High Salt Enhances Reactive Oxygen Species and Angiotensin II Contractions of Glomerular Afferent Arterioles From Mice With Reduced Renal Mass.

High salt, Ang II (angiotensin II), and reactive oxygen species enhance progression of chronic kidney disease. We tested the hypothesis that a high salt intake generates specific reactive oxygen species to enhance Ang II contractions of afferent arterioles from mice with reduced renal mass (RRM). C57BL/6 mice were subjected to surgical RRM or sham operations and received 6% or 0.4% NaCl salt diet for 3 months. Ang II contractions were measured in perfused afferent arterioles and superoxide (O2-) and hydrogen peroxide (H2O2) by fluorescence microscopy. RRM enhanced the afferent arteriolar gene expression for p47phox and neutrophil oxidase (NOX) 2 and high salt intake in RRM mice enhanced gene expression for angiotensin type 1 receptors, POLDIP2 and NOX4 and reduced catalase. High salt in mice with RRM enhanced arteriolar O2- and H2O2 generation and maximal contractions to Ang II (10-6 mol/L) that were dependent on O2- because they were prevented by gene deletion of p47phox and on H2O2 because they were prevented by transgenic smooth muscle cell expression of catalase (tgCAT-SMC) and POLDIP2 gene deletion. Three months of tempol normalized arteriolar reactive oxygen species and Ang II contractions. However, arteriolar contractions to lower concentrations of Ang II (10-8 to 10-11 mol/L) were paradoxically inhibited by H2O2 and POLDIP2. In conclusion, both O2- from p47phox/NOX2 and H2O2 from NOX4/POLDIP2 enhance maximal arteriolar Ang II contractions from RRM mice during high salt, but H2O2 and NOX4/POLDIP2 reduce the sensitivity to lower concentrations of Ang II by >100-fold. Tempol prevents all of these changes in function.

Regulation of fluid reabsorption in rat or mouse proximal renal tubules by asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase 1.

Nitric oxide prevents hypertension yet enhances proximal tubule Na+ reabsorption. Nitric oxide synthase is inhibited by asymmetric dimethylarginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) whose type 1 isoform is expressed abundantly in the proximal tubule (PT). We hypothesize that ADMA metabolized by DDAH-1 inhibits fluid reabsorbtion (Jv) by the proximal tubule. S2 segments of the PT were microperfused between blocks in vivo to assess Jv in anesthetized rats. Compared with vehicle, microperfusion of ADMA or Nω-nitro-l-arginine methyl ester (l-NAME) in the proximal tubule reduced Jv dose dependently. At 10-4 mol/l both reduced Jv by ~40% (vehicle: 3.2 ± 0.7 vs. ADMA: 2.1 ± 0.5, P < 0.01 vs. l-NAME: 1.9 ± 0.4 nl·min-1·mm-1, P < 0.01; n = 10). Selective inhibition of DDAH-1 in rats with intravenous L-257 (60 mg/kg) given 2 h before and L-257 (10-5 mol/l) perfused in the proximal tubule for 5 min reduced Jv by 32 ± 4% (vehicle: 3.2 ± 0.5 vs. L-257: 2.2 ± 0.5 nl·min-1·mm-1; P < 0.01) and increased plasma ADMA by ≈50% (vehicle: 0.46 ± 0.03 vs. L-257: 0.67 ± 0.03 µmol/l, P < 0.0001) without changing plasma symmetric dimethylarginine. Compared with nontargeted control small-interference RNA, knock down of DDAH-1 in mice by 60% with targeted small-interference RNAs (siRNA) reduced Jv by 29 ± 5% (nontargeted siRNA: 2.8 ± 0.20 vs. DDAH-1 knockdown: 1.9 ± 0.31 nl·min-1·mm-1, P < 0.05). In conclusion, fluid reabsorption in the proximal tubule is reduced by tubular ADMA or by blocking its metabolism by DDAH-1. L-257 is a novel regulator of proximal tubule fluid reabsorption.

NRF2 prevents hypertension, increased ADMA, microvascular oxidative stress, and dysfunction in mice with two weeks of ANG II infusion.

