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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. [ABSTRACT FROM AUTHOR]

Fibroblast growth factors (FGFs) participate in organ development and tissue maintenance, as well as the control of vascular function. The paracrine-acting FGFs are stored in the extracellular matrix, and their release is controlled by a secreted FGF-binding protein (FGF-BP, FGFBP1, and BP1) that modulates FGF receptor signaling. A genetic polymorphism in the human FGFBP1 gene was associated with higher gene expression and an increased risk of familial hypertension. Here, we report on the effects of inducible BP1 expression in a transgenic mouse model. Induction of BP1 expression in adult animals leads to a sustained rise in mean arterial pressure by >30 mm Hg. The hypertensive effect of BP1 expression is prevented by candesartan, an angiotensin II (AngII) receptor antagonist, or by tempol, an inhibitor of reactive oxygen species. In vivo, BP1 expression sensitizes peripheral resistance vessels to AngII constriction by 20-fold but does not alter adrenergic vasoconstriction. FGF receptor kinase inhibition reverses the sensitization to AngII. Also, constriction of isolated renal afferent arterioles by AngII is enhanced after BP1 expression and blocked by FGF receptor kinase inhibition. Furthermore, AngII-mediated constriction of renal afferent arterioles is abolished in FGF2-/- mice but can be restored by add-back of FGF2 plus BP1 proteins. In contrast to AngII, adrenergic constriction is not affected in the FGF2-/- model. Proteomics and gene expression analysis of kidney tissues after BP1 induction show that MAPK (mitogen-activated protein kinase) signaling via MKK4 (MAPK kinase 4), p38, and JNK (c-Jun N-terminal kinase) integrates the crosstalk of the FGF receptor and AngII pathways and thus impact vascular tone and blood pressure. [ABSTRACT FROM AUTHOR]

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 ( r2 =0.23; P<0.01), angiotensin II contractions (r2=0.57; P<0.0001), and active wall tension (r2 =0.19; P<0.01), but not with active wall stress (r2=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. [ABSTRACT FROM AUTHOR]

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 p22phox, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ovarian hormone loss increases reactive oxidative species, endothelial dysfunction, and cardiovascular disease. Because perivascular adipose tissue (PVAT) regulates endothelial function, we hypothesized that reactive oxidative species in PVAT mediate adverse microvascular effects of ovarian hormone deficiency. Rats were ovariectomized or sham operated and given vehicle or tempol for 6 weeks. Mesenteric resistance arterioles from ovariectomized compared with sham-operated rats had dysfunctional responses to acetylcholine (ACh) including decreased ACh-induced endothelium-dependent relaxation (50±6% versus 72±2%) and endothelium-dependent relaxation factor (17±4% versus 37±2%) and increased endothelium-dependent contracting factor (27±5% versus 9±3%). OVX rat mesenteric arterioles had increased contractions to the thromboxane/prostanoid receptor agonist U-46 619 (58±3% versus 40±5%) and increased reactive oxidative species (tempo-9-AC fluorescence) with U-46 619 (0.65±0.17 versus 0.14±0.06 Δ unit) or ACh (0.49±0.09 versus 0.09±0.05 Δ unit) and increased p22(phox) protein expression (0.89±0.05 versus 0.18±0.04 Δ unit), whereas nitric oxide activity (DAF-FM [4-amino-5-methylamino-2',7'-difluorofluorescein diacetate] fluorescence) with ACh was reduced (0.39±0.1 versus 0.70±0.10 Δ unit). No differences were found in endothelium-dependent hyperpolarizing factor or contractile responses to phenylephrine. PVAT enhanced ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide only in sham-operated rats. Tempol prevented ovariectomy-induced endothelial dysfunction and restored the enhancing effects of PVAT on ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide in ovariectomized rat vessels, but both tempol and PVAT were required to normalize the enhanced U-46 619 contractions after ovariectomy. In conclusion, ovariectomy redirects endothelial responses from relaxation to contraction by reducing vascular nitric oxide, augmenting thromboxane/prostanoid receptor signaling, and attenuating the vasodilatory effects of PVAT, all of which were dependent on reactive oxidative species. [ABSTRACT FROM AUTHOR]

