Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney

We recently reported that NADH oxidase is one of the major enzymes responsible for superoxide (O 2− ·) production in the rat kidney. However, the functional significance of NADH oxidase-mediated O 2− · production and the mechanisms regulating this enzyme activity are poorly understood. Using fluorescence microscopic imaging analysis, the present study demonstrated that thick ascending limbs of Henle's loop (TALHs) exhibited red fluorescence when incubated with dihydroethidium (DHE), suggesting that O 2− · is produced in this tubular segment. Compared with other nephron segments, TALHs from both renal cortex and medulla showed the highest fluorescence intensity. By incubating cortical TALHs (cTALHs) with the substrates of NADH oxidase, xanthine oxidase, nitric oxide synthase, arachidonic acid-metabolizing enzymes, and intramitochondrial oxidases, NADH oxidase was found to be one of the most important enzymes for O 2− · production in this tubular segment. The NADH oxidase inhibitor diphenyleneiodonium (DPI; 100 μM) completely blocked NADH-induced O 2− · production in cTALHs. Exposure of cTALHs to low Po 2 (5–10 Torr) significantly increased O 2− · production regardless of the absence or presence of NADH. Furthermore, angiotensin II (100 nM) increased NADH oxidase activity by 32%, which was completely blocked by DPI. These results suggest that NADH oxidase is a major enzyme responsible for O 2− · production in the TALHs and that the production of O 2− · via NADH oxidase may be regulated by renal tissue oxygenation and circulating hormones.