Online Hypertension

Lundberg, A. Erik G. Persson and Mattias CarlströmXiang Gao, Ting Yang, Ming Liu, Maria Peleli, Christa Zollbrecht, Eddie Weitzberg, Jon O.

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

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2014 American Heart Association, Inc. All rights reserved.

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1

Hypertension is a major risk factor for stroke, coronary heart disease, and premature death. The prevalence is continu-

ously increasing, and it is estimated that elevated blood pressure affects >1 of every 3 adults or ≈1 billion people worldwide.1 The economic burden to the society is enormous, and inten-sified efforts to prevent and control hypertension are crucial. Nitric oxide (NO) is a key regulator of renal and cardiovascu-lar function, and emerging evidence shows that renal oxidative stress and subsequent NO deficiency are critically associated with development of hypertension and cardiovascular disease.2–5 Therefore, treatment modalities that reduce oxidative stress and increase NO production in the kidney may have important implications for renal and cardiovascular homeostasis.

The kidneys control long-term blood pressure level by adjusting renal peripheral vascular resistance and hence body

fluid volume. The afferent arterioles are the major resistance vessels in the kidney, and increased arteriolar reactivity may reduce glomerular perfusion and filtration, increase extracel-lular volume, and thus contribute to development of hyperten-sion.6 There is a clear relationship between afferent arteriolar resistance and blood pressure, and a reduction in arteriolar diameter may even predict later development of hyperten-sion.7 Preglomerular resistance is determined by the balance between constrictor agents such as angiotensin II (ANG II) and vasodilator pathways, notably NO.8,9 Elevated level of ANG II is a strong stimulus for nicotinamide adenine dinu-cleotide phosphate (NADPH) oxidase–induced superoxide formation in the vasculature, and such oxidative stress signifi-cantly contributes to enhanced arteriolar reactivity and sus-ceptibility to hypertension.10–13

Abstract—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. (Hypertension. 2014;65:00-00.) • Online Data Supplement

Key Words: angiotension II ◼ hypertension, renovascular ◼ kidney ◼ microcirculation ◼ nitrates ◼ nitric oxide ◼ oxidative stress

Received July 7, 2014; first decision July 21, 2014; revision accepted August 26, 2014.From the Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden (X.G., A.E.G.P.); and Department of Physiology and Pharmacology,

Karolinska Institutet, Stockholm, Sweden (T.Y., M.L., M.P., C.Z., E.W., J.O.L., M.C.).*These authors contributed equally to this work.The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.

114.04222/-/DC1.Correspondence to Mattias Carlström, Department of Physiology and Pharmacology, Nanna Svartz Väg 2, S-17177, Stockholm, Sweden. E-mail mattias.

[email protected]

NADPH Oxidase in the Renal Microvasculature Is a Primary Target for Blood Pressure–Lowering

Effects by Inorganic Nitrate and NitriteXiang Gao,* Ting Yang,* Ming Liu, Maria Peleli, Christa Zollbrecht, Eddie Weitzberg,

Jon O. Lundberg, A. Erik G. Persson, Mattias Carlström

© 2014 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.114.04222

Original Article

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2 Hypertension January 2015

Research during the past decade has revealed that the endogenous anions nitrate and nitrite are used to generate NO and that stimulation of this nitrate–nitrite–NO pathway can compensate for disturbances in NO generation from NO synthases (NOS).14–17 This has interesting nutritional impli-cations because nitrate is also a normal constituent of our daily diet, with vegetables being the main source. Oral com-mensal bacteria seem obligatory for the reduction of nitrate to nitrite, whereas several mammalian enzymes are capable of subsequent reduction of nitrite to NO and other bioactive nitrogen oxides.15–18

Recent clinical and experimental studies have indepen-dently shown that supplementation with inorganic nitrate, by dietary means or with nitrate salt, reduces blood pressure in healthy normotensive individuals19–21 as well as in hyperten-sives.22 In addition, these anions can partly compensate for metabolic disturbances in aged endothelial NOS knockout mice,23 reduce hypertension and markers of oxidative stress, and ameliorate organ injuries in models of renal and cardio-vascular disease.24–26 The mechanisms for nitrate- and nitrite-mediated antihypertensive effects along with renal and cardiac protection are not yet clear. In the present study, we tested the hypothesis that stimulation of a nitrate–nitrite–NO path-way, with inorganic nitrate or nitrite, limits afferent arteriolar responsiveness and hypertension in response to ANG II by modulating the balance between superoxide and NO.

MethodsAnimalsThe studies were conducted on male C57BL/6J mice (The Jackson Laboratory, Maine). To specifically investigate the role of bacteria in bioactivation of nitrate, germ-free (n=8) and conventional (n=8) mice were used. To investigate the effects of nitrite in a model with oxidative stress, we used superoxide dismutase 1 (SOD1) knock-out (SOD1−/−; n=15) and wild-type (SOD1+/+; n=19). To investigate the effects of dietary nitrate in a model of renal hypertension, we used Sprague-Dawley rats (Charles River Laboratories, Germany) for blood pressure measurements (n=12) (online-only Data Supplement).

Vascular Studies

Isolated and Perfused Afferent ArteriolesDissection and perfusion of renal cortical afferent arterioles and con-tractility studies were performed as described previously (Figure S1 in the online-only Data Supplement).10 The experiments were digi-tally recorded and then digitized off-line and analyzed as described before.11 Changes in luminal diameters were measured to estimate the effect of vasoactive substances.

Fluorescent Detection of NO Production in Afferent ArteriolesA highly sensitive, photostable cell-permeable fluorescent probe, 4-amino-5-methylamino-2′,7′-difluorescein diacetate (Invitrogen, Life Technologies Europe BV, Stockholm, Sweden), was used for the detection of NO production with nitrite (10−5 mol/L) in the isolated and perfused afferent arteriole (Figure S1).

A

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Figure 1. Nitrite-mediated dilatation of renal microvessels in C57 mice is associated with increased nitric oxide bioavailability. A, Effects of acute administration of nitrite on diameters of afferent arterioles. Nitrite dilated arterioles (n=6) in a concentration-dependent manner, whereas time-controlled placebo treatment (control, n=6) had no significant effects. Each dose was applied for 2 minutes. *P<0.05 compared with control group (2-way ANOVA, Bonferroni multiple comparisons test). B, Effect of nitrite (10−5 mol/L; n=4) or placebo (n=4) on nitric oxide (NO) production in afferent arterioles, determined as changes in fluorescence during a 20-minute period. Administration of nitrite was associated with increased NO production compared with baseline (paired Student t test). C, Effects of acute administration of nitrite on diameters of afferent arterioles during inhibition of NO synthase with Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME; 10−4 mol/L). Administration of nitrite (10−5 mol/L; n=8) was associated with increased arteriolar diameter, whereas l-NAME alone (n=6) reduced diameter. l-NAME–mediated contraction was abolished by simultaneous administration of nitrite (n=6). *P<0.05 compared with baseline; #P<0.05 compared with l-NAME (2-way ANOVA, Bonferroni multiple comparisons test). Values are presented as mean±SEM.


Gao et al NO3–NO2–NO Pathway in Hypertension 3

Myograph StudiesCarotid arteries, renal interlobar arteries, and third branch of mes-enteric arteries from C57 mice were isolated, and arterial rings were mounted in myograph chambers (online-only Data Supplement). Resting tension was set according to normalization procedure, and vessel viability was assessed by the responses to 0.1 mol/L KCl. After washout, cumulative concentration response for ANG II (10−12–10−6 mol/L) was obtained, with or without simultaneous incu-bation with nitrite (10−5 mol/L).

Renal NADPH Oxidase Activity

Preglomerular Vascular Smooth Muscle CellsIsolation and culturing of primary preglomerular vascular smooth muscle cells (PG-VSMCs) were performed as previously described,27 and the phenotype was confirmed as described by Dubey et al.28 After 30-minute incubation with different nitrite concentrations (NaNO

2)

or placebo (NaCl), the cells were transferred into reaction tubes.

Kidney CortexThe tissue was homogenized with bullet blender in ice-cold PBS, and the homogenate was centrifuged at 4°C for 20 minutes at 2000g. Fresh tissue homogenates were incubated for 30 minutes with placebo (NaCl), ANG II (10−6 mol/L), nitrite (10−5 mol/L), or combinations with ANG II and nitrite before the NADPH oxidase activity measurements.

