Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency

BACKGROUND: Chronic renal failure (CRF) is associated with oxidative stress, the mechanism of which remains uncertain. Superoxide is the primary oxygen free radical produced in the body, NAD(P)H oxidase is the major source of superoxide production and superoxide dismutase (SOD) is responsible for removal of superoxide. We hypothesized that CRF-induced oxidative stress may be due to increased production and/or decreased dismutation of superoxide. METHODS: Immunodetectable superoxide dismutase isoforms (Cu Zn SOD and Mn SOD), as well as, NAD(P)H oxidase (gp91 phox subunit) proteins and xanthine oxidase (XO) activity were determined in the kidney and liver of CRF (5/6 nephrectomized) and sham-operated control rats. Subgroups of animals were treated with SOD-mimetic drug, tempol and blood pressure and urinary nitric oxide metabolites (NOx) were monitored. RESULTS: The CRF group showed marked down-regulations of CuZn SOD and Mn SOD and significant up-regulation of gp91 phox in the liver and kidney, which are among the metabolically most active tissues. In contrast, XO activity was depressed in both tissues. Arterial pressure and nitrotyrosine abundance were elevated while urinary NOx excretion was depressed, pointing to increased NO inactivation by superoxide and decreased NO availability in CRF animals. Administration of SOD-mimetic agent, tempol, for one week, ameliorated hypertension, reduced nitrotyrosine abundance and increased urinary NOx excretion in the CRF animals. CONCLUSIONS: CRF is associated with depressed SOD and elevated NAD(P)H oxidase expression, which can contribute to oxidative stress by increasing superoxide. This is evidenced by favorable response to administration of SOD-mimetic drug, tempol, and increased nitrotyrosine that is the footprint of NO interaction with superoxide.     

Superoxide dismutase,catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension

BACKGROUND: Earlier studies from this laboratory have revealed the presence of oxidative stress and its role in the pathogenesis of lead-induced hypertension (HTN). We have further shown evidence of increased hydroxyl radical (.OH) and superoxide production in lead-treated rats and cultured endothelial cells. This study was designed to determine whether oxidative stress in animals with lead-induced HTN is associated with dysregulation of the main antioxidant enzymes namely superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) or increased superoxide producing enzyme nicotinamide adenine dinucleotide (phosphate) oxidase [NAD(P)H]. METHODS: Male Sprague-Dawley rats were randomly assigned to lead-exposed and control groups. Animals in the lead-exposed group were provided with drinking water containing 100 ppm lead acetate for 12 weeks. The control group was provided with regular drinking water. At the conclusion of the experiment, immunodetectable Cu Zn SOD, Mn SOD, CAT, GPX and gp91 phox subunit of NAD(P)H oxidase were determined by Western analysis in the kidney, brain and left ventricle of control and lead-exposed rats. Subgroups of the study animals were treated with IV infusion (180 micromol/kg/h) of the superoxide trapping agent, tempol, and arterial pressure and urinary nitric oxide (NO) metabolite (NOx) excretion were determined. RESULTS: Lead exposure for 12 weeks resulted in a marked rise in systolic blood pressure, a significant reduction in urinary NOx excretion, a significant increase in kidney and brain Cu, Zn SOD, a significant increase in brain and insignificant increase in kidney and heart gp91 phox. In contrast, Mn SOD, CAT and GPX in the kidney, brain and left ventricle were unchanged. Incubation with lead acetate did not alter SOD activity in vitro. Infusion of tempol significantly lowered arterial pressure and raised urinary NOx excretion in the lead-exposed group (but had no effect in the control group) pointing to increased superoxide production in the lead-exposed animals. CONCLUSION: Animals with lead-induced hypertension exhibited oxidative stress which was associated with mild up-regulation of superoxide-generating enzyme, NAD(P)H oxidase, with no evidence of quantitative SOD, CAT or GPX deficiencies.     

