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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 F 2α (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.

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., (© 2015 American Heart Association, Inc.)

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., (© 2015 American Heart Association, Inc.)

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.

The high renal oxygen (O(2) ) demand is associated primarily with tubular O(2) consumption (Qo(2) ) necessary for solute reabsorption. Increasing O(2) delivery relative to demand via increased blood flow results in augmented tubular electrolyte load following elevated glomerular filtration, which, in turn, increases metabolic demand. Consequently, elevated kidney metabolism results in decreased tissue oxygen tension. The metabolic efficiency for solute transport (Qo(2) /T(Na) ) varies not only between different nephron sites, but also under different conditions of fluid homeostasis and disease. Contributing mechanisms include the presence of different Na(+) transporters, different levels of oxidative stress and segmental tubular dysfunction. Sustained hyperglycaemia results in increased kidney Qo(2) , partly due to mitochondrial dysfunction and reduced electrolyte transport efficiency. This results in intrarenal tissue hypoxia because the increased Qo(2) is not matched by a similar increase in O(2) delivery. Hypertension leads to renal hypoxia, mediated by increased angiotensin receptor tonus and oxidative stress. Reduced uptake in the proximal tubule increases load to the thick ascending limb. There, the increased load is reabsorbed, but at greater O(2) cost. The combination of hypertension, angiotensin II and oxidative stress initiates events leading to renal damage and reduced function. Tissue hypoxia is now recognized as a unifying pathway to chronic kidney disease. We have gained good knowledge about major changes in O(2) metabolism occurring in diabetic and hypertensive kidneys. However, further efforts are needed to elucidate how these alterations can be prevented or reversed before translation into clinical practice., (© 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.)

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with &frac56; surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na/)Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na/)Q(O2) (sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical Po(2) (sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.

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

Purpose of Review: Nitric oxide is a potent, endogenous vasodilator that regulates systemic blood pressure and renal function, among other functions. The bioactivity of nitric oxide is reduced by superoxide, a major reactive oxygen species. Overproduction of superoxide and other related reactive oxygen species resulting in oxidative stress reduces the biological effects of nitric oxide. Though both of these highly reactive species have distinct roles in other pathways, their interaction is emerging as a major regulatory factor in normal and pathological renal function. The purpose of this review is to highlight the recent studies on oxidative stress and nitric oxide in the kidney, focusing on their interaction in normal and pathological conditions., Recent Findings: Studies have focused on pro-oxidant pathways and nitric oxide defense systems in normal and pathological conditions. The oxidant potential of uncoupled nitric oxide synthases is gaining interest as a pro-oxidant system. Both animal and clinical studies have attempted to identify strategies to intervene at various stages of the oxidant-nitric oxide pathways to improve function during renal failure., Summary: Several new approaches and provocative findings have emerged over the last year. A regulatory role for nitric oxide in the control of the renal microcirculation and as a participant in tubule function is further described. New information of the cause and possible prevention of acute and chronic renal failure has also been produced in the last year. These advances demonstrate the value of research in the normal and pathological roles of oxidative stress and nitric oxide in the kidney.

