Urinary Nitric Oxide Excretion Research Articles

LPIN1 is a new target gene for essential hypertension.

We previously showed Lipin1 (LPIN1) to be a candidate gene for essential hypertension by genome-wide association studies. LPIN1 encodes the Lipin 1 protein, which contributes to the maintenance of lipid metabolism and glucose homeostasis. However, little is known about the association between LPIN1 and blood pressure (BP). We evaluated the BP of LPIN1-deficient [fatty liver dystrophy (fld)] mice and explored related mechanisms. Fld mice have very low expression of LPIN1 and exhibit fatty liver, hypertriglyceridemia, insulin resistance and peripheral neuropathy. Fld mice had significantly elevated SBP and heart rate (HR) throughout the day as measured by a radiotelemetric method. Diurnal variation of SBP and HR was also absent in fld mice. Furthermore, urinary excretion of adrenaline and noradrenaline by fld mice was significantly higher compared with that of control mice. The BP response of fld mice to clonidine (a centrally acting α2-adrenergic receptor agonist) was greater than that of control mice. However, levels of Angiotensinogen and Renin 1 mRNA and urinary nitric oxide excretion were comparable between the two groups. The decrease in SBP at 8 weeks after fat grafting surgery was significantly greater in the transplant group compared with the sham operated group. The elevated BP in fld mice may result from activation of the sympathetic nervous system through decreased levels of adipose cytokines. These results indicate that LPIN1 plays a crucial role in blood pressure regulation and that LPIN1 is a new target gene for essential hypertension.

Pitavastatin Upregulates Nitric Oxide Synthases in the Kidney of Spontaneously Hypertensive Rats and Wistar–Kyoto Rats

Clinical trials show potent renoprotective effects of pitavastatin (PTV), although the precise mechanism for these renoprotective effects is not fully clarified. The aim of this study was to examine the antihypertensive and renoprotective effects of PTV, focusing on the nitric oxide (NO) system. Male, 6-week-old, spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were randomized to receive vehicle or PTV (2 mg/kg bodyweight) for 8 weeks. Blood pressure and urinary albumin excretion were measured every 2 weeks. After 8 weeks, plasma biochemical parameters and renal histology were examined. NO synthase isoform (neuronal, nNOS; inducible, iNOS; and endothelial, eNOS) expression and eNOS phosphorylation were examined by western blotting. PTV attenuated hypertension and albuminuria development in SHR. PTV decreased glomerular desmin expression and medullary interstitial fibrosis in SHR. PTV tended to increase plasma NO in both strains but significantly increased urinary NO excretion only in WKY. PTV significantly increased nNOS and eNOS expression, enhanced eNOS phosphorylation at serine1177, and inhibited eNOS phosphorylation at threonine495 in the kidney of both strains. PTV treatment led to increased renal NOS expression and upregulated eNOS activity in both SHR and WKY. The antihypertensive and renoprotective effects of PTV may be related to upregulation of the NO system.

Experimental Study of the Effects of Three Types of Meat on Endothelial Function in a Group of Healthy Volunteers

Background: There is a relationship between atherosclerotic risk factors and increased vascular production of reactive oxygen species (ROS). Oxidized LDL and ROS may directly cause endothelial dysfunction by reducing endothelial nitric oxide (NO) bioavailability. The semi-essential amino acid L-arginine is the only substrate for NO synthesis in vascular endothelial cells. Therefore, this amino acid improves endothelial function and plays a role in the prevention and/or treatment of multiple cardiovascular diseases: atherosclerosis, hypertension, diabetes and so on. To determine the effects of three different protein matrices (250 g Fillet of Beef, FB; Chicken Raised on the Ground, CRG; Free-Range Chicken, FRC) with a known content of arginine on the cardiovascular workload, vascular compliance and urinary excretion of some parameters of endothelial function as TGF–Beta, NO (nitrate e nitrite) in a group of healthy volunteers. Materials and methods: We enrolled 10 men to study the behavior of Systolic, Diastolic, Mean, and Pulse Blood Pressure, of Vascular Resistances, of Macro and Micro Vascular Elasticity, of urinary excretion of TGF-β and Nitric Oxide as ratio of creatinine before and after two hours of each meal. The cardiovascular parameters are determined by HDI/Pulse Wave CR 2000 (Hypertension Diagnostic Inc, Eagan, MN); TGF-β is analysed by Elisa method (R&D Systems) and NO by colorimetric method (Cayman). Results and Conclusion: The protein meal packed with CRG causes a significant decrease in diastolic blood pressure mean pressure and vascular resistance in urinary excretion of TGF. FB resulted in a significant decrease in vascular resistance and urinary excretion of NO, while significantly increasing the Pulse Pressure, heart rate and urinary excretion of TGF-β. FRC resulted in a significant reduction of macrovascular elasticity; increase the urinary excretion of TGF and Pulse Pressure. We can conclude that CRG meat looks better both in terms of metabolic and cardiovascular load especially at endothelial level.

