3-nitrotyrosine and Diabetic-Nephropathies

Macro- and microvascular disease are the most common causes of morbidity and mortality in patients with diabetes mellitus. Diabetic cardiovascular dysfunction represents a problem of great clinical importance underlying the development of various severe complications including retinopathy, nephropathy, neuropathy and increase the risk of stroke, hypertension and myocardial infarction. Hyperglycemic episodes, which complicate even well-controlled cases of diabetes, are closely associated with increased oxidative and nitrosative stress, which can trigger the development of diabetic complications. Hyperglycemia stimulates the production of advanced glycosylated end products, activates protein kinase C, and enhances the polyol pathway leading to increased superoxide anion formation. Superoxide anion interacts with nitric oxide, forming the potent cytotoxin peroxynitrite, which attacks various biomolecules in the vascular endothelium, vascular smooth muscle and myocardium, leading to cardiovascular dysfunction. The pathogenetic role of nitrosative stress and peroxynitrite, and downstream mechanisms including poly(ADP-ribose) polymerase (PARP) activation, is not limited to the diabetes-induced cardiovascular dysfunction, but also contributes to the development and progression of diabetic nephropathy, retinopathy and neuropathy. Accordingly, neutralization of peroxynitrite or pharmacological inhibition of PARP is a promising new approach in the therapy and prevention of diabetic complications. This review focuses on the role of nitrosative stress and downstream mechanisms including activation of PARP in diabetic complications and on novel emerging therapeutical strategies offered by neutralization of peroxynitrite and inhibition of PARP.

Topics: Animals; Cardiomyopathies; Diabetes Complications; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Endothelium, Vascular; Humans; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Poly(ADP-ribose) Polymerases; Superoxides; Tyrosine

The oxidative stress resulting from increased production of ROS plays a crucial role in the development of diabetic complications. We aim to explore the relationships between oxidative stress, diabetic nephropathy (DN) and short-term insulin pump intensive therapy (insulin therapy).. Levels of 8-hydroxy-deoxyguanosine (8-OHdG), 3-nitrotyrosine (3-NT), glutathione (GSH), superoxide dismutase (SOD) and Interleukin-6 (IL-6) were estimated before and after 2 weeks of insulin therapy in normal group (NC) and type 2 diabetic (DM) with normal albuminuria (NA), microalbuminuria (MA) and clinical albuminuria (CA).. In DM group, levels of 8-OHdG and 3-NT were higher than those in NC group (P < 0.05); GSH and SOD were lower (P < 0.05). And their levels changed with urine albumin-creatinine ratio (P < 0.05). After insulin therapy, these derangements were significantly ameliorated and the changes in NA and MA groups were more significant than CA group (P < 0.05). Correlation analysis showed glycated hemoglobin, the course of disease, the HOME-IR and fasting plasma glucose were positively correlated with 8-OHdG and 3-NT, but negatively correlated with GSH and SOD.. The oxidative stress gradually increased with the magnitude of DN, and insulin pump intensive therapy can significantly ameliorate the derangements in the early stage of DN. Trial registration NCT03174821.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Blood Glucose; Deoxyguanosine; Diabetic Nephropathies; Female; Glutathione; Hemoglobins; Humans; Insulin; Insulin Infusion Systems; Interleukin-6; Male; Middle Aged; Oxidative Stress; Superoxide Dismutase; Tyrosine

The aim of this study was to assess the effect of the angiotensin II receptor blocker Irbesartan on protein damage by glycation, oxidation and nitration in patients with type 2 diabetes and microalbuminuria. In a double-masked randomised crossover trial of 52 hypertensive type 2 diabetic patients, antihypertensive treatment was replaced with bendroflumethiazide. After 2-months wash-out, patients were treated randomly with Irbesartan 300, 600, and 900 mg o.d., each dose for 2 months in a three-way crossover study. Glycation, oxidation and nitration adduct residues in plasma protein and related urinary free adducts were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Treatment with Irbesartan decreased urinary excretion of advanced glycation endproducts (AGEs)--methylglyoxal- and glyoxal-derived hydroimidazolones, MG-H1 and G-H1. Urinary AGEs were decreased by 30-32%. In plasma protein, treatment with Irbesartan increased content of glycation adducts Nε-fructosyl-lysine, AGEs Nε-carboxymethyl-lysine, Nε-carboxyethyl-lysine and pentosidine, and also increased content of oxidation markers N-formylkynurenine and dityrosine. This was attributed to decreased clearance of plasma protein modified by Nε-fructosyl-lysine and oxidative markers through the glomerular filter tightened by Irbesartan treatment. Treatment of patients with type 2 diabetes with Irbesartan decreased urinary excretion of MG-H1, G-H1 and 3-NT, which may result from decreased exposure to these AGEs. This is likely achieved by blocking angiotensin II signalling and related down-regulation of glyoxalase 1 and may contribute to health benefits of Irbesartan therapy.

