Background and PurposeNO deficiency and oxidative stress are crucially involved in the development or progression of cardiovascular disease, including hypertension and stroke. We have previously demonstrated that acute treatment with the newly discovered organic nitrate, 2-nitrate-1,3-dibuthoxypropan (NDBP), is associated with NO-like effects in the vasculature. This study aimed to further characterize the mechanism(s) and to elucidate the therapeutic potential in a model of hypertension and oxidative stress.Experimental ApproachA combination of ex vivo, in vitro and in vivo approaches was used to assess the effects of NDBP on vascular reactivity, NO release, NADPH oxidase activity and in a model of hypertension.Key ResultsEx vivo vascular studies demonstrated NDBP-mediated vasorelaxation in mesenteric resistance arteries, which was devoid of tolerance. In vitro studies using liver and kidney homogenates revealed dose-dependent and sustained NO generation by NDBP, which was attenuated by the xanthine oxidase inhibitor febuxostat. In addition, NDBP reduced NADPH oxidase activity in the liver and prevented angiotensin II-induced activation of NADPH oxidase in the kidney. In vivo studies showed that NDBP halted the progression of hypertension in mice with chronic angiotensin II infusion. This was associated with attenuated cardiac hypertrophy, and reduced NADPH oxidase-derived oxidative stress and fibrosis in the kidney and heart.Conclusion and ImplicationsThe novel organic nitrate NDBP halts the progression of angiotensin II-mediated hypertension. Mechanistically, our findings suggest that NDBP treatment is associated with sustained NO release and attenuated activity of NADPH oxidase, which to some extent requires functional xanthine oxidase.

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.

BACKGROUND: Src homology 2 domain‐containing protein B (Shb) is an adapter protein which regulates several signal transduction cascades and endothelial cell functions. We used Shb‐knockout (Shb‐/‐) and wild‐type (Shb+/+) mice to investigate the role of Shb in regulating glomerular filtration rate (GFR), vascular resistance and tubuloglomerular feedback.METHODS: GFR was measured in conscious Shb‐/‐ and Shb+/+ mice using FITC‐inulin. Isotonic contractions were measured in isolated and perfused renal afferent arterioles from Shb‐/‐ and Shb+/+ mice. Concentration responses to Ang II (10‐12 to 10‐6M; 2 minutes each) doses, low‐dose Ado (10‐8 mol/l; 15 min) alone or Nω‐nitro‐l‐arginine methyl ester (L‐NAME; 10‐4 mol/l; 15 min) alone, as well as Ado (10‐8 mol/l) or nitric oxide (NO) synthase inhibitor L‐NAME (10‐4 mol/l) in combination with cumulative application of Ang II (10‐12 to 10‐6 mol/l; 2 minutes each) were studied in both genotypes.RESULTS: There was a significantly increased GFR (371 ± 12 µl/min, n=11) in Shb‐/‐ comparing to Shb+/+ (321 ± 11 µl/min, n=8) mice. The maximal arteriolar contraction to Ang II was remarkably larger in Shb‐/‐ (87 %, n=8) than in Shb+/+ (54 %, n=8) mice. Low‐dose Ado contracted afferent arterioles in both genotypes (6% in Shb‐/‐ and 7% in Shb+/+). Ado significantly enhanced Ang II constriction in afferent arterioles in both genotypes (to 93% in Shb‐/‐ and to 72 % in Shb+/+). L‐NAME reduced arteriolar diameters significantly more in Shb‐/‐ (14 %, n=7) than in wild types (9 %, n=7) and remarkably augmented ANG II responses in both genotypes. L‐NAME with Ang II almost fully constricted renal afferent arterioles in Shb‐/‐ (98 %), while contracted 64% in Shb+/+ mice.CONCLUSION: The absence of Shb markedly increases GFR. Both low‐dose adenosine and L‐NAME treatments significantly augment Ang II arteriolar constriction effectiveness, which indicates Ado‐Ang II and NO‐Ang II interactions in both Shb‐/‐ and Shb+/+ mice. The underlying mechanisms remain to be resolved.