Poor Glycemic Control Is Related to Increased Nitric Oxide Activity Within the Renal Circulation of Patients With Type 2 Diabetes

OBJECTIVEExperimental studies have shown that glucose releases endothelial nitric oxide (NO) and that NO contributes to renal hyperperfusion in models of diabetes. To examine whether this translates into the human condition, we studied the relationship between glycemic control and renal NO activity in patients with type 2 diabetes.RESEARCH DESIGN AND METHODSA total of 113 patients with type 2 diabetes and a wide range of HbA1c concentrations were included. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were determined by constant infusion input clearance. Functional NO activity in the renal circulation was determined as change of RPF to infusion of the NO synthase (NOS) inhibitor N(G)-monomethyl-l-arginine (l-NMMA) (4.25 mg/kg). As additional markers, we measured urinary excretion of NO (UNOx) and l-arginine–to–asymmetrical dimethylarginine (ADMA) ratio in plasma.RESULTSSubjects within the highest tertile of HbA1c concentration had increased RPF (low, medium, and high tertiles 576 ± 17 vs. 585 ± 22 vs. 627 ± 33 mL/min/m2, P = 0.05 by one-way ANOVA), while GFR was similar across tertiles. The response of RPF to NOS blockade was augmented in subjects with higher HbA1c levels (−55 ± 7 vs. −64 ± 8 vs. −86 ± 8 mL/min, P = 0.04 by one-way ANOVA). Further, l-arginine–to–ADMA ratio and UNOx were increased in subjects with higher HbA1c levels.CONCLUSIONSIn line with experimental evidence, we could demonstrate in humans that poor glycemic control is related to higher NO activity and hyperperfusion of the kidney. The renal NO system may thus be a novel therapeutic target for improving renal hemodynamics in patients with diabetes.

Role of the eNOS-NO System in Regulating the Anti-Proteinuric Effects of VEGF Receptor 2 Inhibition in Diabetes

Since the original description over a decade ago of the upregulation of the VEGF/VEGFR-2 system in the diabetic kidney, there has been intense interest in the pathogenetic role of this system in renal decline and its amenability to therapeutic interventions. Recently, it has become apparent that subtle perturbations in intraglomerular VEGF/VEGFR-2 signalling or in the influencing microenvironment can profoundly affect renal function, resulting in the apparently paradoxical observation that VEGF blockade attenuates proteinuria development in experimental diabetes despite exerting the opposite effect under other circumstances. In the present study, we sought to explore the role of eNOS-NO activity in regulating the differential response to VEGF blockade in the diabetic and non-diabetic settings. In a rodent model of accelerated renal injury, the transgenic (mRen-2)27 (Ren-2) rat, VEGFR-2 inhibition with the small molecule vandetanib resulted in an increase in urine protein excretion preceding a subsequent rise in systolic blood pressure. When compared to their normoglycemic counterparts, diabetic Ren-2 rats exhibited an increase in the renal expression of eNOS and in urinary excretion of nitric oxide (NO) metabolites. In contrast to the heavy proteinuria observed with vandetanib in non-diabetic TGR(mRen-2)27 rats, VEGFR-2 inhibition reduced urine protein excretion in diabetic animals, despite a comparable magnitude of histological injury. However, proteinuria was markedly increased by concomitant treatment of diabetic Ren-2 rats with vandetanib and the nitric oxide synthase inhibitor L-NAME. These observations highlight the pivotal role that the eNOS-NO system plays in regulating the biological response to VEGF within the glomerulus especially under diabetic conditions.