Chemiluminescence technique was used to determine the NADPH oxidase–mediated superoxide formation. In experiments with both PG-VSMCs and renal cortex, 100 μL of NADPH (10−4 mol/L) and lucigenin (5×10−5 mol/L; Sigma-Aldrich) were injected into the re-action tube (final volume of 1 mL), and NADPH oxidase activity

was determined by measuring lucigenin chemiluminescence every 3 seconds for 3 minutes with an AutoLumat LB953 Multi-Tube Luminometer (Berthold Technologies, Bad Wildbad, Germany). Results were corrected by cell number (VSMC) or by protein quanti-fication (cortex) using Bradford protein assay (Bio-Rad Laboratories, United Kingdom).

Renal NADPH Oxidase and Xanthine Oxidase ExpressionRenal cortex and PG-VSMCs were used for quantitative polymerase chain reaction analysis of NADPH oxidases and xanthine oxidase (XO) expression (online-only Data Supplement).

Plasma Analysis

Cyclic Guanosine MonophosphateThe inhibitor of cAMP/cGMP phosphodiesterases (IBMX [3-isobutyl- 1-methylxanthine]) was added to plasma samples (final concentra-tion, 10−5 mol/L) before freezing and was later analyzed for cGMP content with ELISA method (Biotrak EIA System; Amersham).23

Nitrite and NitratePlasma samples were extracted with methanol and analyzed using a highly sensitive high-performance liquid chromatography technique as previously described.23

Blood Pressure Response to Prolonged ANG II InfusionTelemetric devices (PA-C40, DSI, St Paul, MN) were implanted in adult male Sprague-Dawley rats (n=12; body weight=335±4), and

A B

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Figure 2. Nitrite attenuates renal microvascular responses to angiotensin II (ANG II) via xanthine oxidase (XO)–dependent nitric oxide (NO) formation. A, ANG II concentration response curves (control) in isolated and perfused afferent arterioles of C57 mice (n=6). Simultaneous administration of nitrite (10−5 mol/L) attenuated ANG II–mediated contraction (n=8). B, Effect of nitrite on ANG II–induced contractions (control) during inhibition of NO synthase (NOS) with Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Isolated and perfused arterioles were pretreated with l-NAME alone (10−4 mol/L; n=8) or in combination with nitrite (10−5 mol/L; n=8) for 15 minutes, followed by cumulative doses of ANG II (2 minutes each dose). During NOS inhibition, both sensitivity and contractility to ANG II were strongly enhanced. At the same time, the attenuating effect of nitrite on ANG II–mediated contraction was more pronounced. C, The attenuated contractile response to ANG II+l-NAME in the presence of nitrite was blocked by simultaneous administration of the XO inhibitor oxypurinol (0.5×10−3 mol/L; n=7) or by scavenging of NO with 2- (4- carboxyphenyl)- 4, 5- dihydro- 4, 4, 5, 5- tetramethyl- 1H- imidazolyl- 1- oxy- 3- oxide (cPTIO; n=7). However, the effect of nitrite was only partially blocked by inhibition of soluble guanylyl cyclase (sGC) with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; n=6). Oxypurinol, cPTIO, and ODQ were administered 15 minutes before the ANG II dose response. D, Maximal arteriolar contraction for each group. *P<0.05 compared with ANG II (control; A) or ANG II+l-NAME (control; B); †P<0.05 compared with nitrite in combination with ODQ, cPTIO, and oxypurinol; #P<0.05 compared with nitrite, nitrite+cPTIO, and nitrite+oxypurinol (C; 2-way ANOVA, Bonferroni multiple comparisons test). Values are presented as mean±SEM.



measurements were conducted as previously described.29 After surgery, the animals were allowed to recover for 10 days before measurements were started. Blood pressure and heart rate were measured continu-ously during (1) baseline conditions (72 hours), followed by giving (2) Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) in drinking water (500 mg/L) supplemented with placebo (NaCl) or sodium nitrate (NaNO

3, 10−2 mol/L) for 72 hours. The rats were then anesthetized by

spontaneous inhalation of isoflurane (Forene, Abbot Scandinavia AB, Sweden) in air (≈2.2%), and an osmotic minipump (Alzet, Durect, CA) was implanted subcutaneously, delivering ANG II (Sigma-Aldrich) at 120 ng/kg per 24 hours for 20 days. Two days after implantation, tele-metric measurements were performed continuously for 15 days with ANG II infusion. During the past 4 days, measurements were con-ducted without placebo or nitrate supplementation.

Drugs and ReagentsDrugs and chemicals used for this study were obtained from Sigma, unless otherwise stated. In the arteriolar contraction experiments, all compounds were applied to the bath solution.

Statistical AnalysisValues are presented as mean±SEM. ANOVA, followed by post hoc comparison, was used to test time- or concentration-dependent changes in the arteriolar diameter and to allow for >1 comparison with the same variable among groups. Single comparisons among normally distributed parameters were tested for significance with Student paired or unpaired t test as appropriate. Experiments were evaluated in a blinded fashion. Statistical significance was defined as P<0.05.

EthicsThe experiments were approved by the Uppsala or Stockholm Ethical Committee for Animal Experiments and were conducted in accor-dance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Results

Effect of Nitrite on Renal Afferent Arteriolar Diameter and NO ProductionNitrite dilated arterioles in a concentration-dependent manner, whereas no change in diameter was observed for the time con-trol (Figure 1A). Significant vasodilatation was observed already at 10−7 mol/L nitrite, which is well within the physiological range. The nitrite-evoked dilatation (10−5 mol/L) was paralleled by increased NO formation as measured with 4-amino-5-methylamino-2′,7′-difluorescein fluorescence (Figure 1B). Inhibition of NOS with l-NAME reduced arteriolar diameter, and simultaneous administration of nitrite completely prevented this l-NAME–mediated contraction (Figure 1C).

Nitrite Attenuates ANG II–Induced Contractions in the Renal MicrovasculatureANG II is a strong modulator of preglomerular resistance. Afferent arteriolar contraction to ANG II involves NADPH oxidase–derived superoxide,11,13 but the response to this is dampened by a concomitant increase in NO formation by NOS.10,30,31 ANG II (10−12–10−6 mol/L; 2 minutes each dose) constricted arterioles in a concentration-dependent manner (threshold response, 10−9 mol/L), with a maximum response of 34% (Figure 2A). Simultaneous treatment with nitrite (10−5 mol/L) attenuated both sensitivity (threshold response, 10−8 mol/L) and contractility to ANG II (maximum response, 18%).

To investigate whether the renal microcirculation is espe-cially sensitive to nitrite, we performed similar experimental

protocols in conductance vessels, larger renal vessels, and small extrarenal resistance arteries. The demonstrated attenuating effect of nitrite (10−5 mol/L) on ANG II–induced contractions in preglomerular arterioles was not observed in carotid, renal interlobar, or mesenteric arteries (Figure S2). These results sug-gest the renal microcirculation as a primary target of nitrite.

To investigate the role of NOS-derived NO in offsetting ANG II–induced arteriolar contraction, vessels were treated with l-NAME (10−4 mol/L) for 15 minutes before and during treatment with ANG II. As expected, l-NAME enhanced both sensitivity (threshold response, 10−12 mol/L) and contractility to ANG II (maximum response, 56%; Figure 2B). In arteri-oles exposed to nitrite, these effects were markedly attenuated (threshold response, 10−11 mol/L and maximum response, 26%).

Nitrite Effects Are Mediated by XO-Catalyzed NO FormationNext, we aimed to investigate the mechanism(s) for nitrite-mediated inhibition on ANG II contractility. To further

A

B

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Figure 3. Nitrite attenuates renal microvascular responses to angiotensin II (ANG II) similar to that observed during nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition. A, Inhibition of NADPH oxidase with apocynin (10−4 mol/L) attenuated the contractile response to ANG II+Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) (control) in arterioles from C57 mice (n=6). B, However, the contractile response of arterioles with combined apocynin+nitrite treatment (n=7) was not different from that with apocynin or nitrite alone. C, Maximal arteriolar contraction for each group. *P<0.05 compared with ANG II+l-NAME (control; 2-way ANOVA, Bonferroni multiple comparisons test). Values are presented as mean±SEM.



investigate whether the nitrite-mediated attenuation of arte-riolar contractility was dependent on generation of NO, a NO scavenger (2- (4- carboxyphenyl)- 4, 5- dihydro- 4, 4, 5, 5- tetramethyl- 1H- imidazolyl- 1- oxy- 3- oxide [cPTIO]) was administered together with l-NAME and nitrite. In the pres-ence of cPTIO, the effect of nitrite was abolished (Figure 2C), and the contractile response was indistinguishable from that of ANG II+l-NAME (Figure 2D). A variety of proteins and enzymes including hemoglobin, myoglobin, mitochon-drial complexes, aldehyde oxidase, and XO can catalyze the reduction of nitrite to NO in blood and tissues.17,32 Although oxypurinol alone did not significantly alter the contractile response to ANG+l-NAME (data not shown), it prevented the effects of nitrite on arteriolar responses (Figure 2C and 2D). Interestingly, the effect of nitrite was only partially attenuated during simultaneous inhibition of soluble guany-lyl cyclase with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinox-alin-1-one). Taken together, these findings demonstrate that nitrite can attenuate ANG II–induced arteriolar contraction via XO-dependent NO generation. Moreover, the subse-quent events are partially independent of the NO–soluble guanylyl cyclase–cGMP pathway.