Nuclear factor erythyroid factor 2 (Nrf2) transcribes genes in cultured endothelial cells that reduce reactive oxygen species (ROS) and generate nitric oxide (NO) or metabolize asymmetric dimethylarginine (ADMA), which inhibits NO synthase (NOS). Therefore, we undertook a functional study to test the hypothesis that activation of Nrf2 by tert-butylhydroquinone (tBHQ) preserves microvascular endothelial function during oxidative stress. Wild-type CB57BL/6 (wt), Nrf2 wt (+/+), or knockout (-/-) mice received vehicle (Veh) or tBHQ (0.1%; activator of Nrf2) during 14-day infusions of ANG II (to induce oxidative stress) or sham. MAP was recorded by telemetry. Mesenteric resistance arterioles were studied on isometric myographs and vascular NO and ROS by fluorescence microscopy. ANG II increased the mean arterial pressure (112 ± 5 vs. 145 ± 5 mmHg; P < 0.01) and excretion of 8-isoprostane F2α (2.8 ± 0.3 vs. 3.8 ± 0.3 ng/mg creatinine; P < 0.05) at 12-14 days. However, 12 days of ANG II reduced endothelium-derived relaxation (27 ± 5 vs. 17 ± 3%; P < 0.01) and NO (0.38 ± 0.07 vs. 0.18 ± 0.03 units; P < 0.01) but increased microvascular remodeling, endothelium-derived contractions (7.5 ± 0.5 vs. 13.0 ± 1.7%; P < 0.01), superoxide (0.09 ± 0.03 vs. 0.29 ± 0.08 units; P < 0.05), and contractions to U-46,619 (87 ± 6 vs. 118 ± 3%; P < 0.05), and endothelin-1(89 ± 4 vs. 123 ± 12%; P < 0.05). tBHQ prevented all of these effects of ANG II at 12-14 days in Nrf2+/+ mice but not in Nrf2-/- mice. In conclusion, tBHQ activates Nrf2 to prevent microvascular endothelial dysfunction, remodeling, and contractility, and moderate ADMA and hypertension at 12-14 days of ANG II infusion, thereby preserving endothelial function and preventing hypertension.

Superoxide and hydrogen peroxide counterregulate myogenic contractions in renal afferent arterioles from a mouse model of chronic kidney disease.

Myogenic contractions protect kidneys from barotrauma but are impaired in chronic kidney disease (CKD). Since myogenic contractions are enhanced by superoxide but impaired by hydrogen peroxide, we tested the hypothesis that they are counterregulated by superoxide and H2O2 from NOX2/p47phox and/or NOX4/POLDIP2 in CKD. Myogenic contraction in isolated perfused afferent arterioles from mice with surgical 5/6 nephrectomy or sham operations fed a 6% sodium chloride diet was measured directly while superoxide and H2O2 were measured by fluorescence microscopy. Compared to sham-operated animals, an increase in perfusion pressure of arterioles from CKD mice doubled superoxide (21 versus 11%), increased H2O2 seven-fold (29 versus 4%), and reduced myogenic contractions profoundly (-1 versus -14%). Myogenic contractions were impaired further by PEG-superoxide dismutase or in arterioles from p47phox-/- (versus wild type) mice but became supra-normal by PEG-catalase or in mice with transgenic expression of catalase in vascular smooth muscle cells (-11 versus -1%). Single arterioles from mice with CKD expressed over 40% more mRNA and protein for NOX4 and POLDIP2. Myogenic responses in arterioles from POLDIP2 +/- (versus wild type) mice with CKD had over an 85% reduction in H2O2, but preserved superoxide and a normal myogenic response. Tempol administration to CKD mice for 3 months decreased afferent arteriolar superoxide and H2O2 and maintained myogenic contractions. Thus, afferent arteriolar superoxide generated by NOX2/p47phox opposes H2O2 generated by NOX4/POLDIP2 whose upregulation in afferent arterioles from mice with CKD accounts for impaired myogenic contractions.

Remodeling of Afferent Arterioles From Mice With Oxidative Stress Does Not Account for Increased Contractility but Does Limit Excessive Wall Stress.