Ovarian hormone loss increases reactive oxidative species, endothelial dysfunction, and cardiovascular disease. Because perivascular adipose tissue (PVAT) regulates endothelial function, we hypothesized that reactive oxidative species in PVAT mediate adverse microvascular effects of ovarian hormone deficiency. Rats were ovariectomized or sham operated and given vehicle or tempol for 6 weeks. Mesenteric resistance arterioles from ovariectomized compared with sham-operated rats had dysfunctional responses to acetylcholine (ACh) including decreased ACh-induced endotheliumdependent relaxation (50±6% versus 72±2%) and endothelium-dependent relaxation factor (17±4% versus 37±2%) and increased endothelium-dependent contracting factor (27±5% versus 9±3%). OVX rat mesenteric arterioles had increased contractions to the thromboxane/prostanoid receptor agonist U-46619 (58±3% versus 40±5%) and increased reactive oxidative species (tempo-9-AC fluorescence) with U-46619 (0.65±0.17 versus 0.14±0.06 A unit) or ACh (0.49±0.09 versus 0.09±0.05 A unit) and increased p22phox protein expression (0.89±0.05 versus 0.18±0.04 A unit), whereas nitric oxide activity (DAF-FM [4-amino-5-methylamino-2',7'-difluorofluorescein diacetate] fluorescence) with ACh was reduced (0.39±0.1 versus 0.70±0.10 A unit). No differences were found in endothelium-dependent hyperpolarizing factor or contractile responses to phenylephrine. PVAT enhanced ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide only in sham-operated rats. Tempol prevented ovariectomy-induced endothelial dysfunction and restored the enhancing effects of PVAT on ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide in ovariectomized rat vessels, but both tempol and PVAT were required to normalize the enhanced U-46619 contractions after ovariectomy. In conclusion, ovariectomy redirects endothelial responses from relaxation to contraction by reducing vascular nitric oxide, augmenting thromboxane/prostanoid receptor signaling, and attenuating the vasodilatory effects of PVAT, all of which were dependent on reactive oxidative species. [ABSTRACT FROM AUTHOR]

The article presents a study on the effect of p47phox in superoxide generation, hypertension and renal vasoconstriction. The research concludes the component is necessary for NADPH oxidase activation in kidneys with the introduction of Angiotensin II (Ang II). The investigation also proves Ang II infusion influences the reaction of afferent arterioles.

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. [ABSTRACT FROM AUTHOR]

The article presets a study which hypothesized that stretch generates superoxide that mediates myogenic responses in mice. Basal tone and myogenic contractions were reduced by pegylated superoxide dismutase, Tempol, apocynin, and diphenyleneiodinium, compared with vehicle. The study concluded that increasing the pressure within afferent arterioles resulted to calcium-independent increased vascular superoxide production from nicotinamide adenine dinucleotide phosphate oxidase.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 x 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 x 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 x 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. [ABSTRACT FROM AUTHOR]

Angiotensin II maintains renal cortical blood flow and renal oxygenation in the clipped kidney of early 2-kidney, 1-clip Goldblatt hypertensive (2K,1C) rats. The involvement of Ang II is believed to decline, whereas oxidative stress increases during the progression of 2K,1C hypertension. We investigated the hypothesis that the acute administration of drugs to inhibit reactive oxygen species (Tempol), angiotensin II type 1 receptors (candesartan), or angiotensin-converting enzyme (enalaprilat) lowers mean arterial pressure and increases kidney blood flow and oxygenation in the clipped kidney of chronic 2K,1C rats in contrast to sham controls. Twelve months after left renal artery clipping or sham, mean arterial pressure, renal cortical blood flow, and renal cortical and medullary oxygen tension were measured after acute administration of Tempol followed by enalaprilat or candesartan followed by enalaprilat. The mean arterial pressure of the 2K,1C rat was reduced by candesartan (-9%) and, more effectively, by Tempol (-35%). All of the applied treatments had similar blood pressure-lowering effects in sham rats (average: -21%). Only Tempol increased cortical blood flow (+35%) and cortical and medullary oxygen tensions (+17% and +94%, respectively) in clipped kidneys of 2K,1C rats. Administration of enalaprilat had no additional effect, except for a modest reduction in cortical blood flow in the clipped kidney of 2K,1C rats when coadministered with candesartan (-10%). In conclusion, acute administration of Tempol is more effective than candesartan in reducing the mean arterial blood pressure and improving renal blood perfusion and oxygenation in the clipped kidney of chronic 2K,1C rats. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article focuses on angiotensin (Ang) II and its effect on hypertension and vascular dysfunction. Ang II-generated reactive oxygen species, the source of O2 or superoxides, isoforms of nicotinamide-adenine dinucleotide phosphate oxidase, extracellular signalling, salt-sensitive hypertension, levels of Ang II in the brain and kidney, cyclooxygenase-2 expression, and animal experiments are discussed.