Nitrite Modulates NADPH Oxidase ActivityRecent reports have suggested that nitrate and nitrite may reduce oxidative stress in models of renal and cardiovascular disease.24–26 Here, inhibition of NADPH oxidases with apocynin markedly attenuated the contractile response to ANG II during NOS inhibition (Figure 3A). This supports the notion that ANG II–induced contraction is linked to increased NADPH oxidase activity. Simultaneous administration of nitrite and apocynin had no further attenuating effect compared with that of nitrite alone (Figure 3B and 3C), suggesting that nitrite reduces ANG II–induced vasoconstriction by modulating NADPH oxidase function. Next, we investigated whether nitrite mod-ulates NADPH oxidase activity in renal cortex and primary PG-VSMCs. Nitrite attenuated basal NADPH oxidase–derived superoxide production in cortical tissue compared with control (Figure 4A). Incubation with ANG II significantly increased NADPH oxidase activity, and in the presence of nitrite, this effect was abolished (Figure 4A). In PG-VSMCs, incubation with nitrite reduced basal NADPH oxidase activity in a dose-dependent fashion (Figure 4B). Similar to renal cortex, ANG II increased NADPH oxidase–derived superoxide production in PG-VSMC that was abolished by nitrite (Figure 4C). As

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Figure 4. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is attenuated with nitrite. A, In the renal cortex from C57 mice, incubation with angiotensin II (ANG II; 10−6 mol/L; n=5) for 30 minutes increased NADPH oxidase activity compared with control group. Incubation with nitrite (10−5 mol/L) for 30 minutes attenuated NADPH oxidase–derived superoxide production compared with control and abolished the effect of ANG II. n=6 (kidneys) for each group. B, In preglomerular vascular smooth muscle cells (PG-VSMCs), incubation with nitrite (30 minutes) attenuated NADPH oxidase–derived superoxide production compared with control. The attenuating effect with nitrite was dose dependent. Control: n=14; nitrite: n=11 (10−3), n=13 (10−4), n=14 (10−5), n=10 (10−6 mol/L). *P<0.05 compared with control. C, Similar to that in the renal cortex, ANG II increased NADPH oxidase activity in PG-VSMCs, which was abolished by nitrite. D, Inhibition of xanthine oxidase (XO) with febuxostat (3×10−8 mol/L) abolished nitrite-mediated effects on NADPH oxidase activity under both basal and ANG II–stimulated conditions. Control, n=10; ANG II, n=10, nitrite, n=10; febuxostat, n=10; ANG II+febuxostat, n=10; nitrite+febuxostat, n=10, nitrite+ANG II+febuxostat, n=10 (1-way ANOVA, Bonferroni multiple comparisons test). Values are presented as mean±SEM.



described in Figure 2C and 2D, the nitrite-mediated reduction in arteriolar contractility was dependent on functional XO. In PG-VSMCs, inhibition of XO with febuxostat abolished the ability of nitrite in reducing NADPH oxidase activity (Figure 4D). Expression of XO was confirmed in both renal cortex and PG-VSMCs (online-only Data Supplement). In aggregate, these results suggest that nitrite modulates NADPH oxidase function in the kidney, primarily in preglomerular vessels, and this effect requires functional XO.

Nitrite Restores Contractile Responses in a Mouse Model of Oxidative StressNext, we investigated the effects of nitrite in SOD1-knockout mice, a transgenic mouse model characterized by increased oxi-dative stress and NO deficiency. In SOD1 wild-type mice, the con-tractile response to ANG II (maximal response, 41%) was similar to that observed in C57 mice (compare Figure 2A), and simul-taneous incubation with nitrite attenuated arteriolar contraction (maximum response, 24%; Figure 5A). Arterioles from SOD1 knockouts displayed greatly enhanced contractile responses to ANG II (threshold response, 10−11 mol/L; maximum response, 90%; Figure 5B). Treatment with nitrite markedly attenuated the arteriolar responses (threshold response, 10−9 mol/L; maximum contraction, 44%), which were similar to that observed in placebo-treated C57 (Figure 2) and SOD1 wild-type mice (Figure 5A). Maximal arteriolar responses to ANG II are summarized in Figure 5C. Again, this supports that nitrite interferes with reactive oxygen species generating systems to elicit vasodilatation.

Dietary Nitrate Attenuates NADPH Oxidase Activity and Microvascular Responses to ANG IISupplementation with dietary nitrate can fuel a nitrate–nitrite–NO pathway and increase levels of bioactive nitrogen oxides,14,15,32 and we have reported therapeutic effects with inorganic nitrate in renal and cardiovascular disease models associated with NO deficiency and oxidative stress.23,24 Next, we investigated whether chronic dietary supplementation with nitrate could modulate renal arteriolar function and NADPH oxidase activ-ity. One week of dietary nitrate treatment was associated with increased plasma nitrate but did not significantly (P=0.10) alter circulating nitrite or cGMP levels (Figure S3). However, arte-rioles from mice treated with dietary nitrate displayed reduced ANG II–induced contractions (maximum response, 29%) com-pared with placebo-treated mice (maximum response, 58%; Figure 6A and 6B). Analysis of renal NADPH oxidase activity from the same groups showed that nitrate supplementation was associated with an ≈50% reduction in NADPH oxidase–derived superoxide production (Figure 6C). The effect of dietary nitrate on NADPH oxidase activity was not associated with any signifi-cant changes in mRNA expression of NADPH oxidase isoforms or its subunits (Figure S4).

Nitrate Bioactivation Is Dependent on BacteriaOral commensal bacteria have been suggested to be important in the bioactivation of nitrate by first reducing it to the more reactive nitrite anion.18,33,34 To investigate this, we used germ-free mice that are born and raised under completely sterile conditions. The effect of dietary nitrate supplementation on vascular contractil-ity was abolished in germ-free mice (Figure 6B). Similar to the

arteriolar responses, the inhibitory effect of nitrate supplementa-tion on NADPH oxidase activity was not observed in germ-free mice (Figure 6D). Overall, this demonstrates a crucial role of commensal bacteria in bioactivation of dietary nitrate.

Dietary Nitrate Attenuates Blood Pressure Elevation in a Model of Renal HypertensionFollowing the demonstration of pronounced effects of nitrate supplementation on arteriolar contractile responses ex vivo, together with inhibition of ANG II–induced NADPH oxidase activity, we investigated whether dietary nitrate would also affect cardiovascular function in a model of hypertension. Here,

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Figure 5. Nitrite restores renal microvascular responses to angiotensin II (ANG II) in a model with oxidative stress and exaggerated arteriolar contraction. A and B, ANG II concentration response curves in isolated and perfused afferent arterioles from superoxide dismutase 1 (SOD1) wild-type (SOD1+/+, n=8) and knockout (SOD1−/−, n=6) mice. Similar to that observed with C57 mice (compare with Figure 2A), simultaneous administration of nitrite (10−5 mol/L) attenuated ANG II–mediated contraction in wild-type mice (SOD1+/+ nitrite, n=8). In arterioles from the knockouts, the effect of nitrite was much enhanced (SOD1−/− nitrite, n=8) and the abnormal contractile response was normalized (similar to that in SOD1+/+ without nitrite). *P<0.05 compared with SOD1+/+ (A) or compared with SOD1−/− (B); #P<0.05 compared with corresponding SOD1+/+ mice in (A; 2-way ANOVA, Bonferroni multiple comparisons test). C, Maximal arteriolar contraction to ANG II in SOD1+/+ and SOD1−/− mice, with or without simultaneous nitrite treatment. P<0.05 between indicated groups (1-way ANOVA, Bonferroni multiple comparisons test). Values are presented as mean±SEM. cPTIO indicates 2- (4- carboxyphenyl)- 4, 5- dihydro- 4, 4, 5, 5- tetramethyl- 1H- imidazolyl- 1- oxy- 3- oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; and l-NAME, Nω-nitro-l-arginine methyl ester hydrochloride.



we find that the blood pressure elevation in response to chronic ANG II infusion was markedly attenuated in animals with dietary nitrate supplementation (Figure 7A). The placebo group displayed a characteristic slow pressure response to ANG II with progressive hypertension throughout the study period. No differ-ences in heart rate were observed between the placebo and nitrate groups (Figure S5). Finally, when the nitrate supplementation was stopped, the blood pressure increased in the nitrate group, whereas no change was observed in the placebo group. Maximal changes in blood pressure for the different treatment periods are shown in Figure 7B for the placebo and nitrate groups.