Because superoxide dismutase (SOD) knockout enhances arteriolar remodeling and contractility, we hypothesized that remodeling enhances contractility. In the isolated and perfused renal afferent arterioles from SOD wild type (+/+) and gene-deleted mice, contractility was assessed from reductions in luminal diameter with perfusion pressure from 40 to 80 mm Hg (myogenic responses) or angiotensin II (10(-6) mol/L), remodeling from media:lumen area ratio, superoxide (O2 (·-)) and hydrogen peroxide (H2O2) from fluorescence microscopy, and wall stress from wall tension/wall thickness. Compared with +/+ strains, arterioles from SOD1-/-, SOD2+/-, and SOD3-/- mice developed significantly (P<0.05) more O2 (·-) with perfusion pressure and angiotensin II and significantly increased myogenic responses (SOD1-/-: -20.7±2.2% versus -12.7±1.6%; SOD2+/-: -7.4±1.3% versus -12.6±1.4%; and SOD3-/-: -9.1±1.9% versus -15.8±2.2%) and angiotensin II contractions and ≈2-fold increased media:lumen ratios. Media:lumen ratios correlated with myogenic responses (r(2) =0.23; P<0.01), angiotensin II contractions (r(2)=0.57; P<0.0001), and active wall tension (r(2) =0.19; P<0.01), but not with active wall stress (r(2)=0.08; NS). Differences in myogenic responses among SOD3 mice were abolished by bath addition of SOD and were increased 3 days after inducing SOD3 knockout (-26.9±1.7% versus -20.1±0.7%; P<0.05), despite unchanged media:lumen ratios (2.01±0.09 versus 2.02±0.03; NS). We conclude that cytosolic, mitochondrial, or extracellular O2 (·-) enhance afferent arteriolar contractility and remodeling. Although remodeling does not enhance contractility, it does prevent the potentially damaging effects of increased wall stress.

Thromboxane prostanoid receptors enhance contractions, endothelin-1, and oxidative stress in microvessels from mice with chronic kidney disease.

Cardiovascular disease is frequent in chronic kidney disease and has been related to angiotensin II, endothelin-1 (ET-1), thromboxane A2, and reactive oxygen species (ROS). Because activation of thromboxane prostanoid receptors (TP-Rs) can generate ROS, which can generate ET-1, we tested the hypothesis that chronic kidney disease induces cyclooxygenase-2 whose products activate TP-Rs to enhance ET-1 and ROS generation and contractions. Mesenteric resistance arterioles were isolated from C57/BL6 or TP-R+/+ and TP-R-/- mice 3 months after SHAM-operation (SHAM) or surgical reduced renal mass (RRM, n=6/group). Microvascular contractions were studied on a wire myograph. Cellular (ethidium: dihydroethidium) and mitochondrial (mitoSOX) ROS were measured by fluorescence microscopy. Mice with RRM had increased excretion of markers of oxidative stress, thromboxane, and microalbumin; increased plasma ET-1; and increased microvascular expression of p22(phox), cyclooxygenase-2, TP-Rs, preproendothelin and endothelin-A receptors, and increased arteriolar remodeling. They had increased contractions to U-46,619 (118 ± 3 versus 87 ± 6, P<0.05) and ET-1 (108 ± 5 versus 89 ± 4, P<0.05), which were dependent on cellular and mitochondrial ROS, cyclooxygenase-2, and TP-Rs. RRM doubled the ET-1-induced cellular and mitochondrial ROS generation (P<0.05). TP-R-/- mice with RRM lacked these abnormal structural and functional microvascular responses and lacked the increased systemic and the increased microvascular oxidative stress and circulating ET-1. In conclusion, RRM leads to microvascular remodeling and enhanced ET-1-induced cellular and mitochondrial ROS and contractions that are mediated by cyclooxygenase-2 products activating TP-Rs. Thus, TP-Rs can be upstream from enhanced ROS, ET-1, microvascular remodeling, and contractility and may thereby coordinate vascular dysfunction in chronic kidney disease.

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-γ 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 µmol·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-γ 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-γ 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-γ. Expression of Nrf2 was reduced by knockdown of PPAR-γ, whereas PPAR-γ 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-γ to increase eNOS and its phosphorylation and activity thereby coordinating 3 pathways that enhance endothelial NO generation.

Angiotensin II reduces transport-dependent oxygen consumption but increases transport-independent oxygen consumption in immortalized mouse proximal tubular cells.

