Shakil Aslam

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

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(2alpha) and malonyldialdehyde were measured in groups (n = 6 to 8) of conscious rats during low-salt, normal-salt, or HS diets. NADPH- and NADH-stimulated superoxide anion (O(2)(.-)) 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 two- to threefold with salt intake (P < 0.001), whereas prostaglandin E(2) was unchanged. Renal cortical NADH- and NADPH-stimulable O(2)(.-) generation increased (P < 0.05) 30 to 40% with salt intake. Compared with low-salt diet, HS significantly (P < 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)(.-) accompanied by enhanced renal expression and activity of NAD(P)H oxidase with diminished renal expression of IC- and Mn-SOD.

Roles of Oxidative Stress and AT1 Receptors in Renal Hemodynamics and Oxygenation in the Postclipped 2K,1C Kidney

Abstract—The spontaneously hypertensive rat (SHR) exhibits angiotensin II (Ang II)–dependent oxidative stress and reduced efficiency of renal oxygen usage (QO2) for tubular sodium transport (TNa). We tested the hypothesis that oxidative stress determines the reduced TNa:QO2 ratio in the clipped kidney of the early 2-kidney, 1-clip (2K,1C) Ang II–dependent model. One week after sham operation (Sham) or clip placement, 2K,1C rats received for 2 weeks either a vehicle, the superoxide dismutase mimetic tempol (Temp), or candesartan (Cand). Oxidative stress was assessed from excretion of 8-isoprostaglandin F2&agr; (PGF2&agr;) and malondialdehyde (MDA) and renal oxygenation from pO2 in the renal cortex and from the ratio of calculated TNa and QO2 values. The mean arterial pressure (MAP) of Sham (113±6 mm Hg) was increased in 2K,1C vehicle-treated rats (148±4 mm Hg), but both Temp and Cand restored MAP to Sham levels. The excretions of 8-iso-PGF2&agr; and MDA were higher in 2K,1C vehicle-treated rats compared with Sham and were normalized by Temp. The pO2 of Sham (42±2 mm Hg) was lower in 2K,1C vehicle-treated animals (28±2 mm Hg). This was restored to Sham values by Temp (36±3 mm Hg) but not by Cand (28±2 mm Hg). The TNa:QO2 of Sham (12.9±1.6) was reduced in 2K,1C vehicle-treated rats (9.7±2.8) and was restored to Sham values by Temp (13.7±2.5) but not by Cand (7.5±1.6). We conclude that the correction of oxidative stress in the 2K,1C model partially corrects renal cortical hypoxia and inefficient utilization of O2 for Na+ transport, independent of the fall in blood pressure.

Role of Oxidative Stress in Endothelial Dysfunction and Enhanced Responses to Angiotensin II of Afferent Arterioles from Rabbits Infused with Angiotensin II

The hypothesis that O(2)(.-) enhances angiotensin II (AngII)-induced vasoconstriction and impairs acetylcholine-induced vasodilation of afferent arterioles (Aff) in AngII-induced hypertension was investigated. Rabbits (n = 6 per group) received 12 to 14 d of 0.154 M NaCl (Sham), subpressor AngII (60 ng/kg per min; AngII 60) or slow pressor AngII (200 ng/kg per min; AngII 200). Individual Aff were perfused in vitro at 60 mmHg. AngII 200 increased mean arterial pressure (mean +/- SD; 103 +/- 9 versus 73 +/- 6 mmHg; P < 0.01), plasma lipid peroxides (2.6 +/- 0.3 versus 2.0 +/- 0.3 nM; P < 0.05), renal cortical NADPH- and NADH-dependent O(2)(.-) generation, and Aff mRNA for p22(phox) 5-fold (P < 0.001) but decreased that for AT(1)-receptor 2.4-fold (P < 0.01). AngII 60 increased only NADH-dependent O(2)(.-) generation by renal cortex. Aff from AngII 200 rabbits had diminished acetylcholine relaxations (+50 +/- 4 versus +85 +/- 6%; P < 0.001), but these became similar in the presence of nitro-L-arginine (10(-4) M). Aff from AngII 60 and AngII 200 rabbits had unchanged norepinephrine contractions (10(-7) M) but significantly (P < 0.05) enhanced AngII contractions (10(-8) M: Sham -52 +/- 5 versus AngII 60 to 77 +/- 5 versus AngII 200 to 110 +/- 10%). The superoxide dismutase mimetic tempol (10(-4) M) moderated the AngII responses of Aff from AngII 200 rabbits to levels of AngII 60 rabbits (-64 +/- 7%). The AngII slow pressor response enhances renal cortical O(2)(.-) and p22(phox) expression. Increased O(2)(.-) generation in Aff mediates an impaired nitric oxide synthase-dependent endothelium-derived relaxing factor response and paradoxically enhances contractions to AngII despite downregulation of the mRNA for AT(1) receptors. A subpressor dose of AngII enhances Aff responses to AngII independent of O(2)(.-).