Effects of diabetes,insulin and antioxidants on NO synthase abundance and NO interaction with reactive oxygen species

BACKGROUND: Earlier studies have provided evidence for increased production of reactive oxygen species (ROS) and altered nitric oxide (NO) metabolism in diabetes. This study was intended to explore the effect of type I diabetes and its treatment with insulin alone or insulin plus antioxidant-fortified diet on expression of NOS isoforms and ROS interactions with lipids, glucose and NO. METHODS: Rats with streptozotocin-induced diabetes were divided into once-daily insulin (ultralente)-treated, insulin plus antioxidant (vitamin E and vitamin C)-treated and untreated groups. After four weeks, plasma malondialdehyde (MDA) and tissue endothelial (eNOS), neuronal (nNOS) NO synthases, carboxymethyllysine (CML) and nitrotyrosine were determined. RESULTS: The untreated diabetic animals exhibited severe hyperglycemia, elevated blood pressure, increased plasma MDA, high tissue CML and reduced tissue nitrotyrosine denoting enhanced lipid, glucose and protein oxidation but reduced NO oxidation by ROS. This was coupled with significant reduction of eNOS and nNOS expression in renal cortex and eNOS in the left ventricle. Insulin therapy partially lowered blood pressure, tissue CML, plasma glucose and MDA, but significantly raised eNOS expression and nitrotyrosine abundance to supranormal levels. Combined insulin and antioxidant therapies resulted in normalization of blood pressure, plasma MDA, tissue CML and nitrotyrosine without affecting glucose level or NOS expression. CONCLUSION: Oxidative stress in untreated diabetes is associated with down-regulation of NOS isoforms and increased ROS-mediated oxidation of lipid and glucose, but not NO. Amelioration of hyperglycemia with once-daily insulin administration alone results in up-regulation of NOS isoforms, reduction of lipid and glucose oxidation and increased NO oxidation. However, insulin plus antioxidant supplementation can normalize all three parameters.     

Oxidative stress and antioxidant therapy: their impact in diabetes-associated erectile dysfunction

Oxidative stress is believed to affect the development of diabetic-associated vasculopathy, endothelial dysfunction, and neuropathy within erectile tissue. Our hypothesis is that, given adequate concentrations of the oxygen free radical scavenger vitamin E, enhanced levels of circulating nitric oxide (NO) should improve erectile function with the potential for a synergistic effect with a phosphodiesterase type 5 (PDE5) inhibitor. Twenty adult male Sprague-Dawley streptozotocin-induced (60 mg/kg intraperitoneally) diabetic rats were placed in 4 therapeutic groups (n = 5 per group) as follows: 1) peanut oil only (diabetic control), 2) 20 IU of vitamin E per day, 3) 5 mg/kg of sildenafil per day, and 4) vitamin E plus sildenafil using oral gavage for 3 weeks. In addition, 5 age-matched rats served as normal nondiabetic controls (normal). Erectile function was assessed by measuring the rise in intracavernous pressure (ICP) following cavernous nerve electrostimulation. Penile tissue was evaluated for neuronal NO synthase (nNOS), smooth muscle alpha-actin, nitrotyrosine, and endothelial cell integrity. Urine nitrite and nitrate (NOx) concentration was quantified, and electrolytes were tested by a serum biochemistry panel. A significant decrease in ICP was recorded in the diabetic animals, with improvement measured in the animals receiving PDE5 inhibitors either with or without vitamin E; the controls had a pressure of 54.8 +/- 5.3 cm H2O, the vitamin E group had a pressure of 73.5 +/- 6.6 cm H2O, the sildenafil group had a pressure of 78.4 +/- 10.77 cm H2O, and the vitamin E plus sildenafil group had a pressure of 87.9 +/- 5.5 cm H2O (P <.05), compared with the normal cohorts at 103.0 +/- 4.8 cm H2O. Histoexaminations showed improved nNOS, endothelial cell, and smooth muscle cell staining in the vitamin E plus sildenafil group compared to the control animals. Urine NOx increased significantly in all the diabetic groups but was blunted in the vitamin E and vitamin E plus sildenafil groups. A significant increase in positive staining for nitrotyrosine was observed in the vitamin E plus sildenafil group. Vitamin E enhanced the therapeutic effect of the PDE5 inhibitor in this study, supporting the potential use of oxygen free radical scavengers in salvaging erectile function in diabetic patients.     

