NOx Excretion Research Articles

Nitric oxide and long-term outcomes after kidney transplantation: Results of the TransplantLines cohort study

Impaired endogenous nitric oxide (NO) production may contribute to graft failure and premature mortality in kidney transplant recipients (KTR). We investigated potential associations of 24-h urinary NOx (NO3− + NO2−) excretion (uNOx) with long-term outcomes. uNOx was determined by HPLC and GC-MS in 698 KTR and in 132 kidney donors before and after donation. Additionally, we measured urinary nitroso species (RXNO) by gas-phase chemiluminescence. Median uNOx was lower in KTR compared to kidney donors (688 [393–1076] vs. 1301 [868–1863] before donation and 1312 [982–1853] μmol/24 h after donation, P < 0.001). During median follow-up of 5.4 [4.8–6.1] years, 150 KTR died (61 due to cardiovascular disease) and 83 experienced graft failure. uNOx was inversely associated with all-cause mortality (HR per doubling of uNOx: 0.84 [95% CI 0.75–0.93], P < 0.001) and cardiovascular mortality (HR 0.78 [95% CI 0.67–0.92], P = 0.002). The association of uNOx with graft failure was lost when adjusted for renal function (HR per doubling of uNOx: 0.89 [95% CI 0.76–1.05], P = 0.17). There were no significant associations of urinary RXNO with outcomes. Our study suggests that KTR have lower NO production than healthy subjects and that lower uNOx is associated with a higher risk of all-cause and cardiovascular mortality.

Sex-Dependent Effects of Nephron Ift88 Disruption on BP, Renal Function, and Cystogenesis.

Primary cilia regulation of renal function and BP in health and disease is incompletely understood. This study investigated the effect of nephron ciliary loss on renal physiology, BP, and ensuing cystogenesis. Mice underwent doxycycline (DOX)-inducible nephron-specific knockout (KO) of the Ift88 gene at 2 months of age using a Cre-LoxP strategy. BP, kidney function, and renal pathology were studied 2 and 9 months after DOX (Ift88 KO) or vehicle (control). At 2 months post-DOX, male, but not female, Ift88 KO, compared with sex-matched control, mice had reduced BP, enhanced salt-induced natriuresis, increased urinary nitrite and nitrate (NOx) excretion, and increased kidney NOS3 levels, which localized to the outer medulla; the reductions in BP in male mice were prevented by L-NAME. At 9 months post-DOX, male, but not female, Ift88 KO mice had polycystic kidneys, elevated BP, and reduced urinary NOx excretion. No differences were observed in plasma renin concentration, plasma aldosterone, urine vasopressin, or urine PGE2 between Ift88 KO and control mice at 2 or 9 months post-DOX. Nephron cilia disruption in male, but not female, mice (1) reduces BP prior to cyst formation, (2) increases NOx production that may account for the lower BP prior to cyst formation, and (3) induces polycystic kidneys that are associated with hypertension and reduced renal NO production.

Collecting Duct NOS1 is Necessary to Maintain Sodium Balance in Male, But Not Female Mice

Nitric oxide synthase 1 (NOS1) is a well‐established regulator of fluid and electrolyte balance in the collecting duct (CD). We previously reported that a loss of CDNOS1 in male mice results in reduced sodium excretion, urine volume, and nitric oxide (NO) excretion as well as increased plasma sodium compared to flox control mice after being challenged with 1 day of high salt diet. However, it is not yet known if this genotype difference is conserved in female mice. Therefore, we investigated whether collecting duct NOS1 is critical in maintaining homeostasis in female mice. Female CDNOS1KO and flox control mice were acclimated to metabolic cages and placed on a sodium deficient (LS, 1 week) then challenged with a high sodium (HS, 1 day) diet and urine collected. Female CDNOS1KO mice and flox control mice had similar sodium excretion (Flox: 3.24±0.32, KO: 2.62±0.31mmol/day; n=8–12/group, p&gt;0.05), urine volume (Flox: 7.69±0.75, KO: 6.42±0.82mL/day; n=8–12/group, p&gt;0.05), and NOx excretion (Flox: 1099±205, KO: 1339±389 μmol/day; n=8–12/group, p&gt;0.05) in addition to comparable plasma sodium levels (Flox: 146±1.0, KO: 147±0.5mmol/L; n=6/group, p&gt;0.05) after one day of a high salt challenge. Furthermore, urinary prostaglandin E metabolite excretion was similar between genotypes after a high salt challenge (Flox: 3187±382, KO: 2744±291pg/day; n=8–10/group, p&gt;0.05), suggesting that female mice are able to compensate for a loss of NOS1‐derived NO in the collecting duct. Additionally, we performed RT‐PCR on male and female renal vessels and found that unlike males, female CDNOS1KO mice have significantly elevated vascular endothelin 1 mRNA expression (Male‐ flox: 1.00±0.09, KO: 0.98±0.03; n=6/group, p&gt;0.05; Female‐ flox: 1.00±0.18, KO: 1.75±0.24; n=5–6/group, p=0.03). In conclusion, female mice do not rely on collecting duct NOS1‐derived NO to maintain fluid‐electrolyte balance. We propose that female mice utilize endothelin‐1 and/or NO production in other renal segments.Support or Funding InformationF31DK111067 to RSS, F31HL149235 to LSD, and P01HL136267, AHA 19SRG34930023 to JSP

