3-nitrotyrosine and Proteinuria

Chronic renal fibrosis is the final common pathway of end stage renal disease caused by glomerular or tubular pathologies. Genetic background has a strong influence on the progression of chronic renal fibrosis. We recently found that Rowett black hooded rats were resistant to renal fibrosis. We aimed to investigate the role of sustained inflammation and oxidative/nitrative stress in renal fibrosis progression using this new model. Our previous data suggested the involvement of podocytes, thus we investigated renal fibrosis initiated by doxorubicin-induced (5 mg/kg) podocyte damage. Doxorubicin induced progressive glomerular sclerosis followed by increasing proteinuria and reduced bodyweight gain in fibrosis-sensitive, Charles Dawley rats during an 8-week long observation period. In comparison, the fibrosis-resistant, Rowett black hooded rats had longer survival, milder proteinuria and reduced tubular damage as assessed by neutrophil gelatinase-associated lipocalin (NGAL) excretion, reduced loss of the slit diaphragm protein, nephrin, less glomerulosclerosis, tubulointerstitial fibrosis and matrix deposition assessed by periodic acid-Schiff, Picro-Sirius-red staining and fibronectin immunostaining. Less fibrosis was associated with reduced profibrotic transforming growth factor-beta, (TGF-β1) connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL-1a1) mRNA levels. Milder inflammation demonstrated by histology was confirmed by less monocyte chemotactic protein 1 (MCP-1) mRNA. As a consequence of less inflammation, less oxidative and nitrative stress was obvious by less neutrophil cytosolic factor 1 (p47phox) and NADPH oxidase-2 (p91phox) mRNA. Reduced oxidative enzyme expression was accompanied by less lipid peroxidation as demonstrated by 4-hydroxynonenal (HNE) and less protein nitrosylation demonstrated by nitrotyrosine (NT) immunohistochemistry and quantified by Western blot. Our results demonstrate that mediators of fibrosis, inflammation and oxidative/nitrative stress were suppressed in doxorubicin nephropathy in fibrosis-resistant Rowett black hooded rats underlying the importance of these pathomechanisms in the progression of renal fibrosis initiated by glomerular podocyte damage.

Topics: Aldehydes; Animals; Body Weight; Chemokine CCL2; Connective Tissue Growth Factor; Disease Progression; Disease Resistance; Dose-Response Relationship, Drug; Doxorubicin; Fibrosis; Kidney; Male; Membrane Proteins; Oxidative Stress; Proteinuria; Rats; Reactive Nitrogen Species; Species Specificity; Transforming Growth Factor beta1; Tyrosine

Chronic kidney disease (CKD) patients have a poor prognosis due to cardiovascular disease. Anemia and endothelial dysfunction are important risk factors for cardiovascular events in CKD patients, and treatment with erythropoiesis-stimulating agent (ESA) has been reported to improve the quality of life in CKD patients. In this study, we evaluated the effect of anemia correcting dose of epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.) on endothelial function in 5/6 nephrectomized rats (Nx rats). C.E.R.A. was subcutaneously administered once a fortnight, 5 times in total, from 1 week after nephrectomy. Twenty-four hours after last administration, endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anesthetized Nx rats by ultrasound system. Femoral arteries were harvested for western blot analysis. C.E.R.A. significantly increased FMD of Nx rats. Endothelium-independent vasodilation induced by nitroglycerin injection was not influenced by C.E.R.A treatment. Nox4 expression and nitrotyrosine accumulation were significantly decreased, and phosphorylation of eNOS was significantly enhanced in the femoral arteries of C.E.R.A.-treated rats. C.E.R.A. normalized hemoglobin levels but did not affect body weight, systolic blood pressure, heart rate, urinary protein excretion and plasma creatinine. These results indicate that C.E.R.A. prevented endothelial dysfunction in Nx rats, possibly through reduction of local oxidative stress and enhancement of eNOS phosphorylation in the arteries. This study provides the first evidence that C.E.R.A. prevented endothelial dysfunction in CKD model rats under conditions of amelioration of anemia.