We tested the hypothesis that superoxide anion (O(2)(-).) generated in the kidney by prolonged angiotensin II (ANG II) reduces renal cortical Po(2) and the use of O(2) for tubular sodium transport (T(Na):Q(O(2))). Groups (n = 8-11) of rats received angiotensin II (ANG II, 200 ng.kg(-1).min(-1) sc) or vehicle for 2 wk with concurrent infusions of a permeant nitroxide SOD mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol, 200 nmol.kg(-1).min(-1)) or vehicle. Rats were studied under anesthesia with measurements of renal oxygen usage and Po(2) in the cortex and tubules with a glass electrode. Compared with vehicle, ANG II increased mean arterial pressure (107 +/- 4 vs. 146 +/- 6 mmHg; P < 0.001), renal vascular resistance (42 +/- 3 vs. 65 +/- 7 mmHg.ml(-1).min(-1).100 g(-1); P < 0.001), renal cortical NADPH oxidase activity (2.3 +/- 0.2 vs. 3.6 +/- 0.4 nmol O(2)(-)..min(-1).mg(-1) protein; P < 0.05), mRNA and protein expression for p22(phox) (2.1- and 1.8-fold respectively; P < 0.05) and reduced the mRNA for extracellular (EC)-SOD (-1.8 fold; P < 0.05). ANG II reduced the Po(2) in the proximal tubule (39 +/- 1 vs. 34 +/- 2 mmHg; P < 0.05) and throughout the cortex and reduced the T(Na):Q(O(2)) (17 +/- 1 vs. 9 +/- 2 mumol/mumol; P < 0.001). Tempol blunted or prevented all these effects of ANG II. The effects of prolonged ANG II to cause hypertension, renal vasoconstriction, renal cortical hypoxia, and reduced efficiency of O(2) usage for Na(+) transport, activation of NADPH oxidase, increased expression of p22(phox), and reduced expression of EC-SOD can be ascribed to O(2)(-). generation because they are prevented by an SOD mimetic.

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.

Oxidative stress accompanies angiotensin (ANG) II infusion, but the role of ANG type 1 vs. type 2 receptors (AT1-R and AT2-R, respectively) is unknown. We infused ANG II subcutaneously in rats for 1 wk. Excretion of 8-isoprostaglandin F2alpha (8-Iso) and malonyldialdehyde (MDA) were related to renal cortical mRNA abundance for subunits of NADPH oxidase and superoxide dismutases (SODs) using real-time PCR. Subsets of ANG II-infused rats were given the AT1-R antagonist candesartan cilexetil (Cand) or the AT2-R antagonist PD-123,319 (PD). Compared to vehicle (Veh), ANG II increased 8-Iso excretion by 41% (Veh, 5.4 +/- 0.8 vs. ANG II, 7.6 +/- 0.5 pg/24 h; P < 0.05). This was prevented by Cand (5.6 +/- 0.5 pg/24 h; P < 0.05) and increased by PD (15.8 +/- 2.0 pg/24 h; P < 0.005). There were similar changes in MDA excretion. Compared to Veh, ANG II significantly (P < 0.005) increased the renal cortical mRNA expression of p22phox (twofold), Nox-1 (2.6-fold), and Mn-SOD (1.5-fold) and decreased expression of Nox-4 (2.1-fold) and extracellular (EC)-SOD (2.1-fold). Cand prevented all of these changes except for the increase in Mn-SOD. PD accentuated changes in p22phox and Nox-1 and increased p67phox. We conclude that ANG II infusion stimulates oxidative stress via AT1-R, which increases the renal cortical mRNA expression of p22phox and Nox-1 and reduces abundance of Nox-4 and EC-SOD. This is offset by strong protective effects of AT2-R, which are accompanied by decreased expression of p22phox, Nox-1, and p67phox.

The spontaneously hypertensive rat (SHR) exhibits angiotensin II (Ang II)-dependent oxidative stress and reduced efficiency of renal oxygen usage (QO2) for tubular sodium transport (TNa). We tested the hypothesis that oxidative stress determines the reduced TNa:QO2 ratio in the clipped kidney of the early 2-kidney, 1-clip (2K,1C) Ang II-dependent model. One week after sham operation (Sham) or clip placement, 2K,1C rats received for 2 weeks either a vehicle, the superoxide dismutase mimetic tempol (Temp), or candesartan (Cand). Oxidative stress was assessed from excretion of 8-isoprostaglandin F2alpha (PGF2alpha) and malondialdehyde (MDA) and renal oxygenation from pO2 in the renal cortex and from the ratio of calculated TNa and QO2 values. The mean arterial pressure (MAP) of Sham (113+/-6 mm Hg) was increased in 2K,1C vehicle-treated rats (148+/-4 mm Hg), but both Temp and Cand restored MAP to Sham levels. The excretions of 8-iso-PGF2alpha and MDA were higher in 2K,1C vehicle-treated rats compared with Sham and were normalized by Temp. The pO2 of Sham (42+/-2 mm Hg) was lower in 2K,1C vehicle-treated animals (28+/-2 mm Hg). This was restored to Sham values by Temp (36+/-3 mm Hg) but not by Cand (28+/-2 mm Hg). The TNa:QO2 of Sham (12.9+/-1.6) was reduced in 2K,1C vehicle-treated rats (9.7+/-2.8) and was restored to Sham values by Temp (13.7+/-2.5) but not by Cand (7.5+/-1.6). We conclude that the correction of oxidative stress in the 2K,1C model partially corrects renal cortical hypoxia and inefficient utilization of O2 for Na+ transport, independent of the fall in blood pressure.

