| Objective The protective effects of endothelin-1 mRNA antisense oligodeoxynucleotide (ET-1 AS-ODN) on diabetic nephropathy in early diabetic rats were investigated by inhibiting gene expression with ET-1 AS-ODN.Methods 1. 89 male SD rats were randomly divided into 3 groups: normal control (NC group, intravenous injection of normal saline 0.5ml/rat/wk via the caudal vein), diabetesmenitus control group (DM group, normal saline 0.5ml/rat/wk iv), and treatment group (ET-1 AS-ODN 6 OD/kg/4d iv.) 65 rats of all were used to establish the experimental rat model of diabetes with intraperitoneal injection of STZ at 60mg/kg. Each group was further divided into three subgroups: 2-week group, 4-week group and 8-week group. Body weight, blood glucose and 24-h urinary output were observed in all groups. 2. Renal function was observed in terms of blood urea nitrogen (BUN) and urine creatinine (Ucr) by chemical colorimety at 2, 4 and 8 weeks after successful establishment of the model. 3. To investigate more changes in the renal function, the urinary UAERumin excretion rate (UAER),β2-MG and ET-1 were examined by radioimmunoassay (RIA) at different time points. 4. Morphologic changes in renal cortex were observed by light microscopy and electron microscopy. 5. mRNA expression of TGF-β1 was detected quantitatively by real-time quantitative PCR to see whether DN proliferation and mesangial matrix expansion were affected by ET-1 AS-ODN. Results:1. The level of blood glucose remained within a range of 19.0~40.0mmol/L after the establishment of the diabetic model, and there was no significant difference between DM group and ET-1 AS-ODN treatment group (P>0.05). 24-h urine output in the treatment group increased significantly compared with NC group (P<0.01). The body weight of NC group gradually increased as measured at 2-, 4- and 8-week points, and the body weight at the 8-week point increased by 47.7% compared with that at the 2-week point. The body weight of DM group at the three time points gradually reduced, and by 31.2% at the 8-week point compared with that at the 2-week time point (P<0.01). In the treatment group, the growth ratio of the body weight at the 8-week time point was 17.2% compared with that at the 2-week point (P<0.01). 2. No significant renal dysfunction was observed in DM group during first 2 weeks after the establishment of the diabetic model. The levels of the BUN, Scr and Ccr in DM group increased by 43.8%, 11.4% and 92.8% respectively at the 4-week point, compared with those of NC group (P<0.01). At the 8-week point, BUN, Scr and Ccr levels in DM group increased by 54%, 22.4% and 93.8% respectively compared with those in NC group (P<0.01). After 8-week therapy, BUN and Scr levels in the treatment group decreased by 17.7% and 13.7% respectively but Ccr level in treatment group heightened by 42.4%, compared with those of DM group (P<0.01). 3. The changes of UAER andβ2-MG in different groups: UAER measured at 2, 4 and 8 weeks was 0.34 mg/24h, 1.12 mg/24h and 1.89 mg/24h respectively, all of which were higher than those in NC group. After the administration of ET-1 AS-ODN for 2 weeks and 4 weeks, UAER in the treatment group restored to normal level. After 8 weeks, the level of UAER in the treatment group was lower than that in DM group by 76.8% (P<0.01). Urinaryβ2-MG in the treatment group after 8-week therapy was 65% lower than DM group (P<0.01). The above data suggest that ET-1 AS-ODN had a protective effect against early diabetic kidney damage. 4. The results of electron microscopy of DM group showed that the glomerular basement membrane (GBM) was irregularly thickened; the number of mesangial cells, endothelial cells and foot cells slightly increased; and the foot process partly fused 4 weeks after the establishment of the diabetic model, compared with 2 weeks before. The results of electron microscopy at 8 weeks showed that GBM was irregularly thickened, mesangial stroma was increased, the mesangial area was enlarged, and the foot process fused incompletely. All these microstructural changes were alleviated in the treatment groups at 8 weeks, as represented by a 54.3% decrease in mean thickness of MGBM in the treatment group, compared with DM group(P<0.01)(Fig. 2). 5. The results of RIA and real-time PCR showed that the level of ET-1 protein expression in the treatment group decreased by 60.1% as compared with that in DM group (P<0.01), and the mRNA expression of TGF-β1 of the renal cortex in the treatment group decreased by 2.11-2.55 fold compared with DM group.Conclusion: 1. ET-1 AS-ODN improved renal function by delaying the progression of glomerulosclerosis in diabetic rats. 2. The mechanisms of the protective effects of ET-1 AS-ODN could partly involve the regulation of mRNA expression of TGF-β1 by preventing the expression of ET-1.This is a target point of the ET-1 effect after theETaR again...
|
PDF Full Text Request