Improvement Of Renal Interstitial Fibrosis By PHGF And CTGF Antisense Oligodeoxynucleotide

Objective:To explore the roles of hepatocyte growth-promoting factor(pHGF) and connective tissue growth factor(CTGF) antisense oligodeoxynucleotide(ASODN) in epithelial-to-mesenchymal transition(EMT) of human kidney epithelial cell lines(HKC) and in the process of rat renal interstitial fibrosis.Methods:Cell experiment:Epithelial-to-mesenchymal transition of human kidney epithelial cell lines(HKC) were induced by MCP-1 and AAâ… ,then administered pHGF,CTGF antisense oligodeoxynucleotide(ASODN) respectively and synergistically.Cell apoptosis andα-SMA mRNA were detected by flow cytometry,andα-smooth muscle actin(α-SMA) mRNA was detected by reverse transcriptase-polymerase chain reaction(RT-PCR),α-SMA,Fibronectin(FN) and transforming growth factor-β₁ (TGF-β₁) of HKC cells were assessed by indirect enzyme immunohistochemistry. Animal experiment:48 Sprague-Dawley(SD) rats were randomly divided to sham-operation group(SOR),unilateral ureteral obstruction group(UUO),pHGF treated group(pHGF),CTGF ASODN treated group,CTGF missense oligodeoxynucleotide (MSODN) treated group and Drug combination group(pHGF and CTGF ASODN).All groups of rats were treated with different drugs and raised in the following days after unilateral ureteral obstruction,then killed in the 14th day.The degree of renal fibrosis was assessed by measuring cortex hydroxyproline content, The Collagen distribution was evaluated by Masson stain,α-SMA mRNA was detected by RT-PCR,andα-SMA,FN,TGF-β₁ in the renal cortex were examined by immunohistochemistry.Results:Cell experiment:apoptosis rate for cells treated with 5,150,1500 ng/ml pHGF was 3.3%—4.4%on 24 h by flow cytometry,but increased to 9.1%in model group.The rate was significantly lower in treated groups than that in pHGF group(p＜0.01),but there was no difference between model group and CTGF ASODN group.The rate ofα-SMA/GAPDH in Model group,pHGF,CTGF ASODN and Drug combination group was 0.91±0.002,0.68±0.015,0.61±0.005,0.65±0.010 respectively by RT-PCR.α-SMA mRNA level in treated groups was lower than that in model group(p＜0.01). Antagonistic effect existed in pHGF and CTGF ASODN valuated by Jin Zhengjun's Q-value Probability Statistics(Q=0.572,＜0.85).Proteins ofα-SMA,FN,TGF-β₁ in HKC cells were decreased in pHGF,CTGF ASODN and combined group,detected by immunohistochemistry,compared with that in model group.Animal experiment:Renal tissue hydroxyproline content in UUO model group, pHGF group,CTGF ASODN group,Drug combination group and CTGF MSODN group was 4.85±0.201,3.76±0.134,3.34±0.306,4.06±0.179 and 4.91±0.172μg/mg respectively.Compared wtih UUO model group,pHGF group CTGF ASODN group and Drug combination group had higher content of hydroxyproline(p＜0.01).But there was opposite effects in pHGF and CTGF ASODN valuated by Jin Zhengjun's Q-value Probability Statistics(Q =0.349,＜0.85).Fibrosis degree was weaker in pHGF group,CTGF ASODN group and Drug combination group than that in model group assessed by Masson stain.Rate ofα-SMA/β-actin mRNA in UUO group and CTGF ASODN group was 1.31±0.049,0.77±0.022 by RT-PCR,respectively.The rate in CTGF ASODN group was lower than that in UUO model group(p＜0.05),α-SMA, FN,TGF-β₁ expressed in the renal cortex were weaker in pHGF group,CTGF ASODN group and Drug combination group,compared with that in UUO model group and CTGF MSODN group detected by immunohistochemistry.Conclusions:pHGF and CTGF ASODN antagonize EMT of HKC and inhibited fibrosis development in rat renal interstitium induced by unilateral ureteral obstruction, respectively,but there was no synergistic effect when treated with both.