L-Arginine Reduces Nitro-Oxidative Stress in Cultured Cells with Mitochondrial Deficiency.

L-Arginine (L-ARG) supplementation has been suggested as a therapeutic option in several diseases, including Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like syndrome (MELAS), arguably the most common mitochondrial disease. It is suggested that L-ARG, a nitric oxide (NO) precursor, can restore NO levels in blood vessels, improving cerebral blood flow. However, NO also participates in mitochondrial processes, such as mitochondrial biogenesis, the regulation of the respiratory chain, and oxidative stress. This study investigated the effects of L-ARG on mitochondrial function, nitric oxide synthesis, and nitro-oxidative stress in cell lines harboring the MELAS mitochondrial DNA (mtDNA) mutation (m.3243A>G). We evaluated mitochondrial enzyme activity, mitochondrial mass, NO concentration, and nitro-oxidative stress. Our results showed that m.3243A>G cells had increased NO levels and protein nitration at basal conditions. Treatment with L-ARG did not affect the mitochondrial function and mass but reduced the intracellular NO concentration and nitrated proteins in m.3243A>G cells. The same treatment led to opposite effects in control cells. In conclusion, we showed that the main effect of L-ARG was on protein nitration. Lowering protein nitration is probably involved in the mechanism related to L-ARG supplementation benefits in MELAS patients.

Cupuaçu extract reduces nitrosative stress and modulates inflammatory mediators in the kidneys of experimental diabetes.

BACKGROUND & AIMS: We have previously reported an increased nitrosative stress in the kidneys of diabetic animals, which was reduced by an antioxidant probiotic. The present study evaluated whether the extract of cupuaçu (EC), an antioxidant compound rich in polyphenols and theograndins, when administered at a dose that can be reasonably obtained through daily consumption, could delay the onset of diabetic complications in the kidney. METHODS: Mouse immortalized mesangial cells (MiMC) were placed in medium normal glucose (NG) or high glucose (HG), with or without EC (500, 100, 50 or 10 µg/mL) during 24, 48 or 72 h for analysis of viability, proliferation, nitric oxide (NO) levels and reactive oxygen species or nitrogen (ROS/RNS). Male, adult Wistar rats were distributed in 4 groups: control (CTL) and diabetic (DM) who received water; CTLEC and DMEC who received 1 mL/day of EC (1 g/mL), via gavage for 8 consecutive weeks. After, metabolic profile and renal function were evaluated and, kidneys were collected for analysis of NO, ROS, 3-nitrotyrosine (3-NT), endothelial nitric oxide synthase (eNOS), IL-6, IL-10, TNF-α and NF-κB p65. RESULTS: The MiMC showed normal viability in all groups, demonstrating that EC had no cytotoxic effect at doses on 24, 48 or 72 h. MiMC under HG presented significant increase in proliferation, NO and ROS vs. NG; these parameters were significantly reduced after 72 h of EC treatment (P < 0.05). DMEC showed a significant reduction of feed intake, ROS and NO, 3-NT, IL-6 and eNOS vs. DM (P < 0.05). Supplementation with EC at a dose consumed daily could improve control of nitrosative stress, combined with reduction of inflammatory factors, suggesting the importance of bioactive compounds as non-pharmacological adjuvant therapy to delay kidney complications in diabetic patients.

N-acetylcysteine protects against diabetic nephropathy through control of oxidative and nitrosative stress by recovery of nitric oxide in rats.

