Caspases (ICE/Ced3 proteases) are a recently identified group of closely related cysteine proteases that play key roles in cell death. Our in vitro and in vivo studies provide strong evidence for an important role of caspases in hypoxic injury to renal tubular epithelial cells. In in vitro studies we have demonstrated that specific peptide inhibitors of caspases prevent cell death and DNA damage in renal proximal tubular epithelial cells exposed to hypoxic and oxidant injury. A stably transfected LLC-PK1 cell line developed to overexpress the caspase-3 inhibitor, p35 provided protection to hypoxic as well as oxidant injury. To delineate the role of caspases in renal pathophysiology in vivo it was essential to know which caspases are expressed in the kidney. Since this information was not known, we determined by RT-PCR that rat kidney cortex transcribes the genes for caspases 1, -2, -3, -6, -7, -8 and -9 out of the known family members of caspases. We have also demonstrated by Western and Northern blot analyses, respectively, that in rats subjected to ischemia/reperfusion injury to the kidney, there is both activation and upregulation of caspase-3 and enhanced expression of caspases -1, -2, -3 -6 and -8 mRNAs. To examine the immunohistochemical localization of the executioner caspace-3 in renal ischemia/reperfusion injury we developed a polyclonal antibody to rat recombinant caspase-3 and demonstrated redistribution and translocation of caspase-3 from the cytoplasm to the nuclei in renal tubular epithelial cells during injury. Based on these studies, we hypothesize that subcellular redistribution and activation of caspases plays an important role in cell death and DNA damage in renal ischemia/reperfusion injury and that the inhibition of caspase activation will protect or reduce the ischemic injury. Thus, the objectives of the present proposal are to examine the signaling pathway/s of activation and subcellular redistribution of caspases that regulate DNA damage and cell death in renal ischemic injury. Based on the enhanced mRNA expression, caspase activation and upregulation of caspases during hypoxic and ischemia/reperfusion injury, we will focus our studies on the executioner caspase, caspase-3, and the initiator caspases, caspase-8 and -9. While these studies will focus on ischemic injury, we believe the information derived from these studies will be applicable in future studies which will examine the role of caspases in other forms of acute renal failure.
The specific aims of the proposal are: I.To examine the role of caspases in hypoxia/reoxygenation in vitro and ischemia/reperfusion injury in vivo by modulating their expression and/or activity. II. To examine the subcellular distribution and mechanisms of translocation of caspase-3 in in vitro and in vivo models of ischemia/reperfusion injury. III. To examine the mechanism of activation of caspases in hypoxia/reoxygenation and ischemia/reperfusion injury. IIIA. To examine mitochondrial-mediated activation of caspase-3 in hypoxic and oxidant injury and contribution by nonmitochondrial-dependent pathway: IIIB. To examine the relation between hypoxia- and oxidant-induced alterations in mitochondrial PT in activation of caspases. IV. To identify the role of caspases in endonuclease/s activation. These studies will provide new insights into our understanding of the cellular and molecular mechanisms of ischemic renal injury as well as other forms of acute renal failure.
| Singh, Amar B; Kaushal, Varsha; Megyesi, Judit K et al. (2002) Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury. Kidney Int 62:106-15 |
| Kaushal, G P; Kaushal, V; Hong, X et al. (2001) Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells. Kidney Int 60:1726-36 |
