The Effect Of Atorvastatin On ENOS Expression In Diabetic Rat Kidney And Its Possible Role In Diabetic Kidney

Objective: Diabetic kidney disease(DKD)is one of the major microvascular complications of diabetes mellitus(DM),which will lead to end-stage renal failure,a fatal threat to the patients.Although it is widely accepted that the clinical benefit obtained with statins is a direct result of their lipid-lowering properties,these agents appear to display additional cholesterol-independent or pleiotropic effects on diabetic patients.Nitric oxide may improve the glomerular filtration rate and renal plasma flow through dilating the inferent and efferent arteriole.This study aimed to investigate the effect of atorvastatin on the up-regulation of endothelial nitric oxide synthase(eNOS)and NO level in type 2 diabetic rat kidney and clarify a possible protection of atorvastatin on DKD patients by eNOS-NO pathway,finally to provide new strategies for clinical treatment for diabetic kidney disease.Methods:54 healthy female Sprague-Dawley(SD)rats weighed160-200 g were divided randomly into two groups.The normal control group(NC group)rats received standard chow.The experimental type rats were given a high carbohydrate and high fat diet all through and an intraperitoneal injection with STZ at the dose of 30mg/kg.At week 10,insulin resistance in the diabetic rats was confirmed by the HOMA-IR,we measured the body weight(BW)and biochemical index,including the fasting blood glucose(FBG)? fasting insulin(FINS)? triglycerides(TG)and total cholestero1(TC).Then the rats were randomly taken into three groups:NC group,diabetes mellitus group(DM group)and atorvastatin group(A group).We measured blood glucose and body weight weekly.At week 21,FBG,FINS,TG,TC,alanine transaminase(ALT),aspartate aminotransferase(AST),serum creatine(SCr),UREA,HOMA-IR and BW were measured totally.The kidneys were quickly resected and weighted(kidney weight,KW).Kidney index(KI)(KW/BW)was calculated for each rat.Kidney specimens were prepared for HE-stained and PAS-stained to observe morphology changes.Take part of the renal cortex homogenate to examine NO level.The contents of eNOS m RNA in renal cortex were evaluated by real-time PCR,and the expressions of eNOS in renal cortex were assessed by immunohistochemical staining.Data analysis was performed by SPSS 21.0,and P<0.05 was considered statistically significant difference.Results:1 BW changes At week 10,the BW of the diabetic rats was significantly higher than the NC group(P<0.05).At week 21,compared to the NC group,BW of the rats in DM group and the A group were significantly lower(P<0.01).No significant difference was observed between DM group and the A group(P>0.05).2 Biochemical indexAt week 10,the levels of FBG,TG,TC and FINS of the diabetic rats were significantly increased when compared with NC group(P<0.01).At week 21,the levels of FBG,FINS,TG,TC and Hb Alc of the rats in DM group and A group were increased obviously compared to NC group(P<0.01,P<0.05),but there was no significant difference between the DM group and A group(P>0.05).UREA level in DM group and A group was higher than NC group(P<0.05),no difference was found between the DM group and A group(P>0.05).Among the three groups,the levels of SCr,ALT and AST had no significant difference(P>0.05).3 Sensitivity to insulinAt week 10,the levels of HOMA-IR of the diabetic rats were much higher than those in NC group(P<0.01).At week 21,the levels of HOMA-IR in DM group and A group were increased significantly(P<0.01).No difference was confirmed between DM group and A group(P>0.05).4 Kidney Index(KI)At week 21,KI of rats in DM group was higher(P<0.01)than that in NC group,the KI of rats in A group was also increased(P<0.01),but it waslower(P<0.01)than that in DM group.5 Morphology changes:The rats in NC group had normal glomerulus and renal tubule.The DM group rats showed glomerular hypertrophy,mesangial expansion,irregular thickening of basement membranes,as well as increased deposition of PAS positive substance.Part of the renal tubules appeared cell vacuoles degeneration,and the tube lumen narrowed.Affection to lighten slightly in A group rats,but still had the difference with NC group.6 NO level in renal cortexFor the level of NO in renal cortex of rats,DM group and A group was decreased compared with NC group rats(P<0.01),the level of DM group was much lower than A group(p<0.01),the difference was statistically significant.7 Immumohistochemical stainingThe brown color in the cytoplasm denoted positive staining.Compared to NC group,the protein expression of eNOS in renal cortex was decreased in DM group and A group(P<0.01,P<0.05).The level of eNOS in DM group was lower than that in A group,treated with atorvastatin(P<0.05).8 ENOS m RNA by real-time PCRThe eNOS m RNA level in NC groups was the highest(P<0.01);the level of eNOS m RNA in A group rats was up-regulated than A group(P<0.01).Conclusions:1 The type 2 diabetic rat models were induced by an intraperitoneal injection with STZ at the dose of 30mg/kg and the high carbohydrate and high fat diet.With the progress of the disease,kidney morphology changes of rats were silmilar to DKD patients.2 Compared to the normal rats,the decrease of eNOS and NO in renal cortex was observed in diabetic rats.Atorvastatin could up-regulate the levels of eNOS and NO in type 2 diabetic rats kidneys,with obvious improvement of renal pathological changes,but no significant effect on FBG,Hb A1 C and HOMA-IR,which showed a possible cholesterol-independent protection on kidney.