Topics: Albuminuria; Amino Acids; Angiotensin II Type 1 Receptor Blockers; Antihypertensive Agents; Bendroflumethiazide; Biomarkers; Biphenyl Compounds; Cross-Over Studies; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Dose-Response Relationship, Drug; Female; Glycation End Products, Advanced; Glycosylation; Humans; Hypertension; Irbesartan; Male; Oxidative Stress; Tetrazoles; Tyrosine

Growing evidence indicates that oxidative stress induced by excessive superoxide has a central role in the pathogenesis of diabetic nephropathy (DN). Telmisartan, one of the currently available angiotensin II type 1 receptor blockers (ARBs), has been shown to exert a more powerful proteinuria (albuminuria) reduction in patients with DN, but whether the prominent renoprotective effect of telmisartan is mediated through enhancing antioxidant defense capacity and reducing oxidative stress has not been fully elucidated. The present study first revealed that the serum activity of superoxide dismutase (SOD) responsible for superoxide removal is reduced in the DN stage of microalbuminuria, but not in normoalbuminuria in type 2 diabetic patients. We next examined the alteration of SOD and oxidative stress following an 8-week treatment with telmisartan (40 mg per day) in 12 type 2 diabetic patients with microalbuminuria. Interestingly, the telmisartan treatment not only reduced the circulating levels of two oxidative stress markers, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine (NT), but also enhanced serum SOD activity. Notably, a significant correlation was observed between the increase in serum SOD activity and the reduction in albuminuria. We further compared the anti-oxidative effect of telmisartan with that of losartan, another member of the ARB class, by implementing an 8-week interval crossover treatment with these ARBs in another 12 microalbuminuric type 2 diabetic patients. The patients showed higher serum SOD activity, and lower circulating levels of 8-OHdG and NT, during treatment with telmisartan than with losartan. These results suggest that telmisartan has a more potent antioxidative effect through its ability to enhance SOD activity in type 2 diabetic patients with microalbuminuria.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Albuminuria; Angiotensin II Type 1 Receptor Blockers; Antioxidants; Benzimidazoles; Benzoates; Cross-Over Studies; Deoxyguanosine; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Humans; Japan; Losartan; Male; Middle Aged; Nitric Oxide; Oxidative Stress; Superoxide Dismutase; Telmisartan; Tyrosine

The aim of the present study was to investigate the effects of the iron chelator deferiprone in diabetic nephropathy (DN) rats and the mechanisms involved.. Thirty-two male Wistar rats (180-220 g, 6 weeks old) were randomly divided into a control group, a DN group and two DN groups treated with either 50 or 100 mg/kg per day deferiprone. The DN group was established by feeding of a high-carbohydrate-fat diet and injection of 35 mg/kg streptozotocin into the vena caudalis. The duration of deferiprone treatment was 20 weeks. Histopathological changes were detected by hematoxylin-eosin and Masson staining, as well as transmission electron microscopy. Levels of nuclear factor (NF)-κB, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase (MMP)-9, tissue-specific inhibitor of metalloproteinase (TIMP)-1, cyclo-oxygenase (COX)-2, and nitrotyrosine were determined in kidney tissues using reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry.. Histopathological observations showed that deferiprone treatment alleviated inflammation infiltrates and collagenous fibrosis in DN rats. Results from RT-PCR and western blotting indicated that deferiprone inhibited the expression of NF-κB, MCP-1, COX-2, and nitrotyrosine, which were overexpressed in DN rats. Immunohistochemistry showed that the mechanism of deferiprone action may involve regulation of MMP-9 and TIMP-1. Decreased MMP-9 expression and increased TIMP-1 expression in DN rats were significantly promoted and inhibited by deferiprone, respectively.. Iron chelation by oral deferiprone has a renoprotective effect in DN rats by relieving oxidative stress, inflammation, and fibrosis, which is related to the cytokines NF-κB, MCP-1, MMP-9, TIMP-1, COX-2, and nitrotyrosine.

Topics: Administration, Oral; Animals; Blotting, Western; Chemokine CCL2; Cyclooxygenase 2; Deferiprone; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Dietary Carbohydrates; Dose-Response Relationship, Drug; Gene Expression; Immunohistochemistry; Iron Chelating Agents; Kidney; Male; Matrix Metalloproteinase 9; Microscopy, Electron, Transmission; NF-kappa B; Protective Agents; Pyridones; Random Allocation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinase-1; Tyrosine

Kallistatin is a serine protease inhibitor with anti-inflammatory, anti-angiogenic, and anti-oxidative properties. Since oxidative stress plays a critical role in the pathogenesis of diabetic nephropathy, we studied the effect and mechanisms of action of kallistatin superinduction. Using ultrasound-microbubble-mediated gene transfer, kallistatin overexpression was induced in kidney tubules. In db/db mice, kallistatin overexpression reduced serum creatinine and BUN levels, ameliorated glomerulosclerosis and tubulointerstitial injury, and attenuated renal fibrosis by inhibiting TGF-β signaling. Additionally, downstream PAI-1 and collagens I and IV expression were reduced and kallistatin partially suppressed renal inflammation by inhibiting NF-κB signaling and decreasing tissue kallikrein activity. Kallistatin lowered blood pressure and attenuated oxidative stress as evidenced by suppressed levels of NADPH oxidase 4, and oxidative markers (nitrotyrosine, 8-hydroxydeoxyguanosine, and malondialdehyde) in diabetic renal tissue. Kallistatin also inhibited RAGE expression in the diabetic kidney and AGE-stimulated cultured proximal tubular cells. Reduced AGE-induced reactive oxygen species generation reflected an anti-oxidative mechanism via the AGE-RAGE-reactive oxygen species axis. These results indicate a renoprotective role of kallistatin against diabetic nephropathy by multiple mechanisms including suppression of oxidative stress, anti-fibrotic and anti-inflammatory actions, and blood pressure lowering.