Enhanced nitric oxide inactivation in aortic coarctation-induced hypertension

BACKGROUND: Abdominal aortic coarctation above the renal arteries leads to severe hypertension (HTN) above the stenotic site. We have recently shown marked up-regulations of endothelial nitric oxide synthase (eNOS) in heart and thoracic aorta and of neuronal NOS (nNOS) in the brain of rats with severe aortic coarctation above the renal arteries. We hypothesize that the presence of severe regional HTN in the face of marked up-regulation of NO system may be partly due to enhanced NO inactivation by reactive oxygen species (ROS) leading to functional NO deficiency. METHODS: Tissue nitrotyrosine (which is the footprint of NO interaction with ROS) was determined by Western blot in sham-operated control and aortic-banded (above renal arteries) rats three weeks postoperatively. Intra-arterial pressure and tissue nitrotyrosine (Western blot) were measured. RESULTS: The banded group showed a marked rise in arterial pressure measured directly through a carotid cannula (203 +/- 9 vs. 131 +/- 2 mm Hg, P < 0.01). Compared with the sham-operated controls, the banded animals exhibited significant increases in nitrotyrosine abundance in the heart, brain, and the aorta segment above the stricture. In contrast, nitrotyrosine abundance was unchanged in the abdominal aorta segment below the stricture wherein blood pressure was not elevated. CONCLUSION: Coarctation-induced HTN is associated with increased nitrotyrosine abundance in all tissues exposed to high arterial pressure, denoting enhanced ROS-mediated inactivation and sequestration of NO in these sites. This can, in part, account for severe regional HTN in this model. The normality of nitrotyrosine abundance in the abdominal aorta wherein blood pressure is not elevated points to the role of baromechanical factors as opposed to circulating humoral factors that were necessarily similar in both segments.     

Effects of nitric oxide synthesis inhibition on FK506-induced nephrotoxicity in rats

This study was designed to evaluate the role of nitric oxide (NO) in FK506-induced nephrotoxicity by administering an inhibitor of NO synthesis, N omega-nitro-L-arginine methyl ester (L-NAME) to rats treated with FK506. After one week of treatment with FK506 (3.2 mg/kg/day, intramuscularly) and/or L-NAME (5 mg/100 mL of L-NAME in the drinking water), the arterial pressure, urinary NOx, and parameters for renal function were measured, and histological analysis of the kidney was made. In the L-NAME without FK506 group, L-NAME administration effectively inhibited urinary NOx excretion and increased mean arterial pressure (MAP) without any change in renal function. In the FK506 without L-NAME group, FK506 treatment showed increase in urinary NOx excretion and mild renal dysfunction. In the FK506 with L-NAME group, urinary NOx excretion was decreased by L-NAME administration and renal function was significantly worsened than FK506 without L-NAME group. The plasma creatinine, BUN and urinary N-acetyl-beta-D-glucosaminidase increased 2-, 3-, and 3-fold, respectively and the creatinine clearance was reduced by 50% as compared with that in the FK506 without L-NAME group. Histological analysis revealed severe interstitial fibrosis and tubular atrophy in the FK506 + L-NAME treatment group. Thus, results suggest that NO synthesis is enhanced in the kidney during FK506-induced nephrotoxicity and that NO synthesis inhibition aggravates FK506-induced nephrotoxicity. NO may play a protective role attributable to the balance of vasoactive substances in FK506-induced nephrotoxicity.     

Effect of hereditary obesity on renal expressions of NO synthase, caveolin-1, AKt, guanylate cyclase, and calmodulin

BACKGROUND: Obesity has emerged as a major cause of diabetes, cardiovascular disease, and renal insufficiency worldwide. Obese Zucker rats exhibit hyperphagia, obesity, insulin resistance, hyperlipidemia, and glomerulosclerosis and are frequently used as a model to study hereditary form of metabolic syndrome. Nitric oxide plays a major role in preservation of renal function and structure. The present study was designed to test the hypothesis that renal disease in this model may be associated with down-regulation of endothelial (eNOS) and neuromal NO synthases (nNOS) in the kidney. The study further sought to explore expressions of caveolin-1, phospho AKt, and calmodulin, which regulate activities of constituitive NOS isoforms, as well as soluble guanylate cyclase (sGC), which is involved in NO signaling. METHODS: Twenty-two-week-old male obese and lean Zucker rats were studied. Body weight, serum lipids, urine albumin excretion, and renal tissue abundance of the above proteins were determined. RESULTS: Serum glucose and arterial pressure were unchanged, whereas urinary NO metabolite (NO(chi)) excretion and renal tissue nitrotyrosine abundance were markedly reduced (denoting depressed NO production) in the obese versus lean Zucker rats. This was accompanied by significant glomerulosclerosis, tubulointerstitial damage, renal immune cell infiltration, marked down-regulations of renal tissue eNOS and nNOS, mild reduction of caveolin-1, and unchanged calmodulin, phospho-AKt, and sGC. CONCLUSION: Hereditary obesity can result in down-regulations of kidney eNOS and nNOS, marked reduction of NO production, and glomerulosclerosis prior to the onset of frank diabetes and hypertension.     