Topics: Animals; Blood Pressure; Body Weight; Creatinine; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Femoral Artery; Heart Rate; Hemoglobins; Kidney Function Tests; Male; NADPH Oxidase 4; NADPH Oxidases; Nephrectomy; Nitric Oxide Synthase Type III; Nitroglycerin; Polyethylene Glycols; Proteinuria; Rats; Renal Insufficiency, Chronic; Tyrosine; Ultrasonography; Vasodilation

Metabolic diseases such as hyperlipidemia and diabetes attenuate the cardioprotective effect of ischemic preconditioning. In the present study, we examined whether another metabolic disease, prolonged uremia, affects ischemia/reperfusion injury and cardioprotection by ischemic preconditioning. Uremia was induced by partial nephrectomy in male Wistar rats. The development of uremia was verified 29 wk after surgery. Transthoracic echocardiography was performed to monitor cardiac function. At week 30, hearts of nephrectomized and sham-operated rats were isolated and subjected to a 30-min coronary occlusion followed by 120 min reperfusion with or without preceding preconditioning induced by three intermittent cycles of brief ischemia and reperfusion. In nephrectomized rats, plasma uric acid, carbamide, and creatinine as well as urine protein levels were increased as compared with sham-operated controls. Systolic anterior and septal wall thicknesses were increased in nephrectomized rats, suggesting the development of a minimal cardiac hypertrophy. Ejection fraction was decreased and isovolumic relaxation time was shortened in nephrectomized rats demonstrating a mild systolic and diastolic dysfunction. Infarct size was not affected significantly by nephrectomy itself. Ischemic preconditioning significantly decreased infarct size from 24.8 ± 5.2% to 6.6 ± 1.3% in the sham-operated group and also in the uremic group from 35.4 ± 9.5% to 11.9 ± 3.1% of the area at risk. Plasma ANG II and nitrotyrosine were significantly increased in the uremic rats. We conclude that although prolonged experimental uremia leads to severe metabolic changes and the development of a mild myocardial dysfunction, the cardioprotective effect of ischemic preconditioning is still preserved.

Topics: Angiotensin II; Animals; Biomarkers; Creatinine; Disease Models, Animal; Ischemic Preconditioning, Myocardial; Male; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nephrectomy; Proteinuria; Rats; Rats, Wistar; Stroke Volume; Time Factors; Tyrosine; Ultrasonography; Urea; Uremia; Uric Acid; Ventricular Function, Left

Renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system activation are crucial in the pathogenesis of hypertension, cardiovascular and renal disease. NADPH oxidase-mediated increases in reactive oxygen species (ROS) are an important mediator for RAAS-induced cardiovascular and renal injury. Increased levels of ROS can diminish the bioactivity of nitric oxide (NO), a critical modulator of RAAS effects on the kidney. Thereby, we hypothesized that in vivo nebivolol therapy in a rodent model of activated RAAS would attenuate glomerular damage and proteinuria through its actions to reduce NADPH oxidase activity/ROS and increase bioavailable NO.. We utilized the transgenic Ren2 rat which displays heightened tissue RAAS, hypertension, and proteinuria. Ren2 rats (6-9 weeks of age) and age-matched Sprague-Dawley littermates were treated with nebivolol 10 mg/kg/day (osmotic mini-pump) for 21 days.. Ren2 rats exhibited increases in systolic blood pressure, proteinuria, kidney cortical tissue total NADPH oxidase activity and subunits (Rac1, p67(phox), and p47(phox)), ROS and 3-nitrotyrosine, as well as reductions in podocyte protein markers; each of these parameters improved with nebivolol treatment along with increases in renal endothelial NO synthase expression.. Our data suggest that nebivolol improves proteinuria through reductions in renal RAAS-mediated increases in NADPH oxidase/ROS and increases in bioavailable NO.