Oxidative stress is closely associated with renal dysfunction following diabetes and hypertension. Angiotensin II (Ang II) can activate the NADPH-oxidase, increasing oxidative stress that is thought to blunt proximal tubular electrolyte transport and thereby oxygen consumption (QO2). We investigated the effect of Ang II on QO2 in immortalized mouse proximal tubular cells over-expressing the NADPH oxidase subunit p22(phox); a model of increased oxidative stress. Cultured cells were exposed to either Ang II or H2O2 for 48 h. QO2 was determined during baseline (113 mmol/l NaCl; transport-dependent QO2) and during sodium-free conditions (transport-independent QO2). Ang II reduced transport-dependent QO2 in wild-types, but not in p22(phox) which also displayed increased QO2 at baseline. Transport-independent QO2 was increased in p22(phox) and Ang II had no additional effect, whereas it increased QO2 in wild-type. Addition of H2O2 reduced transport-dependent QO2 in wild-types, but not in p22(phox). Transport-independent QO2 was unaffected by H2O2. The similar effects of Ang II and H2O2 to reduce transport-dependent QO2 suggest a direct regulatory role of oxidative stress. In accordance, the transport-dependent QO2 was reduced in p22(phox) already during baseline. The effects of Ang II on transport-independent QO2 was not replicated by H2O2, indicating direct regulation via Ang II-receptors independently of oxidative stress. However, the Ang II effect was absent in p22(phox), suggesting that oxidative stress also modulates normal Ang II signaling. In conclusion, Ang II affects both transport-dependent and transport-independent QO2 in proximal tubular cells and may be an important pathway modulating renal QO2.

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-ß 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.

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 p47(phox)/neutrophil oxidase-2, whereas it downregulates NOX-4. Therefore, we tested the hypothesis that p47(phox) enhances afferent arteriolar angiotensin contractions. Angiotensin II infusion in p47(phox) +/+ but not -/- mice increased renal cortical NADPH oxidase activity (7±1-12±1 [P<0.01] versus 5±1-7±1 10(3) · RLU · min(-1) · µg 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 p47(phox) -/- 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 µm versus -/-: 9.9±0.6-7.5±0.4 µm). Angiotensin and increased perfusion pressure generated significantly (P<0.05) more reactive oxygen species in p47(phox) +/+ than -/- arterioles. Angiotensin II infusion increased the maximum responsiveness of afferent arterioles from p47(phox) +/+ mice to 10(-6) M angiotensin II yet decreased the response in p47(phox) -/- mice. The angiotensin infusion increased the sensitivity to angiotensin II only in p47(phox) +/+ mice. We conclude that p47(phox) 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.

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 µmol · 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.

Glomerular tubular balance is suppressed in adenosine type 1 receptor-deficient mice.

Glomerular tubular balance maintains a stable fractional solute and fluid reabsorption in the proximal tubule over a range of glomerular filtration rates. The mediators of this process are unknown. We tested the hypothesis that adenosine, produced in proximal tubule cells acting on adenosine type 1 receptors (A(1)-AR) promotes Na(+) and fluid uptake and mediates glomerular tubular balance. Absolute proximal fluid reabsorption (J(v)) was measured by in vivo microperfusion in A(1)-AR knockout and wild-type mice during perfusion of the closed proximal tubule at 2-10 nl/min. J(v) increased with perfusate flow from 2-4 nl/min in both strains, but the fractional increase was lower in A(1)-AR(-/-) mice (A(1)-AR(+/+): 114% vs. A(1)-AR(-/-): 38%; P < 0.001), suggesting reduced glomerular tubular balance (GTB). At higher perfusion rates, J(v) increased modestly in both strains, indicating less GTB at higher flow. The physiological effects of reduced GTB in A(1)-AR(-/-) mice were assessed from the response to an acute volume load (1 ml/2 min). Na(+) excretion and urine flow increased 76 and 73% more in A(1)-AR(-/-) mice than A(1)-AR(+/+) over the following 30 min, accompanied by a higher proximal tubule flow (A(1)-AR(-/-): 6.9 ± 0.9 vs. A(1)-AR(+/+): 5.2 ± 0.6 nl/min; P < 0.05). The expression of the sodium-hydrogen exchanger 3 and sodium phosphate cotransporter-2 were similar between strains. In conclusion, GTB is dependent on adenosine acting on type 1 receptors in the proximal tubule. This may contribute to acute changes in Na(+) and fluid reabsorption.

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 p22(phox) 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 p22(phox)-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 micromol/L (P<0.005) in p22(phox)-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.

Renal proximal tubular reabsorption is reduced in adult spontaneously hypertensive rats: roles of superoxide and Na+/H+ exchanger 3.