Angiotensin-converting enzyme inhibitors (ACEIs) decrease the glomerular filtration rate and renal blood flow in the clipped kidneys of early 2-kidney, 1-clip Goldblatt hypertensive rats, but the consequences for oxygenation are unclear. We investigated the hypothesis that angiotensin II type 1 or angiotensin II type 2 receptors or NO synthase mediate renal oxygenation responses to ACEI. Three weeks after left renal artery clipping, kidney function, oxygen (O(2)) use, renal blood flow, renal cortical blood flow, and renal cortical oxygen tension (Po(2)) were measured after acute administration of an ACEI (enalaprilat) and after acute administration of ACEI following acute administration of an angiotensin II type 1 or angiotensin II type 2 receptor blocker (candesartan or PD-123,319) or an NO synthase blocker (N(G)-nitro-L-arginine methyl ester with control of renal perfusion pressure) and compared with mechanical reduction in renal perfusion pressure to the levels after ACEI. The basal renal cortical Po(2) of clipped kidneys was significantly lower than contralateral kidneys (35+/-1 versus 51+/-1 mm Hg; n=40 each). ACEI lowered renal venous Po(2), cortical Po(2), renal blood flow, glomerular filtration rate, and cortical blood flow and increased the renal vascular resistance in the clipped kidney, whereas mechanical reduction in renal perfusion pressure was ineffective. PD-123,319 and N(G)-nitro-L-arginine methyl ester, but not candesartan, reduced the Po(2) of clipped kidneys and blocked the fall in Po(2) with acute ACEI administration. In conclusion, oxygen availability in the clipped kidney is maintained by angiotensin II generation, angiotensin II type 2 receptors, and NO synthase. This discloses a novel mechanism whereby angiotensin can prevent hypoxia in a kidney challenged with a reduced perfusion pressure. [ABSTRACT FROM AUTHOR]

Background: Remodeling of resistant vessels in hypertension is postulated to enhance their contractility. We showed that knockout of SOD increased superoxide (O2.-), contractility and remodeling of afferent arterioles (Affs). Therefore, we tested the hypothesis that the remodeling causes the enhanced contractility.Methods: Myogenic responses (MRs) were assessed from the slope of the regression of active wall tension (AWT) on perfusion pressure (PP) in wild type and knockout SOD-1,-2 or -3 mice and in mice 3 days after induction of SOD-3 knockout before remodeling had developed. Contractions to 10-6 M Ang II were tested also. O2.- was assessed from PEG-SOD inhibitable ethidium:dihydroethidium fluorescence and remodeling from media:lumen area ratio (M:L).Results: 1. Compared to +/+, increasing PP or 10-6 M Ang II increased O2.- of Affs more in SOD1-/-, SOD2+/- and SOD3-/- (P<0.01). 2. Compared to +/+, MRs (dynes.cm-1.mmHg-1) were significantly (P<0.05) increased in SOD1-/- (3.49 ± 0.30 vs 2.30 ± 0.27), in SOD2+/- (3.85 ± 0.48 vs 1.80 ± 0.34), and in SOD3-/- (3.51 ± 0.20 vs 2.43 ± 0.15). 3. Compared to +/+, Ang II contractions were also stronger in knockout mice (P<0.001). 4. Compared to +/+, M:L ratio of Affs were significantly (P<0.001) greater in SOD1-/- (4.0 ± 0.2 vs 1.8 ± 0.2), in SOD2+/- (3.7 ± 0.4 vs 1.8 ± 0.4), and in SOD3-/- (4.5 ± 0.2 vs 1.9 ± 0.1). 5. Across genotypes, MR (r2 =0.32, P<0.005) and Ang II contractions (r2 = 0.56, P<0.005) were strongly correlated with M:L ratios. 6. Changes in AWT (Tphysiol - Tpassive) during increase in PP were dependent on SOD genotype (P<0.01) yet changes in vessel wall stress (AWT/wall thickness) were independent (P=NS). 7. Compared to controls , MRs and Ang II contractions were increased 3 days after inducing SOD3 knockout (MR, 3.76 ± 0.55 vs 2.26 ± 0.01, P<0.05; Ang II, -66 ± 4% vs -49 ± 4%, P<0.01) and were not significantly different from SOD3-/- mice. However, the M:L ratio of these Affs was unchanged (2.0 ± 0.1 vs 1.8 ± 0.2, NS).Conclusions: lifetime increases in cytosolic, mitochondrial or extracellular O2.- enhances afferent arteriolar contractility and remodeling. The remodeling is not required for the enhanced contractility, but does prevent the damaging effects of increased wall stress. [ABSTRACT FROM AUTHOR]

