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

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

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

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

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

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

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

Western blot analysis of eNOS in samples of renal cortex at the 8^th week of protocol.

<p>eNOS, endothelial nitric oxide synthase.</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