Proximal tubule reabsorption is regulated by systemic and intrinsic mechanisms, including locally produced autocoids. Superoxide, produced by NADPH oxidase enhances NaCl transport in the loop of Henle and the collecting duct, but its role in the proximal tubule is unclear. We measured proximal tubule fluid reabsorption (Jv) in WKY rats and compared that with Jv in the spontaneously hypertensive rat (SHR), a model of enhanced renal superoxide generation. Rats were treated with the NADPH oxidase inhibitor apocynin (Apo) or with small interfering RNA for p22(phox), which is the critical subunit of NADPH oxidase. Jv was lower in SHR compared with Wistar-Kyoto rats (WKY; WKY: 2.3+/-0.3 vs SHR: 1.1+/-0.2 nL/min per millimeter; n=9 to 11; P<0.001). Apo and small interfering RNA to p22(phox) normalized Jv in SHRs but had no effect in WKY rats. Jv was reduced in proximal tubules perfused with S-1611, a highly selective inhibitor of the Na(+)/H(+) exchanger 3, the major Na(+) uptake pathway in the proximal tubule, in WKY rats but not in SHRs. Pretreatment with Apo restored an effect of S-1611 to reduce Jv in the SHRs (SHR+Apo: 2.9+/-0.4 vs SHR+Apo+S-1611: 1.0+/-0.3 nL/min per millimeter; P<0.001). However, because expression of the Na(+)/H(+) exchanger 3 was similar between SHR and WKY rats, this suggests that superoxide affects Na(+)/H(+) exchanger 3 activity. Direct microperfusion of Tempol or Apo into the proximal tubule also restored Jv in SHRs. In conclusion, superoxide generated by NADPH oxidase inhibits proximal tubule fluid reabsorption in SHRs. This finding implies that proximal tubule fluid reabsorption is regulated by redox balance, which may have profound effects on ion and fluid homeostasis in the hypertensive kidney.

Cellular ADMA: regulation and action.

Asymmetric (N(G),N(G)) dimethylarginine (ADMA) is present in plasma and cells. It can inhibit nitric oxide synthase (NOS) that generates nitric oxide (NO) and cationic amino acid transporters (CATs) that supply intracellular NOS with its substrate, l-arginine, from the plasma. Therefore, ADMA and its transport mechanisms are strategically placed to regulate endothelial function. This could have considerable clinical impact since endothelial dysfunction has been detected at the origin of hypertension and chronic kidney disease (CKD) in human subjects and may be a harbinger of large vessel disease and cardiovascular disease (CVD). Indeed, plasma levels of ADMA are increased in many studies of patients at risk for, or with overt CKD or CVD. However, the levels of ADMA measured in plasma of about 0.5micromol.l(-1) may be below those required to inhibit NOS whose substrate, l-arginine, is present in concentrations many fold above the Km for NOS. However, NOS activity may be partially inhibited by cellular ADMA. Therefore, the cellular production of ADMA by protein arginine methyltransferase (PRMT) and protein hydrolysis, its degradation by N(G),N(G)-dimethylarginine dimethylaminohydrolase (DDAH) and its transmembrane transport by CAT that determines intracellular levels of ADMA may also determine the state of activation of NOS. This is the focus of the review. It is concluded that cellular levels of ADMA can be 5- to 20-fold above those in plasma and in a range that could tonically inhibit NOS. The relative importance of PRMT, DDAH and CAT for determining the intracellular NOS substrate:inhibitor ratio (l-arginine:ADMA) may vary according to the pathophysiologic circumstance. An understanding of this important balance requires knowledge of these three processes that regulate the intracellular levels of ADMA and arginine.

Asymmetric dimethylarginine and lipid peroxidation products in early autosomal dominant polycystic kidney disease.

BACKGROUND: Patients with autosomal dominant polycystic kidney disease (ADPKD) with normal renal function have endothelial dysfunction and decreased nitric oxide synthase activity in subcutaneous resistance vessels. We investigated asymmetric dimethylarginine (ADMA) as a marker of an inhibitor of nitric oxide synthase and the lipid peroxidation product 13-hydroxyoctadecadienoic acid (HODE) as a marker of oxidative stress in patients with early ADPKD. STUDY DESIGN: Cross-sectional study. SETTING & PARTICIPANTS: Patients with early ADPKD (n = 27) and age-matched volunteers (n = 30) from a single academic medical center. FACTOR: Patients with ADPKD versus controls. OUTCOMES & MEASUREMENT: Plasma (P) levels, urinary (U) excretion, and urinary clearance (C) of ADMA and HODE. Because of multiple comparisons, P for significance is considered less than 0.0167. RESULTS: Patients with ADPKD had significantly increased P(ADMA) levels (604 +/- 131 versus 391 +/- 67 nmol/L; P < 0.01) and U(ADMA) excretion (22 +/- 4 versus 15.2 +/- 3 nmol/micromol creatinine; P = 0.01), decreased C(ADMA) (25 +/- 3 versus 33 +/- 4 mL/min; P = 0.01), increased P(HODE) levels (316 +/- 64 versus 230 +/- 38 nmol/L; P < 0.01) and U(HODE) excretion (467 +/- 67 versus 316 +/- 40 nmol/micromol creatinine; P < 0.01), and decreased plasma nitrite plus nitrate (P(NOx)) levels (21 +/- 5 versus 32 +/- 6 micromol/L; P < 0.01) and U(NOx) excretion (59 +/- 7 versus 138 +/- 27 micromol/micromol creatinine; P < 0.01). LIMITATIONS: Small sample size, cross-sectional nature of study, and limited number of markers of oxidative stress. CONCLUSIONS: P(ADMA) and P(HODE) levels are increased in patients with early ADPKD. Increased P(ADMA) level is related to decreased C(ADMA) and is accompanied by oxidative stress.

Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems.

Asymmetric (N(G),N(G))-dimethylarginine (ADMA) inhibits nitric oxide (NO) synthases (NOS). ADMA is a risk factor for endothelial dysfunction, cardiovascular mortality, and progression of chronic kidney disease. Two isoforms of dimethylarginine dimethylaminohydrolase (DDAH) metabolize ADMA. DDAH-1 is the predominant isoform in the proximal tubules of the kidney and in the liver. These organs extract ADMA from the circulation. DDAH-2 is the predominant isoform in the vasculature, where it is found in endothelial cells adjacent to the cell membrane and in intracellular vesicles and in vascular smooth muscle cells among the myofibrils and the nuclear envelope. In vivo gene silencing of DDAH-1 in the rat and DDAH +/- mice both have increased circulating ADMA, whereas gene silencing of DDAH-2 reduces vascular NO generation and endothelium-derived relaxation factor responses. DDAH-2 also is expressed in the kidney in the macula densa and distal nephron. Angiotensin type 1 receptor activation in kidneys reduces the expression of DDAH-1 but increases the expression of DDAH-2. This rapidly evolving evidence of isoform-specific distribution and regulation of DDAH expression in the kidney and blood vessels provides potential mechanisms for nephron site-specific regulation of NO production. In this review, the recent advances in the regulation and function of DDAH enzymes, their roles in the regulation of NO generation, and their possible contribution to endothelial dysfunction in patients with cardiovascular and kidney diseases are discussed.

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress.

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.

Response gene to complement 32, a novel regulator for transforming growth factor-beta-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-beta (TGF-beta) induction. Further studies demonstrated that both Smad and RhoA signaling are critical for TGF-beta-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-beta 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 alpha-actin (alpha-SMA), SM22alpha, and calponin. Of importance, the inhibition of RGC-32 expression correlated with the reduction of alpha-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-beta-induced alpha-SMA, SM22alpha, 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.

Chymase-like angiotensin II-generating activity in end-stage human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by exuberant inflammation and fibrosis, a process believed to contribute to progressive loss of normal renal function. Despite early-onset hypertension and intrarenal renin/angiotensin II (AngII) activation, angiotensin-converting enzyme (ACE) inhibition does not consistently confer renal protection in ADPKD. The hypothesis was that mast cells within the inflammatory interstitium release chymase, an enzyme capable of efficient conversion of AngI to AngII, providing an ACE-independent route of AngII generation. End-stage ADPKD renal tissue extracts and cyst fluids were assayed for time-dependent, chymostatin-inhibitable conversion of (125)I-AngI to (125)I-AngII under conditions of ACE and aminopeptidase inhibition by means of HPLC. Thirteen of 14 ADPKD kidney extracts exhibited chymase-like AngII-generating capacity; calculated initial reaction rates averaged 3.9 +/- 2.9 fmol AngII/min/ micro g protein with a mean maximal conversion of 55% +/- 30% of added substrate. AngII-generating activity was both protein and substrate dependent. All five cyst fluid samples were negative. Chymase-like activity was detectable in only three of six non-ADPKD kidney extracts. Immunoreactive chymase protein was present in/around mast cells within the fibrotic renal interstitium in all samples. Findings demonstrate for the first time the presence of mast cells, mast cell-associated immunoreactive chymase protein, and chymase-like AngII generating capacity in ADPKD cystic kidneys. Results support the potential for ACE-independent AngII generation and for mast cell-initiated inflammatory processes in ADPKD, each with therapeutic implications for ADPKD renal progression.