Background: Proximal tubule (PT) fluid reabsorption (Jv) is diminished by knockout of NOS-I or III. ADMA is an endogenous inhibitor of NOS, but its effects on tubular function are unknown. ADMA is degradated by DDAH whose type 1 isoform (DDAH-1) is richly expressed in the PT. SNIPs of DDAH-1 predict the decline of GFR in chronic kidney disease (CKD).Hypothesis: that DDAH-1 metabolizes ADMA in the PT thereby reducing its plasma concentration and enhancing Jv.Methods: Jv was measured in anesthetized rats and mice in S2 segments of the PT by direct in vivo micropuncture and microperfusion of artificial tubular fluid (ATF).Results: Addition of L-257 (10-4 M; DDAH-1 inhibitor) vs vehicle to ATF perfusing the PT of rats for 10 mins did not affect Jv (3.4 ± 0.20 vs 3.1±0.39 nl/min/mm). However, a bolus i.v. injection (60mg/kg) of L-257 2hrs before and 10 mins of PT perfusion significantly (P< 0.005) reduced Jv by 33% to 2.3±0.17 nl/min/mm and increased plasma ADMA (systemic Veh: 0.46 ± 0.030 vs. systemic L-257: 0.67 ± 0.029 μmol/l; P < 0.0001) without changing SDMA. Microperfusion of ADMA (10-4 M) or L-NAME (10-4 M) in rats both reduced Jv significantly by >40% to 2.0±0.24 and 1.8±0.22 nl/min/mm, respectively (P<0.05). An IV 1 ml bolus of saline containing Transit and siRNA targeted to DDAH-1 (hydrodynamic in vivo gene silencing in mice) three days later reduced Jv significantly by 36% compared to scrambled siRNA (2.2 ± 0.16 vs 1.4 ± 0.26 nl/min/mm; P<0.05). Surprisingly, PT cell-specific DDAH-1 knockdown in mice did not affect Jv significantly compared to wild type (PTC-D1WT: 1.7 ± 0.21 vs PTC-D1KO: 2.0 ± 0.15).Conclusions: PT fluid reabsorption is regulated by ADMA and its tubular metabolism by DDAH-1. The two-hour delay for L-257 to diminish Jv likely was required for accumulation of sufficient tubular ADMA to impair PT reabsorption. Whereas systemic DDAH-1 gene knockdown in mice was as effective as systemic DDAH-1 blockade with L-257 in rats in reducing Jv in the PT, lifelong deletion of PT DDAH-1 was ineffective, suggesting robust adaptive mechanisms that restore PT reabsorption. Thus, L-257 is a novel regulator of PT function. SNIPs that alter DDAH-1 in the PT may change tubular function and thereby contribute to CKD progression. [ABSTRACT FROM AUTHOR]

Background: Aging increases cardiovascular morbidity. Aging in vascular smooth muscle cells (VSMC) induces cell senescence, leading to impaired vasodilation with decreased vascular compliance, increased inflammation and oxidative stress. MicroRNA-34a (miR-34a) has been reported as a biomarker of aging.Methods and Results: After screening for a group of miRs, there was a > 2 fold of miR-34a in aortic SMCs isolated from transgenic mice overexpressing Nox1 in SMCs (Nox-1 SMCs). Therefore, we hypothesized that miR34 contributes to VSMC aging. TaqMan miR assay confirmed an increased miR-34a by 2.1 fold in young NOX-1 SMCs (passage 3-7) with an increase by 5.25 fold in activity of senescence-associated β-galactosidase (SA-β-Gal ) compared to WT. miR-34a in old NOX-1 and WT SMCs (passage 14-16) was increased by 3.8 fold and 2.1 fold, respectively, compared to young SMCs (both P< 0.01 ). There was 1.8 fold more miR-34a detected in old NOX-1 vs. old WT SMCs. SA-β-Gal positive cells accounted for 0.8±1% and 4±1.3% in young WT and NOX-1 SMCs (P<0.01), and 5±1.5% and 32±5% in old WT and NOX-1 SMCs (P <0.005). After transfection with miR-34a inhibitor, mature miR-34a was decreased by 75% and SA-β-Gal activity by 58% in old NOX-1 cells with parallel changes in the protein expression for Gamma-H2AX( a DNA damage marker) and p21( an activator for cell senescence). Moreover, phosphatase nuclear targeting subunit (PNUTS, a recently identified anti-age protein and direct miR-34a target) was not changed in young NOX-1 SMCs but reduced by 78% in old NOX-1 SMCs. Interestingly, nuclear factor (erythroid-derived 2)-like 2 (Nrf2, a key transcription factor for antioxidant genes) mRNA and protein were decreased by 55% and 32% in old NOX-1 SMCs (P<0.01 and 0.05). Nrf2 activator tert-butylhydroquinone (tBHQ) reduced miR-34a from 2.9±0.4 to 1.8±0.2 fold in those cells.Conclusion: Increased NOX-1 in SMCs induces miR-34a that accelerates SMC senescence. The pathway may involve downregulation of Nrf2 and increased P21 through P53 pathway. This provides potential targets for protecting against vascular aging. [ABSTRACT FROM AUTHOR]

