Abstract. The hypothesis that a high salt (HS) intake increases oxidative stress was investigated and was related to renal cortical expression of NAD(P)H oxidase and superoxide dismutase (SOD). 8-Isoprostane PGF 2␣ and malonyldialdehyde were measured in groups (n ϭ 6 to 8) of conscious rats during low-salt, normal-salt, or HS diets. NADPH-and NADH-stimulated superoxide anion (O 2 ·Ϫ ) generation was assessed by chemiluminescence, and expression of NAD(P)H oxidase and SOD were assessed with real-time PCR. Excretion of 8-isoprostane and malonyldialdehyde increased incrementally twoto threefold with salt intake (P Ͻ 0.001), whereas prostaglandin E 2 was unchanged. Renal cortical NADH-and NADPHstimulable O 2 ·Ϫ generation increased (P Ͻ 0.05) 30 to 40% with salt intake. Compared with low-salt diet, HS significantly (P Ͻ 0.005) increased renal cortical mRNA expression of gp91 phox and p47 phox and decreased expression of intracellular CuZn (IC)-SOD and mitochondrial (Mn)-SOD. Despite suppression of the renin-angiotensin system, salt loading enhances oxidative stress. This is accompanied by increased renal cortical NADH and NADPH oxidase activity and increased expression of gp91 phox and p47 phox and decreased IC-and Mn-SOD. Thus, salt intake enhances generation of O 2 ·Ϫ accompanied by enhanced renal expression and activity of NAD(P)H oxidase with diminished renal expression of IC-and Mn-SOD.

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Abstract-The angiotensin II (Ang II) slow-pressor response entails an increase in mean arterial pressure and reactive oxygen species. We used double-stranded interfering RNAs (siRNAs) in Sprague Dawley rats in vivo to test the hypothesis that an increase in the p22 phox component of NADPH oxidase is required for this response. Reactive oxygen species were assessed from excretion of 8-isoprostane prostaglandin F 2␣ and blood pressure by telemetry. Two siRNA sequences to p22 phox (sip22 phox ) reduced mRNA Ͼ85% in cultured vascular smooth muscle cells. Rats received rapid (10 second) IV injections (50 to 100 g) Key Words: hypertension, arterial Ⅲ arterioles Ⅲ oxidative stress Ⅲ kidney A ngiotensin II (Ang II) has been assigned a critical role in the generation and complications of human essential hypertension, yet plasma renin activity and plasma concentrations of Ang II are not remarkably elevated. 1 Mice, 2 rats, 3,4 rabbits, 5 or humans 6 infused with Ang II at doses that are initially subpressor develop a "slow-pressor response" in which the blood pressure (BP) increases progressively despite plasma Ang II concentrations that are increased only moderately. 3 The kidney is implicated in the slow-pressor response, because the development of hypertension depends on salt intake. 3 Moreover, rats or rabbits infused with Ang II have enhanced renal vasoconstriction to Ang II 5,7 despite downregulation of Ang II type 1 receptors.Reactive oxygen species (ROS) and superoxide anion (O 2 ⅐Ϫ ) have been implicated in the development of hypertension in the Ang II slow-pressor model, because hypertension is prevented by antioxidant molecules, such as a permeabilized form of superoxide dismutase or tempol, which is an superoxide dismutase mimetic nitroxide. 2,4,5,8 Infusions of Ang II increase the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in blood vessels 9 and the kidney cortex. 4,10,11 This complex enzyme, which was first described in phagocytes and, later, in blood vessels and the kidney, is composed of membrane-associated components of the flavoprotein catalytic core, gp91phox (now named Nox-2) and p22phox . 12 Activation requires phosphorylation of p47 phox 13and its assembly with p67 phox 14 and Rac-1 at the membrane. 