The diabetes mellitus (DM) induces several changes, with substantial increase of reactive oxygen species (ROS). The ROS cause damage to systemic and renal microvasculature, which could be one of the mechanisms involved in the development of diabetic nephropathy (DN). The ROS modulate other substances like the nitric oxide (NO), a vasodilator with important role in the renal function. N-acetylcysteine (NAC) is an antioxidant that acts replenishing intracellular cysteine levels, which is essential for glutathione formation. The aim of this study was to evaluate the effect of early or late NAC treatment on oxidative/nitrosative stress in DN progression. All rats were submitted to unilateral nephrectomy and diabetes was induced with streptozotocin. The animals were allocated into six groups: controls that received water (CTL) or NAC (CTL + NAC); diabetic groups that received early or late, water (DM-E; DM-L) or NAC (DM + NAC-E; DM + NAC-L), started on 5th day (early) or 4th week (late) after diabetes induction, during 8 weeks. After NAC treatment, the rats were placed in individual metabolic cages to obtain urine and blood samples for analysis of metabolic profile, renal function, thiobarbituric acid reactive substances (TBARS) and NO. At the end of the protocol, the renal cortex was removed for TBARS, NOS evaluation, antioxidants markers and histology. The DM-E group compared to CTL showed a significant increase in glycemia and proteinuria and impaired renal function; there was a significant increase of TBARS in plasma, urine and renal tissue, and also a significant decrease in plasma NO, which were reverted after early NAC treatment. The eNOS was decreased and iNOS was increased in DM-E vs. CTL, p < 0.05. The early NAC treatment in DM rats reduced proteinuria, creatinine, urea, TBARS and iNOS and, increased creatinine clearance, NO and eNOS, increasing significantly the antioxidant defenses, promoting elevated catalase and glutathione compared to DM-E group, all p < 0.05. The late NAC treatment in diabetic rats vs.DM-E showed reduced proteinuria and TBARS excretion and higher values of creatinine clearance and NO, all statistically significant. Histological analysis of the animals in DM-E or DM-L showed significant tubular changes with degeneration and vacuolization in tubular cells, dilated tubular lumen, intense glycosidic degeneration, and discreet mesangial expansion with interstitial fibrosis area. The DM + NAC-E group showed moderate glycosidic degeneration, however, did not present tubular degeneration or fibrosis. The DM + NAC-L group showed severe glycosidic degeneration, moderate tubular cell degeneration, light and focal dilatation of the tubules, with no fibrosis. Our study showed that NAC protected the diabetic rats against renal injury, probably due to the control of oxidative stress via recovery of the NO bioavailability, showing that early NAC was more effective than late treatment. All these data suggest that NAC may be useful in the adjuvant treatment in a safe way, in the early phase of the disease. Eventually, prolonged treatment, even if it is started later, could change the natural history of the disease, delaying the complications of diabetes in renal tissue.

Immunomodulation and nitric oxide restoration by a probiotic and its activity in gut and peritoneal macrophages in diabetic rats.

BACKGROUND: The hyperglycemia leads to increased oxidative stress, causing lipid peroxidation and imbalance in the immune system. AIMS: To investigate the effect of Kefir - a probiotic containing different strains - on metabolic parameters, cytokines, nitric oxide (NO) production, phagocytic activity of peritoneal macrophages and intestinal modulation in diabetes. METHODS: Wistar rats received injection of streptozotocin (45 mg/kg, intravenously) and diabetes was defined as glycemia ≥200 mg/dL. The animals were distributed in four groups: control (CTL); control Kefir (CTLK); diabetic (DM); diabetic Kefir (DMK). Kefir was given at 1.8 mL/day by gavage, started on the 5th day of diabetes, during 8 weeks. The animals were allocated in metabolic cages, pre and post treatment with Kefir, for measurement of the metabolic parameters, such as water intake, food intake, diuresis, glycemia, body mass, insulin and lipid profile, these last two were only measured at the end of Kefir protocol. After treatment, the animals were euthanized and the peritoneal cavity was prepared, resident macrophages were collected and cultured for analysis of the phagocytic activity, cytokines (IL-10, TNF-α, IL-17, IL-1ß) and NO. The intestinal modulation was performed by the quantification of Peyer's patches (PP) in the small intestine. The data were presented as mean ± SEM, with significance of p < 0.05. RESULTS: DM when compared to CTL showed increase in water intake (133 ± 7 vs. 28 ± 1 mL, p < 0.0001), food intake (40 ± 2 vs. 16 ± 1 g, p < 0.0001), diuresis (102 ± 5 vs. 13 ± 1 mL, p < 0.0001) and glycemia (567 ± 12 vs. 84 ± 3 mg/dL, p < 0.0001), while in DMK group all these metabolic parameters were decreased (96 ± 14; 36 ± 1; 86 ± 7 and 407 ± 19, respectively, p < 0.0001), presenting increase of body mass (42 ± 5 vs. 16 ± 4Δ, p < 0.0001) and insulin levels (0.3 ± 0.8 vs. 0.1 ± 0.04 ng/mL, p < 0.0001) compared to DM. The lipid profile of the diabetic groups showed tendency to increase compared to the respective controls. In relation to function of peritoneal macrophages, DMK group vs. DM showed improvement in phagocytic capacity (70 ± 5 vs. 51 ± 7%, p = 0,0023) and increased concentration of all the cytokines analyzed (pg/mL), as IL-10 (926 ± 69 vs. 556 ± 92, p = 0.0004), TNF-α (178 ± 20 vs. 109 ± 20, p = 0.005), IL-17 (33 ± 1 vs. 9 ± 1, p = 0.0001) and IL-1ß (102 ± 14 vs. 70 ± 5, p = 0.0129), after 24 h of LPS stimulation; including NO bioavailability after 24 h (102 ± 9 vs. 66 ± 5 µM/mL, p = 0.0029) or 48 h (143 ± 8 vs. 119 ± 4 µM/mL, p = 0.0102) of LPS stimulation. Moreover, the number of PP in the whole small intestine of DMK group was also increased as compared to DM (22 ± 1 vs. 18 ± 1, p = 0.0292). CONCLUSION: These results show that Kefir has a potential to modulate the immune response and activate peritoneal macrophages in diabetic animals, which suggests that it could enhance the immunocompetence of patients affected by diabetes mellitus. The hypoglycemic effect of this probiotic could be used as a tool to control glycemia, reducing or delaying the onset of complications associated with this disease.

