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The monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is involved in peritoneal dialysis-related epithelial-mesenchymal transition of peritoneal mesothelial cells

Other Titles
 MCP-1/CCR2 시스템이 복막중피세포의 복막투석과 관련한 상피-중간엽 이행에 미치는 영향 
Issue Date
Dept. of Medical Science/박사
Background: Peritoneal fibrosis (PF) is a serious complication in long-term peritoneal dialysis (PD) patients, but the underlying mechanism is not well understood. Recently, accumulating evidences has suggested that epithelial-mesenchymal transition (EMT) is involved in a variety of fibrotic disorders, including renal fibrosis, pulmonary fibrosis, and PF. In addition, TGF-1 is known to plays an important role in the process of EMT. Meanwhile, recent studies have shown that monocyte chemoattractant protein-1 (MCP-1) directly increases TGF-1 production via C-C chemokine receptor 2 (CCR2) and the MCP-1/CCR2 system is implicated in high glucose-induced TGF-1 production in cultured mesangial cells. Since human peritoneal mesothelial cells (HPMCs) also synthesize MCP-1, there is a possibility that the interaction between the MCP-1/CCR2 system and the TGF-1 pathway is involved in EMT and extracellular matrix (ECM) synthesis in HPMCs, resulting in PF.Purpose: This study was undertaken to investigate the direct effect of MCP-1 on EMT and fibronectin expression and the role of the MCP-1/CCR2 system in high glucose- and TGF-1-induced EMT in cultured HPMCs. In addition, the impact of MCP-1/CCR2 inhibition on EMT and PF was elucidated in an animal model of PD.Methods: In vitro, HPMCs were incubated in M199 media containing 5.6 mM glucose (normal glucose, NG), NG+94.4 mM mannitol (NG+M), NG+recombinant human MCP-1 (10 ng/ml) (NG+MCP-1), or 100 mM glucose (high glucose, HG) with or without RS102895 (10 M), a specific chemical inhibitor of CCR2. In addition, dominant negative mutant MCP-1 (mMCP-1)-expressing lentivirus-transfected HPMCs were exposed to NG or NG+MCP-1 media. After 72 hours, cells were harvested and conditioned media were collected. In vivo, PD catheters were inserted into 60 Sprague-Dawley rats, and saline (C group, n=30) or 4.25% PD solution (PD group, n=30) were infused for 4 weeks. Twenty rats from each group were treated with empty lentivirus vector or lentivirus vector containing mMCP-1 intraperitoneally, every 5 days for 3 times, and 10 rats from each group were left untreated. After 4 weeks, rats were sacrificed and the peritoneal tissues were removed. E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin protein expression in HPMCs and the peritoneum were evaluated by Western blot analysis, and fibronectin mRNA expression by real-time PCR. MCP-1 and TGF-1 concentrations in conditioned media were determined by ELISA. PF was assessed by Masson’s trichrome (MT) staining.Results: Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA and fibronectin expression were significantly increased in HPMCs exposed to HG and NG+MCP-1, and these changes were significantly abrogated by RS102895 treatment (p<0.05). RS102895 also significantly ameliorated the increase in TGF-1 levels in HG-conditioned media. In addition, TGF-1- and MCP-1-induced EMT were significantly attenuated by RS102895 and TGF-1 neutralizing antibody, respectively. In rats treated with PD solution for 4 weeks, the ratios of E-cadherin/α-SMA protein expression in the peritoneum were significantly decreased, whereas fibronectin mRNA and protein expression was significantly increased compared to C rats (p<0.05). The thickness of mesothelial layer and the intensity of MT staining in the peritoneum of PD rats were also significantly higher relative to C rats (p<0.05). These changes of the peritoneum in PD rats were significantly abrogated by the administration of lentivirus containing mMCP-1.Conclusions: These findings suggest that the MCP-1/CCR2 system is directly involved in PD-related EMT and ECM synthesis partly via TGF-1 and its inhibition may be a potential therapeutic strategy for PF in PD patients.
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2. 학위논문 > 1. College of Medicine (의과대학) > 박사
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