Background: Superoxide (O2.-) is associated with cardiovascular disease (CVD) and inactivating SNIPs of the EC-SOD gene increased CVD, but the mechanisms are unclear. Superoxide dismutases (SODs) have three isoforms: cytoplasmic Cu/ZnSOD (SOD1), mitochondrial MnSOD (SOD2) and extracellular Cu/ZnSOD (SOD3). Complete SOD2-/- was lethal. We tested the hypothesis that these isoforms of SOD modulate angiotensin II (Ang II), myogenic responses (MRs) and remodeling of afferent arterioles (Affs).Methods and Results: MRs were assessed from the slope of the regression of active wall tension on perfusion pressure (PP) and Ang II contractions from incubation with 10-6 M Ang II. O2.- was assessed from PEG-SOD inhibitable ethidium:dihydroethidium fluorescence and remodeling from media:lumen area ratio (M:L).Results: 1. Compared to +/+, increasing PP from 40 to 80 mmHg or applying 10-6 M Ang II in Affs increased O2.- more in SOD1-/- (PP, 8 ± 1% vs 5 ± 1% , P<0.05; Ang II, 29 ± 7 vs 10 ± 2%, P<0.05), in SOD2+/- (PP, 15 ± 2% vs 7 ± 1%, P<0.01; Ang II, 32 ± 5 % vs 7 ± 1%, P<0.01), and in SOD3-/- ( PP, 20 ± 4% vs 7 ± 1%, P<0.05; Ang II, 32 ± 5% vs 12 ± 3%, P<0.05). However, O2.- did not increase in SOD1+/- and SOD3+/-. 2. Compared to +/+, MRs (dynes.cm-1.mmHg-1) were significantly increased in SOD1-/- (3.49 ± 0.30 vs 2.30 ± 0.27, P<0.05), in SOD2+/- (3.85 ± 0.48 vs 1.80 ± 0.34, P<0.05), and in SOD3-/- (3.51 ± 0.20 vs 2.43 ± 0.15, P<0.01), with no increase in SOD1+/- and SOD3+/-. 3. Compared to +/+, Ang II contractions were stronger in SOD1-/- (-68 ± 2% vs -33 ± 3%, P<0.0001), in SOD2+/- (-66 ± 6% vs -42 ± 3%, P<0.01), and in SOD3-/- (-75 ± 3% vs -34 ± 3%, P<0.0001) 4. Compared to +/+, M:L ratio of Affs were greater in SOD1-/- (4.0 ± 0.2 vs 1.8 ± 0.2, P<0.0001), in SOD2+/- (3.7 ± 0.4 vs 1.8 ± 0.4, P<0.001), and in SOD3-/- (4.5 ± 0.2 vs 1.9 ± 0.1, P<0.0001), but were unchanged in SOD1+/- and SOD3+/-. M:L ratio was strongly correlated to MR (r2 =0.32, P<0.005) and Ang II contractions (r2 = 0.56, P<0.005) across genotypes. 5. There were no compensatory changes in expression of other SOD isoforms.Conclusions: Lifetime increases in cytosolic, mitochondrial or extracellular O2.- enhanced afferent arteriolar myogenic and Ang II contractions that were closely related to vascular remodeling. [ABSTRACT FROM AUTHOR]

Background: Impaired autoregulation and systemic hypertension underlie progressive kidney injury in chronic kidney disease (CKD). We reported that the myogenic response (MR) was severely impaired in afferent arterioles (Affs) from mice with reduced renal mass (RRM) and oxidative stress, yet superoxide (O2.-) enhanced MRs of normal mice. Therefore, we tested the hypothesis that an increase in perfusion pressure (PP) enhances the generation of H2O2 selectively in Affs from mice with RRM and that H2O2 causes the impaired MR.Methods and Results: Groups of sham and RRM C57BL/6 mice were fed a high salt (6% NaCl) diet for 3 months. Alternative groups were treated with 2 mM of the redox cycling nitroxide tempol in drinking water for 3 months, which reduced the excretion of both 8-isoprostane (marker of O2.-) and H2O2. MRs were assessed from the % change in luminal diameter of isolated, perfused Affs after increasing PP from 40 to 80 mmHg, O2.- from PEG-SOD inhibitable ethidium:dihydroethidium fluorescence and H2O2 from PEG-catalase (CAT) inhibitable H2DCFDA fluorescence.Results: 1. Compared to sham, increasing PP in Affs from RRM mice increased O2.- more (21 ± 2% vs 11 ± 2%, P<0.01), but this was prevented by tempol, and increased H2O2 more (29 ± 8% vs 4 ± 1%, P<0.01), which also was prevented by tempol. 2. Compared to sham, Affs from mice with RRM mice had severely impaired MRs (sham, -14 ± 4% vs RRM, -1 ± 4%, P<0.05), yet this defect was corrected in mice drinking tempol (sham + tempol, -9 ± 6% vs RRM + tempol, -11 ± 2%, NS). 3. Bath addition of PEG-SOD (200 units/ml) attenuated MRs from sham mice (sham + vehicle, -14 ± 4%; sham + SOD, -6 ± 1%; P<0.05), but did not affect responses in RRM mice. 4. Bath addition of PEG-CAT (1000 units/ml) selectively enhanced the MRs in RRM mice (RRM + vehicle -1 ± 4%; RRM + CAT -19 ± 2%; P< 0.01) without effect in sham mice (sham + vehicle, -14 ± 4%; sham + PEG-CAT, -14 ± 2% NS).Conclusions: Excessive H2O2 is released by increased PP of the afferent arterioles only from mice with RRM and accounted for the impaired myogenic response. Tempol, or other drugs that metabolize H2O2, may preserve renal autoregulation and prevent progressive loss of GFR in CKD. [ABSTRACT FROM AUTHOR]

