| Objective: The present study observed the kidney and retina expression of the vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in streptozotocin-induced diabetic rats and the intervention effects of candesartan to examine the role of VEGF and CTGF in the development in the early stage of diabetic nephropathy and retinopathy,and the mechanism of candesartan's protective effects on diabetic kidney.Methods:Thirty-six male Wistar rats were randomly divided into 3 groups: A group for the normal control group (NC group, n = 12), B group and diabetic group (DM group, n = 12), C group candesartan treatment group (DM-C group, n = 12).DM, DM-C group induced rat model of type 1 diabetes by a single intraperitoneal injection of streptozotocin (STZ, 55mg/kg) after an overnight fast.NC group were intraperitoneal injected with only citric acid buffer fluid. Three days after STZ injection, the rats with blood glucose levels higher than 16.7mmol/l were considered as diabetes.The DM-C group were treated with candesartan 5mg?kg-1?d-1 and other groups were treated with the same volume of normal saline. After 12 weeks of intervention, systolic blood pressure(SBP) was measured by the cuff-tailed method, and all rats were placed in metabolic cages for 24 hours to collect urine samples, which were used to determine urinary albumin excretion(UAE) and urinary creatinine.The rats were weighted and sacrificed to collect plasma samples for the assay of fasting blood glucose(FBG), serum creatinine and calculate creatinine clearance rate(Ccr). Cut left kidney, removed capsule and weighed , and remove the eyes, Glomerular histopathology was observed by microscope and transmission electron microscope,Retinal morphological changeswas observed by microscope.The expression of VEGF and CTGF in glomeruli were determined by immunohistochemistry. Results were analysed by Image-Pro Plus image-analysis system, including the measurement of Mean Glomerular Area(MGA),PAS positive staining area in glomeruli.Mesangial Matrix Fraction(MMF) (the ration of PAS positive staining area to average section area of glomeruli) was calculated. Mean Glomerular Volume(MGV) was caculated by the equation[β/K×(MGA)3/2]. Glomeruli were isolated from renal cortices of rats for examining the expression of VEGF and CTGF by western blotting.Results: At the end of the experiment, 10 rats in NC group, 8 rats in DM group and 8 rats in DM-C group survived.1 FBG, 24-hour UAE, kidney hypertrophy index(kidney weight/body weight, KW/BW) and Ccr in DM and DM-C group were significantly increased compared with NC group( P<0.01, P<0.01). After candesartan intervention, all the above indicators were significantly decreased compared with DM group except FBG(P<0.01).2 After candesartan intervention, there was no significance in SBP among these groups(P>0.05).3 Pathomorphological changes of the glomerulus: HE and PAS staining showed that the Mean Glomerular Area(MGA) and Mean Glomerular Volume(MGV) were significantly augmentated(P<0.01) and there were increased PAS-positive materials, expansion of mesangial area, hyperplasia of mesangial cells in diabetic glomeruli compared with NC group. Also the mesangial matrix fraction(MMF) was significantly increased in DM group(P<0.01). The abnormal changes were alleviated after candesartan intervention compared with DM group(P<0.01), although not normalized. Transmission electron microscope showed an increased thickness of glomerular basement membrane and confluence of foot process of podocyte in diabetic rats; whereas theses abnormalities were partly relieved in DM-C group.4 The glomerular expression of VEGF and CTGF: Immunohistochemis -try and western blot showed that the expression of VEGF and CTGF in NC group was farthing. The expression of VEGF and CTGF in DM group was higher than that of NC group(P<0.01); and the expression in DM-C group was lower than that of DM group(P<0.01), whereas still higher than that of NC group(P<0.01).5 Pathological changes of the retina: HE staining, The retina of DM group rats was thinner than NC group and the number of ganglion cells was significantly reduced than NC group (P <0.01); DM-C group compared with the DM group were significantly improved (P <0.01) ,However, these indicators was still increased compared with the NC group(P <0.01).6 The Retinal expression of VEGF and CTGF: Immunohistochemistry showed that the expression of VEGF and CTGF in NC group was farthing. The expression of VEGF and CTGF in DM group was higher than that of NC group(P<0.01); and the expression in DM-C group was lower than that of DM group(P<0.01), whereas still higher than that of NC group(P<0.01).Conclusions:1 In the early stage of diabetic nephropathy, the protein expression of VEGF and CTGF in glomeruli was significantly increased accompanying with mesangial matrix expansion .2 In the early stage of diabetic retinopathy , the protein expression of VEGF and CTGF in Retinal was significantly increased .3 Candesartan improved renal and retinal function and alleviated glomerular and retinal histopathological damage with no significant change in blood pressure in DM-C group. Our study suggested that candesartan may implement protective effect of renal and retinal through down-regulating the expression of VEGF and CTGF, which is independent of its effect on blood pressure. |