Angiotensin II and NADPH Oxidase Increase ADMA in Vascular Smooth Muscle Cells

Asymmetrical dimethylarginine inhibits nitric oxide synthase, cationic amino acid transport, and endothelial function. Patients with cardiovascular risk factors often have endothelial dysfunction associated with increased plasma asymmetrical dimethylarginine and markers of reactive oxygen species. We tested the hypothesis that reactive oxygen species, generated by nicotinamide adenine dinucleotide phosphate oxidase, enhance cellular asymmetrical dimethylarginine. Incubation of rat preglomerular vascular smooth muscle cells with angiotensin II doubled the activity of nicotinamide adenine dinucleotide phosphate oxidase but decreased the activities of dimethylarginine dimethylaminohydrolase by 35% and of cationic amino acid transport by 20% and doubled cellular (but not medium) asymmetrical dimethylarginine concentrations (P<0.01). This was blocked by tempol or candesartan. Cells stably transfected with p22phox had a 50% decreased protein expression and activity of dimethylarginine dimethylaminohydrolase despite increased promoter activity and mRNA. The decreased DDAH protein expression and the increased asymmetrical dimethylarginine concentration in p22phox-transfected cells were prevented by proteosomal inhibition. These cells had enhanced protein arginine methylation, a 2-fold increased expression of protein arginine methyltransferase-3 (P<0.05) and a 30% reduction in cationic amino acid transport activity (P<0.05). Asymmetrical dimethylarginine was increased from 6±1 to 16±3 &mgr;mol/L (P<0.005) in p22phox-transfected cells. Thus, angiotensin II increased cellular asymmetrical dimethylarginine via type 1 receptors and reactive oxygen species. Nicotinamide adenine dinucleotide phosphate oxidase increased cellular asymmetrical dimethylarginine by increasing enzymes that generate it, enhancing the degradation of enzymes that metabolize it, and reducing its cellular transport. This could underlie increases in cellular asymmetrical dimethylarginine during oxidative stress.

Angiotensin II Infusion Alters Vascular Function in Mouse Resistance Vessels: Roles of O–·2 and Endothelium

We hypothesized that prolonged angiotensin II (AngII) infusion would alter vascular reactivity by enhancing superoxide anion (O–·2) generation. Male C57BL/6 mice were infused with AngII at 400 ng/kg/min (n = 16, AngII mice) or vehicle (n = 16, sham mice) for 2 weeks via subcutaneous osmotic minipumps. Contraction and relaxation of mesenteric resistance vessels (MRVs) were assessed using a Mulvany-Halpern myograph. AngII infusion increased systolic blood pressure, MRV NADPH oxidase activity and expression of p22phox mRNA. Contraction to norepinephrine was unchanged, but AngII infusion increased contractile responses to AngII (41 ± 5 vs. 10 ± 4%, p < 0.001) and endothelin-1 (ET-1; 95 ± 10 vs. 70 ± 9%, p < 0.05), which was normalized by tempol (10–4 M, a stable membrane-permeable superoxide dismutase mimetic) and ebselen [10–5 M, a peroxynitrite (ONOO–) scavenger]. Endothelium removal enhanced MRV contraction to AngII and ET-1 in sham mice but blunted these contractile responses in AngII mice. Relaxation to ACh was impaired in AngII mice (60.1 ± 8.8 vs. 83.2 ± 3.5%, p < 0.01), which normalized by tempol, whereas relaxation to sodium nitroprusside was similar in both groups. N-nitro-L-arginine (NNLA, a nitric oxide synthase inhibitor), partially inhibited acetylcholine relaxation of vessels from sham mice but not from AngII mice. The residual endothelium-dependent hyperpolarizing-factor-like relaxation was not different between groups. In conclusion,the AngII slow pressor response in mouse MRVs consisted of specific contractile hyperresponsiveness and impairment in the NO-mediated component of endothelium-dependent relaxation, which was mediated by O–·2 and ONOO– in the vascular smooth muscle cell.