Mechanisms underlying the effects of propiverine on bladder activity in rats with pelvic venous congestion and urinary frequency.

We investigated the mechanisms by which propiverine hydrochloride influenced bladder activity in rats with pelvic venous congestion (PC) and urinary frequency. To create PC rats, female rats were anesthetized with isoflurane and the bilateral common iliac veins and bilateral uterine veins were ligated. At 4 weeks after ligation, we assessed voiding behaviour, locomotor activity, and urinary 8-hydroxydeoxyguanosine (8-OHdG) and nitric oxide metabolites (NOx). We also performed cystometry and measured mRNAs for nitric oxide synthase (NOS) and several receptors in the bladder wall. PC rats showed a decrease in locomotor activity and an increased frequency of urination. There was a decrease in endothelial NOS (eNOS), M3, and TRPV1 mRNA expression in the bladder wall, as well as an increase in inducible NOS (iNOS) mRNA. Administration of propiverine to PC rats increased locomotor activity to the level in sham rats, improved bladder function, decreased urinary 8-OHdG excretion, and increased urinary NOx excretion. In addition, propiverine increased neuronal NOS (nNOS) mRNA expression, and decreased expression of iNOS, M3 and TRPV1 mRNA in the bladder wall. Therefore, propiverine not only improved bladder dysfunction through its previously reported actions (anti-muscarinic effect, Ca antagonist effect, and inhibition of noradrenaline re-uptake), but also by reducing inflammation.

Renal Nitric Oxide Deficiency and Chronic Kidney Disease in Young Sheep Born with a Solitary Functioning Kidney.

Previously, we demonstrated that renal hemodynamic responses to nitric oxide (NO) inhibition were attenuated in aged, hypertensive sheep born with a solitary functioning kidney (SFK). NO is an important regulator of renal function, particularly, in the postnatal period. We hypothesized that the onset of renal dysfunction and hypertension in individuals with a SFK is associated with NO deficiency early in life. In this study, renal and cardiovascular responses to L-NAME infusion (Nw-nitro-L-arginine methyl ester) were examined in 6-month old lambs born with a SFK, induced by fetal unilateral nephrectomy (uni-x). Renal responses to L-NAME were attenuated in uni-x sheep with the fall in glomerular filtration rate (GFR) and urinary sodium excretion (UNaV) being less in the uni-x compared to sham lambs (%ΔGFR; −41 ± 3 vs −54 ± 4: P = 0.03, %ΔUNaV; −48 ± 5 vs −76 ± 3, P = 0.0008). 24 hour-basal urinary nitrate and nitrite (NOx) excretion was less in the uni-x animals compared to the sham (NOx excretion μM/min/kg; sham: 57 ± 7; uni-x: 38 ± 4, P = 0.02). L-NAME treatment reduced urinary NOx to undetectable levels in both groups. A reduction in NO bioavailability in early life may contribute to the initiation of glomerular and tubular dysfunction that promotes development and progression of hypertension in offspring with a congenital nephron deficit, including those with a SFK.

Formation of nitric oxide metabolites during growth of transplanted tumors with different metastatic potential

The dynamics of the endogenous production of NO metabolites, nitrites, nitrates, and volatile nitrosamines, has been investigated in the body, a tumor tissue, and peritoneal macrophages of mice F1 (C57Bl × CBA), Balb/c and BDF with subcutaneously transplanted tumors: Ehrlich carcinoma (EC) and Lewis metastatic lung carcinoma (LC). It has been shown that during the first three weeks, growth of EC is accompanied by a statistically significant increase in the total concentration of NO metabolites (NOx; nitrites + nitrates) up to levels (7.3 ± 3.49) × 10−6−(7.8 ± 2.57) × 10−5 mol/kg in the tumor tissue and a sharp increase in NOx excretion with urine. At the same time, in the LC tumor tissue the maximal level of NOx (3.6 ± 0.46) × 10−5 mol/kg was registered on day 7, and later it decreased demonstrating negative correlation with the tumor mass. At the stage of intensive LC growth (14, 21 days) there was significant inhibition of nitrite secretion by peritoneal macrophages. In all the controlled time-intervals the EC tumor tissue contained carcinogenic N-nitrosodimethylamine and N-nitrosodiethylamine; however, their concentrations significantly varied. Thus, the ability of the tumor tissue to produce NO metabolites was more pronounced in EC than in LC and depended on the time parameters of tumor growth.