Hydronephrotic rats and mice have impaired renal function and develop salt-sensitive hypertension, which are associated with oxidative stress. Increased sympathetic nerve activity and oxidative stress in the kidney may play important roles in renovascular hypertension. This study aimed to investigate the contribution of renal sympathetic nerve activity in the development of hypertension in hydronephrosis. A partial unilateral ureteral obstruction (PUUO) was created in 3-weeks old rats to induce hydronephrosis. Surgical denervation, or sham operation, of the PUUO kidney was performed at the time of ureteral obstruction and again 4-weeks later during implantation of a telemetric blood pressure device. Hydronephrotic animals had higher blood pressure (115±3 mmHg) compared with controls (87±1 mmHg), and the blood pressure elevation to a high salt diet was more pronounced (15±2 vs 5±1 mmHg) (p<0.05). Hydronephrosis was also associated with increased urine production (40±4 μl/24h/gBW) and lower urine osmolality (1242±109 mOsm/kg H2O) compared with controls (28±3 μl/24h/gBW and 1751±83 mOsm) (p<0.05). Renal denervation in rats with PUUO attenuated hypertension (97±3 mmHg) and normalized salt-sensitivity (5±1 mmHg), urine production (32±2 μl/24h/gBW) and urine osmolality (1586±127 mOsm/kg H2O) (p<0.05). NADPH oxidase activity in renal cortex from PUUO rats was increased compared with controls (4608±396 vs 3373±217 CLU/min/mg protein) (p<0.05). This was associated with increased cortical mRNA expression of Nox2 (2.3±0.43), p22phox (2.65±0.67) and p47phox (1.39±0.23) compared with controls (p<0.05). Remarkably, denervation in PUUO rats normalized both NADPH oxidase activity (3363±258 CLU/min/mg protein) and mRNA expression of Nox2, p22phox and p47phox (p<0.05). Interestingly, also myocardial tissue from PUUO displayed increased mRNA expression of Nox2 (1.68±0.23) and p22phox (2.82±0.51) compared with control rats, and this was normalized by renal denervation (0.81±0.43) (p<0.05). In conclusion, renal denervation of the hydronephrotic kidney attenuates hypertension and salt-sensitivity, and restores renal excretion pattern. Mechanistically, this is associated with reduced renal NADPH oxidase activity and expression.

BACKGROUND: Src homology 2 domain-containing protein B ( Shb ) is an adapter protein which regulates several signal transduction cascades and endothelial cell functions. The adenosine-angiotensin II (Ado-Ang II) interaction plays an important role in the regulation of glomerular filtration rate (GFR), vascular resistance and tubuloglomerular feedback. We used Shb -knockout ( Shb -/- ) and wild-type ( Shb +/+ ) mice to investigate their GFR and effectiveness of Ado and Ang II to constrict renal resistance vessels. METHODS: GFR was measured in conscious Shb -/- and Shb +/+ mice using FITC-inulin. Isotonic contractions were measured in isolated and perfused renal afferent arterioles from Shb -/- and Shb +/+ mice. Concentration responses to Ang II (10 -12 to 10 -6 M; 2 minutes each) doses or low-dose Ado (10 -8 mol/l; 15 min) alone, as well as Ado (10 -8 mol/l) in combination with cumulative application of Ang II (10 -12 to 10 -6 mol/l; 2 minutes each) were studied in both genotypes. RESULTS: There was a significantly increased GFR (371 ± 12 µL/min, n=11) in Shb -/- comparing to Shb +/+ (321 ± 11 µL/min, n=8) mice. The maximal arteriolar contraction to Ang II was significantly larger in Shb -/- (87 %; n=8) than in Shb +/+ (54 %; n=8) mice. Low-dose Ado alone contracted afferent arterioles in both genotypes (6% in Shb -/- and 7% in Shb +/+ ). Ado significantly enhanced Ang II constriction in afferent arterioles in both genotypes (to 93% in Shb -/- and to 72 % in Shb +/+ ). CONCLUSION: Low-dose adenosine augments Ang II arteriolar constriction effectiveness, which indicates Ado-Ang II interaction in both Shb -/- and Shb +/+ mice. The absence of Shb increases GFR The underlying mechanisms remain to be resolved.

