TIMP-1 Small Hairpin RNA Gene Transfer In Vivo And The Effects Of Leflunomide In Diabetic Nephropathy Rats

Objective: To study the effects of TIMP-1 small hairpin RNA (TIMP-1 shRNA) and leflunomide on the renal interstitial fibrosis caused by diabetic nephropathy in rats.Methods: 120 healthy Wister rats were randomly divided into 6 groups: normal control group(N group),diabetes mellitus(DM group),TIMP-1 shRNA group(DM-RNAi group),HKshRNA group(DM-VE group),leflunomide(DM-LEF group),Carboxymethyl Cellulose group(DM-CMC group). Rats' renal function of each group were monitored;The histological changes of renal interstitial tissues were observed by PAS staining. The mRNAs of TIMP-1,MCP-1,ICAM-1,VCAM-1,TGF-β1,Fractalkine and RANTES were reverse transcribed and quantified by RT-PCR .The proteins of TIMP-1,MMP-2,MMP-9,TGF-β1,MCP-1,ICAM-1 and Fractalkine changes were analyzced by Western Blot analysis.The proteins of FN,TIMP-1,MMP-2,NF-κB,TGF-β1,MCP-1 andα-SMA were assessed by immunohistochemistry analysis.Results: After the rat model were established ,with the extension of time, kidney function of the diabetic rats' suffered gradually,urine protein/ urine creatinine,serum creatinine, blood urea nitrogen,relative kidney weight(KW/BW) in DM group were higher than N group (P<0.05); compared with DM group,the DM-RNAi group and the DM-LEF group were decrease remarkably (P<0.05).Nephridial tissue pathomorphology mani- fested that:In DM group, interstitial fibrosis including tubular astrophy,loss and dilation,inflammatory cell infiltration were evident.These changes were all improved by TIMP-1shRNA and leflunomide treatment.Respectively analysising DN rat kidney TIMP-1mRNA at 10,12,14,16 weeks by RT-PCR,with the extension of time,the DM group's expression was higher than the N group's(P<0.05),but the DM-RNAi group and the DM-LEF group were decreased conspicuously than DM group(P<0.05).In the gene level,the expressions of MCP-1,ICAM-1,VCAM-1,TGF-β1,Fractalkine and RANTES mRNA were increased in the DM group,but the DM-RNAi group and the DM-LEF group were decreased.Meanwhile,in the protein level rat TIMP-1 was consonanced with the gene level; compared with N group,MMP-2 and MMP-9 were accrescenced in DM group;yet,the DM-RNAi group and the DM-LEF group descented significantly than DM group(P<0.05) ; either TIMP-1shRNA or leflunomide significantly decreased the levels of TGF-β1,MCP-1,ICAM-1 and Fractalkine,in DM group was inceased dramatically(P<0.05).In nephridial tissue,through the immunohistochemistry,the masc dyeing areas of FN,TIMP-1,MMP-2,NF-κB,TGF-β1,MCP-1,α-SMA enhanced in DM group, compared with the DM group,the DM-RNAi group and the DM-LEF group extracellular matrix diminished (P<0.05).Conclusions:1. TIMP-1 plays an important role in the development of diabetic nephropathy interstitial fibrosis.2. TIMP-1shRNA downregulates the gene expression of TIMP-1,promotes the degradation of extracellular material and improves interstitial fibrosis; in addition,TIMP-1shRNA might inhibit inflammation and improve tubules injury ,therefore protects rat renal function.3. Leflunomide may attenuate interstitial inflammation reaction, suppresse the expressions of inflammatory factors,improve interstitial fibrosis which is probably correlated with myofibroblast proliferation. therefore,it can protect renal function in diabetic nephropathy.