Serelaxin reduces oxidative stress and asymmetric dimethylarginine in angiotensin II-induced hypertension

Recent findings suggest the therapeutic action of relaxin during hypertension is dependent on nitric oxide synthase (NOS) activation; however, the mechanisms underlying the beneficial effects of relaxin on the NOS system have not been fully elucidated. We hypothesized that the protective effects of relaxin include reducing both oxidative stress and the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). We examined the effect of Serelaxin [human recombinant relaxin-2 (RLX)] in male Sprague-Dawley rats given high-dose angiotensin (ANG) II (400 ng·kg(-1)·min(-1) sc) for 6 wk or shams. RLX was administered (4 μg/h sc) to half of the rats in each group after 2 wk of ANG II for the remaining 4 wk. ANG II induced hypertension and proteinuria, reduced NO oxidation products (NOx), and increased oxidative stress (NADPH oxidase activity, thiobarbituric acid-reactive substances, and 8-isoprostane excretion) and plasma ADMA. While RLX had no effect on sham rats, RLX attenuated the ANG II-dependent hypertension (165 ± 5 vs. 135 ± 13 mmHg, P < 0.05) and proteinuria at 6 wk (62 ± 6 vs. 41 ± 4 mg·day(-1)·100 g(-1), P < 0.05) and normalized oxidative stress and circulating ADMA, in association with restored NOx excretion and kidney cortex NOx. We found that RLX had no impact on the ADMA-regulatory enzymes protein arginine methyltransferase and dimethylarginine-dimethylaminohydrolase (DDAH). Furthermore, RLX treatment did not increase DDAH activity in kidney cortex or liver. These data suggest that benefits of RLX treatment include reduced ADMA levels and increased NO bioavailability, possibly due to its antioxidant effects.

Cardiac function and tolerance to ischemia–reperfusion injury in chronic kidney disease

Cardiac dysfunction is an independent risk factor of ischemic heart disease and mortality in chronic kidney disease (CKD) patients, yet the relationship between impaired cardiac function and tolerance to ischemia-reperfusion (IR) injury in experimental CKD remains unclear. Cardiac function was assessed in 5/6 ablation-infarction (AI) and sham male Sprague-Dawley rats at 20 weeks of age, 8 weeks post-surgery using an isolated working heart system. This included measures taken during manipulation of preload and afterload to produce left ventricular (LV) function curves as well as during reperfusion following a 15-min ischemic bout. In addition, LV tissue was used for biochemical tissue analysis. Cardiac function was impaired in AI animals during preload and afterload manipulations. Cardiac functional impairments persisted post-ischemia in the AI animals, and 36% of AI animals did not recover sufficiently to achieve aortic overflow following ischemia (versus 0% of sham animals). However, for those animals able to withstand the ischemic perturbation, no difference was observed in percent recovery of post-ischemic cardiac function between groups. Urinary NOx (nitrite + nitrate) excretion was lower in AI animals and accompanied by reduced LV endothelial nitric oxide synthase and NOx. LV antioxidants superoxide dismutase-1 and -2 were reduced in AI animals, whereas glutathione peroxidase-1/2 as well as NADPH-oxidase-4 and H(2)O(2) were increased in these animals. Impaired cardiac function appears to predispose AI rats to poor outcomes following short-duration ischemic insult. These findings could be, in part, mediated by increased oxidative stress via nitric oxide-dependent and -independent mechanisms.