OBJECTIVEAdenosine (Ado) mediates tubuloglomerular feedback, whereas AngII and NO are important modulators. A1‐deficiency abolish TGF and diminish effectiveness of L‐NAME and AngII to constrict renal resistance vessels. We used A1‐knockout (A1−/−) and wild‐type (A1+/+) mice to investigate the synergism between AngII and low‐dose Ado in regulation of arteriolar responses.METHODSContractions were measured in renal afferent arterioles from nontreated mice and those with 14‐days pretreatment with L‐NAME (10‐4M) or AngII (400 ng/kg/min). Concentration response curves were obtained by Ado (10‐8M; 15 min) alone, or in combination with cumulative application of AngII (10–12 to 10‐6M).RESULTSAdo contracted arterioles from A1+/+ (11%), but had no significant effect in A1−/−. Ado significantly enhanced AngII‐mediated contraction in both genotypes, however, the contractile response was stronger in A1+/+ (56%) than in A1−/− (40%). Prolonged treatment with L‐NAME or AngII did not change the responses to Ado alone. However, arteriolar response to combination with Ado+AngII was enhanced in A1+/+, but was attenuated in A1−/−.CONCLUSIONLow‐dose Ado augments AngII‐induced constriction in both A1+/+ and A1−/−, by non‐receptor‐mediated actions. Underlying mechanisms may involve modulation of NO bioavailability or intracellular effects of Ado, introduced by Ado‐transporters.

Increased tubuluglomerular feedback (TGF) responsiveness has been demonstrated in development of hypertension. Adenosine A1‐receptors are suggested mediators of the TGF, whereas Ang II and NO are important modulators. We used A1‐knockout (A1−/−) and wild‐type (A1+/+) mice to investigate the hypothesis that absence of functional TGF may protect from development of Ang II or L‐NAME‐induced hypertension.

Objective Inorganic nitrite is emerging as a substrate for nitric oxide (NO) synthase-independent in vivo generation of NO. Physiologial and therapeutical effects of nitrite have been demonstrated in renal and cardiovascular disease, but its influence on renal microvascular function is not known. Methods Effects of nitrite (10−5 M) on isotonic contractions to Ang II (10−12 to 10−6 M) and L-NAME (10−4 M) were measured in isolated and perfused renal afferent arterioles. Results Nitrite alone mediated a mild vasodilatation of arterioles (6±2%). Ang II constricted arterioles in a concentration-dependent manner with a maximum response of 40±2%, and simultaneous nitrite treatment reduced the maximal response (15±5%). L-NAME enhanced maximal Ang II-mediated contraction (56±4%), and nitrite attenuated the maximal response (25±2%). The attenuating effect of nitrite on Ang II+L-NAME-induced contractions were abolished by the NO scavenger cPTIO (66±2%), the guanylyl cyclase inhibitor ODQ (58±4%), as well as the xanthine oxidase inhibitor oxypurinol (63±3%). Conclusion Inorganic nitrite undergoes xanthine oxidase-mediated reduction to NO in the renal microcirculation, and hence modulates the contractile behavior. This novel function of nitrite may contribute to the reported effects in renal and cardiovascular health and disease.

Objective Activation of adenosine A1-receptors on the afferent arteriole is suggested to mediate the tubuluglomerular feedback, whereas interaction with Ang II and NO may modulate the response. We used A1-knockout (A1−/−) and wild-type (A1+/+) mice to investigate the hypothesis that A1-receptors influence arteriolar responses to L-NAME and Ang II. Methods Isotonic contractions were measured in isolated/perfused afferent arterioles in response to 1) Ang II (10−12 to 10−6M), 2) L-NAME (10−4M), 3) Ang II+L-NAME, or 4) Ang II+L-NAME+tempol (10−4M). Results Maximal arteriolar contractions to both Ang II and L-NAME alone were significantly reduced in A1−/− (L-NAME 5±1%; Ang II 12±1%) than in A1+/+ (L-NAME 14±2%; Ang II 40±1%). The contractile response to Ang II in the presence of L-NAME was enhanced in both genotypes, but still significantly lower in A1−/− (29±1%) than in A1+/+ (58±2%). Simultaneous tempol treatment attenuated the maximal Ang II+L-NAME response in A1+/+ (30±3%), but had no effect in A1−/− (29±2%). There were no differences in arteriolar media or luminal area, or in media-to-lumen ratio between A1−/− and A1+/+. Conclusion A1-receptors enhance the contractile responses to Ang II and L-NAME in afferent arterioles. The underlying mechanisms for adenosine and Ang II receptor interaction remain to be further investigated, but modulation of NO and oxidative stress may contribute.

