Effects Of Blockage Of Wnt/β-catenin Signaling Pathway On The Epithelial-Mesenchymal Transition In Human Proximal Tubular Epithelial Cells

Background and objective:Progressive renal failure and tubulointerstitial fibrosis are the basic characteristics ofchronic kidney disease development caused of different reasons，and tubulointerstitialfibrosis plays as the final common pathological changes marked by fibroblast proliferationand extracellular matrix deposition. Studies have shown that renal tubular epithelial cells-mesenchymal transdifferentiation (EMT) plays an important role in the process ofinterstitial fibrosis. Under the stimulation of pathological factors, the renal tubular epithelialcells losses epithelial cell characteristics, gets characteristic as myofibroblast, transfers intothe tubulointerstitial, playing fibroblast functionand accelerating fibrosis progression.The Wnt signaling pathway is one of the most extremely conservative signalingpathways in biological evolution which plays an important role in cell adhesion, migration,epithelial-mesenchymal transformation, growth, differentiation, apoptosis, embryonicdevelopment, organogenesis, and maintenance of environmental stability in the tissues andorgans. Recent researches have found that the Wnt/β-catenin signaling pathway proteinsexpression were elevated in the animal models of renal interstitial fibrosis, suggesting itmay be involved in the process of interstitial fibrosis, however，the mechanism ofWnt/β-catenin signaling pathway in interstitial fibrosis is still unclear.In this study, treat the human proximal tubular epithelial cells with the Wnt blockersDickkopf-1(Dkk-1) and the short hairpin RNA of interfering beta-catenin in vitro, toobserve its impacts on the EMT of renal tubular epithelial cells, explore the mechanism ofWnt/beta-catenin signaling pathway on occurrence and development of renal interstitialfibrosis, and provide theoretical basis for finding out a new target in the treatment ofanti-tubular interstitial fibrosis.Methods: 1. Human proximal tubular epithelial cells were cultured in vitro, and divided intothree groups: control group, TGF-β1group (concentration of TGF-β1is20ng/ml),TGF-β1+Dkk-1group (concentration of TGF-β1and Dkk-1are20ng/ml and100ng/ml).The mRNA expressions of Wnt4, β-catenin, E-cadherin, and α-SMA were determined byRT-PCR. E-cadherin, α-SMA expressions were detected by cell immunofluorescence. Theprotein expressions of Wnt4, β-catenin, E-cadherin, and α-SMA were detected by Westernblot.2. According to the shRNA design principles, design three interference targets forβ-catenin gene, construct the shRNA-β-catenin inhibition vector, transfect into HKC cellsinduced by TGF-β1. RT-PCR, Western blot detects mRNA and protein expression levels ofβ-catenin, screen the best inhibitory vector.3. HKCs were divided into four groups: control group, cultured with DMEM/F12medium containing10%FBS; TGF-β1group, cultured with the conventional mediumadded TGF-β1(20ng/ml); TGF-β1+empty vector group, cultured with the conventionalmedium added TGF-β1(20ng/ml) and transfected control blank vector; TGF-β1+inhibitionvector group, cultured with the conventional medium added TGF-β1(20ng/ml) andtransfected shRNA-β-catenin inhibition vector. After treatment according to the packetprocessing for48hours, inverted phase contrast microscope observes cell morphology,RT-PCR and Western blot detect mRNA and protein expression of β-catenin, E-cadherinand α-SMA, immunofluorescence detects expression of E-cadherin and α-SMA.Results:1. Compared with control group, the mRNA expression of Wnt4, and the proteinexpression of Wnt4were significantly increased in TGF-β1group and TGF-β1+Dkk-1group. the β-catenin protein expression was significantly increased in TGF-β1group, butmRNA expression has no different in the three groups. the mRNA and protein expression ofE-cadherin was significantly decreased in group TGF-β1group. α-SMA mRNA and proteinexpression was low in control group, they were significantly increased TGF-β1group, butsignificantly decreased in TGF-β1+Dkk-1group.2. The design fragments were fully inserted into shRNA-β-catenin inhibition vectorsby restriction analysis and sequencing. The transfection efficiency is at the level of60%approximately. β-catenin expressions at the mRNA and protein levels were lower than the empty vector group, and screening shRNA-β-catenin-1into the experimental group.3. transfection of shRNA-β-catenin-1cells β-catenin expression at the mRNA andprotein levels were lower than the TGF-β1-induced group and the empty vector group, andβ-catenin transferred nuclear significantly reduced; compared with TGF-β1-induced groupand the empty vector group, E-cadherin mRNA and protein expression levels weresignificantly increased. On the contrast, compared with TGF-β1-induced group and theempty vector group, α-SMA mRNA and protein expression levels were significantly lower.E-cadherin andα-SMA expressions detected by Immunofluorescence are consistent with theWestern blot.Conclusion:1. Wnt/β-catenin signaling pathway is involved in HKCs epithelial mesenchymaltransdifferentiation, and the Wnt blocker Dkk-1can ameliorate the transdifferentiation.2. shRNA-β-catenin inhibition vectors were successfully transfected into HKCs whichcan inhibit the expression of β-catenin effectively.3. shRNA-β-catenin inhibition vector can attenuate transdifferentiation in humanproximal tubular epithelial cells.