Cupuaçu extract protects the kidneys of diabetic rats by modulating Nrf2/NF-κB p65 and iNOS.

Diabetes is characterized by increased levels of oxidative stress. Its suggested that extract of cupuaçu could improve the antioxidant system in diabetes. The aim was to evaluate the effect of EC on Nrf2/NF-κB p65 in normal and diabetic rats. Male, adult Wistar rats (9-week-old) 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. The diabetes was inducted with a single intravenous dose of 45 mg/kg streptozotocin. Glycemia and body weight were measured at the beginning and end of the protocol, and the renal tissue was analyzed by Western blot for SOD-1, SOD-2, CAT, GSSG, Nrf2, NF-κB p65, iNOS and 3-NT. Glycemia was reduced in DMEC vs. DM after 8 weeks of EC treatment. There was no difference in body weight of DMEC vs. DM; however, DMEC vs. DM presented increased levels of CAT and Nrf2, with a significant reduction of NF-κB p65, iNOS and 3-NT. Therefore, we suggest that EC could be utilized as a complementary therapy to ameliorate the antioxidant profile via Nrf2 and to delay the evolution of diabetic complications in renal tissue by inflammatory pathway inhibition.

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.

P2X(7) receptor in the kidneys of diabetic rats submitted to aerobic training or to N-acetylcysteine supplementation [corrected].

Previous studies in our laboratory showed that N-acetylcysteine supplementation or aerobic training reduced oxidative stress and the progression of diabetic nephropathy in rats. The P2X(7 receptor is up-regulated in pathological conditions, such as diabetes mellitus. This up-regulation is related to oxidative stress and induces tissue apoptosis or necrosis. The aim of the present study is to assess the role of P2X(7) receptor in the kidneys of diabetic rats submitted to aerobic training or N-acetylcysteine supplementation. Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, i.v.) and the training was done on a treadmill; N-acetylcysteine was given in the drinking water (600 mg/L). By confocal microscopy, as compared to control, the kidneys of diabetic rats showed increased P2 × 7 receptor expression and a higher activation in response to 2'(3')-O-(4-benzoylbenzoyl) adenosine5'-triphosphate (specific agonist) and adenosine triphosphate (nonspecific agonist) (all p<0.05). All these alterations were reduced in diabetic rats treated with N-acetylcysteine, exercise or both. We also observed measured proteinuria and albuminuria (early marker of diabetic nephropathy) in DM groups. Lipoperoxidation was strongly correlated with P2X(7) receptor expression, which was also correlated to NO•, thus associating this receptor to oxidative stress and kidney lesion. We suggest that P2X(7) receptor inhibition associated with the maintenance of redox homeostasis could be useful as coadjuvant treatment to delay the progression of diabetic nephropathy.

Kefir administration reduced progression of renal injury in STZ-diabetic rats by lowering oxidative stress.

This study aimed at assessing the effects of Kefir, a probiotic fermented milk, on oxidative stress in diabetic animals. The induction of diabetes was achieved in adult male Wistar rats using streptozotocin (STZ). The animals were distributed into four groups as follows: control (CTL); control Kefir (CTLK); diabetic (DM) and diabetic Kefir (DMK). Starting on the 5th day of diabetes, Kefir was administered by daily gavage at a dose of 1.8 mL/day for 8 weeks. Before and after Kefir treatment, the rats were placed in individual metabolic cages to obtain blood and urine samples to evaluate urea, creatinine, proteinuria, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and C-reactive protein (CRP). After sacrificing the animals, the renal cortex was removed for histology, oxidative stress and NOS evaluation. When compared to CTL rats, DM rats showed increased levels of glycemia, plasmatic urea, proteinuria, renal NO, superoxide anion, TBARS, and plasmatic CRP; also demonstrated a reduction in urinary urea, creatinine, and NO. However, DMK rats showed a significant improvement in most of these parameters. Despite the lack of differences observed in the expression of endothelial NO synthase (eNOS), the expression of inducible NO synthase (iNOS) was significantly lower in the DMK group when compared to DM rats, as assessed by Western blot analysis. Moreover, the DMK group presented a significant reduction of glycogen accumulation within the renal tubules when compared to the DM group. These results indicate that Kefir treatment may contribute to better control of glycemia and oxidative stress, which is associated with the amelioration of renal function, suggesting its use as a non-pharmacological adjuvant to delay the progression of diabetic complications.