Avaliação do tratamento precoce ou tardio com N-acetilcisteína sobre o controle do estresse oxidativo na nefropatia diabética experimental

• Ano de defesa: 2016
• Autor(a) principal: Nogueira, Guilherme Baia [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: N-acetylcysteine; Antioxidant; Nitric oxide; Experimental diabetes mellitus; Diabetic nephropathy and oxidative stress; N-acetilcisteína; Antioxidante; Óxido nítrico; Diabetes mellitus experimental; Nefropatia diabética e estresse oxidativo
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=4699339; http://repositorio.unifesp.br/handle/11600/47341

INTRODUCTION : Diabetes mellitus (DM) induces intra and extracellular changes, with substantial increase in reactive oxygen species (ROS). ROS causes damage to the systemic and renal microvasculature, which could be one of the mechanisms involved in the development of diabetic nephropathy. ROS modulate other substances like the nitric oxide (NO), a powerful vasodilator with important role in the kidney function. N-acetylcysteine (NAC) is an antioxidant that acts replenishing intracellular cysteine levels, which is essencial in the glutathione (GSH) formation. OBJECTIVE : The aim of this study was to evaluate the effect of early or late treatment with N-acetylcysteine on oxidative/nitrosative stress control in experimental diabetic nephropathy. MATERIAL AND METHODS: We used 60 adult male Wistar rats that underwent unilateral nephrectomy. Diabetes was induced with streptozotocin (60 mg / kg, iv) in 40 animals (DM) and the others received vehicle (CTL). Half of CTL animals were supplemented with NAC (600mg/L of water, ad libitum, CTL+NAC) and the others received water as NAC vehicle. 10 diabetic animals received NAC [600mg/L of water, ad libitum, DM+NAC (PRE)] and 10 received the NAC vehicle [water, DM (PRE)] during 8 weeks after diabetes induction. 10 animals received NAC [600mg/L of water, ad libitum, DM+NAC (TAR)] and 10 received the NAC vehicle [water, DM (TAR)] for 8 weeks, beginning after 4 weeks of diabetes. N = 10 each group. Before any procedure and before and after the early or late treatment protocol with NAC, we collected urine and blood and only at the end of the protocol, the kidney tissue was removed for further analysis. Data were expressed as mean±SEM and analyzed by non-parametric statistical analysis and unpaired t test or one-way ANOVA with Tukey's post-test, when appropriate; it was considered statistically significant when p<0.05. RESULTS : DM (PRE) compared to CTL showed a significant increase in glycemia levels (mg/dL): 509.0±19.6 vs 85.0±3.7, impaired renal function with increased plasmatic creatinine (mg/dL): 2.0± 0.1 vs 1.1±0.1 and urea (mg/dL): 92.3± 8.3 vs 51.9±5.2 and proteinuria (mg/24h): 40.1±3.9 vs 17.7±2.9 and also a reduction in creatinine clearance (mL/min): 1.0±0.1 vs 1.8±0.1. DM (PRE) vs CTL showed a significant increase in TBARS, a lipoperoxidation marker in plasma (nmol/mL), urine (nmol/24h) and kidney tissue (nmol/mg of protein): 5.0±0.7 vs 2.9±0.1; 446.7±19.2 vs 97.7±8.5 and 8.0±0.8 vs 5.3±0.5, respectively, and also a significant decrease in plasmatic NO (mM): 35.4±2.1 vs 54.4±3.8. Analysis of these two groups by means of western blotting showed that DM group (PRE) increased iNOS (0.8±0.2 vs 0.3±0.1) and decreased eNOS (0.9±0.1 vs 1.6±0.2), both p<0.05. Early NAC supplementation in DM rats reduced proteinuria (18.2±2.3), plasmatic creatinine (1.5±0.1) and urea (66.1±2.3) and a decrease in TBARS levels, in plasma, urine and renal tissue (2.6±0.1, 326.0±5.7 and 35.9±0.3), as well as increased creatinine clearance (1.9±0.4), plasma NO (59.8±5.2) and eNOS (1.3±0.1), and reduction of iNOS (0.4±0.1) vs DM (PRE), all p<0.05. This early treatment in DM rats significantly increased antioxidant defenses promoting increased catalase (1.3±0.1 vs 0.7±0.1) and glutathione (1.4±0.1 vs 0.9±0.1). In late treatment with NAC in DM rats as compared to DM rats without NAC, we found decreased proteinuria (32.6±6.0 vs 61.6±7.9) and TBARS excretion (284.8±26.0 vs 326.0±35.9) and increased creatinine clearance (1.4±0.1 vs 0.7±0.1) and NO plasmatic (60.1±3.4 vs 41.4±2.3), all statistically significant. Histological analysis of the diabetic rats without treatment with NAC, [DM (PRE) or DM (TAR)] showed significant tubular changes with degeneration and vacuolization in tubular cells, dilated tubular lumen, intense glycosidic degeneration, and discrete mesangial expansion with interstitial fibrosis area. DM + NAC (PRE) also showed intense glycosidic degeneration, but did not present tubular degeneration or fibrosis. DM+NAC (TAR) showed severe glycosidic degeneration, moderate tubular cell degeneration, light and focal dilatation of the tubules, without fibrosis. CONCLUSION : Our study showed that NAC protected the diabetic rats against renal injury, probably due to the control of oxidative stress and/or increased NO bioavailability, showing that early NAC was more effective than late treatment. This suggests that NAC may be useful in the adjuvant treatment of diabetic patients in the early phase of the disease. Eventually, prolonged treatment, even if it is started later, could change the natural history of the disease, improving survival and quality of life in diabetic patients.