Maria Peleli

NADPH Oxidase in the Renal Microvasculature Is a Primary Target for Blood Pressure–Lowering Effects by Inorganic Nitrate and Nitrite

Renal oxidative stress and nitric oxide (NO) deficiency are key events in hypertension. Stimulation of a nitrate–nitrite–NO pathway with dietary nitrate reduces blood pressure, but the mechanisms or target organ are not clear. We investigated the hypothesis that inorganic nitrate and nitrite attenuate reactivity of renal microcirculation and blood pressure responses to angiotensin II (ANG II) by modulating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and NO bioavailability. Nitrite in the physiological range (10−7–10−5 mol/L) dilated isolated perfused renal afferent arterioles, which were associated with increased NO. Contractions to ANG II (34%) and simultaneous NO synthase inhibition (56%) were attenuated by nitrite (18% and 26%). In a model of oxidative stress (superoxide dismutase-1 knockouts), abnormal ANG II–mediated arteriolar contractions (90%) were normalized by nitrite (44%). Mechanistically, effects of nitrite were abolished by NO scavenger and xanthine oxidase inhibitor, but only partially attenuated by inhibiting soluble guanylyl cyclase. Inhibition of NADPH oxidase with apocynin attenuated ANG II–induced contractility (35%) similar to that of nitrite. In the presence of nitrite, no further effect of apocynin was observed, suggesting NADPH oxidase as a possible target. In preglomerular vascular smooth muscle cells and kidney cortex, nitrite reduced both basal and ANG II–induced NADPH oxidase activity. These effects of nitrite were also abolished by xanthine oxidase inhibition. Moreover, supplementation with dietary nitrate (10−2 mol/L) reduced renal NADPH oxidase activity and attenuated ANG II–mediated arteriolar contractions and hypertension (99±2–146±2 mm Hg) compared with placebo (100±3–168±3 mm Hg). In conclusion, these novel findings position NADPH oxidase in the renal microvasculature as a prime target for blood pressure–lowering effects of inorganic nitrate and nitrite.

Cross-talk Between Nitrate-Nitrite-NO and NO Synthase Pathways in Control of Vascular NO Homeostasis

AIMS Inorganic nitrate and nitrite from endogenous and dietary sources have emerged as alternative substrates for nitric oxide (NO) formation in addition to the classic L-arginine NO synthase (NOS)-dependent pathway. Here, we investigated a potential cross-talk between these two pathways in the regulation of vascular function. RESULTS Long-term dietary supplementation with sodium nitrate (0.1 and 1 mmol kg(-1) day(-1)) in rats caused a reversible dose-dependent reduction in phosphorylated endothelial NOS (eNOS) (Ser1177) in aorta and a concomitant increase in phosphorylation at Thr495. Moreover, eNOS-dependent vascular responses were attenuated in vessels harvested from nitrate-treated mice or when nitrite was acutely added to control vessels. The citrulline-to-arginine ratio in plasma, as a measure of eNOS activity, was reduced in nitrate-treated rodents. Telemetry measurements revealed that a low dietary nitrate dose reduced blood pressure, whereas a higher dose was associated with a paradoxical elevation. Finally, plasma cyclic guanosine monophosphate increased in mice that were treated with a low dietary nitrate dose and decreased with a higher dose. INNOVATION AND CONCLUSIONS These results demonstrate the existence of a cross-talk between the nitrate-nitrite-NO pathway and the NOS-dependent pathway in control of vascular NO homeostasis.

Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism

Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O2(∙-)) as part of the innate host defense system, but exaggerated and sustained O2(∙-) generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O2(∙-) and peroxynitrite (ONOO(-)) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O2(∙-) and ONOO(-) production in macrophages, which was significantly reduced by nitrite (10µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O2(∙-) generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response.