Abstract 273: Increased Blood Pressure and Proteinuria and Absence of Increased Nitric Oxide (NO) Production During Pregnancy in the Dahl Salt Sensitive (S) Rat

In normal pregnancy, systemic vasodilation due to increased NO production allows a drop in blood pressure (BP) despite increased volume retention. Little is known about the pathogenesis of preeclampsia, defined by increased BP and proteinuria, due to a lack of animal models that spontaneously develop the disease. Here we tested the hypothesis that the Dahl S rat, a genetic model of hypertension and kidney disease, is also a spontaneous model of preeclampsia. Female Dahl S rats were implanted with a telemetry unit, and baseline BP was recorded. Rats were placed in metabolic cages for 24 hr urine collection while on a low nitrate diet, and urinary protein and NO metabolite concentrations were measured via Bradford and Greiss assays, respectively. There were no differences in baseline BP (152±1 vs 151±4 mmHg) or proteinuria (61±10 vs 60±17 mg/d) in the rats selected for mating vs virgin rats (n=5-7). Pregnancy was confirmed by presence of sperm (day 1). Measurements were made during mid and late pregnancy (days 10-11, 17-18), and terminal measurements were taken on day 19. Pregnant rats exhibited an increase in BP and proteinuria with no change in urinary NOx excretion (Table), while no changes were observed in age-matched virgin rats. Kidney cortex abundance of neither NOS1 nor NOS3 was increased at late pregnancy; however, plasma concentration of the endogenous NOS inhibitor ADMA was increased in late pregnant compared to virgin rats (0.82±0.06 vs. 0.62±0.06 μM, p&lt;0.05). These data suggest that the Dahl S rat cannot upregulate NO production during pregnancy; therefore, this relative NO deficiency may contribute to worsening hypertension and proteinuria during pregnancy in this strain.

Abstract 493: Renal Injury Induced by Nitric Oxide Depletion is Prevented by Concomitant Inhibition of Hydrogen Sulfide and/or Carbon Monoxide Production

Chronic nitric oxide (NO) depletion induces hypertension and renal damage. Hydrogen sulfide (H 2 S) producing cystathionine-γ-lyase (CSE) and carbon monoxide (CO) producing heme oxygenase-1 (HO-1) appear to be protective in mechanically-induced renal injury (e.g. ischemia reperfusion). However, inhibition of CSE can reduce drug-induced renal injury (e.g. cisplatin). The role of renal H 2 S and HO-1 during chronic NO depletion is unknown. We hypothesized that renal injury secondary to NO depletion via inhibition of NO synthase is diminished by additional H 2 S depletion via inhibition of CSE. Rats (n=6/group) were treated with inhibitors of NO synthase (L-nitroarginine; LNNA), CSE (DL-propargylglycine; PAG), or HO-1 (Sn(IV) protoporphyrin IX dichloride; SnPP) for 1 or 4 weeks or with combinations (LNNA+PAG; LNNA+SnPP; PAG+SnPP or LNNA+PAG+SnPP) for 4 weeks. One week LNNA reduced urinary NOx excretion (35±2% vs baseline) and induced hypertension (173±12 vs. 137±3 mmHg; P&lt;0.01) but renal function remained normal. Four weeks of LNNA further reduced NOx (7±1%), worsened hypertension (223±10 mmHg) and caused renal injury; plasma urea (17±4 vs 7±1 mmol/L; P&lt;0.05), proteinuria (144±35 vs 17±2 mg/d; P&lt;0.01). PAG or SnPP had no effect. NOx was reduced by PAG and increased by SnPP. Renal H 2 S production was completely blocked by PAG and enhanced by SnPP at 1 and 4 weeks. Renal HO-1 expression was induced by LNNA at 4 weeks and by PAG and SnPP at 1 and 4 weeks (all P&lt;0.001). Adding PAG, SnPP, or both, to LNNA did not affect hypertension but preserved renal function. Combining PAG and SnPP had no effect on blood pressure or renal function. Reduction of urine NOx by LNNA was not affected by additional PAG (8±2%) but was ameliorated by adding SnPP (37±4%) or PAG+SnPP (42±9%). Renal H 2 S production was completely inhibited with all PAG-combinations (P&lt;0.01), but was twofold enhanced by LNNA+SnPP (P&lt;0.01). Renal HO-1 expression was increased by all combinations. NO depletion resulted in hypertension and progressive renal injury that was prevented by concomitant inhibition of CSE and/or HO-1. Depletion of H 2 S and CO in the absence of NO depletion had no effect on blood pressure and renal function. These data suggest that pathways from NO depletion to renal injury run via H 2 S or CO.