Topics: Animals; Diabetic Nephropathies; Fibrosis; Gene Transfer Techniques; Genetic Therapy; Kallikreins; Kidney; Kidney Function Tests; Male; Mice, Inbred C57BL; Mice, Transgenic; NADPH Oxidase 4; NADPH Oxidases; Neovascularization, Pathologic; NF-kappa B; Oxidative Stress; Reactive Oxygen Species; Receptor for Advanced Glycation End Products; Serpins; Transforming Growth Factor beta; Tyrosine; Vascular Endothelial Growth Factor A

Diabetic nephropathy (DN) is a common and serious clinical complication of diabetes and presently there are no effective ways to prevent its occurrence and progression. Recent studies show that pentoxifylline (PTX) can improve renal hemodynamics, reduce urinary protein excretion, and alleviate or delay renal failure in DN patients. In this study, we focused on the anti-oxidative stress effect of PTX on alleviating renal damages of DN using rat models. DN rats were established with injection of streptozotocin. Blood glucose, urinary protein excretion, serum cystatin C, renal biopsy, superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and renal homogenate and renal nitrotyrosine levels were analyzed before and 12 weeks after the treatment of PTX. Before treatment, all the DN rats had elevated blood glucose, increased urinary protein excretion and elevated serum cystatin C. Morphologically, DN rats exhibited renal tissue damages, including swelling and fusions of foot processes of podocytes under electron microscope. Masson staining revealed blue collagen deposition in glomeruli and renal interstitium. With treatment of PTX, symptoms and renal pathological changes of DN rats were alleviated. Furthermore, the MDA levels were increased and the SOD levels were decreased in the serum and kidneys of DN rats, and these changes were reversed by PTX. The expression of nitrotyrosine was up-regulated in DN rat model and down-regulated by PTX, indicating that PTX was able to inhibit oxidative reactions in DN rats. PTX could alleviate renal damage in DN, which may be attributable to its anti-oxidative stress activity.

Topics: Animals; Biomarkers; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression Regulation; Kidney; Malondialdehyde; Oxidative Stress; Pentoxifylline; Rats; Streptozocin; Superoxide Dismutase; Tyrosine

To observe the effect of nitrotyrosine on renal expressions of nuclear factor-κB (NF-κB), monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-β1 (TGF-β1) in rats with diabetic nephropathy (DN).. Rat DN models established by a single intraperitoneal injection of streptozotocin (STZ) were randomly allocated into model group, nitrotyrosine group and ebselen group, with untreated rats as the normal control group. The rats were given the corresponding drugs for 8 weeks, and after the last administration, the 24-h urinary protein level was measured and the kidneys of the rats were harvested for detecting the protein and mRNA expressions of NF-κB, MCP-1 and TGF-β1 with immunohistochemistry and RT-PCR, respectively. The pathological changes of the kidneys were assessed microscopically.. Compared with those in the model group, the 24-h urinary protein level and expressions of NF-κB, MCP-1 and TGF-β1 mRNA and protein in the renal tissues were significantly increased by nitrotyrosine treatment, which also caused worsened renal pathology, while treatment with ebselen significantly ameliorated these changes.. Nitrotyrosine can up-regulate the mRNA and protein expressions of NF-κB, MCP-1 and TGF-β1 and aggravate the inflammatory reaction in the renal tissue of DN rats to promote the progression of DN.

Topics: Animals; Chemokine CCL2; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Male; NF-kappa B; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1; Tyrosine

The aim of this study was to investigate the effects of apocynin, a NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase inhibitor, in diabetic rats with nephropathy induced by contrast medium (CIN). Diabetes was induced in male Wistar rats by a single dose of streptozotocin (60 mg/kg i.v.). Animals were then divided into the following groups: 1) control group (diabetic rats treated i.v. with saline solution); 2) iomeprol group (iomeprol at 10 ml/kg was injected i.v. 30 min after saline administration); 3) apocynin group (identical to the iomeprol group, except for pre-treatment with apocynin 5mg/kg i.v., 30 min before iomeprol injection) and 4) N-acetylcysteine group (NAC) (same as iomeprol group, except for the treatment with NAC 20 mg/kg i.v. 30 min before iomeprol injection). CIN in animals were assessed 24h after administration of iomeprol. Apocynin significantly attenuates the impaired glomerular function, concentration of Na(+), K(+), alpha glutathione S-transferase levels in urine and neutrophil gelatinase-associated lipocalin levels in plasma caused by iomeprol. In kidney, immunohistochemical analysis of some inflammatory mediators, such as nitrotyrosine, poly-ADP-ribosyl polymerase, tumor necrosis factor-α, interleukin-1β as well as apoptosis (evaluated as terminal deoxynucleotidyltransferase-mediated UTP end labeling assay) revealed positive staining in tissue obtained from iomeprol group. These parameters were markedly reduced in animals treated with apocynin. Similarly, these parameters were also markedly modified by NAC pre-treatment. Here, we have shown that apocynin attenuates the degree of iomeprol-induced nephropathy in diabetic rats.

Topics: Acetophenones; Acetylcysteine; Acute-Phase Proteins; Animals; Apoptosis; Contrast Media; Cytosine; Diabetic Nephropathies; Enzyme Activation; Enzyme Inhibitors; Glutathione Transferase; Iopamidol; Isoenzymes; Kidney Glomerulus; Lipocalin-2; Lipocalins; Male; NADPH Oxidases; Poly(ADP-ribose) Polymerases; Potassium; Proto-Oncogene Proteins; Rats; Rats, Wistar; Sodium; Time Factors; Tyrosine

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca(2+) efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes.