Topics: Animals; Antihypertensive Agents; Benzopyrans; Blood Pressure; Disease Models, Animal; Ethanolamines; Hypertension, Renal; NADPH Oxidases; Nebivolol; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Reactive Oxygen Species; Renin; Renin-Angiotensin System; Tyrosine

We evaluated whether the blockade of the proinflammatory transcription factor NF-kappaB would modify the oxidative stress, inflammation, and structural and hemodynamic alterations found in the kidney as a result of massive proteinuria. Twenty male Sprague-Dawley rats were injected with 2 g of BSA intraperitoneally daily for 2 wk. Ten of them received in addition the inhibitor of NF-kappaB activation pyrrolidine dithiocarbamate (PDTC; 200 mg.kg(-1).day(-1) sc) and the rest received vehicle. Seven rats that received intraperitoneal saline were used as controls. Glomerular hemodynamics were studied after 14 days. Markers of oxidative stress (NF-kappaB subunit p65+ cells, 3-nitrotyrosine, and 4-hydroxynonenal), inflammation (cortical CD68+ cells and NOS-II), and afferent arteriole damage were assessed by immunohistochemistry and morphometry. Activity of antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase was evaluated in renal cortex and medulla. Albumin overload induced massive proteinuria, oxidative stress with reduced activity of antioxidant enzymes, NF-kappaB activation, inflammatory cell infiltration, a significant presence of proteinaceous casts, systemic and glomerular hypertension, as well as arteriolar remodeling. Treatment with PDTC prevented or improved all of these findings. In this model of nephrotic syndrome, we demonstrate a key role for oxidative stress and inflammation in causing systemic and glomerular hypertension and proteinuria. Oxidative stress and inflammation may have a key role in accelerating renal injury associated with intense proteinuria.

Topics: Aldehydes; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood Pressure; Glomerular Filtration Rate; Glutathione Reductase; Hypertension, Renal; Kidney; Kidney Glomerulus; Male; Nitric Oxide Synthase Type II; Organ Size; Oxidative Stress; Peroxidases; Proteinuria; Pyrrolidines; Rats; Rats, Sprague-Dawley; Renal Plasma Flow; Superoxide Dismutase; Thiocarbamates; Transcription Factor RelA; Tyrosine

Oxidative stress has been postulated to be involved in the development of diabetic nephropathy. In the present study, we evaluated the effect of taurine, an endogenous antioxidant, on diabetic nephropathy by mixing it with the daily drinking water (1%w/v) of streptozotocin-induced diabetic rats from the beginning of the fourth month after the induction of diabetes, during which the urinary protein excretion in untreated diabetic rats showed significant increase in comparison with nondiabetic rats. The taurine administration significantly suppressed further increase in urinary protein excretion in diabetic rats, accompanied by the reduction of mesangial extracellular matrix expansion and TGF-beta expression in the renal glomerulus. Immunohistochemical study showed that taurine administration suppressed the intensified stainings to the three different types of oxidative stress markers, such as 8-hydroxyl-2'-deoxyguanosine (8-OHdG), pentosidine, and nitrotyrosine observed in the renal tissues of untreated diabetic rats. These findings suggest that taurine has the ability to suppress the progression of diabetic nephropathy at least in part by its antioxidant property. Since this beneficial effect of taurine was obtained even if its administration was started after the time point when urinary protein excretion already became apparently higher than that of age-matched nondiabetic animals, taurine administration was potentially expected to be applied in clinical field to retard the development of nephropathy in diagnosed diabetic patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biomarkers; Blood Glucose; Body Weight; Deoxyguanosine; Diabetic Nephropathies; Disease Progression; Glycated Hemoglobin; Immunohistochemistry; Male; Oxidative Stress; Proteinuria; Rats; Rats, Sprague-Dawley; Taurine; Tyrosine

The progressive deterioration of renal function and structure resulting from renal mass reduction are mediated by a variety of mechanisms, including oxidative stress and inflammation. Melatonin, the major product of the pineal gland, has potent_antioxidant and anti-inflammatory properties, and its production is impaired in chronic renal failure. We therefore investigated if melatonin treatment would modify the course of chronic renal failure in the remnant kidney model. We studied rats followed 12 wk after renal ablation untreated (Nx group, n = 7) and treated with melatonin administered in the drinking water (10 mg/100 ml) (Nx + MEL group, n = 8). Sham-operated rats (n = 10) were used as controls. Melatonin administration increased 13-15 times the endogenous hormone levels. Rats in the Nx + MEL group had reduced oxidative stress (malondialdehyde levels in plasma and in the remnant kidney as well as nitrotyrosine renal abundance) and renal inflammation (p65 nuclear factor-kappaB-positive renal interstitial cells and infiltration of lymphocytes and macrophages). Collagen, alpha-smooth muscle actin, and transforming growth factor-beta renal abundance were all increased in the remnant kidney of the untreated rats and were reduced significantly by melatonin treatment. Deterioration of renal function (plasma creatinine and proteinuria) and structure (glomerulosclerosis and tubulointerstitial damage) resulting from renal ablation were ameliorated significantly with melatonin treatment. In conclusion, melatonin administration improves the course of chronic renal failure in rats with renal mass reduction. Further studies are necessary to define the potential usefulness of this treatment in other animal models and in patients with chronic renal disease.

