Decreased expression of mitochondrial-derived H2O2 and hydroxyl radical in cytoresistant proximal tubules

Abstract

Increased production of reactive oxygen metabolites (ROM) can contribute to the initiation phase of nephrotoxic and ischemic acute renal failure (ARF). However, whether altered ROM expression also exists during the maintenance phase of ARF has not been adequately assessed. Since diverse forms of tubular injury can initiate a "cytoresistant state," this study tested whether a down-regulation of ROM expression might develop in the aftermath of acute tubular damage, potentially limiting renal susceptibility to further attack. To test this hypothesis, rats were subjected to either mild myohemoglobinuria (glycerol injection) or bilateral ureteral obstruction and 24 hours later, cytoresistant proximal tubular segments (PTS) were isolated to assess ROM expression. PTS from sham operated rats were used to establish normal values. Both sets of cytoresistant PTS manifested approximately 75% reductions in H2O2 levels, as assessed by the phenol red/horseradish peroxidase technique (P < 0.01 to 0.001). A 40% reduction in hydroxyl radical (.OH) levels was also observed (salicylate trap method), thereby substantiating decreased oxidant stress in cytoresistant PTS. Catalase, glutathione peroxidase, and free iron levels were comparable in control and cytoresistant PTS, suggesting that decreased H2O2 production (such as by mitochondria) was the cause of the decreased oxidant stress. To test this latter hypothesis, H2O2 expression by control and cytoresistant PTS was assessed in the presence of respiratory chain inhibitors. Although site 1 and site 3 inhibition markedly suppressed H2O2 production in control PTS, they had no impact on H2O2 production in cytoresistant PTS, implying that production at these sites was already maximally suppressed. Correlates of the decreased mitochondrial H2O2 production were improvements in cell energetics (increased ATP/ADP ratios with Na ionophore treatment) and approximately 40 to 90% increases in PTS/renal cortical glutathione content. We conclude that: (1) proximal tubule H2O2/.OH expression can be downregulated during the maintenance phase of ARF; (2) this seemingly reflects a decrease in mitochondrial ROM generation; and (3) the associated improvements in glutathione content and/or cellular energetics could conceivably contribute to a post-injury cytoresistant state.