DiscussionThe present study provides novel evidence for a role of dietary nitrate and nitrite in long-term blood pressure regulation via effects on the renal microvasculature. Together, our data show that nitrate and nitrite dilate renal afferent arteriole and counteract ANG II–induced vasoconstriction by generating NO-like bioactivity and reducing NADPH oxidase activity.

Blood pressure–lowering effect of dietary nitrate has been demonstrated in healthy humans and rodents, but the mecha-nisms remain uncertain. Systemic vasodilatation by activation of the NO–soluble guanylyl cyclase–cGMP pathway has been sug-gested and is likely of importance in the acute effects of dietary nitrate and nitrite. However, we show that the renal microcircula-tion is exquisitely responsive to nitrite. Indeed, in larger vessels such as the aorta, the threshold for dilatation by nitrite under nor-moxia is >10−5 mol/L,35,36 whereas in renal arterioles, significant

vasodilatation was observed already at 10−7 mol/L, that is, well within the physiological range and actually lower than the mea-sured basal plasma levels of nitrite. Moreover, the other vascular beds (carotid, renal interlobar, and mesenteric arteries) exam-ined in the present study were unresponsive to nitrite under simi-lar conditions. These data suggest that basal circulating levels of nitrite are sufficient to participate in physiological regulation of preglomerular arteriolar tone. The reason for variable responses to nitrite among different vascular beds may be related to the abundance of nitrite reductases in the vascular wall as well as the degree of superoxide generation by NADPH oxidases.

Many of the biological effects of nitrite are thought to pro-ceed via its reduction to NO. Several enzymes and proteins have been implicated in nitrite reduction in the vasculature, including heme proteins such as hemoglobin and myoglobin, enzymes of the respiratory chain, aldehyde oxidase, and XO.15,37 Our find-ings emphasize XO as a major nitrite reducer in the renal micro-vasculature, because XO was highly expressed and inhibitors of the enzyme completely inhibited the nitrite response. The generated NO operates partly by stimulating a NO–soluble gua-nylyl cyclase–cGMP pathway, but more importantly by inhibit-ing vascular NADPH oxidase–dependent superoxide signaling. Evidence for the involvement of NO was supported by the visu-alization of NO formation using 4-amino-5-methylamino-2′,7′-difluorescein probe and more importantly by the functional inhibition of the nitrite response with the NO scavenger cPTIO. In all cases, the nitrite response was evident also in the presence of l-NAME, thereby excluding any involvement of NOS.

A B

DC

Figure 6. Commensal bacteria bioactivate dietary nitrate resulting in attenuated renal microvascular responses to angiotensin II (ANG II). Concentration responses to ANG II in afferent arterioles from mice with nitrate (NaNO3 10−2 mol/L in drinking water) or placebo (NaCl; control) supplementation for 7 to 10 days. Isolated and perfused arterioles were pretreated with Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) alone (10−4 mol/L). A, In conventional mice, dietary nitrate treatment (n=8) was associated with reduced ANG II–induced contractions compared with placebo-treated mice (n=6). This effect with nitrate was abolished in the germ-free mice (curves not shown). *P<0.05 compared with placebo-treated conventional mice (2-way ANOVA, Bonferroni multiple comparisons test). B, Maximal arteriolar contraction to ANG II+l-NAME in conventional and germ-free mice with placebo (conventional, n=6; germ free, n=6) or nitrate treatment (conventional, n=8; germ free, n=8). P<0.05 between indicated groups, 1-way ANOVA, Bonferroni multiple comparisons test). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the renal cortex from conventional (C) and germ-free (D) mice with placebo (conventional, n=10; germ free, n=10) or nitrate treatment (conventional, n=5; germ free, n=5) for 7 to 10 days. P<0.05 between indicated groups (unpaired t test equal SD). Values are presented as mean±SEM.



The renal afferent arterioles are the major resistance ves-sels of the kidneys and play an important role in blood pres-sure regulation.2,6,10,13 Early studies show a relationship between afferent arteriolar diameter and blood pressure and indicate that development of hypertension can be predicted by a reduced renal afferent arteriolar diameter in the prehypertensive state.6,7 The afferent arteriolar tone and resistance are determined by the balance between constrictor agents such as ANG II and vasodilators, particularly NO.8,9 The interactions between the nitrate–nitrite–NO pathway and reactive oxygen species sig-naling were thoroughly examined in the present study. In the renal cortex and PG-VSMCs, ANG II–stimulated NADPH oxidase–dependent superoxide generation was abolished by nitrite. Interestingly, generation of superoxide in nonstimulated PG-VSMCs was dose-dependently reduced by nitrite. The involvement of reactive oxygen species was further investigated in mice with compromised antioxidant capacity (ie, SOD1−/−). In these mice, ANG II–mediated arteriolar contractions were greatly enhanced and pretreatment with nitrite effectively atten-uated the vascular response. Kidneys from mice with dietary nitrate treatment had reduced NADPH oxidase activity, and the arterioles displayed similar attenuation of ANG II–induced contractions as observed with nitrite in vitro. These results dem-onstrate the functional significance of the entire nitrate–nitrite–NO pathway and strongly support the notion that nitrate and nitrite modulate reactive oxygen species signaling in the kid-ney, thereby inhibiting vasoconstriction. The fact that the vaso-dilatory effect of nitrite in nonstimulated vessels was modest in comparison to the degree of inhibition after ANG II stimulation further supports this notion. The functional importance of these findings was further investigated in vivo in animals chroni-cally infused with ANG II. In this model of renal hypertension, ANG II induces a slow pressor response and gradual increase in blood pressure that is preceded by a selective increase in renal vascular resistance.38,39 These responses are accompanied by activation of NADPH oxidase and increased generation of superoxide.10,11,13,38 The essential role of the kidneys in ANG II–induced hypertension has been elegantly demonstrated by Crowley et al.39,40 Wild-type mice became resistant to ANG II–induced hypertension after receiving kidneys from ANG II type 1 receptor–deficient mice, whereas hypertension developed in ANG II type 1 receptor knockout mice receiving wild-type kidneys. In the present study, control animals gradually devel-oped hypertension during ANG II infusion, whereas this blood pressure response was markedly attenuated in nitrate-treated animals. By the end of the study period, blood pressure was 25 mm Hg lower in the nitrate group.

A remaining question that warrants further investigations is the exact mechanism for nitrate/nitrite-mediated reduction in NADPH oxidase–dependent superoxide formation and signaling. Direct scavenging of superoxide by the NO is plausible, but inhibition of NADPH oxidase activity by nitrite-derived reactive nitrogen oxides is also possible, for example, through nitrosation of critical thiols in the catalytic site of the enzyme or via nitration reactions.41–44 Several NADPH oxidase homologs have been described in renal and car-diovascular systems.45,46 We have previously used transgenic mice to illustrate the importance of gp91phox (NADPH oxidase 2)11 and p47phox13 in mediating renal afferent arteriolar contraction and blood pressure responses to ANG II. Mechanistically, reduction of

NADPH oxidase expression by nitrite would represent a third alter-native, although our mRNA expression data of NADPH oxidase and its subunits did not support this.