Role of the eNOS-NO System in Regulating the Antiproteinuric Effects of VEGF Receptor 2 Inhibition in Diabetes

Subtle perturbations in intraglomerular VEGF/VEGFR-2 signaling or in the influencing microenvironment can profoundly affect renal function, resulting in the apparently paradoxical observation that VEGF blockade attenuates proteinuria development in experimental diabetes despite exerting the opposite effect under other circumstances. In the present study, we sought to explore the role of eNOS-NO activity in regulating the differential response to VEGF blockade in the diabetic and nondiabetic settings. In a rodent model of accelerated renal injury, the transgenic (mRen-2)27 (Ren-2) rat, VEGFR-2 inhibition with the small molecule vandetanib resulted in an increase in urine protein excretion preceding a subsequent rise in systolic blood pressure. When compared to their normoglycaemic counterparts, diabetic Ren-2 rats exhibited an increase in the renal expression of eNOS and in urinary excretion of nitric oxide (NO) metabolites. In contrast to the heavy proteinuria observed with vandetanib in nondiabetic TGR(mRen-2)27 rats, VEGFR-2 inhibition reduced urine protein excretion in diabetic animals, despite a comparable magnitude of histological injury. However, proteinuria was markedly increased by concomitant treatment of diabetic Ren-2 rats with vandetanib and the nitric oxide synthase inhibitor L-NAME. These observations highlight the pivotal role that the eNOS-NO system plays in regulating the biologic response to VEGF within the glomerulus.

Altered regulation of renal nitric oxide and atrial natriuretic peptide systems in angiotensin II-induced hypertension

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO) and atrial natriuretic peptide (ANP) systems in the kidney in association with the angiotensin (Ang) II-induced hypertension. Male Sprague–Dawley rats were used. Ang II (100 ng·min − 1 ·kg − 1 ) was infused through entire time course. Thirteenth day after beginning the regimen, kidneys were taken. The protein expression of NO synthase (NOS) and nitrotyrosine was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed hypertension and decreased creatinine clearance in the experimental group. The protein expression of eNOS, nNOS and nitrotyrosine was increased in the cortex, while that of iNOS remained unaltered. The urinary excretion of NO increased in Ang II-induced hypertensive rats. The catalytic activity of soluble guanylyl cyclase was blunted in the glomerulus in Ang II-induced hypertensive rats. The mRNA expression of ANP was increased in Ang II-induced hypertensive rats. Neither the expression of NPR-A nor that of NPR-C was changed. The protein expression of neutral endopeptidase was decreased and the activity of particulate guanylyl cyclase was blunted in the glomerulus and papilla in Ang II-induced hypertensive rats. In conclusion, the synthesis of NO and ANP was increased in the kidney of Ang II-induced hypertension, while stimulated cGMP response was blunted. These results suggest desensitization of guanylyl cyclase in the kidney of Ang II-induced hypertensive rats, which may contribute to the associated renal vasoconstriction and hypertension.

Urinary nitric oxide excretion in infants with eczema

Eczema is characterized by inflammation of the skin and is commonly associated with food allergy. It has been suggested that nitric oxide (NO) is an important player in eczema, food allergy and intestinal inflammation. The aim of this study was to assess the levels of urinary NO breakdown products in infants with eczema and the effect of eczema treatment on NO levels. Ninety-four infants with eczema, 58 boys and 36 girls, with a mean age of 7.5 ± 5.2 months (mean ± s.d.) at inclusion were examined twice with an interval of 6 wk. The sum of nitrite and nitrate was measured colorimetrically in urinary samples from both visits and compared with clinical data concerning eczema severity, nutrition, gastrointestinal symptoms, asthma and skin prick positivity. The levels of NO products increased significantly from the first to the second visit: 289; 374 μm (median; IQR) vs. 457; 678 μm (median; IQR) (p < 0.001) in parallel with a significant improvement of the eczema. After eczema treatment consisting of skin care and elimination diet during the 6-wk interval between evaluations, the NO levels approached the values previously found in healthy children. The results support previous studies indicating that the homeostasis of nitrogen radicals is disturbed in childhood eczema.

Altered regulation of renal nitric oxide, atrial natriuretic peptide and cyclooxygenase systems in aldosterone escape in rats

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO), atrial natriuretic peptide (ANP) and cyclooxygenase (COX) systems in the kidney in association with the aldosterone escape. Male Sprague–Dawley rats were used. Aldosterone (200 μg/day) was infused through entire time course. The control group was kept on a low sodium diet (0.02 mEq/day), and the experimental group was supplied with a higher sodium diet (2.0 mEq/day). Four days after beginning the regimen, the kidneys were taken. The protein expression of NO synthase (NOS) and COX isoforms was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed aldosterone escape in the experimental group. Accordingly, the renal content and the urinary excretion of NO increased. The expression of nNOS was increased in the inner medulla. Neither the expression of eNOS nor that of iNOS was changed. The expression and the catalytic activity of soluble guanylyl cyclase remained unaltered. The mRNA expression of ANP was increased. Neither the expression of NPR-A or NPR-C nor the activity of particulate guanylyl cyclase was altered in the papilla. The protein expression of COX-2 was increased in the inner medulla, while that of COX-1 remained unchanged. In conclusion, the upregulation of nNOS, ANP, and COX-2 may be causally related with the aldosterone escape.

