Christopher S. Wilcox

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.

Asymmetric dimethylarginine, oxidative stress, and vascular nitric oxide synthase in essential hypertension.

We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (HODE), which is a marker of reactive oxygen species. Patients had significantly (P < 0.001) elevated (means +/- SD) plasma levels of ADMA (P(ADMA), 766 +/- 217 vs. 393 +/- 57 nmol/l) and symmetric dimethylarginine (P(SDMA): 644 +/- 140 vs. 399 +/- 70 nmol/l) but similar levels of L-arginine accompanied by significantly (P < 0.015) increased rates of renal ADMA excretion (21 +/- 9 vs. 14 +/- 5 nmol/mumol creatinine) and decreased rates of renal ADMA clearance (18 +/- 3 vs. 28 +/- 5 ml/min). They had significantly increased plasma levels of HODE (P(HODE): 309 +/- 30 vs. 226 +/- 24 nmol/l) and renal HODE excretion (433 +/- 93 vs. 299 +/- 67 nmol/micromol creatinine). For the combined group of normal and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P < 0.001) and inversely correlated with microvascular EDRF/NO and positively correlated with mean blood pressure. In conclusion, elevated levels of ADMA and oxidative stress in a group of hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated 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.

NO generation and action during changes in salt intake: roles of nNOS and macula densa

Micropuncture studies of single nephrons have shown that macula densa solute reabsorption via a furosemide-sensitive pathway activates nitric oxide (NO) generation via neuronal NO synthase (nNOS). This pathway is enhanced during salt loading. We investigated the hypothesis that changes in NO generation via nNOS in the macula densa contribute to changes in whole kidney NO generation and action during alterations in salt intake. Groups of rats (n = 6-10) were equilibrated to high-salt (HS) or low-salt (LS) diets and were administered a vehicle (Veh), 7-nitroindazole (7-NI; a relatively selective inhibitor of nNOS), or furosemide (F; an inhibitor of macula densa solute reabsorption) with volume replacement. Compared with LS, excretion of the NO metabolites, NO2 plus NO3 (NOX) was increased during HS (LS: 9.0 +/- 0.5 vs. HS: 15.7 +/- 0.8 micromol/24 h; P < 0.001), but this difference was prevented by 7-NI (LS: 7.4 +/- 1.3 vs. HS: 9.4 +/- 1.6 micromol/24 h; NS). During nonselective blockade of NOS with NG-nitro-L-arginine methyl ester (L-NAME), renal vascular resistance (RVR) increased more in HS than LS (HS: +160 +/- 17 vs. LS: +83 +/- 10%; P < 0.001). This difference in response to nonselective NOS inhibition was prevented by pretreatment with 7-NI (HS: +28 +/- 6 vs. LS: +34 +/- 8%; NS) or F with volume replacement (HS: +79 +/- 11 vs. LS: +62 +/- 4%; NS). In conclusion, compared with salt restriction, HS intake increases NO generation and renal action that depend on nNOS and macula densa solute reabsorption.Micropuncture studies of single nephrons have shown that macula densa solute reabsorption via a furosemide-sensitive pathway activates nitric oxide (NO) generation via neuronal NO synthase (nNOS). This pathway is enhanced during salt loading. We investigated the hypothesis that changes in NO generation via nNOS in the macula densa contribute to changes in whole kidney NO generation and action during alterations in salt intake. Groups of rats ( n = 6-10) were equilibrated to high-salt (HS) or low-salt (LS) diets and were administered a vehicle (Veh), 7-nitroindazole (7-NI; a relatively selective inhibitor of nNOS), or furosemide (F; an inhibitor of macula densa solute reabsorption) with volume replacement. Compared with LS, excretion of the NO metabolites, NO2 plus NO3(NOX) was increased during HS (LS: 9.0 ± 0.5 vs. HS: 15.7 ± 0.8 μmol/24 h; P < 0.001), but this difference was prevented by 7-NI (LS: 7.4 ± 1.3 vs. HS: 9.4 ± 1.6 μmol/24 h; NS). During nonselective blockade of NOS with N G-nitro-l-arginine methyl ester (l-NAME), renal vascular resistance (RVR) increased more in HS than LS (HS: +160 ± 17 vs. LS: +83 ± 10%; P < 0.001). This difference in response to nonselective NOS inhibition was prevented by pretreatment with 7-NI (HS: +28 ± 6 vs. LS: +34 ± 8%; NS) or F with volume replacement (HS: +79 ± 11 vs. LS: +62 ± 4%; NS). In conclusion, compared with salt restriction, HS intake increases NO generation and renal action that depend on nNOS and macula densa solute reabsorption.

