Renal ischemia targets the proximal tubule epithelial cell and is an important cause of morbidity and mortality. Substantial evidence shows that renal ischemia activates Bax,a pro-apoptotic BCL2 protein that increases mitochondrial membrane permeability and causes apoptosis, an important contributor to organ failure. Metabolic stress disrupts organelle morphology by causing """"""""mitochondrial fission"""""""", a process that promotes apoptosis. Preliminary studies show that metabolic stress, an established model of renal ischemia, targets hexokinase II (HKII), the rate-limiting enzyme in glycolysis and a potential regulator of the Bax-mediated cell death pathway. Metabolic stress causes HKII to dissociate from renal epithelial cell mitochondria and to undergo rapid degradation. Furthermore, HKII interacts with Hsp70, a molecular chaperone, suggesting that stress disrupts HKII conformation and function. Finally, stress causes dynamin-related protein 1 (DRP1), the primary regulator of organelle fission, to accumulate in isolated mitochondria. Selective HKII expression increases mitochondrial HKII content, prevents mitochondrial Bax accumulation, reduces membrane injury and dramatically improves cell survival after stress. The protective mechanism of HKII during stress is presently unknown. We hypothesize that HKII promotes cell viability after stress by: (A) antagonizing Bax-mediated mitochondrial membrane injury and/or (B) preventing mitochondrial fission. Furthermore, we suggest that by interacting with HKII, Hsp70 inhibits HKII degradation and/or facilitates mitochondrial HKII translocation, events that promote cell survival. The effect of increased or decreased HKII or DRP1expression on mitochondrial injury and apoptosis will be studied by measuring AIF leakage, caspase 3 activation, annexin V staining in cells subjected to metabolic stress. Accumulation of mito- chondrial-associated Bax and DRP1and DRP1phosphorylaton will be studied using intact cells and isolated mitochondria. Protein-protein interactions and HKII ubiquitination will be studied by immuno-precipitation. Mitchondrial fission will be evaluated by confocal microscopy using a mitochondrial dye and mitochondrial targeted, photoactivatable GFP. Lastly, HKII ubiquitination and half-life will be correlated with HK content in cells subjected to stress. Renal ischemia, an important cause of morbidity and mortality, damages kidney tubular cells and causes epithelial cell death. The proposed studies will provide molecular insights into the mechanism of renal cell death and may promote the development of therapeutic interventions for treating acute, ischemic renal failure.
|Gall, Jonathan M; Wong, Vincent; Pimental, David R et al. (2011) Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress. Kidney Int 79:1207-16|