Nuclear factor erythroid factor E2-related factor 2 (Nrf2) transcribes antioxidant genes that reduce the blood pressure (BP), yet its activation with tert-butylhydroquinone (tBHQ) in mice infused with angiotensin II (Ang II) increased mean arterial pressure (MAP) over the first 4 days of the infusion. Since tBHQ enhanced cyclooxygenase (COX) 2 expression in vascular smooth muscle cells (VSMCs), we tested the hypothesis that tBHQ administration during an ongoing Ang II infusion causes an early increase in microvascular COX-dependent reactive oxygen species (ROS) and contractility. Mesenteric microarteriolar contractility was assessed on a myograph, and ROS by RatioMaster™. Three days of oral tBHQ administration during the infusion of Ang II increased the mesenteric microarteriolar mRNA for p47phox, the endothelin type A receptor and thromboxane A2 synthase, and increased the excretion of 8-isoprostane F2α and the microarteriolar ROS and contractions to a thromboxane A2 (TxA2) agonist (U-46,619) and endothelin 1 (ET1). These were all prevented in Nrf2 knockout mice. Moreover, the increases in ROS and contractility were prevented in COX1 knockout mice with blockade of COX2 and by blockade of thromboxane prostanoid receptors (TPRs). In conclusion, the activation of Nrf2 over 3 days of Ang II infusion enhances microarteriolar ROS and contractility, which are dependent on COX1, COX2 and TPRs. Therefore, the blockade of these pathways may diminish the early adverse cardiovascular disease events that have been recorded during the initiation of Nrf2 therapy. [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]

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with 5/6 surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O2 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 O2 consumed (TNa/QO2), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F2α and H2O2, renal expression of UCP-2 and renal O2 extraction, and reduced TNa/QO2 (sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical PO2 (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 p22phox, NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf- 2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O2 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. [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.

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 II (ANG II) infusion increases renal superoxide (O2-) and enhances renal vasoconstriction via macula densa (MD) regulation of tubuloglomerular feedback, but the mechanism is unclear. We targeted the p22phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) with small-interfering RNA (siRNA) to reduce NADPH oxidase activity and blood pressure response to ANG II in rats. We compared single nephron glomerular filtration rate (SNGFR) in samples collected from the proximal tubule (PT), which interrupts delivery to the MD, and from the distal tubule (DT), which maintains delivery to the MD, to assess MD regulation of GFR. SNGFR was measured in control and ANG II-infused rats (200 ng·kg-1·min-1 for 7 days) 2 days after intravenous injection of vehicle or siRNA directed to p22phox to test the hypothesis that mediates MD regulation of SNGFR during ANG II. The regulation of SNGFR by MD, determined by PT SNGFR-DT SNGFR, was not altered by siRNA in control rats (control + vehicle, 13 ± 1, n = 8; control + siRNA, 12 ± 2 nI/min, n = 8; not significant) but was reduced by siRNA in ANG II-treated rats (ANG II + vehicle, 13 ± 2, n = 7; ANG II + siRNA, 7 ± 1 nl/min, n = 8; P < 0.05). We conclude that p22phox and NADPH oxidase regulate the SNGFR during ANG II infusion via MD-dependent mechanisms. [ABSTRACT FROM AUTHOR]