MYOGENIC RESPONSES OF MOUSE ISOLATED PERFUSED RENAL AFFERENT ARTERIOLES: EFFECTS OF SALT INTAKE AND REDUCED RENAL MASS

Because defects in renal autoregulation may contribute to renal barotrauma in chronic kidney disease, we tested the hypothesis that the myogenic response is diminished by reduced renal mass. Kidneys from 5/6 nephrectomized mice had only a minor increase in the glomerular sclerosis index. The telemetric mean arterial pressure (108±10 mm Hg) was unaffected after 3 months of high-salt intake (6% salt in chow) or reduced renal mass. Afferent arterioles from 5/6 nephrectomized mice and sham-operated controls were perfused ex vivo during step changes in pressure from 40 to 134 mm Hg. Afferent arterioles developed a constriction and a linear increase in active wall tension above a perfusion pressure of 36±6 mm Hg, without a plateau. The slope of active wall tension versus perfusion pressure defined the myogenic response, which was similar in sham mice fed normal or high-salt diets for 3 months (2.90±0.22 versus 3.22±0.40 dynes · cm−1/mm Hg; P value not significant). The myogenic response was unaffected after 3 days of reduced renal mass on either salt diet (3.39±0.61 versus 4.04±0.47 dynes · cm−1/mm Hg) but was reduced (P<0.05) in afferent arterioles from reduced renal mass groups fed normal and high salt at 3 months (2.10±0.28 and 1.35±0.21 dynes · cm−1/mm Hg). In conclusion, mouse renal afferent arterioles develop a linear increase in myogenic tone around the range of ambient perfusion pressures. This myogenic response is impaired substantially in the mouse model of prolonged reduced renal mass, especially during high salt intake.

Impaired Endothelial Function and Microvascular Asymmetrical Dimethylarginine in Angiotensin II–Infused Rats: Effects of Tempol

Angiotensin (Ang) II causes endothelial dysfunction, which is associated with cardiovascular risk. We investigated the hypothesis that Ang II increases microvascular reactive oxygen species and asymmetrical dimethylarginine and switches endothelial function from vasodilator to vasoconstrictor pathways. Acetylcholine-induced endothelium-dependent responses of mesenteric resistance arterioles were assessed in a myograph and vascular NO and reactive oxygen species by fluorescent probes in groups (n=6) of male rats infused for 14 days with Ang II (200 ng/kg per minute) or given a sham infusion. Additional groups of Ang or sham-infused rats were given oral Tempol (2 mmol · L−1). Ang II infusion increased mean blood pressure (119±5 versus 89±7 mm Hg; P<0.005) and plasma malondialdehyde (0.57±0.02 versus 0.37±0.05 &mgr;mol · L−1; P<0.035) and decreased maximal endothelium-dependent relaxation (18±5% versus 54±6%; P<0.005) and hyperpolarizing (19±3% versus 29±3%; P<0.05) responses and NO activity (0.9±0.1 versus 1.6±0.2 U; P<0.01) yet enhanced endothelium-dependent contraction responses (23±5% versus 5±5%; P<0.05) and reactive oxygen species production (0.82±0.05 versus 0.15±0.03 U; P<0.01). Ang II decreased the expression of dimethylarginine dimethylaminohydrolase 2 and increased asymmetrical dimethylarginine in vessels (450±50 versus 260±35 pmol/mg of protein; P<0.01) but not plasma. Tempol prevented any significant changes with Ang II. In conclusion, Ang redirected endothelial responses from relaxation to contraction, reduced vascular NO, and increased asymmetrical dimethylarginine. These effects were dependent on reactive oxygen species and could, therefore, be targeted with effective antioxidant therapy.