OP26 Renal injury induced by nitric oxide depletion is prevented by concomitant inhibition of hydrogen sulfide and/or carbon monoxide production

Chronic nitric oxide (NO) depletion induces hypertension and renal damage. Chronic kidney disease associates with diminished NO and hydrogen sulfide (H 2 S) availability in the kidney. H 2 S producing cystathionine- γ -lyase (CSE) and carbon monoxide (CO) producing heme oxygenase-1 (HO-1) appear to be protective in mechanical-induced renal injury (e.g. ischemia reperfusion). However, inhibition of CSE can also reduce drug-induced renal injury (e.g. cisplatin or adriamycin). The role of renal H 2 S and HO-1 during chronic NO depletion is unknown. We hypothesized that renal injury secondary to NO depletion caused by inhibition of NO synthase is diminished by additional H 2 S depletion via inhibition of CSE. Rats ( n = 6/group) were treated with inhibitors of NO synthase ( l -nitroarginine; LNNA), CSE (DL-propargylglycine; PAG), or HO-1 (Sn (IV) protoporphyrin IX dichloride; SnPP) for 1 or 4 weeks. Other rats were treated with combinations (LNNA + PAG; LNNA + SnPP; PAG + SnPP or LNNA + PAG + SnPP) for 4 weeks. One week LNNA reduced urinary NOx excretion (35 ± 2%) and induced hypertension (173 ± 12 vs. 137 ± 3 mmHg; P < 0.01) but renal function remained normal. Four weeks of LNNA further reduced urinary NOx (7 ± 1%), worsened hypertension (223 ± 10 mmHg) and caused renal injury, plasma uremia (17 ± 4 vs. 7 ± 1 mmol/L; P < 0.05) and proteinuria (144 ± 35 vs. 17 ± 2 mg/d; P < 0.01). PAG or SnPP had no effect. Interestingly, urine NOx was reduced by PAG and increased by SnPP. Renal H 2 S production was completely blocked by PAG and enhanced by SnPP at 1 and 4 weeks. Renal HO-1 expression was induced by LNNA at 4 weeks and by PAG and SnPP at 1 and 4 weeks (all P < 0.001). Adding PAG, SnPP, or both, to LNNA did not affect hypertension but maintained renal function. Combining PAG and SnPP had no effect on blood pressure and renal function. Strong reduction of urine NOx by LNNA was not affected by additional PAG (8 ± 2%) but was ameliorated by adding SnPP (37 ± 4%) or PAG + SnPP (42 ± 9%). Renal H 2 S production remained completely inhibited with all PAG-combinations ( P < 0.01), but was twofold enhanced by LNNA + SnPP ( P < 0.01). Renal HO-1 expression was increased by all combinations. NO depletion resulted in hypertension and progressive renal injury, while depletion of H 2 S and CO had no effect on blood pressure and kidney function. As hypothesized, renal injury characteristic of NO depletion was prevented by concomitant inhibition of CSE. Interestingly, concomitant inhibition of HO-1 achieved the same result. These data suggest that pathways from NO depletion to renal injury run via H 2 S or CO.

Sex Differences in the Beneficial Cardiac Effects of Chronic Treatment with Atrial Natriuretic Peptide In Spontaneously Hypertensive Rats

IntroductionThe aim of this study was to investigate both the effects of chronic treatment with atrial natriuretic peptide (ANP) on systolic blood pressure (SBP), cardiac nitric oxide (NO) system, oxidative stress, hypertrophy, fibrosis and apoptosis in spontaneously hypertensive rats (SHR), and sex-related differences in the response to the treatment.Methods10 week-old male and female SHR were infused with ANP (100 ng/hr/rat) or saline (NaCl 0.9%) for 14 days (subcutaneous osmotic pumps). SBP was recorded and nitrites and nitrates excretion (NOx) were determined. After treatment, NO synthase (NOS) activity, eNOS expression, thiobarbituric acid-reactive substances (TBARS) and glutathione concentration were determined in left ventricle, as well as the activity of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). Morphological studies in left ventricle were performed in slices stained with hematoxylin-eosin or Sirius red to identify collagen as a fibrosis indicator; immunohistochemistry was employed for identification of transforming growth factor beta; and apoptosis was evaluated by Tunel assay.ResultsFemale SHR showed lower SBP, higher NO-system activity and less oxidative stress, fibrosis and hypertrophy in left ventricle, as well as higher cardiac NOS activity, eNOS protein content and NOx excretion than male SHR. Although ANP treatment lowered blood pressure and increased NOS activity and eNOS expression in both sexes, cardiac NOS response to ANP was more marked in females. In left ventricle, ANP reduced TBARS and increased glutathione concentration and activity of CAT and SOD enzymes in both sexes, as well as GPx activity in males. ANP decreased fibrosis and apoptosis in hearts from male and female SHR but females showed less end-organ damage in heart. Chronic ANP treatment would ameliorate hypertension and end-organ damage in heart by reducing oxidative stress, increasing NO-system activity, and diminishing fibrosis and hypertrophy.

