Protective Effect And Its Mechanism Of Clopidogrel On Renal Ischemia Reperfusion Injury In Mice

Renal ischaemia reperfusion injury(IRI) leads to acute renal failure(ARF) in both native kidneys and renal allografts, which is a complex pathophysiologic process involving cell apoptosis and oxidant damage. Clopidogrel has been subjected to considerable pharmacological investigations that have revealed its protective effects on acute coronary syndromes and acute extremity ischemia. But there is no previously reported study about its effects on renal IRI. The aim of this study was to investigate the possible effects of clopidogrel on renal IRI in mice.Objectives:To explore influences of different renal ischemia time and gender on the renal IRI models, to evaluate possible effects of clopidogrel on renal IRI and explore relating mechanism of action in BALB/c mice.Methods:(1) The IRI was induced in the bilateral kidneys of 156 male and 30 female BALB/c mice. Renal function, serum creatinine and blood urea nitrogen, and pathology of the kidneys were examined at 24 h after IRI. (2) Clopidogrel treated renal IRI:Healthy male BALB/c mice were randomly assigned to one of the following groups:PBS+IRI, clopidogrel+IRI, PBS+sham, clopidogrel+sham. IRI mice were subjected to bilateral renal ischamia for 45 min followed by reperfusion. Kidney function tests, histopathological examination, renal cell apoptosis, renal antioxidant activities and CD41 expression were determined at 24 h after reperfusion and animals' survival was also examined.Results:(1)Renal IRI was generated successfully in 182 of 186 mice with a 97.85% success rate. The levels of serum creatinine and blood urea nitrogen were significantly increased in male mice subjected to 30 min,35 min, or 45 min of renal ischemia and in female mice subjected to 75 min of renal ischemia, compared to the control group at 24 h after operation. In males following 35 min or 45min of ischemia and in females following 75 min of ischemia, typical acute tubular necrosis was found in the areas of corticomedullary junction and the histopathologic scores, which represent the degree of renal tissue injuries, were significantly increased. (2)Pre-treatment with clopidogrel produced reduction in serum levels of blood urea nitrogen and creatinine caused by IRI, had a lower histopathological score and significantly improved animals' survival. Renal cell apoptosis induced by IRI was suppressed in kidneys of clopidogrel pre-treatment mice, involving the increase of bcl-2, bcl-xL activities and the decrease of caspase-3, caspase-8, bax activities by immunoblot analysis. The renal enzymatic activities of superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) were not significantly changed in any of the experimental groups and sham operation groups. However, pre-treatment with clopidogrel before IRI resulted in higher total antioxidant capacity (TAC) level of kidney than PBS pre-treated IRI group.Immunofluorescent studies showed clopidogrel pretreatment offered a noticeable decrease in fluorescence for CD41.Conclusions:Our findings suggest that (1) the kidneys of male are much more susceptible to IRI than those of female. The optimal ischemia time of kidney is 35 45 min in males and 75 min in females for generating a stable model of IRI in mice. (2) inhibition of platelet activation and aggregation by clopidogrel exerts protective effects on the renal IRI in mice by suppressing renal cell apoptosis via proteins of Bcl-2 and caspase family and improving renal antioxidant capacity. The protective effect conferred by clopidogrel reveals that clopidogrel may be a useful therapeutic strategy in renal IRI.