EFFECTS OF NITRIC OXIDE SYNTHESIS INHIBITION ON FK506-INDUCED NEPHROTOXICITY IN RATS

This study was designed to evaluate the role of nitric oxide (NO) in FK506-induced nephrotoxicity by administering an inhibitor of NO synthesis, Nω-nitro-L-arginine methyl ester (L-NAME) to rats treated with FK506. After one week of treatment with FK506 (3.2 mg/kg/day, intramuscularly) and/or L-NAME (5 mg/100 mL of L-NAME in the drinking water), the arterial pressure, urinary NOx, and parameters for renal function were measured, and histological analysis of the kidney was made. In the L-NAME without FK506 group, L-NAME administration effectively inhibited urinary NOx excretion and increased mean arterial pressure (MAP) without any change in renal function. In the FK506 without L-NAME group, FK506 treatment showed increase in urinary NOx excretion and mild renal dysfunction. In the FK506 with L-NAME group, urinary NOx excretion was decreased by L-NAME administration and renal function was significantly worsened than FK506 without L-NAME group. The plasma creatinine, BUN and urinary N-acetyl-β-D-glucosaminidase increased 2-, 3-, and 3-fold, respectively and the creatinine clearance was reduced by 50% as compared with that in the FK506 without L-NAME group. Histological analysis revealed severe interstitial fibrosis and tubular atrophy in the FK506 + L-NAME treatment group. Thus, results suggest that NO synthesis is enhanced in the kidney during FK506-induced nephrotoxicity and that NO synthesis inhibition aggravates FK506-induced nephrotoxicity. NO may play a protective role attributable to the balance of vasoactive substances in FK506-induced nephrotoxicity.     

Kidney immune cell infiltration and oxidative stress contribute to prenatally programmed hypertension

BACKGROUND: Prenatal environment has been shown to modify adult blood pressure profile, but the underlying mechanisms are not well understood. The role of renal immune cell infiltration, oxidative stress, and nitric oxide bioavailability in the pathogenesis was investigated. METHODS: Adult hypertension in rat offspring was induced by maternal low protein diet. Oxidative stress was determined by quantitative immunoblotting for nitrotyrosine, and T-cell and macrophage content by immunostaining, in offspring kidneys before and after the onset of hypertension. Nitric oxide metabolites (NOx) were measured in 24-hour urines. A group of offspring was treated with the immunosuppressive drug mycophenolate mofetil (MMF) to reduce inflammation, or with the superoxide dismutase mimetic Tempol to reduce oxidative stress, for a 3-week period before the onset of hypertension. RESULTS: During the prehypertensive stage, at 4 weeks of age, the low protein diet pups exhibited an increase in kidney nitrotyrosine content and in number of immune cells, both of which persisted in untreated animals after hypertension was established, at 8 weeks of age. Urine NOx was increased at 4 weeks and unchanged at 8 weeks of age. Both MMF and Tempol treatment prevented the immune cell infiltration, the increase in kidney nitrotyrosine abundance, and the development of hypertension. The effect on blood pressure persisted throughout the 4- to 10-week observation period after discontinuation of the treatments. CONCLUSION: Renal oxidative stress and infiltrating immune cells may play a pathogenetic role in prenatally programmed hypertension. Nitric oxide bioavailability does not appear impaired.     

Modulation of urinary kallikrein and plasma renin activities does not affect established hypertension in the fawn-hooded rat

Fawn-hooded (FH) rats develop low-renin hypertension which is preceded by a decrease in urinary kallikrein. We examined urinary excretion of active and inactive kallikrein in hypertensive FH male rats and matched animals of the ancestral, normotensive Wistar strain. To determine the effects of modulation of salt intake on the kallikrein profile, rats were given standard rat chow (0.39% NaCl), a low-salt diet (0.02% NaCl), or a high-salt diet (standard chow plus water with 1% NaCl). Control FH rats excreted less active kallikrein (p less than 0.02), had similar amounts of inactive kallikrein, and had a higher inactive/active kallikrein ratio (p less than 0.02) than control Wistar rats. Low salt intake increased active kallikrein 136% (p less than 0.002) and 54% (p less than 0.035) in FH and Wistar rats, respectively, but did not change the level of inactive kallikrein or the inactive/active kallikrein ratio. High salt intake had no effect on kallikrein excretion in either strain. Low salt intake did not change blood pressure in either strain in spite of significant changes in plasma renin activity, angiotensin II and active kallikrein excretion. The low urinary active kallikrein and the high inactive/active kallikrein ratio in FH rats do not appear to play a role in the established hypertension in the FH rat, since modulation of these parameters did not cause a significant change in the elevated blood pressure.     

