Although substantial evidence implicates Bax and Bcl2, two antagonistic members of the BCL2 family, as a primary cause of mitochondrial membrane injury and cell death after ischemia, the intracellular signals that regulate these BCL2 proteins are not known. Preliminary data from our laboratory show that Hsp70 is a major regulator of Bax and Bcl2 after ATP depletion in vitro and renal ischemia in vivo, insults that dramatically shift the Bax:Bcl2 balance in a pro-apoptotic direction. Using a novel synthetic Bax substrate, we show that ATP depletion causes changes in Akt and GSK3b activity that promote Bax activation. Stress also results in: cytosolic accumulation of nucleophosmin, (NPM) a newly described Bax chaperone;displacement of hexokinase II (HK II) from mitochondria;and inactivation and degradation of Bcl2, a potent Bax antagonist. New evidence shows that Hsp70 interacts with, and potentially regulates each of these key steps in the cell death pathway. In Hsp70 knockout or Hsp70 heterozygous mice, ischemic renal failure is far more severe, tubular injury greater, and Bax activation and Bcl2 degradation are more pronounced than in Hsp70 replete control. Furthermore, proximal tubule cells harvested from these animals and grown in primary culture have parallel sensitivity to ATP depletion as observed after ischemia in vivo. The sensitivity of Hsp70 deficient cells to ATP depletion can be completely rescued by the selective addition of Hsp70. We therefore hypothesize that Hsp70 interferes with Bax activation either by: (1) modifying serine phosphorylation events required for Bax activation;(2) interfering with NPM-mediated Bax activation;(3) re-targeting HK II to the mitochondrial membrane to prevent """"""""Bax attack"""""""";and/or (4) preserving the ability of Bcl2 to inhibit Bax in response to stress. These hypotheses will be tested in established Hsp70 over-expressing transgenic, Hsp 70 knockout and Hsp70 heterozygous mice, in primary culture derived from these animals, in immortalized murine proximal tubule cells, and in isolated mitochondria. In these studies, we will utilize well- characterized Bax mutants with specific phosphomimetic and phospho-deletion changes, nucleophosmin mutants with defective targeting, and Hps70 deletion mutants that either lack the chaperone or ATPase domains or are unable to traffic into nuclei or nucleoli. Agents that either induce Hsp70 (GGA) or directly inhibit the Bax channel (Bci1 and Bci2) will be administered to mice to demonstrate their therapeutic efficacy in preventing or treating ischemia renal failure. By examining Hsp70-regulated steps in the cell injury pathway, these studies will characterize the mechanisms of BCL2 protein-induced cell death and provide new targets for developing therapeutic interventions to prevent or treat acute ischemic renal failure.

Public Health Relevance

Acute kidney failure is a common and often a life-threatening consequence of reduced blood flow (ischemia) to the organ. At present, the mechanisms that cause the kidney to fail are unclear and as a result, there are no specific treatments. We propose to study the role of an intrinsic, cytoprotective protein, Hsp70, in preventing kidney cell death and organ failure. These studies will not only identify some of the mechanisms of acute ischemic kidney injury, but also may provide new targets for therapy.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Pathobiology of Kidney Disease Study Section (PBKD)
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Kimmel, Paul
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Boston Medical Center
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Gall, Jonathan M; Wang, Zhiyong; Bonegio, Ramon G et al. (2015) Conditional knockout of proximal tubule mitofusin 2 accelerates recovery and improves survival after renal ischemia. J Am Soc Nephrol 26:1092-102
Wang, Zhiyong; Gall, Jonathan M; Bonegio, Ramon et al. (2013) Nucleophosmin, a critical Bax cofactor in ischemia-induced cell death. Mol Cell Biol 33:1916-24
Gall, Jonathan M; Wang, Zhiyong; Liesa, Marc et al. (2012) Role of mitofusin 2 in the renal stress response. PLoS One 7:e31074
Wang, Zhiyong; Gall, Jonathan M; Bonegio, Ramon G B et al. (2011) Induction of heat shock protein 70 inhibits ischemic renal injury. Kidney Int 79:861-70
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
Havasi, Andrea; Borkan, Steven C (2011) Apoptosis and acute kidney injury. Kidney Int 80:29-40
Wang, Zhiyong; Havasi, Andrea; Gall, Jonathan et al. (2010) GSK3beta promotes apoptosis after renal ischemic injury. J Am Soc Nephrol 21:284-94
Havasi, Andrea; Wang, Zhiyong; Gall, Jonathan M et al. (2009) Hsp27 inhibits sublethal, Src-mediated renal epithelial cell injury. Am J Physiol Renal Physiol 297:F760-8
Wang, Zhiyong; Havasi, Andrea; Gall, Jonathan M et al. (2009) Beta-catenin promotes survival of renal epithelial cells by inhibiting Bax. J Am Soc Nephrol 20:1919-28
Havasi, Andrea; Li, Zhijian; Wang, Zhiyong et al. (2008) Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism. J Biol Chem 283:12305-13

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