Topics: Animals; Benzoquinones; Calcium; Diabetic Nephropathies; Diet, High-Fat; Disease Models, Animal; HSP90 Heat-Shock Proteins; Kaplan-Meier Estimate; Kidney Function Tests; Kidney Glomerulus; Kidney Tubules; Lactams, Macrocyclic; Male; Mice; Mice, Knockout; Mitochondria; Renal Insufficiency; Tyrosine

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in protecting the kidney from oxidant-mediated damage in the streptozotocin (STZ) rat model of type-1 diabetes mellitus (DM-1). HO-derived metabolites, presumably carbon monoxide (CO), mediate vasodilatory influences in the renal circulation, particularly in conditions linked to elevated HO-1 protein expression or diminished nitric oxide (NO) levels. We tested the hypothesis that diabetes increases oxidative stress and induces HO-1 protein expression, which contributes to regulate renal hemodynamics in conditions of low NO bioavailability. Two weeks after the induction of diabetes with STZ (65 mg/kg iv), Sprague-Dawley rats exhibited higher renal HO-1 protein expression, hyperglycemia, and elevated renal nitrotyrosine levels than control normoglycemic animals. In anesthetized diabetic rats, renal vascular resistance (RVR) was increased, and in vivo cortical NO levels were reduced (P < 0.05) compared with control animals. Acute administration of the HO inhibitor Stannous mesoporphyrin (SnMP; 40 μmol/kg iv) did not alter renal hemodynamics in control rats, but greatly decreased glomerular filtration rate and renal blood flow, markedly increasing RVR in hyperglycemic diabetic rats. Chronic oral treatment with the SOD mimetic tempol prevented the elevation of nitrotyrosine, the HO-1 protein induction, and the increases in RVR induced by SnMP in the diabetic group, without altering basal NO concentrations or RVR. Increasing concentrations of a CO donor (CO-releasing molecule-A1) on pressurized renal interlobar arteries elicited a comparable relaxation in vessels taken from control or diabetic animals. These results suggest that oxidative stress-induced HO-1 exerts vasodilatory actions that partially maintain renal hemodynamics in uncontrolled DM-1.

Topics: Animals; Antioxidants; Boranes; Carbon Monoxide; Carbonates; Cyclic N-Oxides; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Dose-Response Relationship, Drug; Enzyme Inhibitors; Heme Oxygenase (Decyclizing); Hemodynamics; Kidney; Male; Metalloporphyrins; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Renal Circulation; Spin Labels; Time Factors; Tyrosine; Vasoconstriction; Vasodilation

Epidemiological studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease, including diabetic nephropathy. We have previously reported that nicotine promotes mesangial cell proliferation and hypertrophy via activation of nonneuronal nicotinic acetylcholine receptors and that nicotine worsens renal injury in a model of acute glomerulonephritis (Jaimes E, Tian RX, Raij L. Am J Physiol Heart Circ Physiol 292: H76-H82, 2007; Jaimes EA, Tian RX, Joshi M, Raij L. Am J Nephrol 29: 319-326, 2009). These studies were designed to test the hypothesis that nicotine worsens renal injury in db/db mice, a well-established model of diabetic nephropathy, and that reactive oxygen species play an important as mediators of these effects. For these studies, nicotine (100 μg/ml) was administered in the drinking water to control and db/db mice for 10 wk. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. At death, kidneys were collected for histology and molecular biology. The administration of nicotine did not result in significant changes in blood pressure or blood glucose and resulted in cotinine levels similar to those found in the plasma of smokers. In diabetic mice, the administration of nicotine significantly increased urinary protein excretion (1-fold), glomerular hypertrophy, and mesangial area (∼20%). These changes were accompanied by significant increases in NADPH oxidase 4 (∼30%) and increased nitrotyrosine and Akt expression. In vitro, we determined that nicotine has additive effects to high glucose on reactive oxygen species generation and Akt phosphorylation in human mesangial cells. These findings unveil novel mechanisms that may result in the development of novel strategies in the treatment and prevention of diabetic nephropathy in smokers.

Topics: Animals; Blood Glucose; Blood Pressure; Cells, Cultured; Diabetic Nephropathies; Disease Models, Animal; Disease Progression; Humans; Kidney Glomerulus; Male; Mesangial Cells; Mice; Mice, Inbred C57BL; NADPH Oxidase 4; NADPH Oxidases; Nicotine; Nicotinic Agonists; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Smoking; Tyrosine

To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress.. The study was performed using diabetic apolipoprotein E/GPx1 (ApoE(-/-)GPx1(-/-))-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks.. Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways.. Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.