1. The kidney has a unique environment that results in relatively low tissue oxygen tension ( Po2). However, recent studies have shown that renal hypoxia is more severe during hypertension and may reflect changes in the way O2 is used. The present review summarizes studies that explore the relationship between renal oxygen tension ( Po2), oxygen consumption and hypertension. 2. More recent studies suggest that oxidative stress accompanying hypertension, rather than the elevated blood pressure per se reduces Po2. The Po2 in various sections of the kidney often reflects the level of oxygen consumption, which varies depending on the sites of Na+ reabsorption, a process that consumes nearly 90% of total renal oxygen. 3. The efficient use of oxygen for the transport of Na+ in the kidney is reduced during hypertension, which may contribute to the resulting hypoxia. Conversely, the defect in renal oxygen usage due to oxidative stress may exacerbate hypertension in animal models. The goal of many of these studies is to determine the impact of renal hypoxia in the generation of hypertension. [ABSTRACT FROM AUTHOR]

A thromboxane prostanoid receptor (TP-R) agonist U-46,619 enhances tubuloglomerular feedback (TGF). Glomerular expression of TP-R and enhancement of TGF by U-46,619 increase with salt intake. We investigated the hypothesis that 8-isoprostaglandin F2α (8-Iso) activates TGF via TP-R. The maximal TGF response in rats was assessed from the fall in proximal stop flow pressure (PSF; an index of glomerular capillary pressure) during loop of Henle (LH) microperfusion of artificial tubular fluid (ATF) at 40 nl/min. Microperfusion of 8-Iso (10-4 M) into the efferent arteriole (EA) enhanced TGF responses by 20 ± 3% (P < 0.01). TGF response to 8-Iso was independent of dietary salt [ΔTGF%, low salt (LS): 21 ± 5%; normal salt (NS): 17 ± 4%; high salt (HS): 29 ± 8%, not significant (ns)], unlike the salt-dependent effect of U-46,619 (ΔTGF%, LS: 41 ± 5%; NS: 52 ± 4%; HS: 112 ± 21%). Ifetroban, the TP-R antagonist, abolished TGF responses to 8-Iso and U-46,619 at all levels of salt intake. During luminal perfusion of N-monomethyl-L-arginine (L-NMA), the effect of 8-Iso on TGF was enhanced in NS and HS but not in LS (LS: 22 ± 6 vs. LS + L-NMA: 28 ± 6%, ns; NS: 18 ± 4 vs. NS + L-NMA: 40 ± 4, P < 0.01; HS: 27 ± 3 vs. HS + L-NMA: 65 ± 6, P < 0.01). However, U-46,619 did not further increase TGF after L-NMA in all salt groups (LS: 43 ± 7 vs. LS + L-NMA: 51 ± 6, ns; NS: 52 ± 7 vs. NS + L-NMA: 48 ± 8, ns; HS: 114 ± 21 vs. HS + L-NMA: 74 ± 22, ns). In conclusion, activation of TP receptors by U-46,619 and 8-Iso-PGF2α enhances TGF. In addition, the effect of U-46,619 was salt dependent, whereas the effect of 8-Iso-PGF2α was salt independent. However, stimulation of NO by 8-isoprostanes masks its salt-sensitive effect on TGF. [ABSTRACT FROM AUTHOR]

We tested whether chronic ANG II infusion into rats affects descending vasa recta (DVR) contractility, synthesis of superoxide, or synthesis of nitric oxide (NO). Rats were infused with ANG II at 250 ng·kg-1·min-1 for 11–13 days. DVR were loaded with dihydroethidium (DHE) to measure superoxide and 3-amino-4-aminomethyl-2′,7′-difluorofluorescein (DAFFM) to measure NO. Acute constriction of DVR by ANG II (0.1,1, and 10 nM) was diminished, and NO generation rate was raised by chronic ANG II infusion. DHE oxidation by DVR from ANG II infused rats was similar to controls and was significantly higher when NO synthesis was prevented with Nω-nitro-L-arginine methyl ester (L-NAME). The superoxide dismutase mimetic Tempol (1 mM) increased NO generation compared with controls. The increased synthesis of NO by chronic ANG II-treated vessels persisted in the presence of Tempol. DVR endothelial cytoplasmic Ca2+ response to ACh was diminished by chronic ANG II treatment, but the capacity of ACh to increase NO generation was unaltered. We conclude that DVR generation of superoxide is not affected by chronic ANG II exposure but that basal NO synthesis is increased DVR superoxide is unlikely Ill be an important mediator of chronic ANG II slow pressor hypertension in rats. [ABSTRACT FROM AUTHOR]