Studies with the proximal tubule-specific angiotensin type 1 receptor (AT1R) knockout mouse have shown that the proximal tubule (PT) contributes to Ang II-induced hypertension. Ang II levels in the PT fluid are 100- to 1000-fold higher than in plasma, yet the function of the AT1-Rs on the luminal side of the PT is unclear. Moreover, hemodynamic actions of Ang II that raise the filtration fraction and the peritubular fluid uptake forces also stimulate PT fluid uptake. We tested the effects of Ang II and AT1-Rs on PT fluid uptake in rats. Ang II (200 ng/kg/min) delivered for 2 weeks increased MAP (Con: 95±9 vs Ang II: 140±5 mmHg, p<0.001), single nephron GFR (snGFR)(Con: 36±3 vs Ang II: 43±3, p<0.05) and absolute proximal reabsorption (APR) (Con: 20±3 vs Ang II: 34.5±6.5 nl/min, p<0.01), measured by free flow collections in the S2 segments of the PT in Sprague Dawley (SD) rats. These effects were normalized by acute reduction of MAP via a supra-renal aortic clamp. In a separate group of Munich Wistar Frömter (MWF) rats, which have surface glomeruli, Ang II increased snGFR and APR in the S1 segment (Con: 19±4 vs AngII: 44±5 nl/min, p<0.01; +125±9 %) substantially more than in the S2 segment (Con: 25±6 vs Ang II 37±7 nl/min, p<0.01; +48±6 %). In microperfusion and recollection experiments, in which snGFR was controlled, fluid uptake (Jv) in the PT of SD rats was reduced dose-dependently by Ang II delivered directly into the lumen of S2 segments. Ang II (10-9 M) at physiological levels reduced Jv by 60±7%. This effect on Jv was blocked by co-perfusion of an AT1R blocker. Ang II (10-9 M), microperfused directly into the S2 reduced Jv in MWF rats as well by 44±5%. Ang II in cultured PT cells at higher concentrations (10-7 to 10-9 M) also reduced 22Na+ uptake by 20-35%. In conclusion, enhanced PT fluid reabsorption by Ang II is due to hemodynamic enhancement that likely entails peritubular fluid uptake primarily in the S1 segment. However, luminal Ang II acting on AT1-Rs in the S1 and S2 segments of the PT reduces fluid uptake. This effect could be overridden by the hemodynamic effect of systemic Ang II to increase reabsorption especially in the S1 segment. [ABSTRACT FROM AUTHOR]

Perivascular adipose tissue (PVAT) normally promotes vascular endothelium dependent relaxation (EDRs), whereas reactive oxygen species (ROS) impair endothelial function. However, the effects of the menopause on PVAT, and its interaction with ROS, are unexplored. We tested the hypothesis that ovariectomy (OVX) causes oxidative stress that impairs microvascular PVAT signaling. Mesenteric arterioles (MAs) were isolated from rats 4-6 weeks after sham-operation (Sham) or OVX. Alternate rats received vehicle or tempol (2mmol · 1-1 of water) throughout (n=6/group). MAs were preconstricted with 10-7 M norepinephrine for measurement of acetylcholine (ACh)-induced endothelial derived relaxation (EDR), endothelium derived relaxation factor (EDRF) and NO activity (DAF-FM fluorescence) using a Mulvany-Halperin wire myograph and fluorescence RatioMaster system. Other vessels were studied under spontaneous tone with dilator pathways blocked with 10-4M L-NAME, 10-5M apamine and 10-6M charybdotoxin to measure ACh-induced endothelium dependent contraction factor (EDCF) and ROS generation (temp-9AC fluorescence). OVX rats had vascular remodeling (midia: lumen diameter: 4.4± 0.4 vs 1.5 ± 0.3 μm·μm-1, P<0.01). Their MAs without PVAT had diminished EDR (48 ±5 vs 73±3%; P<0.05), EDRF (20±4 vs 41±4%; P<0.01) and NO activity (0.4 ± 0.1 vs 0.7± 0.1 units; P<0.05) and developed an EDCF (23±6 vs 5±3%; P<0.05) and ACh-induced ROS (0.5±0.1 vs 0.1±0.0 units; P<0.01). The presence of PVAT surrounding Sham vessels enhanced EDR (90±3 vs 73±3%; P<0.05), EDRF (57±3 vs 41±3%; P<0.05) and NO (1.2±0.01 vs 0.7±0.1 units; P<0.05). All of these beneficial effects of PVAT were lost in OVX rats. Four to six weeks of tempol drinking in OVX rats restored normal intrinsic vascular endothelial function and NO and restored all the beneficial effects of PVAT while preventing microvascular remodeling. In conclusion, OVX rats had microvascular remodeling and severe endothelial dysfunction ascribed to intrinsic vascular effects of ROS that summated with ROS-dependent interruption of a beneficial microvascular PVAT signaling pathway. Thus, therapeutic targets for menopausal vascular dysfunction should be expanded to include ROS and its extravascular actions on PVAT. [ABSTRACT FROM AUTHOR]