Effect of Losartan Therapy on Endothelial Function in Hypertensive Patients

The aim of the study was to evaluate the effect of losartan therapy on endothelial function by measuring serum nitric oxide (NO) levels and urinary excretion of NO in patients with essential hypertension. A group of 30 untreated stage 2 hypertensive patients (15 males and 15 females; age, 51.3 +/- 1.5 years) were included in the study. Office systolic and diastolic blood pressure (BP) was measured by using a mercury sphygmomanometer according to phase I and V of Korotkoff sounds. NO levels in serum and 24-hour urine were determined at baseline and after 6 weeks of daily dosing with losartan (50-100 mg). Losartan therapy resulted in a significant fall in systolic/diastolic BP (from 169.7 +/- 4.1/105 +/- 1.8 mm Hg at baseline to 146 +/- 2.7/91 +/- 1.9 mm Hg at the end of losartan treatment; P < 0.001). The therapy also caused significant increases in both serum NO level (32.74 +/- 3.01 microM/L at baseline versus 79.04 +/- 5.17 microM/L; P < 0.001 after therapy) and urinary NO excretion (58.21 +/- 3.72 microM/L at baseline versus 113.21 +/- 8.63 microM/L; P < 0.001 after therapy). Losartan therapy also reduced serum malondialdehyde (MDA), which is a measure of oxidative stress, by 0.201 nM (15.3%; P = 0.009). Losartan at a dose of 50 to 100 mg per day was effective in reducing elevated BP. The increase in serum NO levels and urinary NO excretion and a decrease in serum MDA levels by losartan treatment indicate a reduction in oxidative stress and enhances NO availability, both of which improve endothelial function. Thus, losartan therapy improves endothelial function in hypertensive patients with essential hypertension.

Effect of Losartan Plus Hydrochlorothiazide on Nitric Oxide Status in ‘Nondipper’ Hypertensive Patients

The objective of this study was to investigate the effects of losartan (100 mg) plus hydrochlorothiazide (HCTZ; 25 mg) on nitric oxide (NO) production and blood pressure (BP) in "nondipper" severe hypertensive patients. Twelve hypertensive "nondipper patients" (6 of each gender) with sitting systolic/diastolic BP of 188.0 +/- 5.2/116.2 +/- 1.2 mm Hg were studied by 24-hour ambulatory blood pressure monitoring (ABPM) after daily administration of 100 mg losartan plus 25 mg HCTZ for a period of 12 weeks. Office and mean 24-hour, as well as mean awake- and sleep-time systolic/diastolic BP, serum NO levels, and urinary excretion of NO were measured after the placebo period (3 weeks) and after 12 weeks of therapy. At the end of the 12-week treatment period, the mean 24-hour systolic/diastolic BP decreased significantly from 158.6 +/- 4.7/102.2 +/- 2.6 mm Hg (placebo period) to 140.3 +/- 4.8/90.9 +/- 3.3 mm Hg (P = 0.001/< or = 0.002). The mean BP (systolic/diastolic) during the waking period was reduced from 159.3 +/- 4.4/103.0 +/- 2.5 mm Hg to 135.0 +/- 4.4/88.2 +/- 3.1 (P < or = 0.007/P < or = 0.002), whereas the mean BP (systolic/diastolic) during the sleeping hours changed from 154.9 +/- 5.3/98.9 +/- 3.1 to 140.9 +/- 4.6 (P = 0.035)/91.7 +/- 3.2 mm Hg (P = 0.035/P = 0.051). Serum NO levels increased from 40.89 +/- 5.69 microM/L (placebo period) to 67.35 +/- 6.96 microM/L (posttreatment; P < or = 0.007), whereas the 24-hour urinary NO excretion did not change significantly (69.71 +/- 3.68 microM/L [placebo period] vs 79.64 +/- 4.25 microM/L [posttreatment]; P < or = 0.16). Urinary clearance of NO also did not change. Serum NO levels increased significantly without a significant change in urinary NO excretion. BP was significantly reduced but without modifying the nondipper pattern in these patients.