Topics: Actins; Animals; Blood Pressure; Cell Movement; Collagen Type IV; Creatinine; Disease Models, Animal; Hypertension; Hypertrophy; Inflammation; Kidney; Kidney Failure, Chronic; Leukocytes; Male; Malondialdehyde; Melatonin; Nephrectomy; Oxidative Stress; Proteinuria; Rats; Rats, Sprague-Dawley; Transcription Factor RelA; Transforming Growth Factor beta; Tyrosine

Reduction in nitric oxide (NO) levels during kidney failure has been related to the reaction of NO with superoxide anions to yield peroxynitrite which possesses the biological activity responsible for renal damage. However, stimulation of the NO pathway ameliorates the progression of kidney failure. Thus, it is unclear whether NO prevents or acts as the compound responsible for the cytotoxicity observed during kidney failure.. We evaluated the development of kidney failure in animals that were wild type and deficient in endothelial NO synthase (eNOS -/-) and tested the effects of an antioxidant treatment and NO precursors on the generation of superoxide anion and kidney failure parameters.. In wild-type mice, five-sixths nephrectomy increased proteinuria from 3.0 +/- 0.35 to 14.5 +/- 0.76 mg protein/24 h (P < 0.05), blood pressure from 83.1 +/- 1.8 to 126.6 +/- 1.7 mmHg (P < 0.05), and superoxide production from 1.4 +/- 0.6% to 74.3 +/- 0.8% (P < 0.05). The effects of five-sixths nephrectomy on the eNOS -/- mice were greater compared with wild-type mice. Proteinuria increased from 6.7 +/- 0.5 to 22.7 +/- 2.0 mg protein/24 h (P < 0.05), blood pressure increased from 93.3 +/- 0.9 to 151.2 +/- 3.4 mmHg (P < 0.05), and superoxide production increased from 12.9 +/- 0.5% to 99.8 +/- 1.3% (P < 0.05). The nitrotyrosine levels were lower in eNOS -/- mice as compared to wild-type mice. A combination of L-arginine and antioxidant treatment ameliorated renal damage. The effect was improved in wild-type animals.. Our data support the relevance of NO as an antagonist to superoxide in renal tissues and suggest that the loss of this mechanism promotes the progression of kidney failure.

Topics: Animals; Antioxidants; Arginine; Blood Pressure; Disease Models, Animal; Drug Combinations; Kidney; Kidney Failure, Chronic; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nephrectomy; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Proteinuria; Superoxides; Time Factors; Tyrosine

Previous studies have reported that isohumulones, the bitter compounds in beer, improve insulin resistance and hyperlipidemia in several animal models. In this study, we examined whether isohumulones ameliorate renal injury. Dahl salt-sensitive hypertensive rats were fed a low-salt diet (LS), a high-salt diet (HS) or a high-salt diet containing 0.3% isohumulones (HS+IH) for 4 weeks. Urinary nitrite/nitrate (NOx) excretion was measured at 4 weeks along with blood pressure and urinary protein excretion. Renal injury was evaluated histologically and reactive oxygen species (ROS) and nitric oxide (NO) production in the renal cortex was visualized. Oxidative stress and NO synthase (NOS) expression were evaluated by immunohistochemical staining and Western blot analysis. Mean blood pressure was significantly decreased in the HS+IH group compared with the HS group at 4 weeks (158.1+/-8.7 vs. 177.5+/-3.7 mmHg; p<0.05). Isohumulones prevented the development of proteinuria in the HS+IH group compared with the HS group at 2 weeks (61.7+/-26.8 vs. 117.2+/-9.8 mg/day; p<0.05). Glomerulosclerosis and interstitial fibrosis scores were significantly decreased in the HS+IH group compared with the HS group (0.61+/-0.11 vs. 1.55+/-0.23, 23.7+/-6.8 vs. 36.1+/-3.5%; p<0.05 for both). In the HS group, increased ROS and decreased NO were observed in glomeruli in vivo. Isohumulones reduced the ROS production, leading to the restoration of bioavailable NO. Urinary NOx excretion was significantly increased in the HS+IH group compared with the HS group. Furthermore, renal nitrotyrosine was increased in the HS group compared with the LS group, and this effect was prevented by isohumulones. Renal NOS expression did not differ among the three groups. These results suggest that isohumulones may prevent the progression of renal injury caused by hypertension via an anti-oxidative effect.

