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. (C) 2014 Elsevier Inc. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo (FAP-Unifesp)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pathol, BR-04023900 São Paulo, BrazilUniv São Paulo, Coll Publ Hlth, Dept Nutr, São Paulo, BrazilUniv São Paulo, Dept Biochem & Pharmaceut Technol, São Paulo, BrazilUniv São Paulo, Dept Nephrol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pathol, BR-04023900 São Paulo, BrazilWeb of Scienc

P2X(7) Receptor in the Kidneys of Diabetic Rats Submitted to Aerobic Training or to N-Acetylcysteine Supplementation

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 P2X7 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 center dot, 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo (FAP)Universidade Federal de São Paulo, Div Nephrol, Dept Med, São Paulo, BrazilUniversidade Federal de São Paulo, Cardiovasc Div, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Neurol Neurosurg, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Div Mol Biol, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Investigat Pathol Div, Dept Pathol, São Paulo, BrazilUniversidade Federal de São Paulo, Emergency Div, Dept Med, São Paulo, BrazilUniversidade Federal de São Paulo, Div Nephrol, Dept Med, São Paulo, BrazilUniversidade Federal de São Paulo, Cardiovasc Div, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Neurol Neurosurg, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Div Mol Biol, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Investigat Pathol Div, Dept Pathol, São Paulo, BrazilUniversidade Federal de São Paulo, Emergency Div, Dept Med, São Paulo, BrazilWeb of Scienc

Oxidative stress and diabetic nephropathy at the 8^th week of protocol.

<p>Nonlinear regression oxidative stress, NO^• and proteinuria, n = 10 for all groups. a) TBARS urinary excretion and proteinuria are related (<i>p</i><0.001 and r² = 0.713); b) NO^• urinary excretion and proteinuria are also linked (<i>p</i><0.001 and r² = 0.706); c) TBARS and NO^• urinary excretion are dependent (<i>p</i><0.001 and r² = 0.660) (<i>n</i> = 10). CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC; TBARS, thiobarbituric acid reactive substances; NO^•, nitric oxide.</p

Confocal analysis of P2×₇-R in the kidney at the 8^th week of protocol.

<p>a) Intracellular calcium concentration by confocal microscopy. Intensity was quantified using pseudocolor image according to fluorescence intensity by Fluo-4; these images showed the calcium mobilization in renal tissue when exposed to ATP 1 mM. Micrographies were obtained with x400 of magnification. b) ­Graphics of the calcium dynamics in relation to basal fluorescence. c) Quantification after the nonspecific agonist. Each group with n = 5, Two-way ANOVA with Newman-Keuls post-test. p<0.05: a vs. CTL+SE; b vs. DM+SE. CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC; auf, arbitrary unit fluorescence.</p

PAS staining of renal tissue at the 8^th week of protocol.

<p>The black arrows on the micrographies show glycosidic degeneration in the tubules. DM+SE presented these alterations in medulla and renal cortex. There was a reduction in DM+SE+NAC and DM+EX with incidence only in the renal cortex; the DM+EX+NAC was the less affected. Magnification with x400. CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC; PAS, periodic acid-Schiff.</p

Immunohistochemistry analysis of P2×₇-R in the kidney at the 8^th week of protocol.

<p>Two-way ANOVA with Newman-Keuls post-test. <i>p</i><0.05: ^a vs. CTL+SE; ^b vs. DM+SE; ^c vs. DM+SE+NAC; ^d vs. DM+EX. CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC; P2X7-R, P2X7 receptor.</p

Metabolic profile and analysis of renal function at the 8^th week protocol.

<p>Results are represented as mean ±SEM. Albuminuria with n = 5 and all others parameters with n = 10. two-way ANOVA with Newman-Keuls post-test; <i>p</i><0.05: ^a vs. CTL+SE; ^b vs. DM+SE; ^c vs. DM+SE+NAC; ^d vs. DM+EX.</p><p>CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC.</p

P2X₇-R activity and diabetic nephropathy at the 8th week of protocol.

<p>Nonlinear regression between the oxidative stress, P2X₇-R and proteinuria. calcium dynamic by BzATP and proteinuria are also related (<i>p</i><0.001 and r² = 0.526) (<i>n</i> = 5). CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC; TBARS, thiobarbituric acid reactive substances; NO^•, nitric oxide; BzATP, 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5′ –triphosphate; P2X₇-R, P2X₇ receptor.</p

HE staining of renal tissue at the 8^th week of protocol.

<p>The black arrows on the micrographies show that tubular vacuolization, in DM+SE were present at proportion of 10∶12. In DM+SE+NAC this proportion was 6∶12, DM+EX and DM+EX+NAC had 04:12. Magnification of x400. CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC; HE, hematoxylin and eosin.</p

xidative stress analysis at the 8th week protocol.

<p>Results are represented as mean ±SEM. All parameters with n = 10. two-way ANOVA with Newman-Keuls post-test; <i>p</i><0.05: ^a vs. CTL+SE; ^b vs. DM+SE; ^c vs. DM+SE+NAC;^d vs. DM+EX.</p><p>CTL+SE, sedentary control; CTL+SE+NAC, sedentary control plus NAC; CTL+EX, training control; CTL+EX+NAC, training control plus NAC; DM+SE, sedentary diabetic; DM+SE+NAC, sedentary diabetic plus NAC; DM+EX, training diabetic; DM+EX+NAC, training diabetic plus NAC; TBARS, thiobarbituric acid reactive substances; GSR, glutathione reductase enzyme; GPx, glutathione peroxidase enzyme; NO^•, nitric oxide.</p