Background: The spontaneously hypertensive rat (SHR) has oxidative stress and enhanced O2 usage (Q(O2)) relative to tubular sodium transport (TNa). Angiotensin II (Ang II) acting on Type I receptors (AT1-R) causes renal oxidative stress and functional nitric oxide (NO) deficiency that could enhance O2 usage. Therefore, we investigated the hypothesis that AT1-Rs mediate the inefficient renal oxygenation in the SHR. Methods: Groups of SHR and WKY received vehicle (Veh), candesartan (Cand) or hydralazine + hydrochlorothiazide + reserpine (HHR) for two weeks. Results: Compared to WKY + Veh, the elevated BP of SHR + Veh (153 +/- 3 vs 115 +/- 3 mm Hg; P < 0.001) was normalized by Cand (117 +/- 4) or HHR (113 +/- 5 mm Hg). The reduced renal blood flow of SHR + Veh (2.4 +/- 0.3 vs. 4.1 +/- 0.3 mL. min-1. 100 g-1) was increased (P < 0.05) by Cand (3.6 +/- 0.3) and HHR (3.2 +/- 0.2). Compared to WKY + Veh, SHR + Veh had a 50% reduction in TNa: (16.9 +/- 2.0 vs. 7.8 +/- 0.9 micromol: micromol-1, P < 0.01) that was unchanged by HHR (8.6 +/- 1.1), but was increased by Cand (13.2 +/- 1.4; P < 0.01). The pO2 of outer cortex was lower in SHR + Veh than WKY + Veh (31 +/- 3 vs. 41 +/- 2 mm Hg; P < 0.05) and it was not changed significantly by HHR (37 +/- 2) but was normalized by Cand (44 +/- 3 mm Hg; P < 0.01). The pO2 in the deep cortex also was lower in SHR + Veh than WKY + Veh (18 +/- 3 vs. 30 +/- 3 mm Hg; P < 0.005) and was not changed significantly by HHR (19 +/- 2), but was increased by Cand (25 +/- 3 mm Hg; P < 0.05). Conclusions: The reduced pO2 in outer and inner cortex, and inefficient utilization of O2 for Na+ transport in the SHR kidney can be ascribed to the effects of AT1-R, largely independent of blood pressure. [ABSTRACT FROM AUTHOR]

Oxidative stress induced by Ang II or by inhibition of nitric oxide reduces renal efficiency of O2 usage to transported Na+. Furthermore, high Ang II reduced net Na+ uptake in PT cells. We tested the hypothesis that Ang II-induced oxidative stress increases paracellular permeability in PT cells. We measured transepithelial resistance (TER); an index of paracellular Na+ permeability and mannitol permeability; an index of paracellular fluid permeability. Immortalized mouse PT cells and in MDCK-1, a model of distal tubule (DT) cells, in monolayers grown on special plates, were treated with vehicle or Ang II (10 -8 to 10 -10 M). Ang II increased TER in MDCK (35-49 ohm) at all doses but decreased (15-22 ohm) TER in PT cells. Basal mannitol permeability was higher in PT cells compared to MDCK cells (PT: 7.894-0.6 vs. MDCK: 2.68±0.2 %/h, n=22-24, P<0.0001), but was reduced dose-dependently by Ang 1I in both PT and MDCK cells. In summary, physiological doses of Ang II increase paracellular Na+, but not fluid permeability selectively in the PT. An increase in Na+ backftux into the PT lumen may underlie both the reduced net Na+ reabsorption from the PT and the reduced efficiency of O2 use for net Na+ transport caused by Ang II. [ABSTRACT FROM AUTHOR]