Recent studies together with the current in vitro findings suggest that the biological effects of dietary nitrate proceed via intermediate generation of the more reactive nitrite anion. Oral bacteria seem central in this process because they most effi-ciently reduce the nitrate that accumulates in saliva to nitrite.17 Nitrite is then swallowed and further reduced to NO and other reactive nitrogen oxides in blood and tissues.17 To test the involvement of bacteria in this model, we used germ-free mice that are unable to use this prokaryotic pathway for nitrate reduc-tion. Interestingly, dietary nitrate treatment had no effect on contractility in renal arterioles from germ-free mice, whereas vessels from conventionally housed mice displayed reduced contractility to ANG II. Moreover, NADPH oxidase activity in renal tissue was reduced by nitrate in conventional mice, but unaffected in the germ-free animals. This finding confirms

A

B

Figure 7. Supplementation with dietary nitrate attenuates blood pressure elevation in a model of renal hypertension. A, Mean arterial pressure in conscious Sprague-Dawley rats. The telemetric measurements were conducted continuously during control conditions (baseline, 3 days) without any other treatment. This was followed by a period with Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) drinking water (500 mg/L) supplemented with either nitrate (NaNO3, 10−2 mol/L; n=6) or placebo (NaCl, n=6) for 3 days. Blood pressure was then measured with chronic ANG II infusion at a slow-pressor rate (120 ng/kg per minute; 15 days). During the past 4 days, blood pressure was recorded after stopping the placebo and nitrate supplementation. The hypertensive responses to l-NAME and combination of ANG II+l-NAME were attenuated in the nitrate-supplemented rats. Data points indicate day and night blood pressure levels. B, Maximal changes in blood pressure compared with baseline for placebo- or nitrate-supplemented rats. *P<0.05 compared with placebo group (2-way ANOVA [A] or 1-way ANOVA [B], Bonferroni multiple comparisons test). Values are presented as mean±SEM.



what has been previously proposed, namely a central role of oral commensal bacteria for the bioactivation of dietary nitrate.

The increased incidence of hypertension, cardiovascular disease, and diabetes mellitus in developing countries fol-lows the trend of urbanization and lifestyle changes, perhaps most importantly a Western-style diet.47 The present findings may have intriguing dietary implications building on a series of recent studies showing beneficial effects of dietary nitrate on cardiovascular function in health and disease. Inorganic nitrate is predominantly found in green leafy vegetables, which figure prominently in cuisines renowned for their car-dioprotective properties. Future studies will reveal whether the high content of nitrate in vegetables contributes to the blood pressure–lowering effect of this food group.48

We conclude that the renal microvasculature is a primary target for blood pressure regulation by inorganic nitrate and nitrite (Figure 8). Dietary nitrate is reduced to nitrite in vivo by oral commensal bacteria, and nitrite further regulates superox-ide signaling in the renal vasculature through XO-mediated NO formation. Preglomerular arterioles seem particularly sen-sitive to nitrite, where vasodilatation occurred at concentra-tions below basal circulating plasma levels. This indicates a physiological role for nitrate and nitrite in regulation of renal microvascular tone and in extension of blood pressure.

PerspectivesStimulation of a nitrate–nitrite–NO pathway is associated with reduction in blood pressure, but the mechanisms or the target organs have remained obscure. Preglomerular vascular resistance is important for long-term blood pressure control, and emerging evidence suggests renal oxidative stress in hypertension. The profound effects of inorganic nitrate and

nitrite on afferent arteriolar regulation, as demonstrated in this study, may explain their cardiovascular effects. Mechanisms are linked to NO generation and inhibition of NADPH oxi-dase in preglomerular VSMCs. Clinical trials will determine the therapeutic potential of these inorganic anions in prevent-ing development or persistence of hypertension and how they influence glomerular hemodynamic and filtration properties.

AcknowledgmentsWe thank Margareta Stensdotter, Carina Nihlén, and Annika Olsson (Karolinska Institutet, Stockholm) for technical assistance.

Sources of FundingThis work was supported by grants from the Swedish Research Council (521-2011-2639 to M. Carlström, K2009-64X-03522 to E.G. Persson), the Swedish Heart and Lung Foundation (20110589), Jeanssons Foundation (JS2013-00064), the Swedish Society of Medical Research (SSMF), Stockholm City Council (ALF), and KID funding from the Karolinska Institutet.

DisclosuresE. Weitzberg and J.O. Lundberg are codirectors of Heartbeat Ltd., a company that owns patent applications relating to therapeutic uses of inorganic nitrate and nitrite. The other authors report no conflicts.

References 1. Go AS, Mozaffarian D, Roger VL, et al; American Heart Association

Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics–2013 update: a report from the American Heart Association. Circulation. 2013;127:e6–e245.

2. Araujo M, Wilcox CS. Oxidative stress in hypertension: role of the kidney. Antioxid Redox Signal. 2014;20:74–101.

3. Carlström M, Persson AE. Important role of NAD(P)H oxidase 2 in the reg-ulation of the tubuloglomerular feedback. Hypertension. 2009;53:456–457.

4. Ollerstam A, Pittner J, Persson AE, Thorup C. Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. J Clin Invest. 1997;99:2212–2218.

5. Wilcox CS. Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regul Integr Comp Physiol. 2005;289:R913–R935.

6. Smeda JS, Lee RM, Forrest JB. Structural and reactivity alterations of the renal vasculature of spontaneously hypertensive rats prior to and during established hypertension. Circ Res. 1988;63:518–533.

7. Levy BI, Ambrosio G, Pries AR, Struijker-Boudier HA. Microcirculation in hypertension: a new target for treatment? Circulation. 2001;104:735–740.

8. Navar LG, Ichihara A, Chin SY, Imig JD. Nitric oxide-angiotensin II inter-actions in angiotensin II-dependent hypertension. Acta Physiol Scand. 2000;168:139–147.

9. Patzak A, Persson AE. Angiotensin II-nitric oxide interaction in the kid-ney. Curr Opin Nephrol Hypertens. 2007;16:46–51.

10. Carlström M, Lai EY, Ma Z, Steege A, Patzak A, Eriksson UJ, Lundberg JO, Wilcox CS, Persson AE. Superoxide dismutase 1 limits renal microvascular remodeling and attenuates arteriole and blood pressure responses to angiotensin II via modulation of nitric oxide bioavailability. Hypertension. 2010;56:907–913.

11. Carlström M, Lai EY, Ma Z, Patzak A, Brown RD, Persson AE. Role of NOX2 in the regulation of afferent arteriole responsiveness. Am J Physiol Regul Integr Comp Physiol. 2009;296:R72–R79.

12. Datla SR, Griendling KK. Reactive oxygen species, NADPH oxidases, and hypertension. Hypertension. 2010;56:325–330.

13. Lai EY, Solis G, Luo Z, Carlstrom M, Sandberg K, Holland S, Wellstein A, Welch WJ, Wilcox CS. p47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice. Hypertension. 2012;59:415–420.

14. Gladwin MT, Schechter AN, Kim-Shapiro DB, et al. The emerging biol-ogy of the nitrite anion. Nat Chem Biol. 2005;1:308–314.

15. Lundberg JO, Gladwin MT, Ahluwalia A, et al. Nitrate and nitrite in biol-ogy, nutrition and therapeutics. Nat Chem Biol. 2009;5:865–869.

16. Lundberg JO, Weitzberg E, Cole JA, Benjamin N. Nitrate, bacteria and human health. Nat Rev Microbiol. 2004;2:593–602.

Figure 8. Proposed mechanism for nitrate- and nitrite-mediated effects on renal microvasculature and blood pressure regulation. Dietary nitrate (NO3

−) is reduced to nitrite (NO2−) by commensal

oral bacteria. Absorbed and circulating nitrite can be reduced by xanthine oxidases (XO) in the renal microvasculature to form nitric oxide (NO), which may attenuate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase–derived superoxide (O2

−) production or stimulate soluble guanylyl cyclase (sGC)–mediated vasodilatation. Together, this will reduce renal microvascular resistance and lower blood pressure. ANG II indicates angiotensin II.



17. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide path-way in physiology and therapeutics. Nat Rev Drug Discov. 2008;7:156–167.

18. Petersson J, Carlström M, Schreiber O, Phillipson M, Christoffersson G, Jägare A, Roos S, Jansson EA, Persson AE, Lundberg JO, Holm L. Gastroprotective and blood pressure lowering effects of dietary nitrate are abolished by an antiseptic mouthwash. Free Radic Biol Med. 2009;46:1068–1075.

19. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S, Macallister R, Hobbs AJ, Webb AJ, Ahluwalia A. Inorganic nitrate supplementation low-ers blood pressure in humans: role for nitrite-derived NO. Hypertension. 2010;56:274–281.

20. Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med. 2006;355:2792–2793.

21. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia A. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension. 2008;51:784–790.

