement of Bax for cytochrome c leak, the nature of the interaction of Bax with specific proteins related to cell death, and whether changes in the mitochondrial inner membrane permeability also occur. The second specific aim will determine the molecular mechanisms underlying Bax translocation from the cytosol to the mitochondria during hypoxia. Emphasis will be placed on the roles played by phosphorylation, nucleotide binding, and the association of the stress proteins. Deletion mutants of Bax will be used to map protein sequences required for translocation. The third specific aim will investigate molecular mechanisms that underlie Bcl-2 inhibition of cytochrome c release during hypoxia.
This specific aim will determine the sequences of Bcl-2 that are required to inhibit cytochrome release, the requirement of heterodimerization with Bax for protective activity, the association of Bcl-2 with proteins related to death pathways, and the effects of Bcl-2 on Bax turnover. The fourth specific aim will perform studies to extend the observations made on cultured cells to tubules of the kidney in microdissected rat nephrons and rabbit proximal tubules. Additionally, studies will be conducted in ischemia reperfusion injury.
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