Nitric Oxide and Malondialdehyde in Human Hypertension

Nitric oxide (NO), a multifunctional effector molecule that plays a central role in the maintenance of vascular homeostasis, regulates vascular tone and inhibits platelet and leukocyte adhesion to endothelial cells. NO status is related to the endothelial function. Patients with hypertension have lower levels of NO, increased free radical production, higher oxidative stress, augmented platelet aggregation, and a change in the arachidonic acid cascade metabolism, all leading to the acceleration of the atherosclerotic process. The study subjects included a group of 21 normotensive healthy subjects (8 males and 13 females) with a mean age of 39.2 +/- 1.8 years and a body mass index of 27.9 kg/m, and another group of 42 patients (19 males and 23 females) with untreated essential hypertension with a mean age of 47.6 +/- 1.7 years and a body mass index of 28.3 kg/m. Serum levels and urinary excretion of NO determined as combined nitrate/nitrite (NOx) and serum malondialdehyde (MDA) concentrations were measured in the 2 groups of subjects. The serum levels and 24-hour urinary excretion of NOx were significantly higher and the renal clearance of NO was lower in the normotensive group than in the hypertensive patients, indicating decreased NO status in hypertension. There was a negative correlation between serum NO levels and mean arterial pressure, suggesting that a decrease in NO availability is related to increase in blood pressure. Serum concentrations of MDA were higher in the hypertensive patients as compared with the normotensive individuals, suggesting increased oxidative stress in hypertensive patients. These results are in agreement with previous studies showing decreased NO and increased oxidative stress in hypertension. In conclusion, patients with essential hypertension as compared with normotensive individuals have lower NO status, which may contribute to the endothelial dysfunction in hypertension. Increased serum malondialdehyde in hypertensives suggests an association between increased oxidative stress with higher blood pressure.

Effects of Losartan + l-Arginine on Nitric Oxide Production, Endothelial Cell Function, and Hemodynamic Variables in Patients With Heart Failure Secondary to Coronary Heart Disease

The purpose of the present study was to evaluate the effects of losartan and the combination of losartan and L-arginine on endothelial function and hemodynamic variables in patients with heart failure (HF). Endothelium-dependent vasodilation is impaired in patients with HF. It was hypothesized that the administration of losartan and the combination of losartan and L-arginine might increase nitric oxide production and have a beneficial additive effect on endothelial function and hemodynamic variables in patients with HF. Nine patients with HF (ejection fraction<35%) were given losartan 50 mg orally on 2 consecutive days. On the second day, 1 hour after losartan 50 mg administration, L-arginine 20 g was given by intravenous infusion. Endothelial function in the form of endothelium-dependent brachial artery flow-mediated vasodilation (FMV) was measured by ultrasound. Hemodynamic variables were estimated using Doppler echocardiography at baseline and at 2 and 4 hours after losartan alone and after combination therapy. Urinary levels of nitrite (NO2) or nitrate (NO3) were measured. Four hours after losartan administration, significant reductions in systemic vascular resistance and estimated end-systolic elastase were observed. On the second day, 1 hour after L-arginine infusion, an additive hemodynamic effect was observed, with significant increases in the cardiac index and stroke volume and significant reductions in systemic vascular resistance and calculated left ventricular end-diastolic pressure. A trend toward improved FMV was observed with losartan alone, but without statistical significance. Combination therapy significantly improved postintervention FMV compared with baseline. The increase in urinary nitric oxide excretion after losartan treatment and combination therapy was significantly correlated with improved hemodynamic variables and improved FMV. In conclusion, losartan induces significant afterload reduction, reduced contractility, and increased nitric oxide urinary excretion. The combination of L-arginine and losartan seems to have superior effects on hemodynamic variables and endothelium-dependent vasodilation compared with losartan alone.

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). l-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p < 0.05) and a ∼3 fold greater Ca 2+-dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53–7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.

