Nicorandil suppresses urinary protein excretion and activates eNOS in Dahl salt-sensitive hypertensive rats.

BACKGROUND: Hypertension is a risk factor common to both chronic kidney disease and cardiovascular disease. Nicorandil is widely used for the treatment of angina. We investigated the benefits of nicorandil with respect to renal dysfunction in Dahl salt-sensitive hypertensive (DS) rats. METHOD: DS rats were fed a high-salt (HS) diet and nicorandil was administered via the drinking water. Blood pressure and renal function were measured for 4 weeks after starting the rats on the HS diet. RESULTS: In rats fed the HS diet, renal dysfunction was manifested by an increase in urinary protein and N-acetyl-ß-D-glucosaminidase excretion. Nicorandil ameliorated renal function with a concomitant reduction in urinary 8-hydroxy-2'-deoxyguanosine and an increase in urinary NOx. Significant upregulation of endothelial nitric oxide synthase (eNOS) expression and an increase in the eNOS dimer/monomer ratio (reduction of eNOS uncoupling) was demonstrated in glomeruli following nicorandil treatment. The blood pressure of DS rats was increased by salt loading; however, no significant change in blood pressure was observed with nicorandil treatment. CONCLUSION: In DS rats fed a HS diet, nicorandil prevented the development of renal dysfunction, which was accompanied by an increase in eNOS expression in the kidneys.

Role of specific T-type calcium channel blocker R(-) efonidipine in the regulation of renal medullary circulation.

OBJECTIVES: Blockade of the T-type calcium channel (TCC), which is expressed in the renal efferent arterioles of the juxtamedullary nephron and vasa recta, has been shown to protect against renal injury. Studies were designed to determine the effects of a specific TCC blocker, R(-) efonidipine [R(-)EFO], on the regulation of renal circulation. METHODS AND RESULTS: Renal medullary blood flux (MBF) and cortical blood flux (CBF) were simultaneously monitored using laser-Doppler flowmetry in Sprague-Dawley rats. Responses were also determined in rats with angiotensin II (AngII) induced renal ischemia. Intravenous (i.v.) or renal interstitial (r.i.) infusion of R(-)EFO (0.25 mg/h, i.v. or r.i.) significantly increased MBF by 24.0 ± 7.0 and 21.0 ± 4.4%, respectively, but without changing CBF or mean arterial pressure. The nitric oxide (NO) synthase inhibitor NG-nitro-L-argininemethylester (L-NAME, 1 µg/kg per min, i.v. or r.i.) significantly attenuated R(-)EFO-induced increase in MBF. R(-)EFO inhibited the AngII-mediated (50 ng/kg per min, i.v.) reduction of MBF (28.4 ± 1.7%), which was associated with increased urinary NO(2) + NO(3) excretion and decreased urinary hydrogen peroxide (H(2)O(2)) excretion. Intracellular H(2)O(2) fluorescence (real-time fluorescence imaging) in the epithelial cells of isolated medullary thick ascending limb (mTAL) significantly increased following AngII stimulation (1 µmol/L, 235 ± 52 units), which was significantly inhibited by pre and coincubation with R(-)EFO. R(-)EFO stimulation also increased the intracellular NO concentration in the epithelial cells of mTAL (220 ± 62 units). CONCLUSION: These results suggest that TCC blockade with R(-)EFO selectively increases MBF, an effect that appears to be mediated by changes in renal NO and oxidative stress balance, which may protect against ischemic renal injury in the renal medullary region.

Arginine decreases Cryptosporidium parvum infection in undernourished suckling mice involving nitric oxide synthase and arginase.

