It is generally accepted that ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro are characterized by a rapid collapse of internal homeostasis of the cell. However, DNA damage as an early event prior to loss of cell viability has not been previously described in this model. The central hypothesis of the present proposal is that DNA damage is an early event in hypoxia/reoxygenation injury to renal proximal tubules or in ischemia/reperfusion injury to kidney in vivo, and that oxidative stress and endonuclease activation are the major determinants of the DNA damage. Our preliminary studies lend strong support to this hypothesis. DNA damage (measured by the alkaline unwinding assay) occurs with as little as 5 min. of hypoxia followed by reoxygenation, to freshly isolated rat proximal renal tubules (PT). The two well described mechanisms of DNA damage are oxidant stress and endonuclease activation. In our preliminary studies scavengers of reactive oxygen metabolites (ROM) as well as endonuclease inhibitors are protective against the DNA damage induced by hypoxia/reoxygenation. These two mechanisms of DNA damage, oxidative stress and endonuclease activation, are not necessarily mutually exclusive. Indeed, in our recent study we have shown that endonuclease activation is an important mechanism of DNA damage and cell death in LLC-PK1 cells (a renal tubular epithelial cell line) exposed to hydrogen peroxide. The objectives of the present proposal, which will be carried out using freshly isolated rat PT for in vitro studies, and for in vivo studies utilizing rat kidneys subjected to ischemia/reperfusion are the following: I. To characterize DNA damage in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo. II. To examine the role of endonuclease activation in DNA damage and cell injury in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo. III. To examine the role of calcium in hypoxia/reoxygenation-induced DNA damage and cell injury. IV. To examine the role of reactive oxygen metabolites in DNA damage and cell injury in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo. V.To examine the role of iron in DNA damage and cell injury in hypoxia/reoxygenation injury in vitro and ischemia/reperfusion injury in vivo.
