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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK047990-05
Application #
2713387
Study Section
Pathology B Study Section (PTHB)
Project Start
1994-06-01
Project End
2000-05-31
Budget Start
1998-06-01
Budget End
2000-05-31
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Basnakian, Alexei G; Ueda, Norishi; Kaushal, Gur P et al. (2002) DNase I-like endonuclease in rat kidney cortex that is activated during ischemia/reperfusion injury. J Am Soc Nephrol 13:1000-7
Carmago, Simone; Shah, Sudhir V; Walker, Patrick D (2002) Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats. Kidney Int 61:959-66
Ueda, N; Shah, S V (2000) Tubular cell damage in acute renal failure-apoptosis, necrosis, or both. Nephrol Dial Transplant 15:318-23
Ueda, N; Shah, S V (2000) Role of endonucleases in renal tubular epithelial cell injury. Exp Nephrol 8:13-Aug
Ueda, N; Kaushal, G P; Shah, S V (2000) Apoptotic mechanisms in acute renal failure. Am J Med 108:403-15
Ueda, N; Kaushal, G P; Shah, S V (1997) Recent advances in understanding mechanisms of renal tubular injury. Adv Ren Replace Ther 4:17-24
Kaushal, G P; Ueda, N; Shah, S V (1997) Role of caspases (ICE/CED 3 proteases) in DNA damage and cell death in response to a mitochondrial inhibitor, antimycin A. Kidney Int 52:438-45
Hagar, H; Ueda, N; Shah, S V (1996) Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. Am J Physiol 271:F209-15
Hagar, H; Ueda, N; Shah, S V (1996) Endonuclease induced DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. Kidney Int 49:355-61