Topics: Aldehydes; Animals; Antioxidants; Apolipoproteins E; Atherosclerosis; Azoles; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Diabetic Nephropathies; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Isoindoles; Kidney; Male; Mice; Mice, Knockout; Organoselenium Compounds; Reactive Oxygen Species; Tyrosine; Vascular Cell Adhesion Molecule-1

Oxidative stress and inflammation are implicated in the pathogenesis of diabetic nephropathy. Because sildenafil citrate (Viagra) has variable cardiovascular benefits, including antioxidative and immunomodulating effects, we investigated its influence on oxidative stress and inflammation in diabetic rat kidney.. Streptozotocin-induced diabetic rats received sildenafil (3 mg/kg/day in drinking water) or not (undosed water) for 8 weeks and were compared to age-matched nondiabetic animals. We evaluated 8-hydroxydeoxyguanosine (8-OHdG; for oxidative DNA damage), inducible nitric oxide synthase (iNOS) and nitrotyrosine (for excessive NO production and peroxynitrite formation), and representative chemoattractants [monocyte chemotactic protein-1, MCP-1; for inflammation and monocyte/macrophage infiltrations (ED-1)] in the kidney.. Sildenafil-treated rats had a lower kidney-to-body weight ratio than untreated diabetic rats. Urinary albumin excretion in diabetic rats decreased significantly after sildenafil treatment without changes in systolic blood pressure. Sildenafil-treated rats had significantly lower urinary and renal cortical 8-OHdG levels than the nonsildenafil group. Sildenafil administration significantly attenuated the increased renal nitrotyrosine protein expression, positive iNOS and ED-1 staining in glomeruli and tubulointerstitium, and nitrotyrosine staining in tubulointerstitium. Cortical MCP-1 RNA expression in the sildenafil group was significantly lower than in the nonsildenafil group.. Sildenafil treatment may attenuate renal damage by ameliorating oxidative and inflammatory injuries in diabetic rats.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers; Blotting, Western; Chemokine CCL2; Deoxyguanosine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Immunohistochemistry; Inflammation Mediators; Kidney; Male; Nephritis; Nitric Oxide Synthase Type II; Oxidative Stress; Piperazines; Purines; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sildenafil Citrate; Streptozocin; Sulfones; Tyrosine; Vasodilator Agents

It has been reported that urinary oxidative stress markers are higher in diabetic patients with proteinuria. We performed the present study to elucidate the relationship between urinary excretion of oxidative stress markers, albumin excretion, and histological changes, and to confirm the potential utility of oxidative stress markers for clinical treatment.. Diabetic db/db mice or nondiabetic db/m mice were administered candesartan (10 mg/kg/day) or hydralazine (50 mg/kg/day) for 18 weeks.. Thirty-week-old male db/db mice treated with control vehicle revealed elevated urinary excretion and immunohistological levels of 8-hydroxydeoxyguanosine in glomeruli when compared to db/m mice. Treatment with candesartan, but not hydralazine, reduced these values to levels in db/m mice. Increased mesangial expansion, urinary excretion of albumin and 8-isoprostane, and glomerular immunohistological levels of nitrotyrosine in db/db mice were also decreased markedly by candesartan but not hydralazine. Interestingly, correlations between levels of albumin and oxidative stress markers in urine were very high, even when groups undergoing long-term (44 weeks) treatment were included (correlation coefficient 0.767 with respect to 8-hydroxydeoxyguanosine, 0.888 with respect to 8-isoprostane).. It is anticipated that urinary concentrations of oxidative stress markers will be direct barometers of glomerulus-derived oxidative stress and glomerular injury in diabetic nephropathy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Albuminuria; Angiotensin II Type 1 Receptor Blockers; Animals; Antihypertensive Agents; Benzimidazoles; Biomarkers; Biphenyl Compounds; Deoxyguanosine; Diabetes Mellitus; Diabetic Nephropathies; Dinoprost; Disease Models, Animal; Hydralazine; Kidney Glomerulus; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Tetrazoles; Treatment Outcome; Tyrosine

Peroxynitrite, a highly reactive oxidant produced by the reaction of nitric oxide with free radicals superoxide, has been indicated to be involved in many diseases. However, the contributions of peroxynitrite to diabetic nephropathy and the underlying mechanism have not been fully explored.. The present study was designed to evaluate the role and the underlying mechanism of peroxynitrite in glomerular lesion of diabetic rat.. Diabetes was induced in male Wistar rats by i.p. injection of streptozotocin, and urate was used as a specific scavenger of peroxynitrite; the pathological changes of rat glomerulus were evaluated by hematoxylin and eosin, periodic acid-Schiff staining and transmission electron microscopy observation; immunohistochemistry and Western blot were used to detect the content of nitrotyrosine (the marker of peroxynitrite) in renal cortex; the expression levels of tyrosine phosphorylation of JAK2, STAT1, and STAT3 were assessed by Western blot assay; RT-PCR and Western blot were used to assay expression levels of transforming growth factor (TGF)-beta1 and fibronectin; biochemical indicators of renal function were also detected.. The content of nitrotyrosine was increased, consistent with the pathological changes of glomerulus and renal dysfunction in the diabetes group. Urate prevented the formation of nitrotyrosine in rat glomerulus and attenuated the pathological alterations. Furthermore, urate inhibited the activation of JAK2, STAT1, and STAT3. Finally, homogenates from renal cortices demonstrated reduced expression of TGF- beta1 and fibronectin under urate treatment.. Our findings thus provides in vivo evidence that exaggerated peroxynitrite formation mediates the glomerular lesion in, at least, Type 1 diabetes, which may function through JAK/STAT signaling pathway.

