| Objective: The present study observed the glomerular expression of Smad1 in streptozotocin-induced diabetic rats and the intervention effects of candesartan to examine the role of Smad1 in the development of mesangial matrix expansion in the early stage of diabetic nephropathy and the mechanism of candesartan's protective effects on diabetic kidney.Methods:Thirty-six male Wistar rats were randomly divided into 2 groups: normal control rats (NC group, n=12) and test rats (n=24). The test rats were induced diabetes by a single intraperitoneal injection of streptozotocin (STZ, 55mg/kg) after an overnight fast. Three days after STZ injection, the rats with blood glucose levels higher than 16.7mmol/l were considered as diabetes. The diabetic rats were randomly divided into diabetic model rats (DM group) and diabetic model rats treated with candesartan (DM-C group). 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). Glomerular histopathology was observed by microscope and transmission electron microscope. The expression of typeIV collagen (Col-IV) and Smad1 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 and positive index (PI) of type IV collagen expression 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 Smad1 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 Col-IV: Immunohistochemistry showed that the expression of Col-IV in NC group was farthing. The expression of Col-IV 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.The glomerular expression of Smad1: The protein expression of Smad1 was significantly increased in DM group (P<0.01), while almost undetectable in NC group by immunohistochemistry and Western blotting. The expression in DM-C group was reduced compared with DM group (P<0.01), whereas still higher than in NC group (P<0.01). 6. Correlation analysis: The glomerular expression of Smad1 was positively correlated with the glomerular expression of Col-IV (r=0.885, P<0.05) and MMF (r=0.891, P<0.01). 24-hour UAE was not associated with MMF (r=0.345, P>0.05).Conclusions: 1. In the early stage of diabetic nephropathy, the protein expression of Smad1 in glomeruli was significantly increased accompanying with mesangial matrix expansion . 2 . The glomerular expression of Smad1 was positively associated with the glomerular expression of type IV collagen and mesangial matrix expansion, indicating that Smad1 may play an important role for the development of glomerulosclerosis. 3 . Candesartan improved renal function and alleviated glomerular histopathological damage with no significant change in blood pressure in DM-C group. Our study suggested that candesartan may implement a renoprotective effect through down-regulating the glomerular expression of Smad1, which is independent of its effect on blood pressure.
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