Dopamine D1 Receptor Augmentation of D3 Receptor Action in Rat Aortic or Mesenteric Vascular Smooth Muscles

Abstract—Dopamine is an important modulator of blood pressure, in part, by regulating vascular resistance. To test the hypothesis that D1 and D3 receptors interact in vascular smooth muscle cells, we studied A10 cells, a rat aortic smooth muscle cell line, and rat mesenteric arteries that express both dopamine receptor subtypes. Fenoldopam, a D1-like receptor agonist, increased both D1 and D3 receptor protein in a time-dependent and a concentration-dependent manner in A10 cells. The effect of fenoldopam was specific because a D1-like receptor antagonist, SCH23390 (10−7 M/24 h), completely blocked the stimulatory effect of fenoldopam (10−7 M/24 h) (D3 receptor: control=21±1 density units [DU]); SCH23390=23±2 DU; fenoldopam=33±2 DU; fenoldopam+SCH23390=23±2 DU; n=10). D1 and D3 receptors physically interacted with each other because fenoldopam (10−7 M/24 h) increased D1/D3 receptor coimmunoprecipitation (35±5 versus 65±5 DU; n=8). A D3 receptor agonist, PD128907, relaxed mesenteric arterial rings independent of the endothelium, effects that were blocked by a D3 receptor antagonist, U99194A. Costimulation of D1 and D3 receptors led to additive vasorelaxation. We conclude that the D1 receptor regulates the D3 receptor by physical interaction and receptor expression. D1 receptor stimulation augments D3 receptor vasorelaxant effects. An interaction of D1 and D3 receptors may be involved in the regulation of blood pressure.

Response Gene to Complement 32, a Novel Regulator for Transforming Growth Factor-β-induced Smooth Muscle Differentiation of Neural Crest Cells

We previously developed a robust in vitro model system for vascular smooth muscle cell (VSMC) differentiation from neural crest cell line Monc-1 upon transforming growth factor-β (TGF-β) induction. Further studies demonstrated that both Smad and RhoA signaling are critical for TGF-β-induced VSMC development. To identify downstream targets, we performed Affymetrix cDNA array analysis of Monc-1 cells and identified a gene named response gene to complement 32 (RGC-32) to be important for the VSMC differentiation. RGC-32 expression was increased 5-fold after 2 h and 50-fold after 24 h of TGF-β induction. Knockdown of RGC-32 expression in Monc-1 cells by small interfering RNA significantly inhibited the expression of multiple smooth muscle marker genes, including SM α-actin (α-SMA), SM22α, and calponin. Of importance, the inhibition of RGC-32 expression correlated with the reduction of α-SMA while not inhibiting smooth muscle-unrelated c-fos gene expression, suggesting that RGC-32 is an important protein factor for VSMC differentiation from neural crest cells. Moreover, RGC-32 overexpression significantly enhanced TGF-β-induced α-SMA, SM22α, and SM myosin heavy chain promoter activities in both Monc-1 and C3H10T1/2 cells. The induction of VSMC gene promoters by RGC-32 appears to be CArG-dependent. These data suggest that RGC-32 controls VSMC differentiation by regulating marker gene transcription in a CArG-dependent manner. Further studies revealed that both Smad and RhoA signaling are important for RGC-32 activation.

p47 phox Is Required for Afferent Arteriolar Contractile Responses to Angiotensin II and Perfusion Pressure in Mice

Myogenic and angiotensin contractions of afferent arterioles generate reactive oxygen species. Resistance vessels express neutrophil oxidase-2 and -4. Angiotensin II activates p47phox/neutrophil oxidase-2, whereas it downregulates NOX-4. Therefore, we tested the hypothesis that p47phox enhances afferent arteriolar angiotensin contractions. Angiotensin II infusion in p47phox +/+ but not −/− mice increased renal cortical NADPH oxidase activity (7±1–12±1 [P<0.01] versus 5±1–7±1 103 · RLU · min−1 · &mgr;g protein−1 [P value not significant]), mean arterial pressure (77±2–91±2 [P<0.005] versus 74±2–77±1 mm Hg [P value not significant]), and renal vascular resistance (7.5±0.4–10.1±0.7 [P<0.01] versus 7.9±0.4–8.3±0.4 mm Hg/mL · min−1 · gram kidney weight−1 [P value not significant]). Afferent arterioles from p47phox −/− mice had a lesser myogenic response (3.1±0.4 versus 1.4±0.2 dynes · cm−1 · mm Hg−1; P<0.02) and a lesser (P<0.05) contraction to 10−6 M angiotensin II (diameter change +/+: 9.3±0.2–3.4±0.6 &mgr;m versus −/−: 9.9±0.6–7.5±0.4 &mgr;m). Angiotensin and increased perfusion pressure generated significantly (P<0.05) more reactive oxygen species in p47phox +/+ than −/− arterioles. Angiotensin II infusion increased the maximum responsiveness of afferent arterioles from p47phox +/+ mice to 10−6 M angiotensin II yet decreased the response in p47phox −/− mice. The angiotensin infusion increased the sensitivity to angiotensin II only in p47phox +/+ mice. We conclude that p47phox is required to enhance renal NADPH oxidase activity and basal afferent arteriolar myogenic and angiotensin II contractions and to switch afferent arteriolar tachyphylaxis to sensitization to angiotensin during a prolonged angiotensin infusion. These effects likely contribute to hypertension and renal vasoconstriction during infusion of angiotensin II.