Topics: Animals; Blotting, Western; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibronectins; Immunohistochemistry; Janus Kinase 2; Kidney Glomerulus; Male; Microscopy, Electron, Transmission; Peroxynitrous Acid; Phosphorylation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Transforming Growth Factor beta; Tyrosine; Uric Acid

Tubular damage is a major feature in the development of diabetic nephropathy. This study investigates the effects of the thiazolidindione rosiglitazone on angiotensin II and advanced glycation end product-induced tubular activation in human proximal tubular epithelial cells IN VITRO. Angiotensin II and advanced glycation end products, both induced a dose-dependent sustained activation of the redox-sensitive transcription factor, Nuclear Factor KAPPA B (NF-kappaB). Nuclear translocation of NF-kappaB was evident already after one hour and persistent for more than four days. Co-incubation of proximal tubular epithelial cells with rosiglitazone significantly reduced angiotensin II and advanced glycation end product-mediated generation of reactive oxygen species, angiotensin II-dependent advanced glycation end product formation, NF-kappaB activation, and NF-kappaB-dependent pro inflammatory gene expression. Most importantly, rosiglitazone effects on NFkappaB activation were maximal at later time points, indicating that rosiglitazone treatment confers long lasting renoprotective effects.

Topics: Angiotensin II; Cell Nucleus; Cells, Cultured; Diabetic Nephropathies; Epithelial Cells; Gene Expression; Glycation End Products, Advanced; Humans; Hypoglycemic Agents; Kidney Tubules, Proximal; NF-kappa B; Oxidative Stress; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Rosiglitazone; Thiazolidinediones; 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 combination of hypertension and diabetes exacerbates renal oxidative stress. The aim of the present study was therefore to evaluate the pro-oxidant and antioxidant mechanisms responsible for the induction of renal oxidative stress in the presence of hypertension and diabetes mellitus.. Diabetes was induced in spontaneously hypertensive rats (SHR) and their genetically normotensive control Wistar-Kyoto (WKY) rats by streptozotocin at 12 weeks of age. After 10 days, pro-oxidant, antioxidant and oxidative stress parameters were evaluated in the renal tissue.. NADPH oxidase-dependent superoxide generation in the renal cortex was significantly elevated in WKY and SHR diabetic (D) groups compared to the respective control (C) groups (p < 0.005, n = 5). However, the highest level of superoxide generation was observed in the SHR-D group compared to all other groups. The expression of the gp91phox subunit of NADPH oxidase was significantly elevated in the SHR-D (p < 0.05, n = 5), but not in the WKY-D group, compared to the respective control groups. The renal cortical extracellular-superoxide dismutase level was found to be markedly decreased in the SHR groups compared to the WKY groups (p < 0.05, n = 5). The antioxidant glutathione level was found to be lower in the SHR-D (p = 0.03, n = 15), but not in the WKY-D group, compared to the respective control groups. Finally, nitrotyrosine and 8-hydroxy-2'-deoxyguanosine, markers of oxidative stress, were found to be similar in the kidneys of WKY-C and WKY-D, but were elevated in the SHR-D compared to the SHR-C group.. We therefore conclude that hypertension increases pro-oxidant generation and decreases antioxidant defense, and thereby induces renal oxidative stress in early diabetes.

Topics: Animals; Antioxidants; Blood Pressure; Diabetic Nephropathies; Female; Glutathione; Hypertension, Renal; Male; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Oxidants; Oxidative Stress; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Superoxides; Tyrosine

It is generally considered that 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) have renoprotective effects via a pathway independent of their cholesterol-lowering cascade. In the kidneys of diabetic nephropathy, monomeric endothelial nitric oxide synthase (eNOS) is thought to be overexpressed; and its dimerization is suppressed. In the present study, we investigated the expression of eNOS and oxidative stress in type 2 diabetes mellitus KK-Ay/Ta mice treated with pitavastatin, one of the statins. The KK-Ay/Ta mice were divided into 3 groups and given pitavastatin intraperitoneally starting at 8 weeks of age for 8 weeks: pitavastatin 3 mg/(kg d) (n=5), pitavastatin 10 mg/(kg d) (n=5), and a control group (n=10). The urinary albumin-creatinine ratio (ACR), urinary 8-hydroxy-2'-deoxyguanosine, body weight, fasting blood glucose, hemoglobin A1c, total cholesterol, and triglyceride were measured; and the intraperitoneal glucose tolerance test was performed. The eNOS, nitrotyrosine, and p47 phox were evaluated by immunohistochemical analyses and/or Western blot analyses. Guanosine triphosphate cyclohydrolase 1 messenger RNA expression in the kidneys was evaluated using a real-time polymerase chain reaction assay. Pitavastatin improved the levels of urinary ACR and 8-hydroxy-2'-deoxyguanosine, intraperitoneal glucose tolerance test, and hemoglobin A1c. Protein levels of monomeric eNOS, nitrotyrosine, and p47 phox in the kidneys were decreased in the pitavastatin-treated groups. Guanosine triphosphate cyclohydrolase 1 messenger RNA expression was significantly increased in the pitavastatin groups. There were no significant changes in body weight, levels of fasting blood glucose, serum total cholesterol, triglyceride, and blood pressure among all groups. Pitavastatin improved urinary ACR apparently because of suppression of eNOS uncoupling and its antioxidant effect in the kidneys of KK-Ay/Ta mice.

