The Relationship Between Renal Microangiopathy And Lipotoxity In The Rats Fed With High-fat-diet And Its Mechanism

Objective:Male SD rat model of obesity was established by feeding with high-fat-diet to evaluate the peripheral insulin resistance via euglycemic hyperinsulinemia clamp and observe the changes of function and structure in the kidney.The aim was to study the effect of the lipotoxity on renal microangiopathy and the effects of pioglitazone intervention in long-term high-fat-diet fed rat.Methods:45 male SD rats weighed 250g±20g, 8 weeks old, were randomly divided into 3 groups after feeding normally for a week: a normal control group(NC,n=15), a group fed with high-fat-diet(HF,n=15), and a group treated with pioglitazone (HP,n=15,pioglitazone 15mg·kg-1·d-1 and high-fat-diet).Normal control group were fed with standard diet, and high-fat-diet group were fed with standard diet added by pork oil, which mainly contained triglyceride. At the end of 32nd week, we collected 24 hours urine of rats by using metabolic cages for determining 24 hours urinary albumin, and collected rats'blood serum to determine fasting blood glucose(FBG), fasting serum insulin(Ins), triglyceride(TG),free fatty acids(FFA).The glucose infusion rate (GIR) was measured by using euglycemic hyperinsulinemia clamp to evaluate the peripheral insulin resistance. All rats were killed at the end of euglycemic hyperinsulinemia clamp test. The left kidney were isolated for histological examination, which were fixed in 4% formaldehyde for Hematoxylin-eosin (HE) stain, periodic acid-schiff (PAS) stain and immunohistochemical stain to observe the changes of function and structure in the kidney of rats and evaluate the expression levels of vascular endothelial growth factor(VEGF), intercellular adhesion molecule-1(ICAM-1) in renal cortex.The other kidney went through quickly freezen slice by liquid nitrogen for Real-time PCR to detect the expression of VEGF, ICAM-1 gene in renal cortex. The visceral fat in abdominal cavity, capsula renis, didymus were isolated to weight at last .Serum fasting blood glucose, fasting serum insulin, 24 hours urinary albumin, triglyceride and free fatty acid were measured by Olympus AU 400 auto–biochemical analyzer, radio-immune assay,and enzymic method respectively. Euglycemic hyperinsulinemia clamp was used to evaluate the peripheral insulin resistance of rats. The rats were given a continuous intravenous infusion of regular human insulin (Novolin R) at a rate of 12 mU.min-1.kg-1. Blood specimens were obtained from the left carotid arterial catheter at 10-min intervals for measuring plasma glucose levels using a glucose analyzer.Based on these values, 20% glucose solution was variably infused to maintain euglycemia (5.0±0.2mmol/L). The test was maintained for 120 mins and at last calculated glucose infusion rate. HE stain, PAS stain were used for the general pathologic observation of the kidney. Immunohistochemical stain (SP method) was used to measure the expression levels of VEGF, ICAM-1 in renal cortex. They were colorated by DBA method, and semiquantitativly analyzed by IPP5.1 image analytical system. The mean density of VEGF, ICAM-1 per renal glomerulus in each group were compared (total IOD/total area per renal glomerulus).Detecting the expression of VEGF, ICAM-1 gene in renal cortex by Real-time PCR. Extraction of total renal RNA was performed by a one-step protocol using Trizol.The renal cDNA of rats were synthesed by ReverTra Ace-αcDNA kit, and the reliable quantitative real-time PCR amplification was perform via real-time PCR instrument (ABI?PRISM 7300). Housekeeping gene was glyceraldehyde-3-phosphate (GAPDH), and all of the primers were producted by Shenggong company in Shanghai .At last the 2-??CT method was used to work out the result.SPSS10.0 was used to process all data. Quantitative data were expressed as x±s. T-test was for the comparison between two groups. If P<0.05 or p<0.01, it was considered that the data of two group were statistical significant. Result:1 The general feature of rats: Compared with normal control group, the body weight and visceral fat weight of the rats in model group and HP group were remarkably high(P<0.01).The serum insulin, triglyceride, free fatty acids concentration in HF group were higher than in NC group(P<0.01 or P<0.05). The 24 hours urinary albumin was increased in HF group compared with NC group (P<0.05). Pioglitazone intervention can reverse these effects. 2 The glucose infusion rate was decreased significantly in HF group compared with NC group ((4.8±1.2) mg.min-1.kg-1 vs (11.3±1.7) mg.min-1.kg-1, P<0.01), that in HP group was (10.1±1.5) mg.min-1.kg-1. 3 Analysis of HE and PAS stained renal sections revealed that most glomeruli in normal kidney exhibited normal glomerular histologic features, with patent capillary loops, normal basement membranes, a delicate mesangium, and small Bowman's space.The most striking glomerular characteristic in HE and PAS-stained sections of the kidney of rats fed with high-fat-diet was a expansion of Bowman's capsule space, prog- ressive development in mesangial hypercellulary and increased mesangial matrix. All of these in HP group were ameliorated. 4 Image analysis of renal immunohistochemistry showed that mean density of expression levels of VEGF, ICAM-1 per renal glomerulus were significantly higher in HF group than in NC group (0.0797 vs 0.0127, P<0.01; 0.0653 vs 0.0145,P<0.01); and that in HP group were 0.0301 and 0.0148 respectively, which remain higher than NC group. 5 The gene expression of VEGF was significantly increased by 21% in the HF group than in NC group (P<0.01),and the gene expression of ICAM-1 was significantly increased by 43% (P<0.01), while the expression of VEGF, ICAM-1mRNA in HP group decreased by 30% , 15% respectively than that in NC group(P<0.01). Conclusion:1 Obesity model rats fed by high-fat-diet could be established, which possess the character of insulin resistance, hyperlipiodemia and disturbance of carbohydrate metabolism. 2 High-fat-diet induced increased renal microangiopathy and 24 hours urinary albumin, which may related with the high expression of VEGF and ICAM-1 in kidney that were mediated by the increased free fatty acids. 3 Intervention with Pioglitazone can reverse these effects.