OBJECTIVE: This study investigated the role of L-arginine supplementation to undernourished and Cryptosporidium parvum-infected suckling mice. METHODS: The following regimens were initiated on the fourth day of life and injected subcutaneously daily. The C. parvum-infected controls received L-arginine (200 mmol/L) or phosphate buffered saline. The L-arginine-treated mice were grouped to receive NG-nitro-arginine methyl ester (L-NAME) (20 mmol/L) or phosphate buffered saline. The infected mice received orally 10(6) excysted C. parvum oocysts on day 6 and were euthanized on day 14 at the infection peak. RESULTS: L-arginine improved weight gain compared with the untreated infected controls. L-NAME profoundly impaired body weight gain compared with all other groups. Cryptosporidiosis was associated with ileal crypt hyperplasia, villus blunting, and inflammation. L-arginine improved mucosal histology after the infection. L-NAME abrogated these arginine-induced improvements. The infected control mice showed an intense arginase expression, which was even greater with L-NAME. L-arginine decreased the parasite burden, an effect that was reversed by L-NAME. Cryptosporidium parvum infection increased urine NO(3)(-)/NO(2)(-) concentrations compared with the uninfected controls, which was increased by L-arginine supplementation, an effect that was also reversed by L-NAME. CONCLUSION: These findings show a protective role of L-arginine during C. parvum infection in undernourished mice, with involvement of arginase I and nitric oxide synthase enzymatic actions.

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice.

We assessed the relative contribution of the mitochondrial respiratory chain and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to deoxycorticosterone acetate (DOCA)-salt hypertension in mice. The daily mean arterial pressure was monitored by radiotelemetry in DOCA-salt-treated mice given vehicle or the mitochondrial respiratory chain complex I inhibitor rotenone. This treatment produced remarkable attenuation of DOCA-salt hypertension. Similar results were obtained with other inhibitors of mitochondrial function, including 5-hydroxydecanoate (specific for mitochondrial potassium-ATP channels), benzylguanidine (complexes I and III), and the cell-permeable manganese tetrakis (4-benzoic acid) porphyrin (a mimic of mitochondrial superoxide dismutase). In parallel with the blood pressure-lowering effect of rotenone, the DOCA-salt-induced increases in urinary 8-isoprostane excretion and in reactive oxygen species production of isolated kidney mitochondria were both significantly attenuated. Conversely, the DOCA-salt-induced reduction of urinary nitrate/nitrite excretion was significantly elevated. Following DOCA-salt treatment, mice deficient in NADPH oxidase subunits gp91(phox) or p47(phox) exhibited a partial attenuation of the hypertensive response at early but not later time points. Thus, the mitochondrial respiratory chain is a major source of oxidative stress in DOCA-salt hypertension, whereas NADPH oxidase may have a relatively minor role during the early stage of hypertension.

The relationship of oxidative stress and cholesterol with dipping status before and after aerobic exercise training.

OBJECTIVE: The purpose of this study was to examine the effects of aerobic exercise training (AEXT) on dipping status in pre-hypertensive and stage-1 hypertensive individuals. A secondary purpose was to evaluate whether AEXT alters oxidative stress and endothelial biomarkers correlated to dipping status. METHODS: Twenty-three subjects underwent 24-h ambulatory blood pressure monitoring at baseline and after 6 months of AEXT. AEXT consisted of training at 70% VO(2max) 3 days/week for 6 months. Total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein (LDL)-cholesterol, oxidized LDL (ox-LDL), triglycerides, urinary and plasma nitric oxide end-products, superoxide dismutase and 8-iso-PGF(2alpha) were measured before and after AEXT. Statistically, ANOVA and linear regression were used. RESULTS: Before and after AEXT, there were no significant differences between dippers and non-dippers in any of the biomarkers except for total cholesterol following AEXT. In a sub-analysis following AEXT, 14 subjects retained their original dipping status, five subjects changed from dippers to non-dippers and four subjects changed from non-dippers to dippers. Significant differences existed between these groups in changes in total and LDL-cholesterol, ox-LDL, 8-iso-PGF(2alpha) and % Dip. CONCLUSIONS: Changes in cholesterol levels but not oxidative stress or endothelial biomarkers were related to changes in BP variables following AEXT in dippers and non-dippers.

Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia.

Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS), in the regulation of renal ATP-binding cassette (ABC) transporters in vivo during endotoxemia. Wistar-Hannover rats were injected with lipopolysaccharide (LPS(+)) alone or in combination with the iNOS inhibitor, aminoguanidine. Controls received detoxified LPS (LPS(-)). After LPS(+), proximal tubular damage and a reduction in renal function were observed. Furthermore, iNOS mRNA and protein, and the amount of NO metabolites in plasma and urine, increased compared to the LPS(-) group. Coadministration with aminoguanidine resulted in an attenuation of iNOS induction and reduction of renal damage. Gene expression of 20 ABC transporters was determined. After LPS(+), a clear up-regulation in Abca1, Abcb1/P-glycoprotein (P-gp), Abcb11/bile salt export pump (Bsep), and Abcc2/multidrug resistance protein (Mrp2) was found, whereas Abcc8 was down-regulated. Up-regulation of Abcc2/Mrp2 was accompanied by enhanced calcein excretion. Aminoguanidine attenuated the effects on transporter expression. Our data indicate that NO, produced locally by renal iNOS, regulates the expression of ABC transporters in vivo. Furthermore, we showed, for the first time, expression and subcellular localization of Abcb11/Bsep in rat kidney.

In vivo detection of aflatoxin-induced lipid free radicals in rat bile.

Aflatoxin B1 (AFB1), a potent hepatotoxin and hepatocarcinogen, is metabolized in the liver via cytochrome P-450 to an AFB1-8,9-epoxide intermediate. The formation of the AFB1-8,9-epoxide correlates with the pathological changes observed in numerous mammalian species. Oxidative damage has been postulated to play a major role in the mechanisms associated with AFB1-induced cytotoxicity and carcinogenecity in mammalian species. The aim of this study was to detect and identify free radical intermediates from the hepatic metabolism of AFB1 in vivo. Rat bile ducts were cannulated and rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone) (1 g/kg i.p.), and bile was collected over a period of 2 h at 20-min intervals. ESR spectroscopy was used to detect a carbon-centered radical adduct of 4-POBN in rat bile. The effect of metabolic inhibitors, such as deferoxamine mesylate (DFO), an iron chelator, and SKF 525A, a cytochrome P-450 inhibitor, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in free radical formation by pre-treatment with both DFO and SKF 525A. This indicates that oxidation of AFB1 generates free radical species via CYP metabolism and an iron-mediated redox mechanism.

Pharmacokinetics in renally impaired subjects of NXY-059, a nitrone-based, free-radical trapping agent developed for the treatment of acute stroke.

OBJECTIVE: NXY-059 is a nitrone-based, free-radical trapping agent being developed for the treatment of acute ischaemic stroke. Elimination of NXY-059 is primarily renal. The objective of the study was to investigate the pharmacokinetics of NXY-059 in subjects with renal impairment. METHODS: Twenty-four subjects with a glomerular filtration rate (GFR) ranging from 19 ml/min to 100 ml/min received NXY-059 intravenously over a 24-h period. Drug in plasma and urine was measured for 72 h. One-hour loading infusion rates were proportional to body weight, while maintenance infusion rates were proportional to GFR. Target plasma levels were 60 micro mol/l for subjects with mild (GFR 50-100 ml/min) and moderate (GFR 30-49 ml/min) renal impairment, and 30 micro mol/l for subjects with severe renal impairment (GFR <30 ml/min). GFR was measured as sinistrin clearance. RESULTS: The data indicated no tolerability or safety concerns with NXY-059. The half-life, which normally is approximately 2-4 h, was in the order of 10-12 h in subjects with moderate and severe renal impairment. The distribution parameters steady-state volume of distribution (V(ss)) and unbound fraction in plasma at 13-15 l and 0.53-0.58, respectively, were virtually the same as previously observed in healthy subjects. Plasma clearance of NXY-059, which ranged from 9 ml/min to 76 ml/min, was directly proportional to kidney function (GFR) with no discernible contribution by non-renal clearance. The correlation coefficient squared (r(2)) was 0.93, both when the renal function parameter was GFR and when it was creatinine clearance estimated from serum creatinine, age, weight and sex. CONCLUSION: Non-renal elimination of NXY-059 appeared to be insignificant even in subjects with low renal capacity. Patients with renal impairment should have their dose of NXY-059 adjusted for renal function, conveniently assessed from serum creatinine.