Dietary nitrate improves age-related hypertension and metabolic abnormalities in rats via modulation of angiotensin II receptor signaling and inhibition of superoxide generation

Advanced age is associated with increased risk for cardiovascular disease and type 2 diabetes. A proposed central event is diminished amounts of nitric oxide (NO) due to reduced generation by endothelial NO synthase (eNOS) and increased oxidative stress. In addition, it is widely accepted that increased angiotensin II (ANG II) signaling is also implicated in the pathogenesis of endothelial dysfunction and hypertension by accelerating formation of reactive oxygen species. This study was designed to test the hypothesis that dietary nitrate supplementation could reduce blood pressure and improve glucose tolerance in aged rats, via attenuation of NADPH oxidase activity and ANG II receptor signaling. Dietary nitrate supplementation for two weeks reduced blood pressure (10-15mmHg) and improved glucose clearance in old, but not in young rats. These favorable effects were associated with increased insulin responses, reduced plasma creatinine as well as improved endothelial relaxation to acetylcholine and attenuated contractility to ANG II in resistance arteries. Mechanistically, nitrate reduced NADPH oxidase-mediated oxidative stress in the cardiovascular system and increased cGMP signaling. Finally, nitrate treatment in aged rats normalized the gene expression profile of ANG II receptors (AT1A, AT2, AT1A/AT2 ratio) in the renal and cardiovascular systems without altering plasma levels of renin or ANG II. Our results show that boosting the nitrate-nitrite-NO pathway can partly compensate for age-related disturbances in endogenous NO generation via inhibition of NADPH oxidase and modulation of ANG II receptor expression. These novel findings may have implications for nutrition-based preventive and therapeutic strategies against cardiovascular and metabolic diseases.

In adenosine A2B knockouts acute treatment with inorganic nitrate improves glucose disposal, oxidative stress, and AMPK signaling in the liver

Rationale: Accumulating studies suggest that nitric oxide (NO) deficiency and oxidative stress are central pathological mechanisms in type 2 diabetes (T2D). Recent findings demonstrate therapeutic effects by boosting the nitrate-nitrite-NO pathway, which is an alternative pathway for NO formation. This study aimed at investigating the acute effects of inorganic nitrate on glucose and insulin signaling in adenosine A2B receptor knockout mice (A−/−2B), a genetic mouse model of impaired metabolic regulation. Methods: Acute effects of nitrate treatment were investigated in aged wild-type (WT) and A−/−2B mice. One hour after injection with nitrate (0.1 mmol/kg, i.p.) or placebo, metabolic regulation was evaluated by intraperitoneal glucose and insulin tolerance tests. NADPH oxidase-mediated superoxide production and AMPK phosphorylation were measured in livers obtained from non-treated or glucose-treated mice, with or without prior nitrate injection. Plasma was used to determine insulin resistance (HOMA-IR) and NO signaling. Results: A−/−2B displayed increased body weight, reduced glucose clearance, and attenuated overall insulin responses compared with age-matched WT mice. Nitrate treatment increased circulating levels of nitrate, nitrite and cGMP in the A−/−2B, and improved glucose clearance. In WT mice, however, nitrate treatment did not influence glucose clearance. HOMA-IR increased following glucose injection in the A−/−2B, but remained at basal levels in mice pretreated with nitrate. NADPH oxidase activity in livers from A−/−2B, but not WT mice, was reduced by nitrate treatment. Livers from A−/−2B displayed reduced AMPK phosphorylation compared with WT mice, and this was increased by nitrate treatment. Finally, injection with the anti-diabetic agent metformin induced similar therapeutic effects in the A−/−2B as observed with nitrate. Conclusion: The A−/−2B mouse is a genetic mouse model of metabolic syndrome. Acute treatment with nitrate improved the metabolic profile in it, at least partly via reduction in oxidative stress and improved AMPK signaling in the liver.

Abrogation of adenosine A1 receptor signalling improves metabolic regulation in mice by modulating oxidative stress and inflammatory responses.