Effects of the antioxidant drug tempol on renal oxygenation in mice with reduced renal mass

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with ⅚ surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na/)Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na/)Q(O2) (sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical Po(2) (sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.

Activation of Nuclear Factor Erythroid 2–Related Factor 2 Coordinates Dimethylarginine Dimethylaminohydrolase/PPAR-γ/Endothelial Nitric Oxide Synthase Pathways That Enhance Nitric Oxide Generation in Human Glomerular Endothelial Cells

Dimethylarginine dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine, which inhibits nitric oxide (NO) synthase (NOS). Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcriptional factor that binds to antioxidant response elements and transcribes many antioxidant genes. Because the promoters of the human DDAH-1 and DDAH-2, endothelial NOS (eNOS) and PPAR-&ggr; genes contain 2 to 3 putative antioxidant response elements, we hypothesized that they were regulated by Nrf2/antioxidant response element. Incubation of human renal glomerular endothelial cells with the Nrf2 activator tert-butylhydroquinone (20 &mgr;mol·L−1) significantly (P<0.05) increased NO and activities of NOS and DDAH and decreased asymmetric dimethylarginine. It upregulated genes for hemoxygenase-1, eNOS, DDAH-1, DDAH-2, and PPAR-&ggr; and partitioned Nrf2 into the nucleus. Knockdown of Nrf2 abolished these effects. Nrf2 bound to one antioxidant response element on DDAH-1 and DDAH-2 and PPAR-&ggr; promoters but not to the eNOS promoter. An increased eNOS and phosphorylated eNOS (P-eNOSser-1177) expression with tert-butylhydroquinone was prevented by knockdown of PPAR-&ggr;. Expression of Nrf2 was reduced by knockdown of PPAR-&ggr;, whereas PPAR-&ggr; was reduced by knockdown of Nrf2, thereby demonstrating 2-way positive interactions. Thus, Nrf2 transcribes HO-1 and other genes to reduce reactive oxygen species, and DDAH-1 and DDAH-2 to reduce asymmetric dimethylarginine and PPAR-&ggr; to increase eNOS and its phosphorylation and activity thereby coordinating 3 pathways that enhance endothelial NO generation.

Acute Knockdown of Uncoupling Protein-2 Increases Uncoupling via the Adenine Nucleotide Transporter and Decreases Oxidative Stress in Diabetic Kidneys

Increased O2 metabolism resulting in chronic hypoxia is common in models of endstage renal disease. Mitochondrial uncoupling increases O2 consumption but the ensuing reduction in mitochondrial membrane potential may limit excessive oxidative stress. The present study addressed the hypothesis that mitochondrial uncoupling regulates mitochondria function and oxidative stress in the diabetic kidney. Isolated mitochondria from kidney cortex of control and streptozotocin-induced diabetic rats were studied before and after siRNA knockdown of uncoupling protein-2 (UCP-2). Diabetes resulted in increased UCP-2 protein expression and UCP-2-mediated uncoupling, but normal mitochondria membrane potential. This uncoupling was inhibited by GDP, which also increased the membrane potential. siRNA reduced UCP-2 protein expression in controls and diabetics (−30–50%), but paradoxically further increased uncoupling and markedly reduced the membrane potential. This siRNA mediated uncoupling was unaffected by GDP but was blocked by ADP and carboxyatractylate (CAT). Mitochondria membrane potential after UCP-2 siRNA was unaffected by GDP but increased by CAT. This demonstrated that further increased mitochondria uncoupling after siRNA towards UCP-2 is mediated through the adenine nucleotide transporter (ANT). The increased oxidative stress in the diabetic kidney, manifested as increased thiobarbituric acids, was reduced by knocking down UCP-2 whereas whole-body oxidative stress, manifested as increased circulating malondialdehyde, remained unaffected. All parameters investigated were unaffected by scrambled siRNA. In conclusion, mitochondrial uncoupling via UCP-2 regulates mitochondria membrane potential in diabetes. However, blockade of the diabetes-induced upregulation of UCP- 2 results in excessive uncoupling and reduced oxidative stress in the kidney via activation of ANT.