Nitric oxide production is more prominent in off-pump than in on-pump coronary artery bypass surgery

The aim of our study was to elucidate the extent to which cardiopulmonary bypass contributes to endogenous nitric oxide (NO) production in patients undergoing coronary artery bypass grafts (CABG). One-hundred-and-sixteen patients undergoing elective CABG with (on-pump, n=66) and without cardiopulmonary bypass (off-pump, n=50) were included. Urinary nitrite/nitrate (NOx) excretion was measured as an index of endogenous NO production during the first two postoperative days. Haemodynamic profiles, serum CK-MB and C-reactive protein (CRP) concentrations were measured after the operation. There was no significant difference in urinary NOx/creatinine (Cr) excretion on day one post CABG. The mean urinary NOx/Cr excretion ratio significantly (P < 0.01) decreased from days one to two in the on-pump group, but not in the off-pump group. The mean urinary NOx/Cr excretion ratio was significantly (P < 0.01) higher in the off-pump group (0.51 +/- 0.26 micromol/mg) than in the on-pump group (0.38 +/- 0.20 micromol/mg) on day two. The mean serum CRP concentration was also significantly (P < 0.01) higher in the off-pump group than in the on-pump group on day two. There was no significant difference in the mean cardiac index or the mean systemic vascular resistance index between the two groups after surgery. The mean serum CK-MB concentration was significantly (P < 0.05) lower in the off-pump group than in the on-pump group on days one and two. These findings suggest that endogenous NO production is stimulated by a surgical inflammatory response and that the cardiopulmonary bypass procedure per se is not the inciting stimulus for NO production in patients undergoing CABG.     

Effects of angiotensin-converting enzyme inhibitor and α1-adrenoceptor antagonist on hypertension induced in rats by long-term inhibition of nitric oxide production

Background. We aimed to elucidate the involvement of the renin-angiotensin system (RAS) and sympathetic nervous system in hypertension induced by the long-term inhibition of nitric oxide (NO) production. Methods. We compared the effects of 9-week treatment with an angiotensin-converting enzyme inhibitor (ACEI), cilazapril (10 mg/kg per day), to that with an α1-adrenoceptor antagonist, doxazosin (10 mg/kg per day), on systemic blood pressure and renal histological changes in Sprague-Dawley rats continuously treated with oral N^G-nitro-L-arginine (L-NA). Results. L-NA induced renal damage associated with a significant fall in urinary nitrate and nitrite (NOx) excretion and a significant rise in systolic blood pressure. Although cilazapril and doxazosin restored urinary NOx to a similar level, only cilazapril treatment significantly suppressed the hypertensive effect of L-NA. Urinary protein excretion in L-NA-treated rats was also significantly reduced by cilazapril treatment. Histologically, treatment with cilazapril, but not doxazosin, significantly inhibited the glomerular injury of mesangial expansion and glomerular sclerosis induced by L-NA treatment. Furthermore, cilazapril significantly reduced urinary aldosterone level. Conclusion. Our findings indicate that the hypertension and hypertensive glomerular injury induced by long-term L-NA treatment were abrogated by an ACEI but not by an α1-adrenoceptor antagonist, and that the fall in high blood pressure induced by treatment with the ACEI was independent of urinary NOx excretion in this model. Received: August 17, 1999 / Accepted: December 8, 1999     

Association of renal injury with nitric oxide deficiency in aged SHR: prevention by hypertension control with AT1 blockade

