Effects Of Valsartan Prevent Renal Interstitial Fibrosis In Diabetic Nephropathy Rats

Background and ObjectivesDiabetic nephropathy (DN),as a serious chronic microvascular complications of diabetes,has become the major cause of end-stage renal disease(ESRD).Renal interstitial fibrosis is a common pathological features of ESRD.Thus the treatment of delaying or even reversing renal interstitial fibrosis in DN as early as possible are very important. Valsartan is an angiotensin receptor antagonist, has a protective effect in kidney deseases,but the mechanisms of prevention the interstitial renal fibrosis are not yet fully understood. The experiment had induced the rat model of diabetic nephropathy by streptozotocin(STZ), and intervented with Valsartan (ARB). We detected HIF-la,TIMP-1 and MMP-9 expressions in kidney tissue, measured urinary protein and other biochemical indicators,and illustrated the pathogenesis of diabetic nephropathy interstitial fibrosis, the possible mechanism for angiotensinⅡreceptor antagonist(valsartan) to protect renal function was also investigated,in order to provide theoretical basis for the prevention and treatment of diabetic nephropathyMethodsFifty-four healthy male Sprague-Dawley (SD) rats were were randomly divided to 3 groups:normal control group(C group), diabetes nephropathy group(D group), treatment group with valsartan (T group) (valsartan 40mg/kg·d). The rats of D and T groups received a signal intraperitoneal injection of STZ at a dose of 60mg/kg WT and the rats of group C only received an injection of the same volume of sodium citrate. The diabetic model was considered to be successful when the blood glucose was 16.7mmol/L after 3 days of STZ injection. After 3 weeks, collecting the urine of 24 hours through metal metabolism cages, and measuring urinary protein of 24 hours. The rats of urine protein of 24 hours≥30mg were succeed.The treatment group rats were treated with valsartan(40mg/kg·d) and the rats of group C and D were treated with the same volume of tap water. At 4th,8th and 12th weeks, serum creatinine, serum albumin, blood glucose,24-hour urine protein, kidney index (kidney weight /body weight) were measured; then six rats were sacrificed for each group respectively.The methods of immunohistochemistry and reverse transcriptase-PCR (RT-PCR) were used to detecct the expressions of HIF-1α, TIMP-1, MMP-9 in renal interstitial. All rats were allowed free access to food and water during the experimcnt,whereas insulin and other hypoglycemic drugs were not supplied.Results1. In the whole time of experiment, blood glucose of the diabetic nephropathy rats kept at high level (Table 1). The serum albumin,blood glucose,serum creatinine, kidney index,24-hour urinary protein in the D and T groups were significantly different with those of the normal control group. (P<0.05).In the treatment group, from the end of 8th week to 12th week serum creatinine,kidney index and 24-hour urinary protein were significantly lower than those in the diabetic nephropathy group,serum albumin was more than that of the diabetic nephropathy group (P<0.05) (Table 2-5).2. ImmunohistochemistryIn normal control group, HIF-1α, TIMP-1 expression was weakly positive, MMP-9 expression was positive.In diabetic nephropathy group,HIF-1α, TIMP-1 and MMP-9 were expressed in the renal tubular epithelial cells cytoplasm. At the 8th and 12th weeks, compared with the D group, the expressions of TIMP-1 and HIF-la in the T group were reduced, MMP-9 expression was relatively increased. 3.Masson stainingIn diabetic nephropathy group and treatment group with valsartan the area of kidney interstitial fibrosis was significantly larger than that of the control group (P <0.05), from the end of 8th week the area in the treatment group less than the area in diabetic nephropathy group (P<0.05). (Table 6, figure4.1-4.9)4.RT-PCRIn normal control group, HIF-1α, TIMP-1, MMP-9 mRNA expressions in 4th week,8th week,12th week had no significant difference.In diabetic nephropathy group and treatment group HIF-1α, TIMP-1mRNA expressions were significantly higher than the normal control group (P<0.05), and gradually increased with time, After 8weeks and 12weeks,the HIF-1α, TIMP-1 expressions level in valsartan treatment group were lower than the diabetic nephropathy group (P<0.05); MMP-9 mRNA expression in diabetic nephropathy group and the theatment group were significantly lower than the normal control group (P<0.05), gradually reducing over time. At the end of 8weeks and 12weeks,the MMP-9 expression level in treatment group was higher than that in diabetic nephropathy group (P<0.05). (Figure A,1,2,3)5. Correlation analysis:In diabetic nephropathy rats model, HIF-1α, TIMP-1 and the area of interstitial fibrosis (RIF) have positively relationship, MMP-9 and the area of renal interstitial fibrosis have negative correlation.Conclusion1. In diabetic nephropathy rats HIF-la, TIMP-1 expressions increased and the expression of MMP-9 decreased.2. Valsartan may reduce the HIF-1αexpression, regulating the expression of MMP-9/TIMP-1 balance, reducing the degree of renal interstitial fibrosis, played a role in renal protection.