12 Homologues of Nox-2 include Nox-1, which has been characterized in vascular smooth muscle cells (VSMCs), 9 and Nox-4, which has been characterized in the kidney. 15 VSMCs and kidneys have the phagocytic NADPH oxidase components. 12 p22 phox is expressed in the thick ascending limb, macula densa, distal convoluted tubule, collecting ducts, vasculature, and interstitial fibroblasts of the kidney. 16 It is believed to dock the enzyme complex in the cell membrane and stabilize Nox proteins. 12 There is colocalization of p22 phox and O 2 ⅐Ϫ generation in atherosclerotic plaques from human blood vessels. 17 The p22 phox component is upregulated in the

Abstract-Low rates of angiotensin II (Ang II) infusion raise blood pressure, renal vascular resistance (RVR), NADPH oxidase activity, and superoxide. We tested the hypothesis that these effects are ameliorated by extracellular superoxide dismutase (EC-SOD). EC-SOD knockout (Ϫ/Ϫ) and wild type (ϩ/ϩ) mice were equipped with blood pressure telemeters and infused subcutaneously with Ang II (400 ng/kg per minute) or vehicle for 2 weeks. During vehicle infusion, EC-SOD Ϫ/Ϫ mice had significantly (PϽ0.05) higher MAP (ϩ/ϩ: 107Ϯ3 mm Hg versus Ϫ/Ϫ: 114Ϯ2 mm Hg; nϭ11 to 14), RVR, lipid peroxidation, renal cortical p22 phox expression, and NADPH oxidase activity. Ang II infusion in EC-SOD ϩ/ϩ mice significantly (PϽ0.05) increased MAP, RVR, p22 phox , NADPH oxidase activity, and lipid peroxidation. Ang II reduced SOD activity in plasma, aorta, and kidney accompanied by reduced renal EC-SOD expression. During Ang II infusion, both groups had similar values for MAP (ϩ/ϩ Ang II: 125Ϯ3 versus Ϫ/Ϫ Ang II: 124Ϯ3 mmHg; P value not significant), RVR, NADPH oxidase activity, and lipid peroxidation. SOD activity in the kidneys of Ang II-infused mice was paradoxically higher in EC-SOD Ϫ/Ϫ mice (ϩ/ϩ: 8.8Ϯ1.2 U/mg protein Ϫ1 versus Ϫ/Ϫ: 13.7Ϯ1.6 U/mg protein Ϫ1 ; PϽ0.05) accompanied by a significant upregulation of mRNA and protein for Cu/Zn-SOD. In conclusion, EC-SOD protects normal mice against oxidative stress by attenuating renal p22 phox expression, NADPH oxidase activation, and the accompanying renal vasoconstriction and hypertension. However, during an Ang II slow pressor response, renal EC-SOD expression is reduced and, in its absence, renal Cu/Zn-SOD is upregulated and may prevent excessive Ang II-induced renal oxidative stress, renal vasoconstriction, and hypertension. Key Words: oxidative stress Ⅲ hypertension Ⅲ renal Ⅲ kidney Ⅲ renal circulation Ⅲ nitric oxide Ⅲ endothelium A n increase in reactive oxygen species (ROS) in the blood vessels and kidneys is reported in several experimental animal models of hypertension and in human essential and renovascular hypertension. 1 Infusions of angiotensin II (Ang II) increase blood pressure (BP), markers of oxidative stress, and renal expression of the p22 phox and Nox-1 components of renal NADPH oxidase. 2 These effects seem specific for Ang II, because similar pressor infusions of norepinephrine into rats do not induce oxidative stress in blood vessels. 3 An increased production of superoxide (O 2 ⅐Ϫ ) reduces bioactive NO 4 and contributes to vascular and renal injury in chronic hypertension. 5,6 O 2 ⅐Ϫ dismutase (SOD) metabolizes O 2 ⅐Ϫ to H 2 O 2 , which is further metabolized to inactive products by peroxidases. Hypertension can be moderated or prevented by gene transfer of extracellular (EC)-SOD 7 or by administration of tempol, 8 which is a nitroxide SOD mimetic.The 3 isoforms of SOD are localized to the kidney. 9 EC-SOD is located on cell membranes of endothelial cells and vascular smooth muscle cells. 10 EC-SOD Ϫ/Ϫ mice have endothelial dysfunction in conduit blood vessels th...