BACKGROUND: Aged spontaneously hypertensive rats (SHR) develop end-stage renal disease resembling that of uncontrolled essential hypertension in humans. Nitric oxide (NO) and angiotensin II (Ang II) play an important role in the regulation of blood pressure and the growth of vascular smooth muscle and renal mesangial cells. The relationship between renal NO system, Ang II activity and renal injury in aged SHR is not fully understood. METHODS: The 8-week-old SHR were randomized into losartan-treated (30 mg/kg/day for 55 weeks) and vehicle treated groups. The age-matched Wistar-Kyoto rats (WKY) served as controls. Renal histology and tissue expressions of endothelial and inducible NO synthases (eNOS and iNOS) and nitrotyrosine were examined at 63-weeks of age. RESULTS: Compared to the WKY group, untreated SHR showed severe hypertension, proteinuria, renal insufficiency, a twofold decrease in renal tissue eNOS and iNOS expressions and massive nitrotyrosine accumulation. This was associated with severe glomerulosclerosis, tubular atrophy and interstitial fibrosis. Losartan therapy normalized blood pressure, prevented proteinuria and renal insufficiency, abrogated the fall in renal eNOS and iNOS protein contents, mitigated renal nitrotyrosine accumulation, and prevented the histological abnormalities found in the untreated SHR. CONCLUSIONS: Aged SHR exhibit severe renal lesions with acquired NO deficiency that are prevented by hypertension control with AT1 blockade. These findings point to the possible role of NO deficiency in the pathogenesis of renal lesions in aged SHR.     

Prevention of renal injury and endothelial dysfunction by chronic L-arginine and antioxidant treatment

We evaluated the effects of vitamins with antioxidant properties (a combination of vitamins C and E) and L-arginine treatment on renal failure in mice by measuring survival rate. The molecular changes were elucidated by determining endothelial tetrahydrobiopterin (BH4) levels and nitric oxide synthase (eNOS) mRNA expression in mice with renal ablation. Previous studies have shown that endothelial dysfunction in 5/6 nephrectomized mice is associated with decreased nitric oxide (NO) bioavailability and increased vascular superoxide production. WTC57 mice were divided into three groups: Group 1 was the sham-operated group (C); Group 2 was the 5/6 nephrectomized group (Nfx); and Group 3 was a group of 5/6 nephrectomized mice, treated with L-arginine and vitamins with antioxidant properties (NfxTx; 200 mg/kg L-arginine, 83 mg/kg vitamin C, and 46.6 mg/kg vitamin E). After 20 weeks of treatment, urinary protein excretion, blood pressure, BH4 and dihydrobiopterin (BH2) levels, eNOS mRNA, oxidative stress, and survival rate were determined. An increase in urinary protein excretion, blood pressure, and oxidative stress was prevented in the NfxTx group, but not in the Nfx group. BH4 and eNOS mRNA expression was increased by 32% and 78%, respectively, in the NfxTx group. Furthermore, the treatment increased the survival rate by 33%. Our results indicate that under normal conditions, NO appears to protect renal function. However, this NO-dependent protection is lost during kidney failure, probably due to increased reactive oxygen species synthesis. The treatment restores the viability of NO and prevents the BH4 oxidation. Therefore, this treatment may represent a therapeutic approach for the management of kidney disease.     

Roles of nitric oxide and oxidative stress in the regulation of blood pressure and renal function in prehypertensive Ren-2 transgenic rats

AIMS: The present study was performed to evaluate the role of nitric oxide (NO) and its interaction with superoxide anion (O2-) in the regulation of blood pressure (BP) and renal function during the developmental phase of hypertension in Ren-2 transgenic rats (TGR). The first aim was to compare BP and renal functional responses to acute NO synthase (NOS) inhibition achieved by intravenous (i.v.) infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) in prehypertensive heterozygous TGR and in transgene-negative Hannover Sprague-Dawley (HanSD) rats. The second aim was to evaluate whether scavenging of O2- by infusion of the superoxide dismutase mimetic tempol increases NO bioavailability which therefore should augment BP and renal functional responses to L-NAME. Methods: Rats were anesthetized, prepared for clearance experiments and BP and renal functional responses were evaluated in response to i.v. L-NAME administration (20 microg.100 g(-1).min(-1)) without or with tempol pretreatment (i.v., 300 microg.100 g(-1).min(-1)). In renal cortical tissue, nitrotyrosine protein expression was assessed by immunoblotting as marker of O2- production and urinary 8-epi-PGF(2alpha) excretion as marker of intrarenal oxidative stress was assessed by enzyme immunoassay. Results: BP, glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion were similar in TGR and HanSD. L-NAME infusion induced greater increases in BP in TGR than in HanSD (+42 +/- 4 vs. +25 +/- 3 mmHg, p < 0.05). In the absence of a significant change in GFR, L-NAME caused similar decreases in RPF (-32 +/- 6 and -25 +/- 4%, p < 0.05) in TGR and HanSD. Despite significantly higher renocortical expression of nitrotyrosine and urinary 8-epi-PGF2alpha excretion in TGR than in HanSD, pretreatment with tempol did not augment the rise in BP and the decrease in RPF induced by L-NAME. CONCLUSIONS: The greater BP response to L-NAME in TGR suggests that prehypertensive TGR exhibit an enhanced NO activity in the systemic vasculature as compared with HanSD. Despite increased intrarenal oxidative stress in TGR, the dependency of the intrarenal vascular tone on NO appears to be similar in TGR and HanSD. The lack of a compensatory increase in renal NO activity may partially account for the enhanced renal vascular response to ANG II present in TGR.     

