Effect Of Metformin And Repaglinide On The Expression Of TGF-β1 MRNA In Diabetic Mouse Kidney

Diabetic nephropathy (DN) is a common microvascular complications of diabetes, which is the main reason of renal failure at the final stage. The percentage of diabetes patients who suffer from DN in the whole diabetes patients has reached 20% -40%. The early pathological features of DN are glomerular hypertrophy, thickening basement membrane and accumulating Extracellular matrix (ECM) at glomerular mesangial. The late pathological features of DN are glomerulosclerosis and interstitial fibrosis. The etiology and pathogenesis of DN have not yet fully been understood. Transforming growth factor-β1 (TGF-β1) is considered to be the "final common pathway" of diabetic nephropathy, and if this pathway has been blocked off, the kidneyslesions can be blocked. Metformin and repaglinide are common blood glucose-lowering drugs. 90% of metformin are excreted through renal in prototype and the amount of repaglinide through renal is less than 8%. Few researches have been reported about the influence of these two drugs on kidney and TGF-β1mRNA expression when they are used in the diabetic patients. In this study, we compared metformin to repaglinide in the influence on expression of renal TGF-β1mRNA as well as renal histopathology of diabetic mice.Purpose:To compare metformin to repaglinide in kidney damagement and the influence on expression of kidney TGF-β1mRNA of diabetic mouse.Method:1. 40 healthy male C57 mice were randomly divided into normal control group and model group with 10 rats and 30 rats in control one and model one respectively. Mice in normal control group were fed with normal diet and mice in model group were fed with high fat diet. After 8 weeks, the abdominal of mice were injected with streptozotocin (STZ) 80mg/kg which was deployed with 0.1mol / l sodium citrate buffer; and Equal volume citric acid - sodium citrate buffer was injected into the abdominal of mice in normal group with the dose of 0.1mol/l after 12h fasting. After 72 h, we measured the tail vein blood glucose by blood glucose test strips and the diabetic mice whose random blood sugar≥16.7mmol / l were defined as diabetic model group. 30 diabetic mice were randomly divided into diabetic model group with 10 rats, metformin treatment group with 10 rats and repaglinide treatment group with 10 rats after the model was successful maded.2. After all the mice were divided into groups, we poured the drugs which were dissolved in distilled water into the stomach of mice at 9 am each day. The dose of metformin solution was 300mg/kg·d in metformin treatment group while the dose of repaglinide solution in repaglinide treatment group was 2mg/kg·d. All the drugs were diluted with 0.1ml distilled water.The same volume of distilled water were poured into the stomach of mice in normal and diabetic group. One rat in each drug-used group became dead during the course of pouring drugs into stomach. We observed the diet, water intake, mental state, activity, stool and urine output each day and the blood glucose and weigh of mice were measured per week. All the mice were put to death after 8 weeks and then 100mg renal cortex were cut from the left kidney, which would immediately be put into liquid nitrogen. We detected the expression of mouse kidney TGF-β1 mRNA levels by using real time quantitative PCR method. The rest were stained in hematoxylin - eosin (HE), VG and PAS method after being made into frozen sections and paraffin sections. The changes of kidney tissue of mice were observed by light microscope and collagen surface density of each group were analyzed by using the semi-automatic image analysis system. We taked the same kidney section of each specimen and got 30 glomeruli in different horizons. Then PAS staining level of each glomerular was determined. 3. Statistical Methods: Data was analyzed by SPSS13.0 statistical software. Comparison of data was analyzed by repeated measures analysis of variance, One-Way ANOVA and Chi-square test. We defined statistically significant by P <0.05.Results:1. The changes of blood glucose and body weight: The blood glucose and body weight were significantly higher in diabetes model group, metformin and repaglinide group than those in normal group before treatment (P<0.01). The blood glucose in metformin and repaglinide group decreased significantly than those in the model group after treatment(P <0.01). And blood glucose was significantly lower in metformin group than that in repaglinide group(P<0.01). The body weight in diabetes group significantly decreased after treatment(P<0.05). The decrease of body weight in metformin and repaglinide group was significantly lower than those in diabetes group (P<0.01). And the decrease of body weight in repaglinide group was significantly higher than that in metformin group(P <0.01).2. Pathological observation by light microscope: Mice in normal group showed normal glomerular structure, no proliferation of mesangial cells and matrix, no PAS-positive material at glomerular basement membrane and mesangial and capillary cavity opening in normal group. However, the number of glomerulus cells and matrix inherent increased and capillary lumen narrowed and some glomerular appeared leaf hardening and narrow apparently in diabetes model group. VG staining showed bright red collagen fibers increased and disordered in basement membrane and renal interstitial. Capillary walls and mesangial areas indicated strongly positive for PAS staining, suggesting that the glomerular basement membrane thickening, large accumulation of mesangial matrix, oppression capillary lumen, and even atresia. Compared with model group, the structures of glomerular in metformin and repaglinide group were significantly improved. The proliferation of glomerular cell reduced. The mesangial proliferation and basement membrane thickening significantly reduced. Collagen fibers had different degrees of reduction and PAS positive material was significantly reduced while improvement in repaglinide group was the most obvious.3. Real time-PCR analysis: Renal expression of TGF-β1 mRNA increased significantly in diabetic model group than that in the normal group(P <0.01).While Renal expression of TGF-β1 mRNA significantly reduced in metformin and repaglinide group than that in diabetic group (P<0.01) and it decreased significantly in repaglinide group than that in the metformin group (P<0.01).Conclusion:1. Metformin reduced blood glucose in diabetic mice more significantly than repaglinide did.2. Repaglinide was superior to metformin in improving renal pathological changes in diabetic mice.3. The expression of renal TGF-β1mRNA in diabetic mice was significantly higher than that in the normal mice and the repaglinide is superior to metformin in reducing the expression of TGF-β1 mRNA. Keywords:...