1 Molecular and Cellular Biochemistry 205: 1–11, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Kidney ischemia-reperfusion: Modulation of antioxidant defenses K. Dobashi, 1 B. Ghosh, 1 J.K. Orak, 1 I. Singh 1 and A.K. Singh 2 1 Department of Pediatrics, Medical University of South Carolina; 2 Department of Pathology, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA Received 5 February 1999; accepted 5 May 1999 Abstract Reactive oxygen species (ROS; O 2 – , H 2 O 2 , and OH · ), normal by-products of cellular metabolic processes, are kept in control by antioxidant enzymes, such as catalase, glutathione peroxidase (GPX) and superoxide dismutases (SODs). To understand the role of antioxidant enzymatic defenses against ROS injury following ischemia-reperfusion, we examined the effect on kidney exposed to varying periods (30, 60 or 90 min) of ischemia followed by different periods of reperfusion. The enzymatic activities and protein levels of catalase, GPX, CuZnSOD and MnSOD were relatively unaffected at 30 min of ischemia followed by 0, 2 or 24 h reperfusion. However, 60 or 90 min of ischemia followed by 0, 2 or 24 h of reperfusion resulted in a decrease in activities and protein levels which paralleled the duration of ischemic injury. MnSOD activity tended to recover towards normal during reperfusion. Examination of the mRNA levels of these antioxidant enzymes demonstrated a severe decrease in mRNA levels of catalase and GPX at a time point of minimal ischemic injury (30 min of ischemia followed by reperfusion) suggesting that loss of mRNA of catalase and GPX may be the first markers of alterations in cellular redox in ischemia-reperfusion injury. Greater loss of mRNA for catalase, GPX and CuZnSOD was observed following longer periods (60 or 90 min) of ischemia. The mRNA for MnSOD was upregulated at all time points of ischemia-reperfusion injury. Actually, the greater decrease in mRNAs for catalase, GPX and CuZnSOD in the acute phase (within 24 h) subsequently showed a further decrease in these enzyme activities in the subacute phase (72 or 120 h after ischemia). These enzyme activities in the 30 min ischemia group, (but not in the 90 min group), already showed tendencies for normalization at 120 h after ischemia. To understand the molecular basis of the loss of mRNA of these antioxidant enzymes during ischemia-reperfusion injury, we examined the rate of transcription by nuclear run-on assays. The similar rates of transcription in control and kidney exposed to ischemia-reperfusion indicates that the loss of mRNA for catalase, GPX and CuZnSOD is possibly due to the increased rate of turnover of their mRNAs. These studies suggest that expression of antioxidant genes during ischemia-reperfusion are not coordinately expressed and that the differential loss of antioxidant enzymes may be the contributing factor(s) towards the heterogeneous renal tissue damage as a result of ischemia-reperfusion induced oxidative stress. (Mol Cell Biochem 205: 1–11, 2000) Key words: kidney, ischemia-reperfusion injury, free radicals, reactive oxygen species, gene expression, antioxidant enzymes Introduction Increasing evidence has accumulated over the last decade indicating that reactive oxygen species (ROS: O 2 – , H 2 O 2 and OH · ) play a crucial role in the pathophysiology of various disease states including ischemia-reperfusion injury [1–5]. When a tissue/organ is injured it produces excessive amounts of superoxide (O 2 – ) and hydrogen peroxide (H 2 O 2 ). This excessive production of ROS, during ischemia and reperfusion, causes oxidative stress that results in changes in mitochondrial oxidative phosphorylation, depletion of ATP, an increase in intracellular calcium, and activation of Address for offprints: K. Singh, Department of Pathology, Ralph Johnson Veterans Affairs Medical Center, Charleston, SC 29425, USA