The role of nitric oxide in deoxycorticosterone acetate-salt hypertensive rats]

The relationship between nitric oxide (NO) and blood pressure (BP) in deoxycorticosterone acetate-salt hypertensive rats (DOC) was investigated. Although urinary NO2- + NO3- (NOx) excretion (UNOxV) increased 2 weeks after surgery (2W-DOC), UNOxV decreased 4 weeks after surgery (4W-DOC) compared with that of the control. BP and UNOxV did not change in DOC after treatment with L-arginine (Arg-DOC). Aorta from 4W-DOC and Arg-DOC had significantly decreased relaxation responses to acetylcholine. Deendothelialized aorta from 4W-DOC and Arg-DOC had significantly decreased relaxation responses to lipopolysaccharide. These data suggest that: 1) transient increase of NO synthesis is accompanied by elevation of BP, but long-term elevation of BP decreases NO synthesis in endothelium and smooth muscle cells; 2) L-arginine supplement has no effect on the development of hypertension nor on NO production by endothelium and smooth muscle cells in DOC.     

Increased nitric oxide synthase-3 expression in kidneys of deoxycorticosterone acetate-salt hypertensive rats.

In addition to its hemodynamic effects, nitric oxide (NO) may play a role in the renal tubular handling of sodium. Experiments were conducted to determine possible changes in renal nitric oxide synthase-3 (NOS3) expression in rats treated with deoxycorticosterone acetate (DOCA) and high salt. All rats were uninephrectomized, and either a placebo or DOCA pellet was implanted subcutaneously. Placebo-treated rats were then given tap water to drink ad libitum, and DOCA-treated rats received a 0.9% NaCl solution to drink. Once a week, rats were placed in metabolic cages so that a 24-h urine sample could be collected. After 3 wk, the animals were sacrificed and the kidneys removed and prepared for subsequent immunohistochemical or Western blot analysis. Urinary excretion of nitrate and nitrite (NOx) was measured to provide an indication of the intrarenal production of NO. DOCA-salt hypertensive rats exhibited increased urinary NOx excretion (2.43 +/- 0.48 micromol NOx/mg creatinine) compared with the placebo control animals (1.17 +/- 0.06 micromol NOx/mg creatinine). Western blot analysis revealed that NOS3 protein levels in both the cortex and medulla were greater in DOCA-salt rats compared with placebo-treated animals. Immunohistochemical analysis of kidneys revealed that NOS3 expression in placebo rats was localized in vascular endothelial cells with slight, but detectable, immunoreactivity in medullary collecting ducts. In DOCA-salt rats, a very large increase in the intensity of immunostaining was detected in tubular epithelia of the proximal tubule, thick ascending limb of Henle's loop, and cortical and medullary collecting duct; immunoreactivity in endothelial cells appeared unchanged. These data suggest that increased tubular expression of NOS3 is responsible, at least in part, for the increased renal production of NO in DOCA-salt hypertension, and are consistent with a role for NO in the renal tubular response to salt loading.     

Increased tyrosine nitration of the brain in chronic renal insufficiency: reversal by antioxidant therapy and angiotensin-converting enzyme inhibition