Acute methanol intoxication generates free radicals in rats: an ESR spin trapping investigation.

Electron spin resonance (ESR) spectroscopy has been used to investigate free radical generation in rats with acute methanol poisoning. The spin trapping technique was used where a spin trapping agent, alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), reacted with the corresponding alcohol-derived or alcohol-dependent radical to form radical adducts. One radical adduct was detected in both bile and urine samples 2 h after acute methanol poisoning in male Sprague Dawley rats. The hyperfine coupling constants for the radical adduct from [(13)C]-labeled methanol detected in the bile were a(N) = 15.58, a(beta)(H) = 2.81 G, and a(beta)(13C) = 4.53 G, which unambiguously identified this species as POBN/*CH@OH. The same radical adduct was detected in urine. The identification of a methanol-derived radical adduct in samples from bile and urine provided strong direct evidence for the generation of the alcohol-derived radicals during acute intoxication by methanol. Simultaneous administration of the alcohol dehydrogenase inhibitor 4-methylpyrazole and methanol resulted in an increase in the generation of the free radical metabolite detected in the bile. This is the first ESR evidence of methanol-derived free radical generation in an animal model of acute methanol intoxication.

Effect of renal function on serum nitrogen oxide concentrations.

Nitric oxide is too short-lived to measure in vivo, but its production can be estimated by measuring its stable oxidation products, nitrites and nitrates, in serum. Renal elimination of these ions has been demonstrated, but the effect of renal function on their concentrations in serum is currently unknown. We evaluated serum and urine nitrates + nitrites as serum nitrogen oxides (sNOx), nitrogen oxide (NOx) clearance, and creatinine clearance in 71 patients on the Intensive Therapy Unit. The correlation between sNOx and plasma creatinine was strong and highly significant (P <0.001). These results suggest that renal function has a significant effect on sNOx concentrations. Studies in which the sNOx concentration is used as an index of nitric oxide production can therefore be interpreted only if renal function has been taken into account.

L-arginine infusion induces hypotension and diuresis/natriuresis with concomitant increased urinary excretion of nitrite/nitrate and cyclic GMP in humans.

1. The vascular endothelium produces endothelium-derived relaxing factor (EDRF) or nitric oxide (NO), which exerts vasodilation through cyclic guanosine monophosphate (cGMP) as a second messenger. To determine whether EDRF has any vasodilating and natriuretic action in man, the present study examined the effects of L-arginine (L-Arg), a substrate for NO, on the responses of mean blood pressure (MBP) and heart rate (HR); plasma concentrations of cGMP, atrial natriuretic factor (ANF) and nitrite/nitrate (NOx); urinary excretion of sodium, cGMP and NOx; and urinary flow in eight normal male subjects. These parameters were compared with those following saline infusion in the same subjects. Clearance of para-aminohippuric acid (PAH) and inulin was studied in five normal subjects. 2. Infusion of L-Arg (30 g) caused a significant fall in MBP (-8 mmHg) with a concomitant rise in HR (10 beats/min), while saline infusion had no effects on these parameters. 3. Neither L-Arg nor saline infusion caused appreciable changes in plasma concentrations of ANF or NOx. Plasma cGMP concentrations increased significantly during (1.7-fold) and after (1.9-fold) L-Arg infusion, but only slightly (1.3-fold) during saline infusion. 4. Urine flow increased more remarkably following L-Arg infusion than that following saline infusion. Remarkable increases in urinary excretion of sodium and fractional excretion of sodium were observed after L-Arg infusion compared with those after saline infusion. Natriuresis was associated with enhanced urinary excretion of cGMP and Nox. Urinary Nox excretion showed positive correlations with urinary flow (r = 0.69, P less than 0.001) and with urinary cGMP excretion (r = 0.60, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)