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

Abstract

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. 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 microM) 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.

Redox Control of Renal Function and Hypertension

“…NAD(P)H oxidase, as well as other metabolic oxidases such as xanthine oxidase and mitochondrial respiratory chain enzymes (I-IV), are functional in the kidney (278). However, we and others have identified NAD(P)H oxidase-induced ROS production as a primary initiator of oxidative stress within the kidney (183,353,355,356 onists that bind to D1-like receptors, and to H ϩ fluxes (28,89,146,184,259,353,377,384) (Figs. 6 through 9).…”

NADPH Oxidase in the Renal Medulla Causes Oxidative Stress and Contributes to Salt-Sensitive Hypertension in Dahl S Rats

“…We have demonstrated previously that O 2 ⅐Ϫ production in the kidney of normotensive SD rats is predominately produced by NAD(P)H oxidase localized in thick ascending limb segments of the cortex and the outer medulla. 29,30 Immunohistochemistry of the NADH oxidase subunit gp91 phox has shown that the highest level of staining is in the outer medulla, with less staining in the cortex and no signal in the papilla. 30 In contrast, the cortex appears to have a higher antioxidant capacity than the medulla, because SOD, catalase, and GPx activities were highest in the cortex of kidneys from Wistar rats.…”

Salt Intake, Oxidative Stress, and Renal Expression of NADPH Oxidase and Superoxide Dismutase

“…NAD(P)H oxidase has been assigned a major role in ROS generation in vascular and cardiac tissue with contributions from other sources such as xanthine oxidase and mitochondrial respiration (8). In the kidney, NAD(P)H oxidase mediates production of O 2 ·Ϫ in mesangial cells (27), thick ascending limb cells (16), and rat renal medullary homogenates (15). Thus, this oxidase may play a critical role in producing ROS in the kidney.…”

“…The p47 phox is a critical regulatory subunit required for assembly and activation of NADPH oxidase (2). Both gp91 phox and p47 phox are highly expressed in the thick ascending limb and distal nephron (14,16), where ROS can enhance tubular Na ϩ reabsorption (16). All components of phagocyte NAD(P)H oxidase are expressed in the macula densa, where they may impair the effects of NO on tubuloglomerular feedback responses (13).…”

“…NAD(P)H oxidase has also been implicated in O 2 ·Ϫ signaling in the vasculature (12) and in the kidney cortex (13,14) and medulla (15). Activation of NAD(P)H oxidase mediates generation of O 2 ·Ϫ induced by Ang II (12,16) and is implicated in the pathogenesis of several models of hypertension (2,14). Phagocytic NAD(P)H oxidase (2) is composed of a glycosylated flavoprotein, gp91 phox , associated with p22 phox , and three cytosolic components, p40 phox , p47 phox , and p67 phox , and a small guanosine triphosphate (GTP)-binding protein rac 1 .…”