Aims/hypothesisAdenosine is an important regulator of metabolism; however, the role of the A1 receptor during ageing and obesity is unclear. The aim of this study was to investigate the effects of A1 signalling in modulating metabolic function during ageing.MethodsAge-matched young and aged A1 (also known as Adora1)-knockout (A1−/−) and wild-type (A1+/+) mice were used. Metabolic regulation was evaluated by body composition, and glucose and insulin tolerance tests. Isolated islets and islet arterioles were used to detect islet endocrine and vascular function. Oxidative stress and inflammation status were measured in metabolic organs and systemically.ResultsAdvanced age was associated with both reduced glucose clearance and insulin sensitivity, as well as increased visceral adipose tissue (VAT) in A1+/+ compared with A1−/− mice. Islet morphology and insulin content were similar between genotypes, but relative changes in in vitro insulin release following glucose stimulation were reduced in aged A1+/+ compared with A1−/− mice. Islet arteriolar responses to angiotensin II were stronger in aged A1+/+ mice, this being associated with increased NADPH oxidase activity. Ageing resulted in multiple changes in A1+/+ compared with A1−/− mice, including enhanced NADPH oxidase-derived O2− formation and NADPH oxidase isoform 2 (Nox2) protein expression in pancreas and VAT; elevated levels of circulating insulin, leptin and proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-12); and accumulation of CD4+ T cells in VAT. This was associated with impaired insulin signalling in VAT from aged A1+/+ mice.Conclusions/interpretationThese studies emphasise that A1 receptors regulate metabolism and islet endocrine and vascular functions during ageing, including via the modulation of oxidative stress and inflammatory responses, among other things.

Enhanced XOR activity in eNOS-deficient mice: Effects on the nitrate-nitrite-NO pathway and ROS homeostasis

Xanthine oxidoreductase (XOR) is generally known as the final enzyme in purine metabolism and as a source of reactive oxygen species (ROS). In addition, this enzyme has been suggested to mediate nitric oxide (NO) formation via reduction of inorganic nitrate and nitrite. This NO synthase (NOS)-independent pathway for NO generation is of particular importance during certain conditions when NO bioavailability is diminished due to reduced activity of endothelial NOS (eNOS) or increased oxidative stress, including aging and cardiovascular disease. The exact interplay between NOS- and XOR-derived NO generation is not fully elucidated yet. The aim of the present study was to investigate if eNOS deficiency is associated with changes in XOR expression and activity and the possible impact on nitrite, NO and ROS homeostasis. Plasma levels of nitrate and nitrite were similar between eNOS deficient (eNOS-/-) and wildtype (wt) mice. XOR activity was upregulated in eNOS-/- compared with wt, but not in nNOS-/-, iNOS-/- or wt mice treated with the non-selective NOS inhibitor L-NAME. Following an acute dose of nitrate, plasma nitrite increased more in eNOS-/- compared with wt, and this augmented response was abolished by the selective XOR inhibitor febuxostat. Livers from eNOS-/- displayed higher nitrite reducing capacity compared with wt, and this effect was attenuated by febuxostat. Dietary supplementation with nitrate increased XOR expression and activity, but concomitantly reduced superoxide generation. The latter effect was also seen in vitro after nitrite administration. Treatment with febuxostat elevated blood pressure in eNOS-/-, but not in wt mice. A high dose of dietary nitrate reduced blood pressure in naïve eNOS-/- mice, and again this effect was abolished by febuxostat. In conclusion, eNOS deficiency is associated with an upregulation of XOR facilitating the nitrate-nitrite-NO pathway and decreasing the generation of ROS. This interplay between XOR and eNOS is proposed to play a significant role in NO homeostasis and blood pressure regulation.

Nitrite-mediated renal vasodilatation is increased during ischemic conditions via cGMP-independent signaling.