Pioglitazone Lowers Systemic Asymmetric Dimethylarginine by Inducing Dimethylarginine Dimethylaminohydrolase in Rats

Peroxisome proliferator activated receptor-gamma (PPARgamma) ligands increase nitric oxide (NO) production and reduce systemic blood pressure. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which has two isoforms, DDAH-I and -II. In order to elucidate the mechanism whereby PPARgamma ligands affect NO metabolism, their effects on the DDAH-ADMA pathway were investigated. Six-week-old male Wister-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were maintained with or without pioglitazone (PIO), a PPARgamma ligand. After 4 weeks, serum ADMA levels and urinary daily NO excretion were analyzed. Tissue DDAH expression was examined by real-time polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry. The results showed that PIO decreased serum ADMA and increased urinary NO excretion in both WKY and SHR. Also in both strains, the expression level of DDAH-II in the kidney was increased at transcriptional levels, although the DDAH-I level was unaffected. PIO lowered blood pressure in SHR, but not in WKY. We also demonstrated that PIO induced DDAH-II protein expression in Marbin-Dubin Canine Kidney (MDCK) cells, a renal tubular cell line. In conclusion, a PPARgamma ligand was here found to increase NO production partly by upregulating tissue DDAH-II expression and decreasing systemic ADMA levels. This mechanism constitutes a direct action on renal tubular cells, but is less likely to be responsible for the blood pressure-lowering effects of PPARgamma ligands. Since ADMA is one of the risk factors for cardiovascular events, this study provides compelling evidence that PPARgamma ligands have the potential for reducing cardiovascular risks.

Macrophages act as effectors of tissue damage in acute renal allograft rejection.

Macrophages constitute 38% to 60% of infiltrating cells during acute renal allograft rejection. Their contribution to tissue damage during acute rejection was examined by depleting macrophages in a rat model. Lewis rats underwent bilateral nephrectomy and then received a Dark Agouti renal allograft and liposomal-clodronate, control phosphate-buffered saline liposomes, or saline intravenously (n=7 per group) on days 1 and 3 postsurgery. Grafts were harvested on day 5. Liposomal-clodronate treatment resulted in a 70% reduction in blood ED1+ monocytes and 60% reduction in intragraft ED1+ macrophages (both P<0.01). Half of all remaining interstitial ED1+ cells were undergoing apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling+/ED1+), and thus functional depletion of more than 75% of macrophages was achieved. Histologic and functional parameters of acute rejection were attenuated: interstitial infiltrate, tubulitis, and glomerulitis (P<0.01); tubular cell apoptosis (P<0.001); tubular cell proliferation (P<0.001); and serum creatinine (P<0.01). Production of inducible nitric oxide synthase by infiltrating cells and urinary nitric oxide excretion was reduced by 90% (P<0.001). In contrast, no reduction in the number of other leukocytes was seen (CD3+, CD4+, CD8+, and natural killer cells). Activation of lymphocytes (CD25+) and production of lymphocyte effector molecules (granzyme B) were unaltered. This study demonstrates that macrophages contribute to tissue damage during acute rejection.

Urinary nitric oxide levels are increased and correlated with plasma concentrations in patients with Behçet's disease: is it a new urinary activity marker?

Nitric oxide (NO) is a free radical and serves many functions within the kidney. Excess NO causes glomerular injury. Behçet's disease (BD) is a systemic immunoinflammatory vasculitis, affecting every organ in the body including the kidneys (subclinic glomerulonephritis). We investigated the role of urinary total nitrite levels (end product of NO) in BD and evaluated whether urinary concentrations were correlated with its plasma levels or disease activity. Thirty-six consecutive Behçet's patients (19 men, 17 women; 35.9 years), and 20 age- and sex-matched healthy control volunteers (12 men, eight women; 33.2 years) were divided into an active (n = 16) and inactive (n = 20) period. Urinary and serum NO levels ( micromol/mg urinary creatinine) were higher in BD patients (4.1 +/- 0.3) than control subjects (1.7 +/- 0.2; P < 0.001). Serum NO levels in Behçet's patients and control subjects were 51.3 +/- 9.8 and 21.7 +/- 7.3 micromol/L, respectively (P < 0.001). Active patients had higher urinary NO excretion (4.9 +/- 0.3) than inactive patients (3.3 +/- 0.3; P < 0.01). Urinary NO levels were correlated with its serum levels (r2 = 0.69, P < 0.001). Higher urinary NO levels found in BD may be produced by the kidney as a result of an inflammatory stimulation. As excess NO is toxic to the tissues, increased NO levels may play a role in mediating subclinic glomerular injury of such patients. However, we could not determine the exact site(s) of NO synthesis by the kidney, such as the glomeruli, blood vessels and/or the tubular cells. Whatever the source, urinary NO levels may be used as a new activity marker in the diagnosis and follow up of BD by serial measurements.