BACKGROUND: Reduced NO bioavailability in the kidney increases preglomerular reactivity, which may contribute to hypertension. Inorganic nitrate and nitrite can be reduced in vivo and emerge as sub...

A high protein intake is associated with increased glomerular filtration rate (GFR), which has been suggested to be mediated by reduced signaling of the tubuloglomerular feedback (TGF) mechanism. Nitric oxide (NO) has been shown to contribute to high protein-induced glomerular hyperfiltration, but the specific NO synthase (NOS) isoform responsible is not clear. In this study, a model for high-protein-induced hyperfiltration in conscious mice was developed. Using this model, we investigated the role of TGF using adenosine A 1 -receptor knockout mice lacking the TGF mechanism. Furthermore, the role of the different NOS isoforms was studied using neuronal-, inducible-, and endothelial-NOS knockout mice, and furthermore, wild-type mice acutely administered with the unspecific NOS inhibitor N ω -nitro-l-arginine methyl ester (100 mg/kg). GFR was measured consecutively in mice given a low-protein diet (8% casein) for 10 days, followed by a high-protein diet (50% casein) for 10 days. All mice developed high protein-induced hyperfiltration to a similar degree. These results demonstrate that high protein-induced glomerular hyperfiltration is independent of the TGF mechanism and NOS isoforms.

Dietary Inorganic Nitrate Attenuates Oxidative Stress and Hypertension, and Prevents Cardiorenal injury in a Model of Renal and Cardiovascular Disease

Hydronephrosis causes renal dysfunction and salt-sensitive hypertension, which is associated with nitric oxide deficiency and abnormal tubuloglomerular feedback (TGF) response. We investigated the role of oxidative stress for salt sensitivity and for hypertension in hydronephrosis. Hydronephrosis was induced in superoxide dismutase 1-transgenic (SOD1-tg), SOD1-deficient (SOD1-ko), and wild-type mice and in rats. In mice, telemetric measurements were performed during normal (0.7% NaCl) and high-sodium (4% NaCl) diets and with chronic tempol supplementation. The 8-iso-prostaglandin-F(2alpha) (F2-IsoPs) and protein excretion profiles and renal histology were investigated. The acute effects of tempol on blood pressure and TGF were studied in rats. In hydronephrosis, wild-type mice developed salt-sensitive hypertension (114 +/- 1 to 120 +/- 2 mmHg), which was augmented in SOD1-ko (125 +/- 3 to 135 +/- 4 mmHg) but abolished in SOD1-tg (109 +/- 3 to 108 +/- 3 mmHg). SOD1-ko controls displayed salt-sensitive blood pressure (108 +/- 1 to 115 +/- 2 mmHg), which was not found in wild types or SOD1-tg. Chronic tempol treatment reduced blood pressure in SOD1-ko controls (-7 mmHg) and in hydronephrotic wild-type (-8 mmHg) and SOD1-ko mice (-16 mmHg), but had no effect on blood pressure in wild-type or SOD1-tg controls. SOD1-ko controls and hydronephrotic wild-type and SOD1-ko mice exhibited increased fluid excretion associated with increased F2-IsoPs and protein excretion. The renal histopathological changes found in hydronephrotic wild-type were augmented in SOD1-ko and diminished in SOD-tg mice. Tempol attenuated blood pressure and normalized TGF response in hydronephrosis [DeltaP(SF): 15.2 +/- 1.2 to 9.1 +/- 0.6 mmHg, turning point: 14.3 +/- 0.8 to 19.7 +/- 1.4 nl/min]. Oxidative stress due to SOD1 deficiency causes salt sensitivity and plays a pivotal role for the development of hypertension in hydronephrosis. Increased superoxide formation may enhance TGF response and thereby contribute to hypertension.