Topics: Animals; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Glycated Hemoglobin; GTP Cyclohydrolase; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunohistochemistry; Mice; Mice, Inbred C57BL; NADPH Oxidases; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Protein Kinase C; Quinolines; RNA, Messenger; Tyrosine

Advanced glycation end products (AGEs) from the Maillard reaction contribute to the pathogenesis of diabetes-associated complications such as diabetic nephropathy. In therapeutic interventions for reducing AGEs, many compounds have been reported as AGE inhibitors. The objective of the present study was to examine the effect of pyridoxamine (K-163), an AGE inhibitor, in type 2 diabetic KK-A(y)/Ta mice. KK-A(y)/Ta mice were given pyridoxamine (200 or 400 mg/kg per day) starting at 8 weeks of age for 12 weeks. They were divided into 3 groups as follows: pyridoxamine 200 mg/kg per day treatment group (n = 10), pyridoxamine 400 mg/kg per day treatment group (n = 10), and a tap water group as the control group (n = 20). The urinary albumin/creatinine ratio (ACR), body weight (BW), levels of fasting and casual blood glucose, blood glycated hemoglobin (HbA(1c)), fasting serum insulin, triglyceride (TG), total cholesterol (T-Cho), and 3-deoxyglucosone (3DG), and systemic blood pressure were measured as biochemical parameters. N(epsilon)-(Carboxymethyl)lysine (CML) and nitrotyrosine accumulations in glomeruli were evaluated by immunohistochemical analyses. Transforming growth factor beta1 (TGF-beta1) and laminin-beta1 messenger RNA expressions in the kidneys were evaluated by real-time polymerase chain reaction. Pyridoxamine, especially at 400 mg/kg per day, improved the levels of urinary ACR, fasting serum TG, and 3DG. CML and nitrotyrosine accumulations in glomeruli were decreased. Furthermore, large doses of pyridoxamine prevented not only urinary ACR but also increases of BW, casual blood glucose, and HbA(1c). TGF-beta1 and laminin-beta1 messenger RNA expressions in kidneys were significantly lower than those in the controls. There were no significant changes in the levels of fasting blood glucose, serum T-Cho, and systemic blood pressure among all groups. It appears that pyridoxamine improved urinary ACR by its anti-AGE and anti-oxidant effects in the kidneys of KK-A(y)/Ta mice.

Topics: Animals; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Dose-Response Relationship, Drug; Glycation End Products, Advanced; Immunohistochemistry; Laminin; Lysine; Male; Mice; Pyridoxamine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Schiff Bases; Transforming Growth Factor beta1; Tyrosine

Accelerated formation of advanced glycation/lipoxidation and endproducts (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several natural and synthetic compounds have been proposed and tested as inhibitors of AGE/ALE formation. We have previously reported the therapeutic effects of several new AGE/ALE inhibitors on the prevention of nephropathy and dyslipidemia in streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the effects of various concentrations of a compound, LR-90, on the progression of renal disease and its effects on AGE and receptor for AGE (RAGE) protein expression on the kidneys of diabetic STZ-rats. Diabetic male Sprague-Dawley rats were treated with or without LR-90 (0, 5, 20, 25, and 50 mg/l of drinking water). After 32 weeks, body weight, glycemic status, renal function, and plasma lipids were measured. Kidney histopathology and AGE/ALE accumulation and RAGE protein expression in tissues were also determined. In vitro studies were also performed to determine the possible mechanism of action of LR-90 in inhibiting AGE formation and AGE-protein cross-linking. LR-90 protected the diabetic kidneys by inhibiting the increase in urinary albumin-to-creatinine ratio and ameliorated hyperlipidemia in diabetic rats in a concentration-dependent fashion without any effects on hyperglycemia. LR-90 treatment also reduced kidney AGE/ALE accumulation and RAGE protein expression in a concentration-dependent manner. In vitro, LR-90 exhibited general antioxidant properties by inhibiting metal-catalyzed reactions and reactive oxygen species (OH radical) and reactive carbonyl species (methlyglyoxal, glyoxal) generations without any effect on pyridoxal 5' phosphate. The compound also prevents AGE-protein cross-linking reactions. These findings demonstrate the bioefficacy of LR-90 in treating nephropathy and hyperlipidemia in diabetic animals by inhibiting AGE accumulation, RAGE protein expression, and protein oxidation in the diabetic kidney. Additionally, our study suggests that LR-90 may be useful also to delay the onset and progression of diabetic atherosclerosis as the compound can inhibit the expression of RAGE and inflammation-related pathology, as well as prevent lipid peroxidation reactions.

Topics: Animals; Ascorbic Acid; Body Weight; Butyrates; Cholesterol; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Dyslipidemias; Glycation End Products, Advanced; Glycosylation; Hydroxyl Radical; Hypoglycemic Agents; Kidney; Lipid Metabolism; Lipoproteins, LDL; Male; Molecular Structure; Oxidation-Reduction; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Triglycerides; Tyrosine

Eicosapentaenoic acid (EPA) has been reported to have beneficial effects on the progression of various renal diseases including diabetic nephropathy; however, the precise mechanisms are not completely understood. We examined the effects of EPA on the early stage of type 2 diabetic nephropathy in KKA(y)/Ta mice and the possible role of inflammation, oxidative stress, and growth factor in this process. KKA(y)/Ta mice were divided into 2 groups. The treatment group was injected with EPA ethyl ester at 1 g/kg per day intraperitoneally from 12 to 20 weeks of age and the control group was injected with saline. Renal morphologic examinations were performed after 8 weeks of treatment. Glomerular macrophage infiltration and expression of monocyte chemoattractant protein 1, malondialdehyde (MDA), nitrotyrosine, transforming growth factor beta1 (TGF-beta1), and type I collagen were evaluated. Eicosapentaenoic acid decreased the levels of urinary albumin, serum triglyceride and MDA, and improved glucose intolerance in KKA(y)/Ta mice. Morphometric analysis showed that accumulation of extracellular matrix and the tubulointerstitial fibrosis area were significantly decreased after treatment. Immunohistochemistry revealed that glomerular macrophage infiltration and the expression of MDA and nitrotyrosine in KKA(y)/Ta mice were increased and were inhibited by EPA treatment. Protein and gene expression levels of monocyte chemoattractant protein 1, TGF-beta1, and type I collagen, which were evaluated by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction, were down-regulated in the EPA treatment group. In conclusion, EPA improves type 2 diabetic nephropathy in KKA(y)/Ta mice. This beneficial effect might be mediated by attenuation of metabolic abnormalities and inhibition of renal inflammation, oxidative stress, and TGF-beta expression.

