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Mechanisms underlying the renal toxicity induced by chronic ethanol consumption: the role of inducible nitric oxide synthase (iNOS)

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Carla Brigagão Pacheco da Silva
Total Authors: 1
Document type: Doctoral Thesis
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Carlos Renato Tirapelli; Bruno Spinosa de Martinis; Rita de Cassia Aleixo Tostes Passaglia; Jose Eduardo Tanus dos Santos
Advisor: Carlos Renato Tirapelli

Chronic ethanol consumption is related to enhancement of oxidative stress and inflammation that can induce progression and development of diseases. Despite some studies described that ethanol leads to renal function disorders, the mechanisms underlying this response have not been fully elucidated. Chronic ethanol intake can induce expression of the inducible nitric oxide synthase (iNOS) isoform, leading to an increase of nitric oxide (NO) levels, and improve reactive oxygen species (ROS) production, mainly superoxide anion (O2.-). The reaction between NO and O2.- results in peroxynitrite (ONOO-) formation, a powerful oxidant molecule that contributes to the pathophysiology of renal dysfunction. Thus, the objective of the present study was to evaluate the role of iNOS in the renal toxicity induced by chronic ethanol consumption. To this purpose, C57BL/6 (WT) and iNOS knockout (iNOS-/-) mice were randomly distributed into 4 groups: control WT and iNOS-/- with free access to filtered water; ethanol WT and ethanol iNOS-/- with access to ethanol 20% (vol./vol.) per 10 weeks. Our results showed that long-term ethanol consumption decreased liquid and solid intake, weight gain, and induced increased systolic blood pressure (SBP) in WT and iNOS-/- animals treated with ethanol. We observed increased iNOS expression in cortex renal from WT mice after chronic ethanol exposure. There was increase of creatinine serum levels and morphological alterations, as interstitial edema and increased glomerular diameter in renal cortex of both WT and iNOS-deficient mice chronically treated with ethanol, implying renal dysfunction induced by ethanol. In the renal cortex, chronic ethanol treatment increased oxidative stress characterized by augmented of O2.- levels, as well as fluorescence intensity of dihydroethidium (DHE) probe, protein expression of Nox4, in addition to the increase of lipoperoxidation. These responses were attenuated or blocked in iNOS-/- animals. Regarding the antioxidant system, ethanol exposure decreased catalase expression in parallel with the increase of reduced glutathione (GSH) levels, without affect the enzymatic activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) nor oxidized glutathione (GSSG) levels in WT and iNOS-/- animals. Ethanol did not alter nitrate/nitrite (NOx) levels, but the renal cortex of iNOS-/- mice showed lower concentrations than WT. Chronic ethanol consumption increased the levels of proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukins (IL)-1β, besides the neutrophil recruitment into the renal cortex. Both the increased TNF-α levels and myeloperoxidase (MPO) activity observed in ethanol WT mice have been attenuated in knockout animals, suggesting that the inflammatory response in the kidney is mediated by iNOS. Matrix metalloproteinases (MMP)-2 and -9 activity, Nox1, Nox2, MMP-2, MMP-9, TIMP-1, TIMP-2, eNOS, nNOS, NF-κB and Nrf2 protein expression nor hydrogen peroxide (H2O2), K+ and Na+ ions, urea, IL-6 and IL-10 levels did not differ among the groups after treatment with ethanol. We concluded that iNOS has not been involved in all effects induced by ethanol, however we verified the role of this enzyme in the renal toxicity, as an important mediator of inflammation and oxidative stress induced by chronic ethanol consumption in renal cortex from mice. (AU)

Grantee:Carla Brigagão Pacheco da Silva
Support Opportunities: Scholarships in Brazil - Doctorate