ObjectiveContrast induced nephropathy (CIN) is a frequent complication of coronary angiography. Outer medullary descending vasa recta (DVR) are contractile microvessels supplying blood flow to the renal medulla ‐ the area of kidney at risk for CIN. Our objective was to test whether perfusion of DVR with a contrast medium (CM) modifies DVR vasoactivity.MethodsDVR from rats were microscopically isolated and perfused with iodixanol (23 mg iodine/ml, calculated after usual doses of CM). DVR luminal diameter was determined by microscopy and nitric oxide (NO) was measured by fluorescent techniques.ResultsCM led to 50% reduction of luminal diameter, and increased vasoconstriction of DVR by angiotensin II (ANG II). Dismutation of superoxide by Tempol prevented these effects. CM also decreased NO bioavailability by >60 percent.DiscussionOur data suggest that CM may lead to renal circulatory derangements described in CIN through a direct effect on DVR. This effect seems to happen via increased oxidative stress. Scavenging of reactive oxygen species might exert a therapeutic effect on CIN by mildering such derangements.Supported by the German Research Foundation, Werner Jackstaedt Foundation, and NIH

Superoxide (O 2 − ), main reactive oxygen species in the vasculature, plays a major role in both renal hemodynamic and blood pressure control. O 2 − levels are limited by superoxide dismutase (SOD) isoforms. Their functional significance in renal and systemic hemodynamics is not clear. The role of SOD1 in afferent arteriolar responsiveness and in angiotensin II (Ang II)-induced hypertension was investigated in SOD1-deficient (SOD1-ko), SOD1-transgenic (SOD1-tg) mice and in littermate controls (wild-type). Arteriolar constrictions to Ang II (10 −14 –10 −6 mol/l) were weaker in SOD1-tg (−14%) and stronger in SOD1-ko (−89%) compared with wild-types (−41%). Unspecific nitric oxide synthase (NOS) inhibition with N ω -Nitro-L-arginine methyl ester hydrochloride (L-NAME; 10 −4 mol/l) reduced basal diameters in wild-types by −8%, in SOD1-ko by −2%, and in SOD1-tg by −38%. Simultaneous application of L-NAME and Ang II caused a similar response in all groups. SOD-mimetic (Tempol; 10 −4 mol/l) had no significant effect in wild-types or SOD1-tg, but caused vasodilatation in SOD1-ko (11%). Simultaneous application of tempol and Ang II attenuated the contractile response only in SOD1-ko (−46%), compared with Ang II alone. Basal blood pressures were similar; however the hypertensive response to chronic Ang II infusion was exaggerated in SOD1-ko (27%) compared with wild-types (9%). The different SOD1-levels were not associated with changes in nitrate/nitrite (NOx) excretion or in renal mRNA expression for NOS-, NADPH oxidase-, SOD-isoforms, or Ang II receptors. In conclusion, SOD1 plays an important role in the control of afferent arteriolar tone, mainly by modulating the NO bioavailability. SOD1-deficiency aggravates Ang II-induced hypertension, which underscores the importance of oxidative stress in this model of hypertension.

Adenosine and angiotensin II (Ang II) interact in the control of renal blood perfusion and glomerular filtration rate. Here we test the hypothesis that Ang II in low concentrations enhances adenosine vasoconstrictor effects. Further, we investigated cytosolic calcium transients and the role of adenosine and Ang II receptors in this context. Bolus application of Ang II (10−12 and 10−10mol/l) induced negligible vasoconstrictions in isolated perfused afferent arterioles of mice, while Ang II 10−8mol/l reduced diameters by about 35% within 2 min. The arteriolar response to cumulative adenosine applications (10−11 to 10−4 mol/l) was very small, but clearly enhanced by Ang II at 10−12,10−10, 10−8 mol/l, respectively. In Fura‐2 AM loaded arterioles, adenosine bolus application induced an increase of the calcium concentration in the arteriolar wall which was greater at 10−5 mol/l compared with 10−8 mol/l. Ang II (10−11 to 10−6 mol/l) induced a concentration dependent increase of cytosolic calcium concentration. Ang II receptor type 1 (AT1AR) antagonist ZD7155 (10−7 mol/l) nearly prevented this Ang II effect. Simultaneous application of Ang II enhanced the calcium concentration to 10−8 and 10−5 mol/l adenosine. The results show that Ang II enhances adenosine induced vasoconstriction even at low concentrations and that this effect goes along with increased calcium transients.

Neuronal nitric oxide synthase inhibition restores the tubuloglonerular feedback response after volume expansion.