Interaction of reactive oxygen species with nitric oxide promotes nitric oxide inactivation and generation of cytotoxic reactive nitrogen species that attack DNA, lipids, and proteins. Nitration of free tyrosine and tyrosine residues of proteins results in production of nitrotyrosine, which can lead to excitotoxicity and frequently is found in the brain of patients and animals with various degenerative, ischemic, toxic, and other neurologic disorders. According to earlier studies, reactive oxygen species activity is increased and neuronal NO synthase expression in the brain is elevated in animals with chronic renal failure (CRF). It was hypothesized, therefore, that tyrosine nitration must be increased in the uremic brain. This hypothesis was tested, through determination of nitrotyrosine abundance (by Western blot analysis), as well as distribution (by immunohistology), in the cerebrum of rats with CRF 6 wk after 5/6 nephrectomy. The results were compared with those of sham-operated controls and antioxidant (lazaroid)-treated and captopril-treated rats with CRF. Western blot analysis revealed a significant increase in nitrotyrosine abundance in the cerebral cortex of rats with CRF. This was accompanied by an intense nitrotyrosine staining of the neuronal processes, including proximal segments of dendrites, axons, and axon terminals of the cortical neurons. Both antioxidant therapy and captopril administration alleviated oxidative stress (as evidenced by normalization of plasma lipid peroxidation product malondialdehyde) and significantly reduced nitrotyrosine abundance in the cerebral cortex of the treated CRF group. In conclusion, CRF resulted in oxidative stress and increased tyrosine nitration in the cerebral cortex. Antioxidant therapy and angiotensin-converting enzyme inhibition alleviated the CRF-induced oxidative stress and mitigated tyrosine nitration in the rats with CRF.     

Vitamin E therapy prevents hyperoxaluria-induced calcium oxalate crystal deposition in the kidney by improving renal tissue antioxidant status

OBJECTIVE: To determine whether vitamin E prevents hyperoxaluria-induced stone formation, using a new animal model of calcium oxalate stone disease, as our previous in- vitro and in-vivo studies showed that oxalate and hyperoxaluria induce free-radical generation, which results in peroxidative injury to renal tubular cells. MATERIALS AND METHODS: Ethylene glycol (EG) was administered at 150 mg/day by gavage for 3 weeks to rats fed on diets with adequate (group 1), excess (group 2) or deficient (group 3) vitamin E. Several indicators of peroxidation, free radicals and enzymatic activity were then assessed. RESULTS: EG treatment in group 1 lead to increased lipid peroxidation, protein thiol, excretion of urinary enzymes, oxalate and decreases in urinary calcium, antioxidant enzymes and altered glutathione redox balance. Although renal function was not altered, there was increased water intake, urine volume and lowered urinary pH in these rats. These changes were more intense, with extensive calcium-oxalate crystal deposition, in rats in group 3, and prevented in rats in group 2, except for urinary oxalate levels, which remained high. Histopathological examination showed that there was no deposition of calcium oxalate crystals in rats in group 2. CONCLUSION: This is the first study to demonstrate in-vivo evidence that hyperoxaluria-induced peroxidative injury induces individual calcium oxalate crystal attachment in the renal tubules. In addition, excess vitamin E completely prevented calcium oxalate deposition, by preventing peroxidative injury and restoring renal tissue antioxidants and glutathione redox balance. Therefore, vitamin E therapy might provide protection against the deposition of calcium oxalate stones in the kidney of humans.     

Remnant liver injury after hepatectomy with the pringle maneuver and its inhibition by an iNOS inhibitor (ONO-1714) in a pig model

BACKGROUND: Although hepatectomy is often performed with the Pringle maneuver, the problem of remnant liver injury is not fully solved. We examined the remnant liver injury of hepatectomy under the Pringle maneuver and its relation to inducible nitric oxide synthase (iNOS) in a pig hepatectomy model. MATERIALS AND METHODS: Pigs were subjected to a total of eight Pringle maneuvers followed by re-perfusion. The pigs were divided into the following three groups: Control group; only Pringle maneuver, liver resection (LR) group; hepatectomy under the Pringle maneuver, and ONO group; and hepatectomy under the Pringle maneuver with an iNOS inhibitor (ONO-1714). We investigated the changes in serum aminotransferase (AST), lactate dehydrogenase (LDH), NO(2)(-)+NO(3)(-) (NOx), the hepatic tissue blood flow (HTBF), the cellular distribution of endothelial and inducible nitric oxide synthase, nitrotyrosine, infiltration of neutrophils, and thrombocyte-thrombi by immunohistochemistry. RESULTS: The serum AST, LDH, NOx levels in the LR group were significantly higher than those in the Control group. The formation of iNOS, nitrotyrosine, thrombocyte-thrombi, and infiltration of neutrophils were recognized in the LR group. These findings were inhibited in the ONO group. CONCLUSIONS: These results indicate that remnant liver injury appeared after hepatectomy with the Pringle maneuver. iNOS was involved in these injuries and the iNOS inhibitor attenuated the injury.