The E2-related factor 2 (Nrf2) binds to AREs and transcriptionally activates several genes that protect against oxidative stress. DDAH phosphorylates and activates eNOS and degrades ADMA which is an endogenous inhibitor of eNOS. We detected 2-3 putative AREs in the promoter regions of the human genes for DDAH-1 and -2, peroxisome proliferator-activated receptor-γ (PPAR-γ) and eNOS. PPAR-γ can increase eNOS expression and eNOS phosphorylation. Therefore, we hypothesized that AREs regulate the DDAH/eNOS/PPAR-γ/ADMA/ NO pathway. Tert-butylhydroquinone (tBHQ) activates Nrf2 by translocation to the nucleus. Incubation of HRGECs for 24 hours with tBHQ enhanced cell proliferation dose-dependently (5-20μM). tBHQ (20μM; n=4) increased medium nitrite by 69 ± 3.7% (P<0.01), intracellular NO (from DAF-FM fluorescence ) by 62 ± 17.1% (P<0.05), and NOS and DDAH activities( from conversion of [3H]-arginine or [14C]-ADMA to [3H]- or [14C]-citrulline) by 58 ± 3.6% and 47 ± 8.5% (P<0.05) and decreased ADMA (by capillary zone electrophoresis) from 1.2±0.03 vs. 2.3±0.16μM/L, (P<0.001, n=6). tBHQ increased the mRNAs and proteins for eNOS by 79 ± 9% and 43 ± 17%, for DDAH-1 by 129 ± 10% and 62 ± 21%, and for DDAH-2 by 119 ± 25% and 48 ± 18% (all P<0.05) and increased the protein expression of PPAR-γ and eNOS phosphorylation and increased tBHQ nuclear translocation markedly. Knockdown of the cell Nrf2 by transfection with a specific siRNA abolished all these effects and attenuated cell proliferation. Chromatin immunoprecipitation (CHIP)-based PCR assays demonstrated that tBHQ enhanced the binding of Nrf2 to one ARE region in the promoters for DDAH-1 and -2 but there was no binding to the eNOS promoter. In conclusion, activation of a specific ARE in the promoter region of the DDAH-1 and -2 genes causes their transcriptional activation. The ensuing enhancement of DDAH activity enhances ADMA metabolism. Nrf2 also increases eNOS expression, NOS activity and NO generation without binding to the ARE for eNOS, likely by upregulation of PPAR-γ. Thus, Nrf2 improves endothelial cell function by coordinating a diverse set of pathways that enhance NO generation. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) stimulates proximal tubule (PT) Na+ and fluid reabsorption. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) but its effects on tubular function are unknown. ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) whose type 1 isoform is predominant in the PT. SNIPs of DDAH-1 predicted the rate of decline of renal function in patients with chronic kidney disease (CKD). Therefore, we tested the hypothesis that DDAH-1 metabolizes ADMA in the proximal tubule and thereby enhances PT fluid reabsorption (Jv). Jv was measured in anesthetized rats by direct in vivo microperfusion and recollection of artificial tubular fluid (ATF) in S2 segments of the PT isolated between oil blocks. Addition of a selective inhibitor of DDAH-1, L-257 (10-4M) to ATF did not effect Jv when compared to addition of vehicle (3.40.6 vs 3.10.3 nl/min/mm). However, L-257 administered as a bolus intravenous injection (60mg/kg) 2hrs before microperfusion of the PT with L-257 significantly reduced Jv to 1.80.2 nl/min/mm, (P<0.05) and significantly enhanced urine flow rate (2.1±0.3 vs 9.0±1.8 μl/min; P<0.05). Microperfusion of ADMA (10-4 M) or L-NAME (10-4 M) into the PT both reduced Jv significantly to 2.00.2 and 1.80.2 nl/min/mm, respectively (P<0.05). Rats pretreated with a rapid intravenous injection of siRNA targeted to DDAH-1 that reduced its renal mRNA expression significantly also had reduced Jv (3.1±0.2 vs 2.2±6.2 nl/min/mm; P<0.05) and had a further reduction in Jv with 10-4M ADMA added to ATF to 1.4±0.1 nl/min/mm; (P<0.005). In conclusion, PT fluid reabsorption is regulated by ADMA and its tubular metabolism by DDAH-1. The two hour delay in the effects of a bolus injection of L-257 to diminish Jv indicated the time required after inhibition of DDAH-1 for accumulation of sufficient tubular ADMA to impair PT reabsorption. Thus, L-257 is a novel regulator of PT function and is a proximal tubule diuretic. SNIPs that alter DDAH-1 in the PT may change renal function and contribute to CKD progression. [ABSTRACT FROM AUTHOR]

