Twist Promotes Epithelial-to-mesenchymal Transition Of Human Peritoneal Mesothelial Cells Under HG Stimulation

【Background】Peritoneal dialysis (PD) is one method of therapy for End Stage Renal Disease (ESRD), but long-term PD causes functional and structural alterations of the peritoneal membrane, and finally that results in peritoneal fibrosis. One of the most important challenges in PD is the long-term preservation of the peritoneal membrane integrity. The presence of EMT in the peritoneum of PD patients was first demonstrated in a landmark paper that published in 2003. Their results indicated that new myofibroblastic cells may arise from local conversion of MC by EMT during the repair responses that take place in PD. Epithelial-to-mesenchymal transition (EMT) or mesothelial-to-mesenchymal transition of human peritoneal mesothelial cells has been regarded as an early mechanism of peritoneal fibrosis. Previous studies indicated that Notch ,snail and NF-κB induced EMT of HPMCs and promoted peritoneal fibrosis. The most likely factors in peritoneal dialysis solutions responsible for peritoneal deterioration are glucose and glucose degradation products (GDPs), which stimulate TGF-βand vascular endothelial growth factor (VEGF) production. In previous study, researchers thought that high glucose (HG) per se could induce EMT of HPMCs, which suggests HG-induced alteration in cell phenotype may be one of the earliest phenomena of peritoneal damage. However, the molecular mechanisms by which HG-induced Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelium remain largely unknown. Twist is a highly conserved basic helix-loop-helix transcription factor that was newly identified as a key regulator of promoting EMT involved in embryogenesis and cancer metastasis. There have been few studies about the molecular mechanism of high glucose-induced EMT of human peritoneal mesothelial cells. Given the known features of Twist, we hypothesized that high glucose in human peritoneal mesothelial cells might affect Twist activity and further mediate EMT of HPMCs.【Objective】1. To explore the role of Twist on epithelial-to-mesenchymal (EMT) of human peritoneal mesothelial cells(HPMCs)under HG stimulation in vitro.2. Further to investigate the expression of Twist in HG-induced peritoneal fibrosis model SD rate in vivo.【Methods】1. HPMCs were treated with different glucose concentration (30,40,50,60,120mmol/L) and with different treatment duration(24h, 48h,72h), in order to make sure of the concentration of high glucose.2. Twist was detected by protein and mRNA level after the treated cells with different treatment duration (0h, 6h, 12h, 24h, 48h, 72h) under HG culture.3. Human peritoneal mesothelial cells were cultured with 50mmol/L (HG) and 5.6mmol/L (NG) D-glucose for 48h. The expression of Twist, E-cadherin andα-SMA were detected by western blotting and immuocytochemistry.4. Twist, E-cadherin andα-SMA were tested by qRT-PCR and western blotting after transfecting the plasmids pcDNA3.1-Twist and empty vector pcDNA3.1 into HPMCs with Lipofectamine 2000 in vitro .5. Twist, E-cadherin andɑ-SMA were tested by western blotting after silencing the expression Twist by siRNA and empty vector pSlience.6. SD rat SD rats were randomly divided into peritoneal fibrosis model group (PD group) and control group. The PD group was injected intraperitoneally with 4.25% PD solution, and control group by intraperitoneal injection with 0.9% sodium chloride . Twist,E-cadherin,α-SMA were detected in PD group and control group by immunohistochemistry. The thick of peritoneal membrane was tested by Light microscope with HE stain.7. Twist was detected by western blotting in PD group compared with control group.【Results】1. The results of experiments with different glucose concentration (30,40,50,60 , 120mmol/L) and with different treatment duration were Twist activation increased significantly at 50mmol/L D-glucose without a significant cell toxicity evaluated by cell morphology. Therefore, we used 50mmol/L D-glucose for the rest of the experiment regarding HG-induced EMT.2. It was shown that Twist was significantly upregulated 12h after high glucose stimulation. Twist remained upregulated for more than 48h . In contrast, the expression of Twist was little in normal glucose cells.3. The overexpression of Twist protein after treatment with 50mmol/L D-glucose for 48h compared to 5.6mmol/L suggested by decreased expression of E-cadherin and increased expression ofα-smooth muscle actin by western blotting and immuocytochemistry (P<0.05).4. The Overexpression of Twist significantly reduced after with the epithelial marker E-cadherin and enhanced after with the fibroblastic markerα-SMA in mRNA level and protein level(P<0.05).5. Silencing the expression of Twist by Si-Twist significantly enhanced the expression of E-cadherin and down-regulated the expression ofα-SMA by western blotting, and finally reversed the phenotype of EMT.6. Light microscopic examination verified that peritoneal membrane of peritoneal fibrosis rat was more thicker than control group. Immunohistochemistry staining showed that Twist was expressed in the cytoplasm of peritoneal mesothelial cells in high glucose PDF rats, whereas was almost no staining in control group peritoneal membrane. The staining of E-cadherin was decreased in rat peritoneal mesothelial cells, whereas the staining ofα-SMA was increased in control group.7. Twist was significantly increased in PD group than control group.【Conclusion】1. Overexpression of Twist promotes epithelial-to-mesenchymal transition of HPMCs under HG stimulation in vitro.2. Twist expression was increased in high glucose PDF rats and was negatively correlated to expression of E-cadherin. That indicated Twist might have direct regulation on the initiation of peritoneal fibrosis in vivo.