Renal Collecting Duct NOS1 Maintains Fluid–Electrolyte Homeostasis and Blood Pressure

Nitric oxide is a pronatriuretic and prodiuretic factor. The highest renal NO synthase (NOS) activity is found in the inner medullary collecting duct. The collecting duct (CD) is the site of daily fine-tune regulation of sodium balance, and led us to hypothesize that a CD-specific deletion of NOS1 would result in an impaired ability to excrete a sodium load leading to a salt-sensitive blood pressure phenotype. We bred AQP2-CRE mice with NOS1 floxed mice to produce flox control and CD-specific NOS1 knockout (CDNOS1KO) littermates. CDs from CDNOS1KO mice produced 75% less nitrite, and urinary nitrite+nitrate (NOx) excretion was significantly blunted in the knockout genotype. When challenged with high dietary sodium, CDNOS1KO mice showed significantly reduced urine output, sodium, chloride, and NOx excretion, and increased mean arterial pressure relative to flox control mice. In humans, urinary NOx is a newly identified biomarker for the progression of hypertension. These findings reveal that NOS1 in the CD is critical in the regulation of fluid-electrolyte balance, and this new genetic model of CD NOS1 gene deletion will be a valuable tool to study salt-dependent blood pressure mechanisms.

Effect of a calcineurin inhibitor on Lipopolysaccharide‐induced loss of vascular tone and blood pressure decrease in conscious mice

The immunosuppressive drug cyclosporin A (CsA) inhibits the production of nitric oxide (NO) through inhibition of the NO synthases. Especially iNOS is involved in the synthesis of large amounts of NO seen in sepsis, which leads to hypotension and multiple organ dysfunction. We hypothesized that CsA counteracts the LPS‐induced blood pressure decrease and reduced vasoreactivity in isolated murine aorta rings in a model of septic shock.To test this, blood pressure and heart rate was measured in conscious mice with indwelling catheters given a bolus (20 or 40 mg/kg) of CsA followed by continuous infusion (20 or 40 mg/kg/day) for one day before an LPS bolus was given (2 mg/kg). In vitro, studies were performed with aortic rings incubated with LPS (50 μg/ml) w/wo CsA, with CsA alone or vehicle.CsA (20 and 40 mg/kg) induced a significant increase in blood pressure (≈11 mmHg), with no effect on heart rate. After an LPS bolus, blood pressure and heart rate decreased, but there was no difference in delta blood pressure and heart rate compared to LPS alone. iNOS mRNA and protein expression was significantly reduced by CsA as well as plasma NOx excretion. CsA significantly attenuated the LPS‐induced decrease in vasoreactivity. It is concluded that CsA improve vasoreactivity in vitro while in a murine model of septic shock in vivo, CsA at 20 and 40 mg/kg did not counteract the effect of LPS on blood pressure.

Deletion of cyclooxygenase-2 in the mouse increases arterial blood pressure with no impairment in renal NO production in response to chronic high salt intake

Experiments were designed to test the hypothesis that cyclooxygenase-2 (COX-2) activity attenuates the blood pressure increase during high NaCl intake by stimulation of endothelial nitric oxide synthase (eNOS)-mediated NO synthesis in the kidney medulla. COX-2(-/-) (C57BL6) an COX-2(+/+) mice were fed a diet with 0.004% (low salt, LS) or 4% (high salt, HS) NaCl for 18 days. Arterial blood pressure was recorded continuously using indwelling catheters. Food and water intake and diuresis were measured in metabolic cages. Urine osmolality and excretion of electrolytes, cGMP, cAMP, and NOx were determined, as well as plasma NOx and cGMP. There was a significant dependence of blood pressure on salt intake and genotype: COX-2(-/-) exhibited higher blood pressure than COX-2(+/+) both on HS and LS intake. COX-2(+/+) littermates displayed an increase in blood pressure on HS versus LS (102.3 ± 1.1 mmHg vs. 91.9 ± 0.9 mmHg) day and night. The mice exhibited significant blood pressure increases during the awake phase (night) that were larger in COX-2(-/-) on HS diet compared with COX-2(+/+). Water intake, diuresis, Na(+), and osmolyte excretions and NOx and cGMP excretions were significantly and similarly elevated with HS in COX-2(-/-) and COX-2(+/+). In summary, C57BL6 mice exhibit a salt intake-dependent increase in arterial blood pressure with increased renal NO production. COX-2 activity has a general lowering effect on arterial blood pressure. COX-2 dampens NaCl-induced increases in arterial blood pressure in the awake phase. In conclusion, COX-2 activity attenuates the changes in nocturnal blood pressure during high salt intake, and COX-2 activity is not necessary for increased renal nitric oxide formation during elevated NaCl intake.