Topics: Animals; Antioxidants; Blood Pressure; Blotting, Western; Cyclopentanes; Humulus; Hypertension; Kidney Diseases; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Oxidative Stress; Proteinuria; Rats; Rats, Inbred Dahl; Reactive Oxygen Species; Sodium Chloride; Tyrosine

Progressive renal damage and hypertension are associated with oxidative and nitrosative stress. On the other hand, S-allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract (AG), has antioxidant properties. The effects of SAC and AG on blood pressure, renal damage, and oxidative and nitrosative stress were studied in five-sixths nephrectomized rats treated with SAC (200 mg/kg ip) and AG (1.2 ml/kg ip) every other day for 30 days. Proteinuria and serum creatinine and blood urea nitrogen concentrations were measured on days 0, 5, 10, 15, and 30, and systolic blood pressure was recorded on days 0, 15, and 30. The degree of glomerulosclerosis and tubulointerstitial damage, the immunostaining for inducible nitric oxide synthase, 3-nitrotyrosine, poly(ADP-ribose), and the subunits of NADPH oxidase p22phox and gp91phox, and the activity of SOD were determined on day 30. SAC and AG reduced hypertension, renal damage, and the abundance of inducible nitric oxide synthase, 3-nitrotyrosine, poly(ADP-ribose), p22phox, and gp91phox and increased SOD activity. Our data suggest that the antihypertensive and renoprotective effects of SAC and AG are associated with their antioxidant properties and that they may be used to ameliorate hypertension and delay the progression of renal damage.

Topics: Animals; Antihypertensive Agents; Antioxidants; Blood Pressure; Blood Urea Nitrogen; Creatinine; Cysteine; Cytoprotection; Garlic; Hypertension; Kidney; Male; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Nephrectomy; Nitric Oxide Synthase Type II; Plant Extracts; Poly Adenosine Diphosphate Ribose; Proteinuria; Rats; Rats, Wistar; Superoxide Dismutase; Systole; Tyrosine

Accumulation of interstitial collagen (fibrosis) between the endothelium and myocytes is one of the hallmarks of cardiac failure in renovascular hypertension (RVH). Renal insufficiency increases plasma homocysteine (Hcy), and levels of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) are inversely related to plasma Hcy levels. We hypothesize that in RVH, accumulation of collagen between the endothelium and myocytes leads to endothelial-myocyte disconnection and uncoupling, in part, by hyperhomocysteinemia. Furthermore, we hypothesize that Hcy increases reactive oxygen species, generates nitrotyrosine, activates latent matrix metalloproteinase, and decreases the levels of endothelial nitric oxide in response to antagonizing PPAR-gamma. To create RVH in mice, the left renal artery was clipped with 0.4-mm silver wire for the 2 kidney, 1 clip (2K1C) method. Sham surgery was used as a control. To induce PPAR-gamma, 8 microg/mL ciglitazone (CZ) was administered to drinking water 2 days before surgery and continued for 4 weeks. Mice were grouped as 2K1C, sham, 2K1C+CZ, or sham+CZ (n = 6 in each group). Plasma Hcy increased 2-fold in the 2K1C-treated group (p < 0.05) as compared with the sham, and CZ had no effect on Hcy levels as compared to the 2K1C-treated group. Hcy binding in cardiac tissue homogenates decreased in the 2K1C-treated group but was substantially higher in the CZ-treated group. Cardiac reactive oxygen species levels were increased and endothelial nitric oxide were decreased in the 2K1C-treated group. Matrix metalloproteinase-2 and -9 activities were increased in the 2K1C-treated group compared with the control. Levels of cardiac inhibitor of metalloproteinase were decreased, whereas there was no change in tissue inhibitor of metalloproteinase-1 expression in the 2K1C-treated group vs. the sham-treated group. Collagen and nitrotyrosine levels were increased in the 2K1C-treated group, but mice treated with CZ showed lower levels comparatively. Cardiac transferase deoxyuridine nick-end labeling-positive cells were increased, and muscle cells were impaired in the 2K1C-treated mice vs. the sham-control mice. This was associated with decreased acetylcholine and bradykinin responses, which suggests endothelial-myocyte uncoupling in 2K1C-treated mice. Our results suggest that fibrosis between the endothelium and myocytes leads to an endothelial-myocyte disconnection and uncoupling by Hcy accumulation secondary to increased reactive oxygen s