22. Ghosh SM, Kapil V, Fuentes-Calvo I, Bubb KJ, Pearl V, Milsom AB, Khambata R, Maleki-Toyserkani S, Yousuf M, Benjamin N, Webb AJ, Caulfield MJ, Hobbs AJ, Ahluwalia A. Enhanced vasodilator activity of nitrite in hypertension: critical role for erythrocytic xanthine oxidoreduc-tase and translational potential. Hypertension. 2013;61:1091–1102.

23. Carlström M, Larsen FJ, Nyström T, Hezel M, Borniquel S, Weitzberg E, Lundberg JO. Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice. Proc Natl Acad Sci U S A. 2010;107:17716–17720.

24. Carlström M, Persson AE, Larsson E, Hezel M, Scheffer PG, Teerlink T, Weitzberg E, Lundberg JO. Dietary nitrate attenuates oxidative stress, prevents cardiac and renal injuries, and reduces blood pressure in salt-induced hypertension. Cardiovasc Res. 2011;89:574–585.

25. Tsuchiya K, Tomita S, Ishizawa K, Abe S, Ikeda Y, Kihira Y, Tamaki T. Dietary nitrite ameliorates renal injury in L-NAME-induced hypertensive rats. Nitric Oxide. 2010;22:98–103.

26. Montenegro MF, Amaral JH, Pinheiro LC, Sakamoto EK, Ferreira GC, Reis RI, Marçal DM, Pereira RP, Tanus-Santos JE. Sodium nitrite down-regulates vascular NADPH oxidase and exerts antihypertensive effects in hypertension. Free Radic Biol Med. 2011;51:144–152.

27. Luo Z, Chen Y, Chen S, Welch WJ, Andresen BT, Jose PA, Wilcox CS. Comparison of inhibitors of superoxide generation in vascular smooth muscle cells. Br J Pharmacol. 2009;157:935–943.

28. Dubey RK, Roy A, Overbeck HW. Culture of renal arteriolar smooth muscle cells. Mitogenic responses to angiotensin II. Circ Res. 1992;71:1143–1152.

29. Carlström M, Sällström J, Skøtt O, Larsson E, Persson AE. Uninephrectomy in young age or chronic salt loading causes salt-sensitive hypertension in adult rats. Hypertension. 2007;49:1342–1350.

30. Patzak A, Lai EY, Mrowka R, Steege A, Persson PB, Persson AE. AT1 receptors mediate angiotensin II-induced release of nitric oxide in afferent arterioles. Kidney Int. 2004;66:1949–1958.

31. Patzak A, Mrowka R, Storch E, Hocher B, Persson PB. Interaction of angiotensin II and nitric oxide in isolated perfused afferent arterioles of mice. J Am Soc Nephrol. 2001;12:1122–1127.

32. Lundberg JO, Carlström M, Larsen FJ, Weitzberg E. Roles of dietary inorganic nitrate in cardiovascular health and disease. Cardiovasc Res. 2011;89:525–532.

33. Govoni M, Jansson EA, Weitzberg E, Lundberg JO. The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide. 2008;19:333–337.

34. Kapil V, Haydar SM, Pearl V, Lundberg JO, Weitzberg E, Ahluwalia A. Physiological role for nitrate-reducing oral bacteria in blood pressure con-trol. Free Radic Biol Med. 2013;55:93–100.

35. Modin A, Björne H, Herulf M, Alving K, Weitzberg E, Lundberg JO. Nitrite-derived nitric oxide: a possible mediator of ‘acidic-metabolic’ vasodilation. Acta Physiol Scand. 2001;171:9–16.

36. Crawford JH, Isbell TS, Huang Z, Shiva S, Chacko BK, Schechter AN, Darley-Usmar VM, Kerby JD, Lang JD Jr, Kraus D, Ho C, Gladwin MT, Patel RP. Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood. 2006;107:566–574.

37. Castiglione N, Rinaldo S, Giardina G, Stelitano V, Cutruzzolà F. Nitrite and nitrite reductases: from molecular mechanisms to significance in human health and disease. Antioxid Redox Signal. 2012;17:684–716.

38. Kawada N, Imai E, Karber A, Welch WJ, Wilcox CS. A mouse model of angiotensin II slow pressor response: role of oxidative stress. J Am Soc Nephrol. 2002;13:2860–2868.

39. Crowley SD, Gurley SB, Herrera MJ, Ruiz P, Griffiths R, Kumar AP, Kim HS, Smithies O, Le TH, Coffman TM. Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proc Natl Acad Sci U S A. 2006;103:17985–17990.

40. Crowley SD, Gurley SB, Oliverio MI, Pazmino AK, Griffiths R, Flannery PJ, Spurney RF, Kim HS, Smithies O, Le TH, Coffman TM. Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system. J Clin Invest. 2005;115:1092–1099.

41. Wolin MS. Reactive oxygen species and the control of vascular function. Am J Physiol Heart Circ Physiol. 2009;296:H539–H549.

42. Qian J, Chen F, Kovalenkov Y, Pandey D, Moseley MA, Foster MW, Black SM, Venema RC, Stepp DW, Fulton DJ. Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation. Free Radic Biol Med. 2012;52:1806–1819.

43. Yun BW, Feechan A, Yin M, Saidi NB, Le Bihan T, Yu M, Moore JW, Kang JG, Kwon E, Spoel SH, Pallas JA, Loake GJ. S-nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature. 2011;478:264–268.

44. Selemidis S, Dusting GJ, Peshavariya H, Kemp-Harper BK, Drummond GR. Nitric oxide suppresses NADPH oxidase-dependent superoxide pro-duction by S-nitrosylation in human endothelial cells. Cardiovasc Res. 2007;75:349–358.

45. Lassègue B, San Martín A, Griendling KK. Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system. Circ Res. 2012;110:1364–1390.

46. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxi-dases: physiology and pathophysiology. Physiol Rev. 2007;87:245–313.

47. Ramachandran A, Ma RC, Snehalatha C. Diabetes in Asia. Lancet. 2010;375:408–418.

48. Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM, Bray GA, Vogt TM, Cutler JA, Windhauser MM, Lin PH, Karanja N. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med. 1997;336:1117–1124.

What Is New?

•The renal microcirculation is exquisitely responsive to nitrate–nitrite–nitric oxide signaling, as demonstrated by vasodilatation and reduced contractility to angiotensin II.

•The observed effects of inorganic nitrite are dependent on functional xanthine oxidase, and nicotinamide adenine dinucleotide phosphate oxi-dase in the vasculature is a primary target.

What Is Relevant?

•Dietary supplementation with inorganic nitrate or nitrite has proven ben-eficial effects in models of renal and cardiovascular disease.

•Our findings demonstrate that boosting this alternative pathway for nitric oxide production modulates oxidative stress and preglomerular resis-

tance, which may help to explain the antihypertensive effects of inor-ganic nitrate and nitrite.

•Clinical trials are warranted to determine the therapeutic potential of these inorganic anions.

Summary

In contrast to other vascular beds (eg, carotid, renal interlobar, and mesenteric arteries), the microvasculature of the kidney is particu-larly sensitive to inorganic nitrite, as demonstrated by vasodila-tation at physiological levels. Moreover, stimulation of a nitrate–nitrite–nitric oxide pathway attenuated angiotensin II–mediated arteriolar contraction and hypertension by lowering nicotinamide adenine dinucleotide phosphate oxidase activity.

Novelty and Significance


Online Data Supplement ‐ Expanded Methods and Results

1

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

Short title: Gao & Yang – The NO3-NO2-NO Pathway in Hypertension

Xiang Gao PhD1*, Ting Yang MD, PhD2*, Ming Liu2 MD, Maria Peleli MSci2, Christa Zollbrecht PhD2, Eddie Weitzberg MD, PhD 2, Jon O. Lundberg MD, PhD 2, A. Erik G.