Lithium increases nitric oxide levels in subjects with bipolar disorder during depressive episodes.

BACKGROUND: Altered nitric oxide (NO) signaling has been associated with the pathophysiology of Bipolar Disorder (BD), directly affecting neurotransmitter release and synaptic plasticity cascades. Lithium has shown to regulate NO levels in preclinical models. However, no study has addressed peripheral NO levels in unmedicated BD. Also, lithium's effects on NO levels have not been studied in humans. METHODS: Plasma NO was evaluated in subjects with BD I and II during a depressive episode (n = 26). Subjects had a score of ≥18 in the 21-item Hamilton Depression Rating Scale and were followed-up during a 6-week trial with lithium. Plasma NO levels were also compared to matched healthy controls (n = 28). NO was determined by chemiluminescence method. RESULTS: Lithium treatment significantly increased plasma NO levels after 6 weeks of treatment in comparison to baseline levels in bipolar depression (p = 0.016). Baseline NO levels during depressive episodes showed no difference when matching up to healthy controls (p = 0.66). CONCLUSION: The present findings suggest that lithium upregulates NO signaling in unmedicated BD with short illness duration. Further studies with larger samples are needed to confirm the effects of lithium on NO pathway and its association with synaptic plasticity and therapeutics of BD.

Ascorbic acid reduces gentamicin-induced nephrotoxicity in rats through the control of reactive oxygen species.

BACKGROUND & AIM: Oxidative stress has been implicated in the pathophysiology of many forms of acute renal failure. The aim was examine the effect of vitamin C on oxidative stress and its relationship with nitric oxide on gentamicin-induced nephrotoxicity in rats. METHODS: We utilized 32 Wistar rats allocated in four groups of eight animals each: control (CTL), vitamin C (VIT C), gentamicin (GENTA), and GENTA + VIT C; all groups were treated during seven days. RESULTS: Serum urea and creatinine, serum and renal tissue malondialdehyde, blood superoxide anion and hydrogen peroxide in GENTA were increased vs CTL and vs VIT C, and decreased in GENTA + VIT C vs GENTA (all P < 0.05). Serum nitric oxide increased in GENTA vs CTL and vs VIT C, and reduced in GENTA + VIT C vs GENTA (P < 0.001). Urinary nitric oxide was reduced in GENTA vs CTL and vs VIT C and increased in GENTA + VIT C vs GENTA (P < 0.001). Severe degeneration of proximal tubules was present in GENTA, but only mild lesions were observed in GENTA + VIT C. CONCLUSION: This study suggests that VIT C is a valuable tool to protect against GENTA-induced nephrotoxicity, by reducing reactive oxygen species and increasing the nitric oxide.

Effects of training and nitric oxide on diabetic nephropathy progression in type I diabetic rats.

The aim of the paper is to assess nitric oxide (NO) production during aerobic training and its role on the progression of diabetic nephropathy in rats. Induction of diabetes mellitus (DM) was achieved in adult male Wistar rats with streptozotocin. Half of the animals underwent training on a treadmill and the others (sedentary) stayed on a turned-off treadmill for the same period according to the following groups: sedentary control (CTL + SE); training control (CTL + EX); sedentary diabetic (DM + SE); and training diabetic (DM + EX) (n = 9 for all groups). The training on treadmill was carried out at a work rate of 16 m/min, 60 min/d, 5 d/week for eight weeks. Before and after the exercises, rats were placed in individual metabolic cages with standard chow and water ad libitum, for 24-h urine collection, followed by three hours' fasting blood sample withdrawal from the retro-orbital plexus, under anesthesia. Diabetic animals showed reduction of body weight, creatinine and urea depurations and NO excretion, increased blood glucose concentrations, albuminuria and thiobarbituric acid reactive substance (TBARS) excretion, when compared with the respective controls. All these alterations induced by DM were attenuated in the DM + EX versus DM + SE group. Analysis of insulin concentrations at the end of the protocol showed no significant change between the DM + SE and DM + EX groups. In conclusion, our data show that a routine physical exercise resulted in a better control of glycemia with an increased NO bioavailability and oxidative stress control, associated with an amelioration of renal function. We suggest aerobic training and the control of oxidative and nitrosative stress as useful non-pharmacological tools to delay the progression of diabetic nephropathy.