Topics: Acetylcholine; Animals; Blotting, Western; Collagen; Endothelin-1; Endothelium, Vascular; Fibrosis; Homocysteine; Hypertension, Renovascular; Hypoglycemic Agents; Kidney; Male; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocytes, Cardiac; Nitroprusside; PPAR gamma; Proteinuria; Thiazolidinediones; Tissue Inhibitor of Metalloproteinase-1; Tyrosine; Vasodilator Agents; Ventricular Remodeling

It has been shown that reactive oxygen species are involved in chronic puromycin aminonucleoside (PAN) induced nephrotic syndrome (NS) and that a 20% soy protein diet reduces renal damage in this experimental model. The purpose of the present work was to investigate if a 20% soy protein diet is able to modulate kidney nitrotyrosine formation and the activity of renal antioxidant enzymes (catalase, glutathione peroxidase, Cu,Zn- or Mn-superoxide dismutase) which could explain, at least in part, the protective effect of the soy protein diet in rats with chronic NS induced by PAN. Four groups of rats were studied: (1) Control rats fed 20% casein diet, (2) Nephrotic rats fed 20% casein diet, (3) Control rats fed 20% soy protein diet, and (4) Nephrotic rats fed 20% soy protein diet. Chronic NS was induced by repeated injections of PAN and rats were sacrificed at week nine. The soy protein diet ameliorated proteinuria, hypercholesterolemia, and the increase in serum creatinine and blood urea nitrogen observed in nephrotic rats fed 20% casein diet. Kidney nitrotyrosine formation increased in nephrotic rats fed 20% casein diet and this increase was ameliorated in nephrotic rats fed 20% soy protein diet. However, the soy protein diet was unable to modulate the antioxidant enzymes activities in control and nephrotic rats fed 20% soy protein diet. Food intake was similar in the two diet groups. The protective effect of a 20% soy protein diet on renal damage in chronic nephropathy induced by PAN was associated with the amelioration in the renal nitrotyrosine formation but not with the modulation of antioxidant enzymes.

Topics: Animals; Antimetabolites; Antioxidants; Blood Urea Nitrogen; Body Weight; Catalase; Cholesterol; Creatinine; Diet; Eating; Glutathione Peroxidase; Glycine max; Immunohistochemistry; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Lipids; Male; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Wistar; Soybean Proteins; Superoxide Dismutase; Tyrosine