Topics: Animals; Anti-Inflammatory Agents; Chemokine CCL2; Collagen Type I; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Eicosapentaenoic Acid; Lipid Peroxidation; Macrophages; Male; Mice; Oxidative Stress; Phenotype; Transforming Growth Factor beta1; Tyrosine

Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or L-arginine and was partially reduced by the addition of diphenylene iodonium or N(G)-nitro-L-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2',7'-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker) with L-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (BH4), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of BH4 availability. These mechanisms are potential key targets for therapeutic interventions.

Topics: Animals; Antioxidants; Biopterins; Diabetic Nephropathies; Gene Expression Regulation, Enzymologic; Kidney Glomerulus; Male; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidative Stress; Rats; Rats, Sprague-Dawley; Superoxides; Tyrosine

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.. 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.. 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.. 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.

Topics: Animals; Antioxidants; Blood Pressure; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Insulin; Lysine; Male; Malondialdehyde; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tyrosine

Renal accumulation of advanced glycation end products (AGEs) has been linked to the progression of diabetic nephropathy. Cleavage of pre-formed AGEs within the kidney by a cross-link breaker, such as ALT-711, may confer renoprotection in diabetes. STZ diabetic rats were randomized into a) no treatment (D); b) treatment with the AGE cross-link breaker, ALT-711, weeks 16-32 (DALT early); and c) ALT-711, weeks 24-32 (DALT late). Treatment with ALT-711 resulted in a significant reduction in diabetes-induced serum and renal AGE peptide fluorescence, associated with decreases in renal carboxymethyllysine and RAGE immunostaining. Cross-linking of tail tendon collagen seen in diabetic groups was attenuated only by 16 weeks of ALT-711 treatment. ALT-711, independent of treatment duration, retarded albumin excretion rate (AER), reduced blood pressure, and renal hypertrophy. It also reduced diabetes-induced increases in gene expression of transforming growth factor beta1 (TGF-beta1), connective tissue growth factor (CTGF), and collagen IV. However, glomerulosclerotic index, tubulointerstitial area, total renal collagen, nitrotyrosine, protein expression of collagen IV, and TGF-beta1 only showed improvement with early ALT treatment alone. This study demonstrates the utility of a cross-link breaker as a treatment for diabetic nephropathy and describes effects not only on renal AGEs but on putative mediators of renal injury, such as prosclerotic cytokines and oxidative stress.

Topics: Albuminuria; Animal Population Groups; Animals; Blood Pressure; Collagen; Connective Tissue Growth Factor; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibrosis; Glycation End Products, Advanced; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Kidney; Kinetics; Lysine; Models, Biological; Rats; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Solubility; Thiazoles; Transforming Growth Factor beta; Transforming Growth Factor beta1; 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

Proximal tubular cells produce nitric oxide (NO.). We have shown that under hyperglycemic conditions, cultured proximal tubular cells express cytochrome P450 2E1, which is capable of producing superoxide (O2.). NO. and O2. react to form peroxynitrite (ONOO.), a powerful oxidant. ONOO. nitrosylates tyrosine moieties on proteins causing tissue damage. Our hypothesis is that ONOO. plays a role in early diabetic tubular damage and perhaps disease progression.. Renal biopsies from patients with diabetic nephropathy (DM), acute allograft rejection (AAR), acute allograft tubular necrosis (ATN), and glomerulonephritis (GN) were obtained. Normal kidney specimens were taken from nephrectomy samples (N = 10 for each group). The tissues were examined for the presence of nitrotyrosine using an immunoperoxidase technique with a polyclonal antibody. Samples were then arbitrarily scored, and the results analyzed (analysis of variance and Student's t-test for unpaired data). The number of apoptotic cells in a sample of tubules in each biopsy was also assessed.. The DM biopsies showed increased staining for nitrotyrosine in proximal tubules (P = 0.0001) and in the thin limb of the loop of Henle (P = 0.0006) compared with all other groups. There was increased staining in the ascending and distal tubules in GN as compared to DM and ATN (P = 0.01). Nitrotyrosine was also found in all distal tubules and collecting ducts, including normals. There was no difference in the number of apoptotic tubular cells in diabetics compared with controls.. To our knowledge, these data provide the first evidence for the presence of nitrotyrosine in both normal and diseased kidneys. The significance of the findings in normals is unclear, but could be due to activation of constitutive NOS. However, the study clearly demonstrates increased production of ONOO. in proximal tubules of patients with DM, and suggests that oxidant injury of the proximal tubules plays an important part in the pathogenesis of DM.

Topics: Adolescent; Adult; Aged; Child; Diabetic Nephropathies; Female; Humans; Immunohistochemistry; Kidney; Male; Middle Aged; Nitrates; Tyrosine