P2X7 siRNA targeted to the kidneys increases klotho and delays the progression of experimental diabetic nephropathy.

Previous studies in our laboratory have suggested that P2X7 could contribute to the progression of diabetic nephropathy and modulated klotho expression. The aim of this study was to investigate if P2X7 receptor is related to the expression of klotho in the onset of diabetic nephropathy in rats. Seven-week-old male Wistar rats weighing 210 g were all uninephrectomized; two-third of the animals were induced to diabetes with 60 mg/kg streptozotocin i.v., and one-third received its vehicle (control rats). At 4th day of the fifth week of the protocol, half of the diabetic rats received a small interfering RNA targeting for P2X7 mRNA, and the other half received its vehicle. Euthanasia was made at the eighth week. Diabetic animals reproduced all classic symptoms of the disease; besides, they showed reduced renal function and low NO bioavailability; also, SOD1, SOD2, and catalase were increased, probably due to the oxidative stress which was elevated in this situation. Metabolic data of diabetic rats did not change by silencing P2X7 receptor. For the other hand, silencing P2X7 was able to contribute to balance oxidative and nitrosative profile, ultimately improving the renal function and increasing plasma and membrane forms of klotho. These findings suggest that the management of P2X7 receptor can benefit the kidneys with diabetic nephropathy. Further studies are needed to show the therapeutic potential of this receptor inhibition to provide a better quality of life for the diabetic patient.

Esculin reduces P2X7 and reverses mitochondrial dysfunction in the renal cortex of diabetic rats.

AIMS: To evaluate the effects of esculin treatment on P2X7 receptor and mitochondrial dysfunction in the renal cortex of diabetic rats. MAIN METHODS: Male Wistar rats, 7 weeks old, were unilaterally nephrectomized. Part of these animals were induced to diabetes using streptozotocin (60 mg/kg). Diabetes was confirmed 48 h after induction, with blood glucose levels ≥200 mg/dL. Part of control and diabetic animals were selected to receive daily doses of esculin (50 mg/kg), during 8 weeks. The animals were placed in metabolic cages at the eighth week of protocol for 24 h urine collection and a small aliquot of blood was collected for biochemical analysis. After this procedure, the animals were euthanized and the remaining kidney was stored for histopathological analysis, Western blotting and mitochondrial high-resolution respirometry. KEY FINDINGS: Although esculin did not change metabolic parameters, renal biochemical function, neither TBARS in DM rats, esculin reduced P2X7 levels in these animals and restored mitochondrial function via glycolysis substrates and ß-oxidation. Besides, at the histological analysis, we observed that esculin reduced inflammatory infiltrates and collagen IV deposits as compared to diabetic group. SIGNIFICANCE: Esculin attenuated the development of renal injuries caused by hyperglycemia, proinflammatory and oxidative mechanisms mediated by P2X7 receptor, as seen by histological findings and improved mitochondrial function in diabetic animals. This suggests that esculin could be used as an adjuvant therapy to prevent the diabetic nephropathy.

Inhibition of the P2X7 receptor improves renal function via renin-angiotensin system and nitric oxide on diabetic nephropathy in rats.

AIM: To evaluate the effects of P2X7 receptor blockade on renin-angiotensin system (RAS) in rats with diabetic nephropathy (DN). MAIN METHODS: Wistar rats were unilaterally nephrectomized and received streptozotocin for diabetes mellitus (DM) induction; control animals (CTL) received the drug vehicle. The animals were submitted to P2X7 receptor silencing, forming the group (DM + siRNA). The animals were placed in metabolic cages for data collection and evaluation of renal function; at the end of the protocol, the kidney was removed for analysis of P2X7, renin, angiotensin-converting enzyme (ACE), ACE2, angiotensin, thiobarbituric acid reactive substance levels (TBARS), nitric oxide (NO) and qualitative histological. KEY FINDINGS: The metabolic profile was attenuated in DM + siRNA vs. DM and there was a significant improvement in creatinine, urea and proteinuria levels in the same group. Renin expression was significantly decreased in DM + siRNA vs. DM. ACE and ACE2 were significantly reduced in DM + siRNA vs. DM. TBARS levels were decreased and NO showed an increase in DM + siRNA vs. DM, both significant. All histological alterations were improved in DM + siRNA vs. DM. SIGNIFICANCE: Data have shown that although silencing of the P2X7 receptor did not decrease fasting glucose, it promoted an improvement in the metabolic profile and a significant recovery of renal function, revealing a protective action by the inhibition of this receptor. This effect must have occurred due to the inhibition of RAS and the increase of NO, suggesting that the use of P2X7 receptors inhibitors could be used as adjuvant therapy against DN progression.

Moderate aerobic exercise on the recovery phase of gentamicin-induced acute kidney injury in rats.