This study was carried out to elucidate whether the protective activity of (-)-epicatechin 3-O-gallate (ECg) against excessive peroxynitrite (ONOO(-)) production, is distinct from the activity of several well-known free radical inhibitors, the ONOO(-) inhibitors ebselen and uric acid, the superoxide anion (O(2)(-)) scavenger copper zinc superoxide dismutase (CuZnSOD) and the selective inducible nitric oxide synthase inhibitor L-N(6)-(1-iminoethyl)lysine hydrochloride (L-NIL). To generate ONOO(-), male Wistar rats (n = 6/group) were subjected to ischaemia-reperfusion process together with lipopolysaccharide (LPS) injection. Although ECg did not scavenge the ONOO(-) precursors nitric oxide (NO) and O(2)(-), it reduced the 3-nitrotyrosine level, a property similar to that of uric acid, but distinct from L-NIL. In addition, the elevation in myeloperoxidase activity was reversed by the administration of ECg, uric acid and SOD, but not by that of L-NIL. Furthermore, ECg was the more potent scavenger of the ONOO(-) decomposition product, the hydroxyl radical (*OH), than any other free radical inhibitor tested. The LPS plus ischaemia-reperfusion process resulted in renal dysfunction, estimated by measuring the parameters of renal function--serum urea nitrogen and creatinine levels. However, administration of ECg ameliorated renal dysfunction more than that of the other free radical inhibitors. Moreover, ECg reduced the excessive uric acid level, while the others did not, suggesting a property of ECg distinct from the others. Furthermore, proteinuria, which was demonstrated by the low- and high-molecular weight (LMW and HMW) protein bands of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, caused by LPS plus ischaemia-reperfusion, was attenuated by administration of ECg and L-NIL, after which the HMW band intensities decreased and LMW protein bands were absent. This study indicates that, in an in-vivo model of ONOO(-) generation, ECg, L-NIL and uric acid exert stronger protective activity against ONOO(-)-induced oxidative damage than SOD and ebselen, and that the mechanism whereby ECg protects against ONOO(-) is distinct from that of L-NIL or uric acid.

Topics: Animals; Azoles; Blood Urea Nitrogen; Catechin; Copper; Creatinine; Disease Models, Animal; Dose-Response Relationship, Drug; Free Radical Scavengers; Isoindoles; Japan; Kidney; Lipopolysaccharides; Lysine; Male; Nitric Oxide; Organoselenium Compounds; Peroxidase; Phytotherapy; Plant Extracts; Plant Roots; Proteinuria; Rats; Rats, Wistar; Reperfusion Injury; Rheum; Superoxide Dismutase; Tyrosine; Uric Acid; Zinc

To test the hypothesis that endothelial dysfunction in hyperhomocysteinemia was due to increased levels of nitrotyrosine and matrix metalloproteinase (MMP) activity in response to antagonism of peroxisome proliferator-activated receptor-alpha (PPAR-alpha), cystathionine beta-synthase (CBS) -/+ mice were bred, tail tissue was analyzed for genotype by PCR, and tail vein blood was analyzed for homocysteine (Hcy) by spectrofluorometry. To induce PPAR-alpha, mice were administered 8 microg/ml of ciprofibrate (CF) and grouped: 1) wild type (WT), 2) WT + CF, 3) CBS, 4) CBS + CF (n = 6 in each group). In these four groups of mice, plasma Hcy was 3.0 +/- 0.2, 2.5 +/- 1.2, 15.2 +/- 2.6 (P < 0.05 compared with WT), 11.0 +/- 2.9 micromol/l. Mouse urinary protein was 110 +/- 11, 86 +/- 6, 179 +/- 13, 127 +/- 9 microg.day(-1). kg(-1) by Bio-Rad dye binding assay. Aortic nitrotyrosine was 0.099 +/- 0.012, 0.024 +/- 0.004, 0.132 +/- 0.024 (P < 0.01 compared with WT), 0.05 +/- 0.01 (scan unit) by Western analysis. MMP-2 activity was 0.053 +/- 0.010, 0.024 +/- 0.002, 0.039 +/- 0.009, 0.017 +/- 0.006 (scan unit) by zymography. MMP-9 was specifically induced in CBS -/+ mice and inhibited by CF treatment. Systolic blood pressure (SPB) was 90 +/- 2, 88 +/- 16, 104 +/- 8 (P < 0.05 compared with WT), 96 +/- 3 mmHg. Aortic wall stress [(SPB. radius(2)/wall thickness)/2(radius + wall thickness)] was 10.2 +/- 1.9, 9.7 +/- 0.2, 16.6 +/- 0.8 (P < 0.05 compared with WT), 13.1 +/- 2.1 dyn/cm(2). The results suggest that Hcy increased aortic wall stress by increasing nitrotyrosine and MMP-9 activity.