The kidney is vulnerable to hypoxia, and substantial efforts have been made to ameliorate renal ischemic injury secondary to pathological conditions. Stimulation of the nitrate-nitrite-nitric oxide pathway is associated with renal and cardiovascular protection in disease models, but less is known about the vascular effects during renal ischemia. This study was aimed at investigating the vascular effects of nitrite in the kidney during normoxic and ischemic conditions. Using a multiwire myograph system, we assessed nitrite-mediated relaxation (10(-9)-10(-4)mol/L) in isolated and preconstricted renal interlobar arteries from C57BL/6 mice under normal conditions (pO2 13kPa; pH 7.4) and with low oxygen tension and low pH to mimic ischemia (pO2 3kPa; pH 6.6). Xanthine oxidoreductase expression was analyzed by quantitative PCR, and production of reactive nitrogen species was measured by DAF-FM DA fluorescence. During normoxia significant vasodilatation (15±3%) was observed only at the highest concentration of nitrite, which was dependent on NO-sGC-cGMP signaling. The vasodilatory responses to nitrite were greatly sensitized and enhanced during hypoxia with low pH, demonstrating significant dilatation (11±1%) already in the physiological range (10(-8)mol/L), with a maximum response of 27±2% at 10(-4) mol/L. In contrast to normoxia, and to that observed with a classical NO donor (DEA NONOate), this sensitization was independent of sGC-cGMP signaling. Moreover, inhibition of various enzymatic systems reported to reduce nitrite in other vascular beds, i.e., aldehyde oxidase (raloxifene), aldehyde dehydrogenase (cyanamide), and NO synthase (L-NAME), had no effect on the nitrite response. However, inhibition of xanthine oxidoreductase (XOR; febuxostat or allopurinol) abolished the sensitized response to nitrite during hypoxia and acidosis. In conclusion, in contrast to normoxia, nitrite exerted potent vasorelaxation during ischemic conditions already at physiological concentrations. This effect was dependent on functional XOR but independent of classical downstream signaling by sGC-cGMP.

Adenosine signaling in diabetes mellitus and associated cardiovascular and renal complications.

Diabetes mellitus is characterized by abnormal glucose and lipid metabolism, and subsequent hyperglycemia and dyslipidemia, which results from defects in pancreatic islet beta-cells insulin secretion and/or decreased insulin sensitivity in metabolically active organs (i.e. liver, skeletal muscle and adipose tissue). Accumulating evidence highlights a critical role for the adenosine system in the regulation of insulin and glucose homeostasis and the pathophysiology of type 2 diabetes (T2D). Adenosine is a key diverse extracellular signaling molecule that regulates several aspects of tissue function by activating four G-protein-coupled receptors (i.e. A1, A2A, A2B and A3 receptors). Moreover, adenosine receptor signaling plays a critical role in inflammation, immune system, and oxidative stress, factors that are also important in metabolic disorders. This review discusses the role of the adenosine receptor system in the development or progression of diabetes mellitus, with specific focus on T2D, and associated complications linked to the cardiovascular and renal systems.

Renal denervation attenuates NADPH oxidase-mediated oxidative stress and hypertension in rats with hydronephrosis

Hydronephrosis is associated with the development of salt-sensitive hypertension. Studies have suggested that increased sympathetic nerve activity and oxidative stress play important roles in hypertension and the modulation of salt sensitivity. The present study primarily aimed to examine the role of renal sympathetic nerve activity in the development of hypertension in rats with hydronephrosis. In addition, we aimed to investigate if NADPH oxidase (NOX) function could be affected by renal denervation. Partial unilateral ureteral obstruction (PUUO) was created in 3-wk-old rats to induce hydronephrosis. Sham surgery or renal denervation was performed at the same time. Blood pressure was measured during normal, high-, and low-salt diets. The renal excretion pattern, NOX activity, and expression as well as components of the renin-angiotensin-aldosterone system were characterized after treatment with the normal salt diet. On the normal salt diet, rats in the PUUO group had elevated blood pressure compared with control rats (115 ± 3 vs. 87 ± 1 mmHg, P < 0.05) and displayed increased urine production and lower urine osmolality. The blood pressure change in response to salt loading (salt sensitivity) was more pronounced in the PUUO group compared with the control group (15 ± 2 vs. 5 ± 1 mmHg, P < 0.05). Renal denervation in PUUO rats attenuated both hypertension (97 ± 3 mmHg) and salt sensitivity (5 ± 1 mmHg, P < 0.05) and normalized the renal excretion pattern, whereas the degree of renal fibrosis and inflammation was not changed. NOX activity and expression as well as renin and ANG II type 1A receptor expression were increased in the renal cortex from PUUO rats and normalized by denervation. Plasma Na(+) and K(+) levels were elevated in PUUO rats and normalized after renal denervation. Finally, denervation in PUUO rats was also associated with reduced NOX expression, superoxide production, and fibrosis in the heart. In conclusion, renal denervation attenuates hypertension and restores the renal excretion pattern, which is associated with reduced renal NOX and components of the renin-angiotensin-aldosterone system. This study emphasizes a link between renal nerves, the development of hypertension, and modulation of NOX function.