Cyclooxygenase-1–Deficient Mice Have High Sleep-to-Wake Blood Pressure Ratios and Renal Vasoconstriction

We used cyclooxygenase-1 (COX-1)–deficient mice to test the hypothesis that COX-1 regulates blood pressure (BP) and renal hemodynamics. The awake time (AT) mean arterial pressures (MAPs) measured by telemetry were not different between COX-1+/+ and COX-1−/− (131±2 versus 126±3 mm Hg; NS). However, COX-1−/− had higher sleep time (ST) MAP (93±1 versus 97±2 mm Hg; P<0.05) and sleep-to-awake BP ratio (+8.6%; P<0.05). Under anesthesia with moderate sodium loading, COX-1−/− had higher MAP (109±5 versus 124±4 mm Hg; P<0.05), renal vascular resistance (23.5±1.6 versus 30.7±1.7 mm Hg · mL−1 · min−1 · g−1; P<0.05) and filtration fraction (33.7±2.1 versus 40.2±2.0%; P<0.05). COX-1−/− had a 89% reduction (P<0.0001) in the excretion of TxB2, a 76% reduction (P<0.01) in PGE2, a 40% reduction (P<0.0002) in 6-ketoPGF1&agr; (6keto), a 27% reduction (P<0.02) in 11-&bgr;PGF2&agr; (11&bgr;), a 35% reduction (P<0.01) in nitrate plus nitrite (NOx), and a 52% increase in metanephrine (P<0.02). The excretion of normetanephrine, a marker for sympathetic nervous activity, was reduced during ST in COX-1+/+ (6.9±0.9 versus 3.2±0.6 g · g−1 creatinine · 10−3; P<0.01). This was blunted in COX-1−/− (5.1±0.9 versus 4.9±0.7 g · g−1 creatinine · 10−3; NS). Urine collection during ST showed lower excretion of 6keto, 11&bgr;, NOx, aldosterone, sodium, and potassium than during AT in both COX-1+/+ and COX-1−/−, and there were positive correlations among these parameters (6keto versus NOx; P<0.005; 11&bgr; versus NOx; P<0.005; and NOx versus sodium; P<0.005). In conclusion, COX-1 mediates a suppressed sympathetic nervous activity and enhanced NO, which may contribute to renal vasodilatation and a reduced MAP while asleep or under anesthesia. COX-1 contributes to the normal nocturnal BP dipping phenomenon.

β1 Receptors Protect the Renal Afferent Arteriole of Angiotensin-Infused Rabbits from Norepinephrine-Induced Oxidative Stress

Renal afferent arterioles (Aff) from angiotensin II (AngII)-infused rabbits have enhanced contractions to AngII that are normalized by tempol (superoxide dismutase mimetic), whereas contractions to norepinephrine (NE) are normal and unaffected by tempol. Tested was the hypothesis that β-receptor stimulation with NE prevents enhanced reactivity and superoxide generation. Preconstricted Aff from AngII- or vehicle-infused rabbits were perfused at physiologic pressure. Aff from vehicle-infused rabbits had strong, endothelium-independent relaxations to dobutamine (β 1 -receptor agonist; 78 ± 6%; P 2 -receptor agonist; 13 ± 3%; P 3 -receptor agonist; 14 ± 3%; P versus −34 ± 3%; NS) and were unaffected by tempol (−32 ± 4%; NS). In contrast, phenylephrine contractions (α 1 agonist) were enhanced in Aff from AngII-infused rabbits (−59 ± 6 versus −46 ± 4%; P P 1 -receptor antagonist; −61 ± 9%), or Rp-cAMP (competitive inhibitor of cAMP; −56 ± 4%); were normalized by tempol; but were unaffected by ICI-118,551 (selective β 2 -receptor antagonist) or SR-59,230A (selective β 3 -receptor antagonist). Superoxide generation in Aff from AngII-infused rabbits that were assessed from ethidium:dihydroethidium was enhanced by addition of CGP-20,712A to NE but was normalized by tempol. Aff have robust α 1 -receptor contraction and β 1 -receptor dilation. NE elicits β 1 signaling via cAMP that moderates oxidative stress and contractions in Aff from AngII-infused rabbits.