Persson MD, PhD 1, Mattias Carlström PharmD, PhD 2

1Dept Medical Cell Biology, Uppsala University, Uppsala, Sweden, 2Dept Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden

*Equal contribution to this work

Correspondence:

Mattias Carlström

Dept. of Physiology and Pharmacology, Nanna Svartz Väg 2, S-17177, Stockholm, Sweden

Phone: +46-(0)8-52487924

Fax: +46-(0)8-332278

E-mail: [email protected]


2

SUPPLEMENTAL METHODS

Animals

The studies were conducted on male C57BL/6J mice (The Jackson Laboratory, Maine, USA). To specifically investigate the role of bacteria in bioactivation of nitrate, germ free (n=8) and specific pathogen free (conventional) NMRI mice (n=8) were used. The GF status was checked weekly by culturing faecal samples, both aerobically and anaerobically at +20 and +37°C.1 To investigate the effects of nitrite in a model with oxidative stress we used homozygous littermates (SOD1-/-; n=15 and SOD1+/+; n=19) from heterozygous breeding pairs of SOD1-ko mice (B6;129S7-Sod1tm1Leb/J (stock# 002972) from The Jackson Laboratory, Maine, USA. Genotyping of the offspring was performed as previously described.2 To investigate the effects of dietary nitrate in a model of renal hypertension we used Sprague-Dawley rats (Charles River Laboratories, Germany) for telemetric blood pressure measurements (n=12). All animals, housed in temperature-controlled rooms with 12hr light/dark cycles, were fed standardized rodent chow (R36, Lactamin, Kimstad, Sweden) and tap water ad libitum. Body weights were between 20-27g for mice and 310-350 g for rats.

Vascular Studies

Isolated and Perfused Afferent Arterioles

Dulbecco’s modified Eagle’s medium (DMEM/F12) with 0.01 mol/L HEPES (Invitrogen AB, Lidingö, Sweden) was used for dissection, bath, and perfusion. The pH was adjusted to 7.4 after addition of bovine serum albumin (BSA:SERVA Electrophoresis Heidelberg, Germany). The concentration of BSA was 0.1% in dissection and bath solutions, and 1% in the perfusion solution. Dissection of renal cortical afferent arterioles was performed at 4°C. Single isolated arterioles with their glomeruli were perfused in a thermo-regulated chamber (37°C) using a micromanipulator perfusion system (Vestavia Scientific, Vestavia Hills, AL, USA), which allowed adjustment of outer holding and inner perfusion pipettes. The chamber and the perfusion system were fixed to the stage of an inverted microscope (Nikon, Badhoevedorp, Netherlands). The perfusion pipette, with a diameter of the tip of 5-µm, was connected to a reservoir containing the perfusion solution. The pressure in the pressure head was 100 mmHg, which corresponds to physiological pressure of 60 mmHg and flow of about 50 nl/min in the connected arteriole. The criteria for the use of an arteriole were: an intact myogenic response and a satisfactory, remaining basal tone. Increasing perfusion pressure rapidly and assessing the change in the luminal diameter, which produced a constriction, tested both criteria. A further criterion was a fast and complete constriction in response to KCl (100 mmol/l) solution. The experiments were digitally recorded and then digitized off-line and analyzed as described before.3 Changes in luminal diameters were measured to estimate the effect of vasoactive substances. The equipment used allowed a resolution of 0.2 µm of the vessel structures.4 In all series, the last 10 seconds of a control or treatment period were used for statistical analysis of steady state responses. Each experiment used a separate dissected afferent arteriole.

Fluorescent detection of NO production in afferent arterioles

A highly sensitive, photo-stable cell-permeable fluorescent probe, 4-Amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM DA) (Invitrogen, Life Technologies Europe BV, Stockholm, Sweden), was used to measure NO production in the isolated and perfused afferent arteriole. DAF-FM DA is diacetylated intracellular by esterases to DAF-FM. It reacts


3

with NO in presence of oxygen to form green-fluorescent triazolofluoresceins. The fluorescent signal provides a temporally integrated estimate of intracellular NO bioavailability. DAF-FM DA was loaded into afferent arterioles by adding the esterified form to the bath (2x10-5 mol/L, 45 min, room temperature). DAF-FM was excited with light at 480nm from a Thiel monochromator using a Nikon eclipse, TE300 microscope (Thiel, Munich, Germany, Nikon Stockholm, Sweden), and the emission isolated at 535nm detected with back-illuminated EMCCD camera (DU-887, Andor Technology, Belfast, Northern Ireland) under software control by MetaFluor (Molecular Devices Corporation, Downington, PA, USA). Digital pictures were acquired every 30 seconds at a magnification of x1300. Change in fluorescence intensity of DAF-FM with nitrite (10-5 mol/L), as an indicator for NO production was quantified as the percent change from the initial value as previously described.5 Myograph Studies in Carotid, Renal Interlobar and Mesenteric Arteries

Carotid arteries, renal interlobar arteries (ILA) and third-order branch of mesenteric resistance vessels (MRV) from C57BL6 mice were isolated and dissected in ice-cold Krebs solution (composition in mmol/L: NaCl 119; KCl 4.7; CaCl2 1.6; KH2PO4 1.2; MgSO4·7H2O 1.2; NaHCO3 25.1; glucose 5.5; EDTA 0.026). Arterial rings (2mm) were mounted on 40µm stainless steel wires (carotid artery) or 25µm tungsten wires (ILA and MRV) in a small vessel myograph (Model 620M, Danish Myo Technology, Denmark) for recording isometric force by transducers (PowerLab 4/30, AD Instruments, Australia). The chambers were filled with Krebs solution (37 °C, pH 7.4) aerated with Carbogen (95% O2, 5% CO2). Resting tension of arteries was set according to the normalization procedure described by Mulvany and Halpern,6 and vessel viability was assessed by the responses to 0.1 mol/L KCl. After washout, cumulative concentration response for ANG II (10-12-10-6 mol/L) was obtained, with or without simultaneous incubation with nitrite (10μM). Contractile responses were expressed as percentage of constriction to 0.1 mol/L KCl (% of KCl).

Renal NADPH Oxidase Activity

Chemiluminescence technique was used to determine the NADPH oxidase-mediated superoxide formation in in renal cortex or in preglomerular vascular smooth muscle cells (PG-VSMC). The isolation and culturing of primary PG-VSMC were performed as previously described,7 and the phenotype was confirmed as described by Dubey and colleagues.8 Experiments were conducted between passage 5 and 15 and PG-VSMC were cultured in DMEM/F12 supplemented with fetal bovine serum (10%), penicillin (100 U/mL), streptomycin (100 mg/mL) and glutamine (200 mg/mL) at 37°C (5% CO2 and 95% air) at 98% humidity. Equal numbers of cells were seeded into 6–well plates (2 mL of DMEM/F-12 medium per well), incubated and left overnight to attach. After 24 h, the cells were rinsed twice and incubated with serum free medium for another 48 h. After incubation, and before the experiment, random plates were used for cell count (in quadruplicate) and cellular viability was evaluated with Trypan Blue Solution, 0.4% (Gibco®) exclusion test. After 30 min incubation with different concentrations of nitrite (NaNO2) or placebo (NaCl) the cells were collected with 300 μL/well dissociation buffer (Invitrogen) and 500 μL/well DPBS and transferred into reaction tubes. In experiments with kidney cortex, the renal tissue was homogenized with bullet blender in ice cold PBS, and the homogenate was centrifuged at 4°C for 20 min at 2000g. Fresh tissue


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homogenates were incubated for 30 min with placebo (NaCl), ANG II (10-6 mol/L), Nitrite (10-5 mol/L) or combinations with ANG II and nitrite. In experiments with both PG-VSMC or renal cortex, 100 μL of NADPH (100μM) and lucigenin (50μM) (Sigma-Aldrich) were injected into the reaction tube (final volume of 1 mL), and NADPH oxidase activity was determined by measuring lucigenin chemiluminescence every 3 seconds for 3 minutes with an AutoLumat LB953 Multi-Tube Luminometer (Berthold Technologies, Bad Wildbad, Germany). Results were corrected by cell number (VSMC) or by protein quantification (cortex) using Bradford protein assay (Bio-Rad Laboratories, UK).

Renal NADPH Oxidase and Xanthine Oxidase Expression

Renal cortex and PG-VSMC were used for expression studies of NADPH oxidases and xanthine oxidase (XO). Infusion of cold PBS (Phosphate Buffered Saline) was started once the vena cava was cut to remove the blood. The kidneys were explanted, blotted and weighed. The renal cortex was separated, homogenized, and quantitative PCR analysis was performed. PG-VSMC were washed with PBS and harvested in RLT buffer for RNA extraction. Total RNA of tissue or cells was isolated using RNeasy Mini Kit (QIAGEN, Valencia, CA), and cDNA was synthesized with High Capacity cDNA Reverse transcription kit (Applied Biosystems). Quantitative PCR analysis was performed regarding to the Applied Biosystems 7500 protocol. Power SYBR Green Master mix (Applied Biosystems) was used for amplification and detection of DNA. PCR reaction was performed in 96-well plates with 20μl mixer/well (0.25 μmol/L of each primer and 5 μL of cDNA corresponding to 25 ng (tissue) and 62.5 ng (cells) of RNA, Applied Biosystems). The efficiency of PCR was calibrated according to the standard curve and the mRNA level was normalized with β-actin by the ΔCt method. Primer sequences and amplification profiles used for Nox2, Nox4, p22phox, p47phox, p67phox, XO and β-actin are described as Table S1.