Topics: Animals; Aorta; Blood Pressure; Clofibric Acid; Cystathionine beta-Synthase; Endothelium, Vascular; Enzyme Activation; Fibric Acids; Genotype; Homocysteine; Hyperhomocysteinemia; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Peroxisome Proliferators; Phenotype; Proteinuria; Tyrosine

Angiotensin II (Ang II) can up-regulate nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase, whose product superoxide anion (O2-) can interact with nitric oxide (NO) to form peroxynitrite (ONOO-). We tested the hypothesis that Ang II subtype 1 (AT1) receptor activation enhances oxidative stress and nitrotyrosine deposition in the kidneys of rats with diabetes mellitus (DM).. After two weeks of streptozotocin-induced DM, rats received either no treatment, an angiotensin-converting enzyme inhibitor (ACEI) or an angiotensin receptor blocker (ARB) for two weeks. At four weeks, renal expression of the p47phox component of NAD(P)H oxidase, endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and nitrotyrosine were evaluated by Western blot and immunohistochemistry and related to plasma lipid peroxidation products (LPO), hydrogen peroxide production in the kidney and 24-hour protein excretion.. Immunoreactive expression of p47phox and eNOS were increased in DM with an increase in plasma LPO, renal hydrogen peroxide production and nitrotyrosine deposition. Expression of nNOS was unaltered. Treatment with either ACEI or ARB prevented all these findings and also prevented significant microalbuminuria. The treatments did not affect the elevated blood sugar, nor did DM or its treatment affect the blood pressure or the creatinine clearance.. Early proteinuric diabetic nephropathy increases renal expression of the p47phox component of NAD(P)H oxidase and eNOS with increased indices of systemic and renal oxidative/nitrosative stress. An ACEI or an ARB prevents these changes and prevents the development of proteinuria, independent of blood pressure or blood sugar. This finding indicates a pathogenic role for AT1 receptors in the development of oxidative damage in the kidneys during early DM.

Topics: Albuminuria; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Benzimidazoles; Biphenyl Compounds; Diabetic Nephropathies; Female; Hydrogen Peroxide; Isoquinolines; Kidney; Lipid Peroxidation; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphoproteins; Proteinuria; Quinapril; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Superoxides; Tetrahydroisoquinolines; Tetrazoles; Tyrosine

In the present paper we studied the role of nitric oxide radicals (NO) on platelet aggregation, fibrinogen deposition, superoxide formation, peroxynitrite formation, hemodynamics, and leukocyte migration in the Thy-1 model of glomerulonephritis. To first study the baseline kinetics of these parameters, groups of anti-Thy-1-treated rats were sacrificed at 1 h, 4 h, 24 h, 3 days, 7 days, and 14 days and compared to controls. Urinary protein excretion was significantly elevated in Thy-1 nephritis at 3 and 7 days. Glomerular macrophages, PMNs, and superoxide anion-positive cells were significantly increased in Thy-1 nephritis. Nitrotyrosine immunoreactivity was absent during the entire study period. Glomerular platelet aggregation was significantly increased in anti-Thy-1 injected rats at 1 h, 4 h, 24 h, and 3 days. Glomerular fibrinogen deposition was significantly elevated at all time points. To elucidate the role of NO in this process, additional groups of anti-Thy-1-injected rats were treated with the NOS inhibitor l-NAME and studied at 24 h. Urinary protein excretion was significantly higher in l-NAME treated Thy-1 rats compared to nontreated Thy-1 rats. Plasma and urine nitrite/nitrate levels were significantly lower in l-NAME-treated Thy-1 rats compared to nontreated Thy-1 rats. Compared to nontreated Thy-1 rats, there were no differences in intraglomerular leukocyte accumulation after treatment with l-NAME. In contrast, we observed a marked increase in platelet aggregation following l-NAME treatment. From these data we conclude that the inflammatory infiltrate in Thy-1 nephritis develops independent of NO radical production, whereas NO radicals prevent the accumulation of platelet aggregates.

Topics: Animals; Blood Pressure; Enzyme Inhibitors; Immunohistochemistry; Isoantibodies; Kidney Glomerulus; Nephritis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Platelet Aggregation; Proteinuria; Rats; Tyrosine