| Objective: To observe the change of CTGF expression in the kidneys of diabetic rats with the Combination therapy of angiotension II receptor antagonist Losartan and tetramethylpyrazine, analyze the relationship with the fibrous degeneration correlate factor COL-IV and study the effects of CTGF on the progression of diabetic nephropathy and the Reno-protective mechanism of Losartan and tetramethylpyrazine on Streptozotocin-induced diabetic Wistar rats.Methods: After uninephrectomy and the injection of streptozotocin (STZ) induced rats model with diabetes, the 40 Wistar rats selected were randomly divided into following 4 groups: normal control (n=10, NC group), diabetes control (n=10, DM group), diabetic rats treated with Losartan (20mg·kg'-d-1, by gavage, n=10, DM1 group) and diabetic rats treated with Losartan and tetramethylpyrazine (20 mg·kg-1·d-1 and 2 mg·kg-1·d-1, by gavage, n=10,DM2 group). Following food deprivation for 10h, DM, DM1 and DM2 groups were treated respectively with peritoneal injection of 50mg/kg STZ for one time, but NC group was intraperloneally injected with the equal citric acid buffer. After 72 hours, the level of blood glucose was detected in venous blood of tails. The rats with fasting blood glucose (FBG) equal to or more than 16.7mmol/L served as diabetic rats. All rats of diabetic groups were not treated with insulin or other anti-diabetes drugs in experimental procedures. After raising rats of four groups for 8 weeks, the rats in the four groups were killed. The expressions of CTGF and COL-IV in kidney were identified by immunohistochemistry and the mRNA expression levels of CTGF and COL-IV in kidney were detected by revese transcription polymerase chain reaction (RT-PCR). In addition, 24-hour urine albuminuria excretion rate (UAER), glycosylated hemoglobin (HbAlc), urinary β2-MG excretion and creatinine clearance (Ccr) were observed by light microscopy after 8 weeks.Results: There were no significant differences among all groups before this experiment (p>0.05). After 8 weeks, the levels of Plasma glucose, HbAlc, Ccr, UAE, β2-MG and expressions of CTGF, COL-IV in DM, DM1 and DM2 groups were increased more significantly than NC group (p<0.05). The immunoreactive positive products of CTGF and COL-IV were mainly distributed in kidney proximal tuble cytoplasma compared to glomeruler. The results of immunohistochemistry and RT-PCR showed that the expressions of CTGF and COL-IV in DM1 and DM2 groups were much lower than DM group (p<0.01). It means that Both Losartan and tetramethylpyrazine could significantly inhibit the expressions of CTGF and COL-IV in diabetic renal tissue (p<0.05), but not make it recovery. Treatment with Losartan and tetramethylpyrazine can inhibit the expressions of CTGF and COL-IV further. The correlation analysis showed that there was significant positive correlation of expression between CTGF and COL-IV, 24h-urinary protein excretion. Conclusion:1. Expression of CTGF, COL-IV were upregulated in glomeruli of diabetic rat and urinary protein excretion of 24h was also increased. The treatment of Losartan and tetramethylpyrazine can downregulate the abnormal expression of CTGF and COL-IV partly at least and reduce urinary protein excretion of 24h.2. Expression of COL-IV have been demonstrated to increase in glomeruli of diabetic rat and have close relationship with CTGF. The use of Losartan and tetramethylpyrazine has been shown to improve partly the expression levels of ECM and CTGF. Further, it can benefit to delay the progression of diabetic nephropathy.3. CTGF and COL-IV play an important role on the progression of diabetic nephropathy, but it shouldn't be ignored that they may injure the kidney tuble. We have demonstrated that both Losartan and tetramethylpyrazine have many significant effects on diabetic nephropathy. Moreover, Treatment combined with Losartan and tetramethylpyrazine can protect kidney from diabetic injury. But their exact mechanism of associated-renoprotective effects remains to be further studied.
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