Nicorandil Ameliorated Hypertensive Renal Injury without Lowering Blood Pressure in Spontaneously Hypertensive Rats

Proteinuria, a symptom of hypertensive renal injury, is a powerful predictor of mortality in chronic kidney disease patients with hypertension. The present study investigated whether a nonhypotensive dose of nicorandil could decrease hypertensive renal injury in male spontaneously hypertensive rats (SHR). Nicorandil (15 mg/kg/day, for 20 weeks) was administered in the drinking water to rats from 11 weeks old. Heart size, kidney size, and β₂-microglobulin occurring with tubular histopathological damage were each significantly greater in SHR than in Wistar-Kyoto (WKY) rats, as was 24-hour excretion of urinary protein (SHR: 33.1 ± 3.5 mg/day, WKY: 5.4 ± 0.3 mg/day). Nicorandil significantly decreased urinary protein (21.7 ± 2.8 mg/day), glomerular cell density, and histopathological score without affecting systolic blood pressure. Nicorandil increased expression of endothelial nitric oxide synthase (eNOS) protein in the renal cortex in SHR without affecting expressions of mRNA for endothelin or genes involved in tissue damage or fibrosis. eNOS expression was negatively correlated with glomerular cell density. In addition, nicorandil increased urinary excretion of NOx, but did not change the eNOS dimer-to-monomer ratio or the decreased level of renal heparan sulfate in SHR. In conclusion, in SHR, long-term administration of nicorandil can ameliorate hypertensive proteinuria, without lowering blood pressure, possibly through an increase in eNOS expression.

Abstract 93: Hypertension and Renal Injury Induced by Nitric Oxide Depletion are Ameliorated by Concomitant Depletion of Hydrogen Sulfide

Chronic nitric oxide (NO) depletion induces hypertension and renal damage. Chronic kidney disease is associated with decreased NO availability and less renal H 2 S production. We hypothesized that combined depletion of NO and H 2 S aggravates hypertension and renal injury. Male 8-wk old Sprague Dawley rats were treated with vehicle, NO synthase inhibitor L-NG-nitroarginine (LNNA; 125 mg/L in drinking water), cystathionine-γ-lyase (CSE) inhibitor propargylglycine (PAG; 37.5 mg/kg BW ip daily) or LNNA + PAG for 1 and 4 weeks (6 rats/group). LNNA after 4w increased systolic blood pressure (SBP; 223±10 vs . 137±3 mmHg in controls; P&lt;0.01), proteinuria (144±35 vs. 17±2 mg/d; P&lt;0.01), uremia (16.6±4.2 vs . 7.0±0.4 mmol/L; P&lt;0.05) and tubulo-interstitial injury (P&lt;0.01). LNNA reduced urinary NO metabolite (NOx) excretion by ∼85% after 1w and 4w. PAG alone had no effect on SBP, renal function or injury, but did reduce urinary NOx excretion. Co-treatment with PAG ameliorated LNNA-induced hypertension (182±10 mmHg; P&lt;0.01) and prevented proteinuria (27±3 mg/d), uremia (8.3±0.4 mmol/L) and tubulo-interstitial injury, but did not further reduce urinary NOx excretion. Renal H 2 S production was almost absent in all PAG groups after 1w and 4w (P&lt;0.01) and was reduced in LNNA-treated rats after 4w (4.6±1.4 vs . 9.2±0.5 μmol/hr/mg; P&lt;0.01). Renal HO-1 gene expression was strongly induced in all PAG-treated groups after 1w and 4w (4 to 19-fold; P&lt;0.01) whereas LNNA only increased HO-1 gene expression at 4w (P&lt;0.01). Immunohistochemistry showed that renal HO-1 protein was primarily interstitial in all PAG-treated groups at 1w and 4w. In contrast, LNNA only showed HO-1 in tubular epithelium in conjunction with protein casts. Depleting NO caused hypertension and renal damage followed by reduced renal H 2 S production and increased renal HO-1 expression. Surprisingly, concomitant inhibition of CSE ameliorated hypertension and prevented renal injury. PAG almost completely blocked renal H 2 S production and caused strong induction of renal HO-1, independently of injury, suggesting that H 2 S suppresses renal HO-1 expression. In conclusion, concomitant upregulation of HO-1 expression by inhibition of H 2 S production, prevents LNNA-induced hypertension and renal injury.

