Effect Of Wnt/β-catenin Signaling On Human Peritoneal Mesothelial Cells Epithelial-mesenchymal Transition

BackgroundPeritoneal dialysis (PD) is an attractive treatment for patients with end-stage renal disease (ESRD). However, continuously exposure to non-physiological peritoneal dialysis solution (PDS), it will inevitably lead to peritoneal fibrosis, which ultimately cause to ultrafiltration failure and increased morbidity as well as mortality. The establishment of peritoneal fibrosis has been associated with the epithelial to mesenchymal transition (EMT) of the peritoneal mesothelial cells monolayer. Many cytokines and pathways were found take part in this process, and transforming growth factor-β1(TGF-β1) is generally considered to play the important role. However, the molecular mechanism is still not well defined yet and no effective treatment has been found.The Wnt pathway, originally characterized as an essential component in early development helps control aspects of cell differentiation and polarity by signaling through at least three distinct pathways. Of the three Wnt pathways, the canonical Wnt pathway acting through activation of beta-catenin is the best understood. In recent years, the role of Wnt/β-catenin signaling in regulating EMT during organ fibrosis and tumor metastasis has been established. More importantly, recently the cross-talk between TGF-β and Wnt/β-catenin signaling in the process of EMT and fibrosis has been demonstrated. Therefore, we hypothesize that Wnt/β-catenin signaling maybe involve in the progression of peritoneal dialysis induced peritoneal mesothelial cells EMT and peritoneal fibrosis. To this end, experimental research is carried out as follow. Chapter I Aberrant expression of Wnt/p-catenin signaling related to epithelial-mesenchymal transition in human peritoneal mesothelial cells (HPMC) isolated from peritoneal dialysis effluentsObjectiveTo investigate the expression of Wnt/β-catenin signaling and the EMT status in HPMC isolated from peritoneal dialysis effluentsMethods24patients undergoing continuous ambulatory peritoneal dialysis (CAPD) were enrolled into this study, including12new patients and12patients undergoing PD over lyear. The cells isolated from peritoneal dialysis effluents were cultured and identified by flow cytometry. The expression of Wnt(1,2,3,3a,4,5a,7b,8a,10a),β-catenin, DKK1, LEF1, a-SMA, E-Cadherin, COL-1, FN and MMP-7were tested by realtime PCR and western blot.Results(1) Cells isolated from peritoneal dialysis effluents were identified as HPMC, and had markedly varied morphologic features, ranging from a cobblestone-like appearance to fibroblast-like cells.(2) Compared with new patients, the expression of E-cadherin was lower and the expression of a-SMA and COL-I were higher in cells from patients undergoing PD over1year.(3) The expression of Wntl, Wnt5a, Wnt7b, Wnt8a and β-catenin was significantly increased in patients undergoing long-term PD.(4) The mRNA level of DKK1, LEF1, FN and MMP-7were higher in patients undergoing long-term PD.ConclusionIncreased expression of Wnt/p-catenin signaling along with EMT of HPMC and ECM accumulation after long-term PD indicated that Wnt/β-catenin signaling may be involved in the process of peritoneal fibrosis. Chapter Ⅱ The role of Wnt/β-catenin signaling in high glucose induced epithelial-mesenchymal transition of HPMC cellsObjectiveInvestigate the effect of Wnt/β-catenin signaling in the process of high glucose induced EMT and ECM accumulation in HPMC cells.MethodsHMrSV5cells were exposed to different concentrations of high glucose with different duration. The expressions of E-cadherin、α-SMA、 COL-Ⅰ、FN and β-catenin、Wntl,5a、LEF1were examined by Western blot, realtime PCR and immunofluorescence. Furthermore, HMrSV5cells were transfected with the plasmid of DKK1and empty vector. Then, The expressions of E-cadherin、α-SMA、COL-Ⅰ、FN and β-catenin、LEF1were detected.ResultsStimulation of HPMC cells with high glucose resulted in a significant reduce in the expression of E-cadherin and increase in the expression of α-SMA、COL-Ⅰ and FN. Meanwhile, high glucose increased the expression of Wnt1,5a,β-catenin and LEF1, in time and concentration-dependent manner. Compared with high glucose group, overexpression of DKK1down regulated the level of β-catenin, along with the upregulation of E-cadherin and downregulation of a-SMA and COL-Ⅰ.ConclusionHigh glucose increased the expression of Wnt/β-catenin signaling in HPMCs. Blockade of the Wnt/β-catenin signaling with DKK1inhibited high glucose-induced EMT of HPMCs. ChapterⅢ The role of Wnt/β-catenin signaling in TGFβ1induced epithelial-mesenchymal transition of HPMCsObjectiveInvestigate the effect of Wnt/β-catenin signaling in the process of TGF-β1induced EMT and ECM accumulation in HPMC cells.MethodsHMrSV5cells were exposed to different concentrations of TGF-β1. The expressions of E-cadherin、α-SMA、COL-Ⅰ、FN and β-catenin、 Wntl,5a、LEF1were examined by Western blot, realtime PCR and immunofluorescence. Furthermore, HMrSV5cells were transfected with the plasmid of DKK1. Then, The expressions of E-cadherin、α-SMA、 COL-Ⅰ、FN and β-catenin、LEF1were detected.ResultsStimulation of HPMCs with TGF-β1resulted in a significant reduce in the expression of E-cadherin and increase in the expression of a-SMA> Col-1and FN. Meanwhile, TGF-β1increased the expression of Wntl,5a, β-catenin and LEF1, in time and concentration-dependent manner. Compared with TGF-βi group, overexpression of DKK1down regulated the level of β-catenin, along with the upregulation of E-cadherin and downregulation of α-SMA and Col-ⅠConclusionTGF-β1increases the expression of Wnt/β-catenin signaling in HPMCs. Blockade of the Wnt/β-catenin signaling with DKK1inhibited TGF-β1-induced EMT of HPMCs.