Production and Actions of Superoxide in the Renal Medulla

Abstract

The present study characterized the biochemical pathways responsible for superoxide (O2−·) production in different regions of the rat kidney and determined the role of O2−·in the control of renal medullary blood flow (MBF) and renal function. By use of dihydroethidium/DNA fluorescence spectrometry with microtiter plates, the production of O2−·was monitored when tissue homogenate from different kidney regions was incubated with substrates for the major O2−·-producing enzymes, such as NADH/NADPH oxidase, xanthine oxidase, and mitochondrial respiratory chain enzymes. The production of O2−·via NADH oxidase was greater (P<0.05) in the renal cortex and outer medulla (OM) than in the papilla. The mitochondrial enzyme activity for O 2−·production was higher (P<0.05) in the OM than in the cortex and papilla. Compared with NADH oxidase and mitochondrial enzymes, xanthine oxidase and NADPH oxidase produced much less O 2−·in the kidney under this condition. Overall, the renal OM exhibited the greatest enzyme activities for O 2−·production. In anesthetized rats, renal medullary interstitial infusion of a superoxide dismutase inhibitor, diethyldithiocarbamate, markedly decreased renal MBF and sodium excretion. Diethyldithiocarbamate (5 mg/kg per minute by renal medullary interstitial infusion [RI]) reduced the renal medullary laser-Doppler flow signal from 0.6±0.04 to 0.4±0.03 V, a reduction of 33%, and both urine flow and sodium excretion decreased by 49%. In contrast, a membrane-permeable superoxide dismutase mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (TEMPOL, 30 μmol/kg per minute RI) increased MBF and sodium excretion by 34% and 69%, respectively. These effects of TEMPOL on renal MBF and sodium excretion were not altered by pretreatment with NG-nitro-l-arginine methyl ester (10 μg/kg per minute RI). We conclude that (1) renal medullary O 2−·is primarily produced in the renal OM; (2) both NADH oxidase and mitochondrial enzymes are responsible for the O 2−·production in this kidney region; and (3) O 2−·exerts a tonic regulatory action on renal MBF.