Angiotensin II receptor blockade reduces salt sensitivity of blood pressure through restoration of renal nitric oxide synthesis in patients with diabetic nephropathy

We have previously demonstrated the increased salt sensitivity of blood pressure (BP) in diabetic patients with early nephropathy. Here, we examined the effects of an angiotensin II receptor blocker (ARB) on salt sensitivity and renal oxidative stress or nitric oxide (NO) in those patients. Type 2 diabetic patients with (n = 6) and without (n = 6) microalbuminuria were studied on a high-salt diet for one week and on a salt-restricted diet for one week. The study was repeated in the patients with microalbuminuria during treatment with an ARB, valsartan (80 mg/day). Salt sensitivity was assessed from the BP/sodium excretion curve. Urinary excretion rates of NOx, 8-hydroxy-2-deoxyguanosine as a marker of oxidative stress, and plasma tetrahydrobiopterin as a cofactor for NO synthase were measured. Compared with diabetic patients without microalbuminuria, patients with microalbuminuria showed greater salt sensitivity and lower urinary excretion of NOx. In the patients with microalbuminuria, treatment with valsartan reduced salt sensitivity in association with increased NOx excretion, reduced 8-hydroxy-2,-deoxyguanosine excretion, and increased plasma tetrahydrobiopterin levels. These data support the hypothesis that ARBs reduce the salt sensitivity of BP by decreasing renal oxidative stress and restoring NO activity in diabetic patients with microalbuminuria.

Nebivolol does not protect against 5/6 ablation/infarction induced chronic kidney disease in rats — Comparison with angiotensin II receptor blockade

AimsNitric oxide (NO) deficiency contributes to chronic kidney disease progression. Nebivolol, a beta adrenergic receptor antagonist, may enhance endogenous NO. Here, we investigated whether Nebivolol attenuates hypertension and renal injury after 5/6 ablation/infarction (A/I). Efficacy was compared to the AT1 receptor antagonist Olmesartan. Main methodsKidney disease and hypertension were induced by right kidney ablation and ~2/3 infarction of the left kidney. Rats were treated orally with vehicle (placebo), Nebivolol (5mg/kg b.i.d.), or Olmesartan (2.5mg/kg/day) for 6weeks after A/I. Key findingsWith placebo, glomerular sclerosis and tubulointersititial fibrosis developed with increased blood pressure and proteinuria, and a fall in NOx excretion. Olmesartan prevented these changes, but Nebivolol had no effect on these measures but lowered heart rate. Neither treatment reduced systemic oxidative stress (urinary hydrogen peroxide and TBARS). Compared to controls, renal cortex abundance of nNOSα decreased and nNOSβ increased in rats after 5/6 A/I, with no changes in eNOS. Neither treatment restored nNOSα; however, both reduced nNOSβ. Activity of DDAH was decreased by 5/6 A/I but restored by both treatments despite no increase in DDAH protein abundance. Kidney cortex abundance of manganese SOD fell after 5/6 A/I and was restored by treatment with Olmesartan but not Nebivolol. Extracellular and copper/zinc SOD abundances were not changed. SignificanceIn conclusion, Nebivolol showed no benefit after 6weeks in rapidly progressing, ANG II-dependent 5/6 A/I model of chronic kidney disease. This contrasts to the protection seen with 6month treatment of Nebivolol in the slowly progressing 5/6 ablation model.

Effects of bench step exercise on arterial stiffness in post-menopausal women: Contribution of IGF-1 bioactivity and nitric oxide production

ObjectiveTo examine the effect of bench step exercise on arterial pulse wave velocity (PWV) and the associated contribution of insulin-like growth factor (IGF)-1 bioactivity and nitric oxide (NO). DesignTwenty-six elderly (post-menopausal) women were randomly allocated to a bench step exercise group or a control group. The participants in the bench step exercise group practiced a 12-week home-based bench step exercise for 10–20min, 3 times daily (i.e., for a total of 140min/week at the intensity level of lactate threshold (LT)). In addition to conventional risk factors of atherosclerosis, PWV, IGF-1/IGF binding protein (IGFBP)-3 molar ratio (an index for IGF-1 bioactivity), and urinary nitrite/nitrate (NOx) excretion were measured before and after the intervention. ResultsBMI, systolic blood pressure, fasting plasma glucose, low-density lipoprotein cholesterol, LT, and PWV were significantly improved in the bench step exercise group. A significant positive correlation between changes in PWV and IGF-1/IGFBP-3 molar ratio, and a significant negative correlation between changes in IGF-1/IGFBP-3 molar ratio and urinary NOx excretion were found in the bench step exercise group. ConclusionThe bench step exercise leads to improvements in not only the classical risk factors of atherosclerosis but also the arterial stiffness in elderly women, partly through NO production via IGF-1 bioactivity.