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SUPPLEMENTAL RESULTS

Renal Xanthine Oxidase Expression

We have previously demonstrated XO activity in the kidney.1 In the present study we showed that the relative expression of xanthine oxidase to beta-actin is high, with ∆Ct value in renal cortex of -0.56±0.13 (Mean Ct(XO) = 24.2±0.1; Mean Ct(beta-actin) = 24.8±0.0) and ∆Ct value in PG-VSMC of -1.79±0.02 (Mean Ct(XO) = 24.3±0.0; Mean Ct(beta-actin) = 26.1±0.0).


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SUPPLEMENTAL REFERENCES

1. Jansson EA, Huang L, Malkey R, Govoni M, Nihlen C, Olsson A, Stensdotter M, Petersson J, Holm L, Weitzberg E, Lundberg JO. A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis. Nat Chem Biol. 2008;4:411-417.

2. Carlstrom M, Lai EY, Ma Z, Steege A, Patzak A, Eriksson UJ, Lundberg JO, Wilcox CS, Persson AE. Superoxide dismutase 1 limits renal microvascular remodeling and attenuates arteriole and blood pressure responses to angiotensin ii via modulation of nitric oxide bioavailability. Hypertension. 2010;56:907-913.

3. Carlstrom M, Lai EY, Ma Z, Patzak A, Brown RD, Persson AE. Role of nox2 in the regulation of afferent arteriole responsiveness. Am J Physiol Regul Integr Comp Physiol. 2009;296:R72-79.

4. Patzak A, Bontscho J, Lai E, Kupsch E, Skalweit A, Richter CM, Zimmermann M, Thone-Reineke C, Joehren O, Godes M, Steege A, Hocher B. Angiotensin ii sensitivity of afferent glomerular arterioles in endothelin-1 transgenic mice. Nephrol Dial Transplant. 2005;20:2681-2689.

5. Liu R, Pittner J, Persson AE. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal nacl concentration. J Am Soc Nephrol. 2002;13:2688-2696.

6. Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res. 1977;41:19-26

7. Luo Z, Chen Y, Chen S, Welch WJ, Andresen BT, Jose PA, Wilcox CS. Comparison of inhibitors of superoxide generation in vascular smooth muscle cells. Br J Pharmacol. 2009;157:935-943.

8. Dubey RK, Roy A, Overbeck HW. Culture of renal arteriolar smooth muscle cells. Mitogenic responses to angiotensin ii. Circ Res. 1992;71:1143-1152.


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Table S1. Primers for PCR Analysis

Number Primer Name Primer Sequence

NM_007807 Nox2-fw 5’- GCA CCT GCA GCC TGC CTG AAT T -3’

Nox2-rev 5’- TTG TGT GGA TGG CGG TGT GCA -3’

NM_015760 Nox4-fw 5’- GGC TGG CCA ACG AAG GGG TTA A-3’

Nox4-rev 5’- GAG GCT GCA GTT GAG GTT CAG GAC A-3’

NM_007806 p22phox-fw 5’- CTG GCG TCT GGC CTG ATT CTC ATC -3’

p22phox-rev 5’- CCG AAA AGC TTC ACC ACA GAG GTC A -3’

NM_010876 p47phox-fw 5’- CAG CCA TGG GGG ACA CCT TCA TT -3’

p47phox-rev 5’- GCC TCA ATG GGG AAC ATC TCC TTC A -3’

NM_010877 p67phox-fw 5’- AAG ACC TTA AAG AGG CCT TGA CGC A -3’

p67phox-rev 5’- TCG GAC TTC ATG TTG GTT GCC AA -3’

NM_011723 XO-fw 5’- CAG CAT CCC CAT TGA GTT CA -3’

XO-rev 5’- GCA TAG ATG GCC CTC TTG TTG -3’


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Figure S1. Vascular Studies – Effects of nitrite on vascular reactivity and NO formation. Photomicrographs demonstrating the set-up for vascular reactivity (Panel A) and NO measurements (Panel B) in isolated and perfused renal afferent arterioles.

Glomerulus

Afferent Arteriole

Holding Pipettes

Perfusion Pipette

ø

10 µm

A B

Baseline

Nitrite


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Figure S2. Myograph Vascular Studies – Nitrite do not change vascular response to angiotensin II in carotid, renal interlobar or mesenteric arteries. Angiotensin II concentration response curves (ANG II) in isolated carotid arteries (Panel A), renal interlobar arteries (Panel B) and mesenteric resistance arteries (Panel C) of C57 mice. Simultaneous administration of nitrite (10-5 mol/L) did not change ANG II-mediated contraction, as observed in renal afferent arterioles. Values are presented as mean ± SEM of n=8-12 in each experimental group

Carotid Arteries

B 10-12 10-11 10-10 10-9 10-8 10-7 10-6 -10

0

10

20

30

ANG II (mol/L)

Co

ntr

ac

tion

(% o

f KC

l)

ANG II

ANG II+Nitrite

Interlobar Arteries

B 10-12 10-11 10-10 10-9 10-8 10-7 10-6 -10

0

10

20

30

ANG II (mol/L)

Co

ntr

ac

tion

(% o

f KC

l) ANG II+Nitrite

ANG II

Mesenteric Arteries

B 10-12 10-11 10-10 10-9 10-8 10-7 10-6 -20

0

20

40

60

80

100

120

ANG II (mol/L)

Co

ntr

ac

tion

(% o

f KC

l) ANG II+Nitrite

ANG II

A

B

C


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Figure S3. Plasma Levels of Nitrate, Nitrite & cGMP Circulating nitrate (NO3

-), nitrite (NO2-) and cGMP levels in conventional mice (n=6 per

group) following dietary nitrate supplementation (NaNO3 10-2 mol/L, in drinking water) or placebo treatment (NaCl; Control) for 7-10 days in. Values are presented as mean ± SEM

0

50

100

150P

lasm

a N

O3- (

um

ol/L

)

Control Nitrate

P<0.05

0.0

0.5

1.0

1.5

Pla

sm

a N

O2- (

um

ol/L

)

Control Nitrate

P = 0.10

0

50

100

150

Pla

sm

a c

GM

P (

nm

ol/L

)

Control Nitrate

A

B

C


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Figure S4. Expression of NADPH Oxidase in the Kidney NADPH oxidase and subunit expressions in renal cortex of conventional mice (n=6 per group) following dietary nitrate supplementation (NaNO3 10-2 mol/L, in drinking water) or placebo treatment (NaCl; Control) for 7-10 days in. Values are presented as mean ± SEM

Nox 2

Control Nitrate0.0

0.5

1.0

1.5

2.0

Re

lativ

e m

RN

A e

xp

res

sio

n

p22phox

Control Nitrate0.0

0.5

1.0

1.5

2.0

Re

lativ

e m

RN

A e

xp

res

sio

n

p67phox

Control Nitrate0.0

0.5

1.0

1.5

2.0

Re

lativ

e m

RN

A e

xp

res

sio

nNox 4

Control Nitrate0.0

0.5

1.0

1.5

2.0

Re

lativ

e m

RN

A e

xp

res

sio

n

p47phox

Control Nitrate0.0

0.5

1.0

1.5

2.0

Re

lativ

e m

RN

A e

xp

res

sio

n

A B

B C

D


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Figure S5. Supplementation with dietary nitrate does not influence heart rate in a model of renal hypertension. Heart rates measured with telemetry in conscious Sprague-Dawley rats. The telemetric measurements were conducted continuously during control conditions (Baseline, 3 days), without any other treatment. This was followed by a period with L-NAME drinking water (500 mg/L) supplemented with either nitrate (NaNO3, 10-2 mol/L, n=6) or placebo (NaCl, n=6) for 3 days. Heart rate was then measured with chronic ANG II infusion at a slow-pressor rate (120 ng/kg/min, 15 days). During the last 4 days, heart rate was recorded after stopping the placebo and nitrate supplementation. Values are presented as mean ± SEM.

0

250

300

350

400

450

500

Time (days & night)

He

art

Ra

te(b

pm

)

Baseline L-NAME L-NAME + ANG II

StopPlacebo

or Nitrate

StartPlacebo

or Nitrate

PlaceboNitrate

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