Adenosine, acting on type 1 receptors in the preglomerular vessels, reduces RBF. Since adenosine release is stimulated in the renal medulla during acute oxidative stress, we tested the hypothesis that chronic Ang II-induced oxidative stress increases adenosine release in the renal cortex. We measured interstitial adenosine by intrarenal dialysis in the renal cortex and medulla of rats treated with vehicle (Veh, n=8) or Ang II (200 ng/kg/min, n=8) for 2 weeks, before and after injection of tempol (T, 72 mmol/kg). MAP was higher in Ang II rats (Ang II:132±7 vs Veh: 102±6, p<0.001). Acute T normalized MAP in Ang II rats, but had no effect in Veh rats. Cortical dialysate adenosine was higher in Ang II rats (Ang II:11.5±1.2 vs Veh: 6.1±1.2 pmol/µl, p<0.01), whereas medullary adenosine was not different (Ang II: 4.2:t:0.7 vs Veh: 3.5±1.0 pg/µl, ns). T reduced cortical adenosine levels in both groups (Ang II+ T: 6.2±0.5 pg/µl, p<0.05; Veh+ T: 2.6±0.4 pg/µI, p<0.01) and had no effect on medullary adenosine. Excretion of 8-isoprostaglandin PGF2sub<α; (8-iso) was higher in Ang II rats (Ang II:27.5±1.3 vs Veh: 19.4±1.5 pg/min, p<0.05). T lowered 8-iso excretion only in Ang II rats (Ang II + T: 14.2±2.1 pg/min, p<0.01). These results demonstrate that Ang II-induced oxidative stress stimulates adenosine release in the renal cortex, suggesting that adenosine contributes to oxidative stress increases in RVR. [ABSTRACT FROM AUTHOR]

We studied inhibition of Ang II-induced superoxide (O2*-) generation in primary cultures of SHR PGVSMCs by catalytic antioxidants (SOD, PEG-SOD, Tempol and Cat-1), by uncharacterized antioxidants (Fe-TPPS, -Epicatechin and Ebselen), by an O2*- spin trap (Nitroblue tetrazolium,NBT) or by vitamins (ascorbic acid, α-tocopherol and Trolox). Ang II (1 µM) increased O2*- generation by 94% determined by lucigenin-derived chemiluminescence (P<0.01, N=3). This was prevented by diphenyleneiodonium (DPI) and apocynin. Antioxidant drugs (10-4- 10-8M) over 4 hours caused dose-dependent decreases in O2*- over 4 hours with a maximum inhibition of 91±1; 82±0; 78±0; 65±2; 61±5; 57±2 and 54±13% for PEG-SOD, SOD, Tempol, Ebselen, NBT, -Epicatechin and Fe-TPPS, respectively (P<0.01 vs. control). The rank order of sensitivity (ED50) was PEG-SOD > SOD > Tempol > Ebselen > Fe-TPPS > -Epicatechin > NBT. Cat-1, ascorbic acid, α-tocopherol and Trolox were minimally effective even over 20 hours (17-31% inhibition). In conclusion, Ang II stimulates NADPH-oxidase-dependent O2*- generation in PGVSMCs. The sensitivity and effectiveness of catalytic antioxidants (SOD, PEG-SOD and tempol) are high, whereas that of Fe-TPPS, -Epicatechin, Ebselen and NBT are moderate, and Cat-1, ascorbic acid, α-tocopherol and Trolox are almost ineffective. These results may explain the efficacy of Tempol and SOD in reversing oxidative stress and the disappointing results with vitamins C and E. [ABSTRACT FROM AUTHOR]

