The Renoprotective Role Of Rosiglitazone And Metformin In Rat Model Of Type 2 Diabetes Mellitus

Objective: Diabetic nephropathy(DN) is one of the major microvascular complications of type 2 diabetes mellitus and clinically defined by persistent proteinuria more than 500 mg/24 hours in a person with diabetic retinopathy without other renal disease. The major clinical character of DN is microalbuminuria, which is associated with hyperglycemia, elevated BP, smoking, and hyperlipidemia. With the development of economy and the improvement of people's living standard, morbidity of diabetic nephropathy is escalating year by year. Rosiglitazone belongs to thiazolidinediones (TZDs)which is a kind of novel insulin sensitizer.TZDs are believed to work through binding and modulating the activity of a family of nuclear transcription factors termed peroxisome proliferator-activated receptor γ(PPARγ). It is associated with slow improvement in glycemic control in parallel with an improvement in insulin sensitivity and reduction of FFA levels. TZDs have renoprotective function which are independent of insulin/glucose effects and are associated with regulation of messangial cell proliferation and hypertrophy. The major action of Metformin is to reduce hepatic insulin resistance and thereby gluconeogenesis and glycogenolysis.Metformin not only consistently can demonstrate effects to improve insulin sensitivity in peripheral tissues,but also plays significant role in decreasing hyperlipemia and hypertension and in improving hemodynamic changs and the diastolic function of vascular smooth muscles,which is beneficial to diabetic microvascular. Therefore, in this study rats model of type 2 diabetic was set up by feeding female SD rats with a high-sucrose-high-fat diet and by injecting them streptozotin intrapenitoneally. Rosiglitazone and metformin were given to type 2 diabetic rats respectively after 8 weeks. On this condition, we observe the abnormal metabolism linked to insulin resistance and investigate the pathophysiological mechanism of diabetic nephropathy. Morever,we want to make it clear what roles rosiglitazone and metformin play in preventing the evolvement of diabetic nephropathy. Methods: Female SD rats were randomly assigned into two groups: normal control group(NC) and diabetic group(DM). Normal control group rats were fed with the normal diet, while diabetic group were fed with the diets containing 10%sucrose, 15%lard, 2%cholesterol and 0.25%cholic acid to induce insulin resistance. Hyperglycemia was developed by intrapenitoneal injection STZ (30mg·kg^-1) in diabetic group rats after 4 weeks on this diets . At 8 weeks diabetic rats were randomly divided into three groups: diabetic control group (DM-C),diabetic group treated with rosiglitazone (3mg·kg^-1d^-1) (DM-R) and diabetic group treated with metformin (625mg·kg^-1d^-1) (DM-M). Body weight, fasting blood glucose, fasting serum insulin, serumtriglyceride and serum cholestrol were measured respectively after 4weeks,8weeks and 18weeks. Serum transforming growth factor β₁ (TGF-β₁),serum leptin ,endogenous creatinine clearance rate(Ccr)and urinary albumin excretion rate (UAER) were respectively observed after 18 weeks. Kidney specimens were prepared for H.E.-stained, PAS-stained and immunohistochemistry. The protein expression of TGF-β1 was examined by immunohistochemistry.The kidney tissue was observed by light microscope and electron microscope.Mean glomerular area(MGA) was calculated by analysis system of image. Morever, TGF-β1mRNA expression in renal cortinal was analysed with reverse transcription polymerase chain reaction(RT-PCR).The correlation between the risk factors of diabetic nephropathy linked to insulin resistance and insulin sensitivity index were respectively analyzed. Results 1. In diabetic group, fed with high-sucrose-high -fat diet for 4 weeks, body weight was markedly increased compared with normal control group (P<0.01); serum triglyceride, serum cholestrol and serum insulin level were increased compared with normal control group (P<0.05, P<0.05, P<0.01); fasting blood glucose had no difference with normal control group (P>0.05); Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was markedly increased compared with normal control group (P<0.01).So insulin resistance was induced indiabetic group . 2. After diabetic group was injected a low dose of STZ intrapenitoneally, at 8 weeks fasting blood glucose level was markedly increased compared with normal control group (P<0.01),and serum insulin level was decreased compared with 4 weeks before, but had no difference with normal control group (P>0.05); HOMA-IR was markedly increased compared with normal control group (P<0.01).So insulin resistance still existed in diabetic group . Body weight was increased compared with normal control group, but no markedly significant(P<0.05). In addition, serum triglyceride and serum cholestrol level were also increased compared with normal control group (P<0.01,P<0.01). Now the rat model of type 2 diabetes mellitus with insulin resistance had been set up successfully. 3. After 18 weeks, in DM-C, kidney weight and kidney weight/body weight were markedly increase compared with NC (P<0.01,P<0.01). Compared with DM-C there were significant decrease in kidney weight and kidney weight/body weight in DM-R and DM-M (P<0.05,P<0.05; P<0.01, P<0.01). Compared with DM-C there were significant decreases in fasting blood glucose and serum insulin in DM-R and DM-M (P<0.05, P<0.01; P<0.01, P<0.05); compared with DM-C, HOMA-IR was markedly decrease in DM-R and DM-M (P<0.01,P<0.05). Compared with DM-C there was a significant decrease in serum triglyceride in DM-R and DM-M (P<0.01,P<0.05). 4. Compared with DM-C, there were significant decreasesin urinary albumin excretion rate (UAER) , serum TGF-β1 and leptin in DM-R (P<0.05,P<0.05,P<0.05), but these were not markedly decreased in DM-M(P>0.05). 5. Compared with NC, protein and mRNA expression of TGF-β1 was markedly increased in DM(P<0.01,P<0.01). Compared with DM-C there were significant decreases in the protein expression of TGF-β₁ and TGF-β₁mRNA in DM-R but not in DM-M (P<0.05,P<0.01; P>0.05, P>0.05). 6. Light microscopy shows: Compared with NC, Glomerular hypetrophy, mesangial expansion, thickening of glomerular basement membrance in DM-C. Electron microscope shows: an increase in mesangial matrix, With progression, capillary wall thickening and mesangial widening lead to capillary narrowing.it was improved in DM-C and DM-M. 7. HOMA-IR was significantly correlation with kidney weight/body weight, serum TGF-β1, serum leptin and serum TG. UAER was significantly correlation with kidney weight/body weight ,serum TGF-β1, serum leptin, serum TG. Conclusions 1. Fed with high-sucrose-high -fat diet for 4 weeks, then rats were injected a low dose of STZ intrapenitoneally, by which the rat model of type 2 diabetes mellitus can be established. This rat model of type 2 diabetes mellitus with hyperglycemia, dyslipidemia, IR and typical renal leisions. It is similar to type 2 diabetes of human being. So this model may be used to study...