The influence of nitric oxide on basal and cholecystokinin-8-induced proliferation and apoptosis in the rat pancreas

Nitric oxide (NO) is formed by different cell types in the pancreas. In this study, inhibition of endogenous nitric oxide by N(omega)-nitro-L-arginine (L-NNA) reduced the urinary excretion of NO(2)/NO(3) and raised serum L-arginine and the NO donator S-nitroso-N-acetylpenicillamine (SNAP) increased the urinary excretion of NO(2)/NO(3). The peptide cholecystokinin-8 (CCK-8) has a strong influence on exocrine pancreatic proliferation. Rat pancreas was excised and studied with regard to tissue weight, protein and DNA contents after 3 days of treatment with saline, L-NNA or SNAP given separately or combined with CCK-8. Further, proliferation of different pancreatic cells was studied with [3H]-thymidine incorporation and apoptotic activity was studied by analysing caspase-3 activity and histone-associated DNA fragments. The effects of L-NNA indicate that endogenous nitric oxide formation has a tonic inhibition on apoptosis in the pancreas during both basal condition and growth stimulation by CCK-8. In CCK-induced hyperplasia, NO inhibits the proliferation of acinar cells but stimulates ductal cells. Endogenous NO may regulate the balance between proliferation and apoptosis and in a situation of growth stimulation by CCK-8, it has a tonic inhibition on both mitogenesis and apoptosis thus slowing down the acinar cell turnover in the pancreas.

Role of Nitric Oxide in the Early Renal Changes Induced by High Fructose Diet in Rats

Background: Early glomerular hypertrophy and late glomerulosclerosis have been observed in rats fed high fructose diet (HFD), comparable with those of diabetic rats. Several studies suggest a role for nitric oxide (NO) in the pathogenesis of renal damage in diabetes. This study investigated the possible role of NO in the pathogenesis of HFD-induced glomerular changes. Methods: Three study protocols were adopted. In the first, 20 rats were divided into two groups to evaluate the effect of HFD on glomerular size and on the urinary excretion of NO. In the second, the glomerular size was evaluated in 40 rats divided into four groups receiving: (1) standard diet (SD); (2) HFD; (3) HFD + L-NAME, and (4) SD + L-NAME for 1 month. In the third, the renal expression of inducible enzyme (iNOS) was compared in 10 rats on HFD and in 10 controls after a 1-month diet. Results: The results showed: (1) increased urinary excretion of NO and glomerular size, both induced by HFD; (2) prevention by L-NAME of the HFD-increased glomerular size, and (3) increased iNOS expression in the kidneys of rats fed HFD. Conclusion: These results suggest a role for NO in the pathogenesis of the early renal changes induced by HFD.

Inhibition of nitric oxide synthase does not influence urinary nitrite plus nitrate excretion after renal ischemic injury.

Whether renal nitric oxide production caused by ischemia/reperfusion (I/R) influences the urinary excretion of nitric oxide (NO) metabolites (nitrite and nitrate) is far from being elucidated. In the present study, we evaluated the role of NO synthase inhibition using N(G)-nitro- L-arginine methyl ester ( L-NAME) in a model of experimental renal I/R injury. Male Wistar rats were used in our experiments, and renal I/R injury was achieved after a 30-min occlusion of the bilateral renal artery followed by a 60-min period of reperfusion. Renal function including nitrite plus nitrate excretion and hemodynamics in reperfused kidneys were measured in the presence and absence of L-NAME. Intravenous application of L-NAME (5 mg/kg body weight) resulted in a marked reduction of urine flow, renal plasma flow (0.7 +/- 0.3 ml/min), and the glomerular filtration rate (0.1 +/- 0.01 ml/min), but a significant increase in NO excretion (FENOx, 67.9 +/- 10.5%). In addition we found after L-NAME injection a significant increase of the fractional excretion of sodium (FENa, 49.3 +/- 7.7%) and lithium (FELi, 70.2 +/- 1.6%), as well as the renal vascular resistance compared with animals with renal I/R but non-treated with L-NAME ( P<0.001). Furthermore, we observed a high correlation between FENOx and FELi (r(2)=0.80, P<0.01). Our results suggest that renal excretion of NO derivatives is not influenced by NO production during renal I/R injury, although NO contributes to the tubular transport capacity in the ischemia/reperfused kidney.