SHR have decreased PT reabsorption of electrolytes and water. Ang II AT-1 receptor blockade corrects the tubular electrolyte and water handling, but the exact mechanism is presently unknown. Since a substantial part of the reabsorption in PT is going through the paracellular route, we investigated if SHR have altered paracellular fluid permeability, and if so, if this is related to Ang II-induced oxidative stress. PT permeability was measured by infusion of 14C-mannitol in PT in vivo. Fractional recovery of 14C was measured in the urine from the contralateral kidney (unifying excretion of reabsorbed matter). Groups (n=8) of SHR received vehicle, the AT-1 receptor antagonist candesartan or equal blood pressure lowering treatment (HHR) and were compared to corresponding Wistar Kyoto (WKY). The oxidative stress marker 8-isoprostane was measured in kidney tissue and plasma. SHR had lower leakage compared to WKY (7.9±1.2 vs. 11.9±0.6%), but increased plasma and kidney isoprostane levels. Both candesartan and HHR restored leakage in SHR (20.2±2.1 and 13.4±1.4%, respectively) and related to isoprostane levels. In conclusion, the paracellular permeability in PT is reduced by Ang II due to AT-1 receptor activation and engagement of oxidative stress that is also due to the increase in blood pressure. These findings display a novel mechanism for regulation of PT permeability, which gives an explanation for the reduced PT reabsorption in SHR. [ABSTRACT FROM AUTHOR]

Mechanisms of antioxidant and pro-oxidant effects ofα-lipoic acid in the diabetic and nondiabetic kidney.Background.α-Lipoic acid is a potent antioxidant that improves renal function in diabetes by lowering glycemia, however, the mechanisms by whichα-lipoic acid exerts its antioxidant effects are not completely understood.Methods.Metabolic parameters, renal function, and morphology, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and subunit expression were analyzed in nondiabetic and streptozotocin-induced diabetic rats fed normal rat chow (control) with or withoutα-lipoic acid (30 mg/kg body weight) for 12 weeks.Results.Blood glucose was increased with diabetes (nondiabetic+ control 89± 3 mg/dL and diabetic+ control 336± 28 mg/dL) and was similar withα-lipoic acid treatment (diabetic+α-lipoic acid 351± 14 mg/dL). In contrast,α-lipoic acid attenuated albuminuria (nondiabetic+ control 8.9± 1.3 mg/day; diabetic+ control 28.1± 4.6 mg/day; and diabetic+α-lipoic acid 17.8± 1.2 mg/day) associated with diabetes. Similarly,α-lipoic acid attenuated glomerulosclerosis (nondiabetic+ control 0.22± 0.01; diabetic+ control 0.55± 0.04; diabetic+α-lipoic acid 0.36± 0.03), tubulointerstitial fibrosis (nondiabetic+ control 0.42± 0.18; diabetic+ control 1.52± 0.05; diabetic+α-lipoic acid 1.10± 0.05), superoxide anion (O·−2) generation (nondiabetic+control 15.8± 1.7; diabetic+control 87.1± 3.5; diabetic+α-lipoic acid 25.5± 3.3 RLU/mg protein), and urine 8-isoprostane (8-iso) excretion (nondiabetic+ control 7.4± 1.4; diabetic+ control 26.0± 4.5; diabetic+α-lipoic acid 19.6± 5.6 ng/day) associated with diabetes.α-Lipoic acid also reduced kidney expression of NADPH oxidase subunits p22phox and p47phox. Surprisingly,α-lipoic acid appears to cause pro-oxidant effects in nondiabetic animals, resulting in increased albuminuria (nondiabetic+α-lipoic acid 14.2± 1.2 mg/day), increase in plasma creatinine levels (nondiabetic+ control 59± 6; diabetic+ control 68± 6; nondiabetic+α-lipoic acid 86± 9; diabetic+α-lipoic acid 69± 7μmol/L), exacerbated glomerulosclerosis and tubulointerstitial fibrosis, increased O·−2 generation, up-regulated p22phox and p47phox expression and increased 8-iso excretion.Conclusion.We conclude thatα-lipoic acid improves albuminuria and pathology in diabetes by reducing oxidative stress, while in healthy animals,α-lipoic acid may act as a pro-oxidant, contributing to renal dysfunction. [ABSTRACT FROM AUTHOR]