Asymmetric dimethylarginine (ADMA), which inhibits NO synthase, is inactivated by N(G),N(G)-dimethylarginine dimethylaminohydrolase (DDAH). We tested whether DDAH-1 or -2 regulates serum ADMA (S(ADMA)) and/or endothelium-derived relaxing factor (EDRF)/NO. Small inhibitory (si)RNAs targeting DDAH-1 or -2, or an siRNA control were given intravenously to rats. After 72 hours, EDRF/NO was assessed from acetylcholine-induced, NO synthase-dependent relaxation and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate for NO activity in isolated mesenteric resistance vessels (MRVs). Expression of mRNA for DDAH-1 versus -2 was 2- and 7-fold higher in the kidney cortex and liver, respectively, whereas expression of DDAH-2 versus -1 was 5-fold higher in MRVs. The proteins and mRNAs for DDAH-1 or -2 were reduced selectively by 35% to 85% in the kidney cortex, liver, and MRVs 72 hours following the corresponding siRNA. S(ADMA) was increased only after siDDAH-1 (266+/-25 versus 342+/-39 [mean+/-SD] nmol x L(-1); P<0.005), whereas EDRF/NO responses and NO activity were not changed consistently by siDDAH-1 but were greatly reduced after siDDAH-2. Mean arterial pressure was not changed significantly by any siRNA. In conclusion, S(ADMA) is regulated by DDAH-1, which is expressed at sites of ADMA metabolism in the kidney cortex and liver, whereas EDRF/NO is regulated primarily by DDAH-2, which is expressed strongly in blood vessels. This implies specific functions of DDAH isoforms.

Triglyceride-rich lipoproteins (chylomicrons and very low-density lipoproteins) bind endotoxin (lipopolysaccharide [LPS]), forming lipoprotein-LPS complexes, and protect against endotoxic shock and death in rodent models of gram-negative sepsis. Hepatocytes play a central role in the protective process, as demonstrated by the increased uptake of chylomicron (CM)-bound LPS by these cells. We have previously reported that CM-LPS complexes inhibit nitric oxide (NO) production by hepatocytes as compared with LPS or CM alone. Herein, we report that CM-LPS selectively inhibits NF-kappaB in hepatocytes. Pretreating cultured primary hepatocyte spheroids with CM-bound LPS inhibited cytokine-induced NF-kappaB activation by approximately 60% vs. untreated control cells (P < 0.03). The lipoprotein-mediated inhibition of NF-kappaB was non-toxic, selective, and associated with inhibition of IkappaB degredation. These data indicate that the mechanism by which CM protect against LPS involves inhibition of the hepatocellular response to proinflammatory stimulation and also support a role for triglyceride-rich lipoproteins as components of the innate host immune response to infection.

Background: Experimental studies have shown that hemorrhagic shock is associated with the expression of inducible heat proteins, especially heat shock protein (Hsp) 72, in liver, brain, heart, and kidney. Moreover, induction of Hsp 72 by various stressors before the onset of shock has been associated with the attenuation of organ injury caused by hemorrhage. However, it is not known whether Hsp 72 is expressed after severe trauma in humans. The purpose of this study was therefore to determine whether Hsp 72 could be detected in the serum of patients early after severe trauma and whether serum levels of Hsp 72 might correlate with survival of trauma patients or the severity of the postinjury inflammatory response., Methods: Clinical data were collected prospectively over a 3-year period for trauma patients mechanically ventilated for more than 2 days who met the following inclusion criteria: Injury Severity Score > or = 16, and age > 18 years. Physiologic data for quantitative assessment of organ dysfunction were collected for each patient. Hsp 72 and nitrate and nitrite levels were measured in the serum of trauma patients collected at or 12 to 48 hours after the admission to the emergency department., Results: Sixty-seven patients with severe trauma were enrolled in the study. Hsp 72 was detected in the serum of all trauma patients. All patients with high initial serum levels of Hsp 72 (serum levels > 15 ng/mL) survived, whereas 29% of the patients with low Hsp 72 serum levels died from their traumatic injuries (p = 0.01). The overall mortality was 21%, occurring within 5 to 7 days. Patients who died were older (mean age, 54 +/- 15 years) than those who survived (mean age, 36 +/- 15 years) (p < 0.0.05). The cause of death was attributable to head injury in 79%, although the severity of head injury (Abbreviated Injury Scale score) was not statistically different between survivors with high serum values of Hsp 72 and patients who died. There was no correlation between the initial serum Hsp 72 values and the severity of organ dysfunction or clinical indicators of the inflammatory response., Conclusion: Hsp 72 can be detected in the serum of severely traumatized patients within 30 minutes after injury. Elevated initial serum levels of Hsp 72 (serum levels > 15 ng/mL) are associated with survival after severe trauma, but are not related to the incidence or severity of the postinjury inflammatory response or organ dysfunction.