INTRODUCTION: Acute kidney injury is a serious public health problem, especially in intensive care units, where patients may require dialysis support, resulting in 50% mortality. AIM: To evaluate the effects of moderate aerobic exercise on the recovery phase of acute kidney injury induced by gentamicin in rats. MAIN METHODS: Male adult Wistar rats were allocated into 4 groups: W10+R30, G10+R30, W10+EX30 and G10+EX30; W10 received water (gentamicin vehicle) and G10 received gentamicin for 10days; R30 remained resting and EX30 made exercise for 30days after gentamicin suspension. Training was performed on treadmill. Blood, 24h urine and kidneys were collected for renal function and oxidative stress, antioxidant, TGF-ß and histological analysis. KEY FINDINGS: Gentamicin treatment caused decreased renal function significant oxidative stress, reduced urinary nitric oxide and increased TGF-ß. G10+R30 presented partial recovery of metabolic data, renal function and lipoperoxidation levels, although they were still altered compared to W10+R30. Besides, we observed the presence of lymphomononuclear infiltrate in the kidneys of G10+R30. G10+EX30 vs G10+R30 showed additional improvement of all the mentioned parameters, showing at histology, regeneration of the tubule epithelium. SIGNIFICANCE: Our data suggest that moderate exercises could help in the recovery of metabolic parameters, renal function and structure on gentamicin-induced AKI, perhaps due to restoration of redox balance. This could protect the kidneys from further insults like challenges with nephrotoxic drugs or the aging per se.

Soybean concentrated extract counteracts oxidative stress in the uterus of rats.

OBJECTIVE: To evaluate the effects of soy isoflavone extract in the pro-oxidant/antioxidant balance in the uterus of ovariectomized rats. METHODS: Twenty 3-month-old adult female Wistar rats were divided into four equal groups: GI: sham-operated (estrous phase); GII: control ovariectomized rats; GIII: ovariectomized rats treated with genistein (50 µg/kg/day) by gavage; GIV: ovariectomized rats subcutaneously treated with estrogen (10 µg/kg/day). After 30 consecutive days of treatment, the rats were euthanized and the uterus removed. The distal thirds of the uterine horns were processed for histomorphometric analyses of endometrial and myometrial thicknesses and glandular area. Other regions of the uteri were kept in liquid nitrogen and subsequently processed for analysis of reactive species quantification (DCF), total antioxidant capacity (TAC) and lipid oxidation status (TBARS). Data were statistically analyzed by one-way ANOVA, complemented by the Tukey-Kramer test (p < 0.05). RESULTS: GII and GIII exhibited lower endometrial thickness, glandular area and myometrial thickness than GI and GIV, while a higher myometrial thickness was observed in GIV compared with the other groups. Moreover, the isoflavone-treated group showed lower DCF and TBARS compared to GII, and also an improvement of TAC compared to GI and GIV. Despite the significant decrease in TBARS, no significant difference in DCF nor a decrease in TAC were observed in GIV when compared to GII. CONCLUSION: Our data show that isoflavones improve antioxidant status and counteract oxidative stress, without promoting a trophic effect in the uterus of rats.

Nitric oxide levels in the intestines of mice submitted to ischemia and reperfusion: L-arginine effects.

OBJECTIVE: Usually an experimental necrotizing enterocolitis experimental model, we Investigated nitric oxide levels in intestinal tissues of newborn mice with or without l-arginine therapy during sessions of ischemia and reoxygenation. METHODS: Twenty-six newborn mice from the Wistar EPM-1 lineage, weighing from 4.5 to 6.2 g, were randomly assigned to three groups: G-I/R, hypoxia and reoxygenation; G-Arg, l-arginine treatment I/R; and G-CTL, controls. G-I/R and G-Arg mice underwent twice a day during their first 3 days of life exposure to gas chambers with 100% CO(2) for 5 minutes at 22 degrees C before reoxygenation with 100% O(2) for another 5 minutes. After 12 hours, all animals were sedated, laparotomized, and had samples of ileum and colon taken and- either formalin fixed histopathologic examinations or frozen to -80 degrees C for estimation of tissue nitric oxide levels. Intestinal injuries were classified according to the criteria of Chiu et al. RESULTS: The G-I/R and G-Arg groups showed injuries characteristic of necrotizing enterocolitis (NEC) with an improved structural preservation rate in G-Arg. The concentration of nitric oxide in the Ileum was much higher with G-Arg (16.5 +/- 4.9; P = 0.0019) G-I/R (7.3 +/- 2.0). This effect was not observed in the colon: G-I/R = 10.7 +/- 4.6 versus G-Arg = 15.5 +/- 8.7 (P = .2480). CONCLUSION: Supply of L-arginine increased tissue levels of nitricoxide and reduced morphologic intestinal injury among mice undergoing I/R.