The Epithelial-To-Mesenchymal And ECM Accumulation Of Human Peritoneal Mesothelial Cell In Response To LDL And Glucose In Vitro

BACKGROUND AND OBJECTIVESUltrafiltration failure due to dysfunction of the peritoneum as a dialyzing organ is a major clinical limitation of peritoneal dialysis. It is increasingly clear that mesothelial cells play an important role in fibrogenesis and vasculopathy that underlie peritoneal membrane dysfunction. New and extensively studied aspects of peritoneal mesothelial cell biology include epithelial-to-mesenchymal transition and cellular senescence. In these processes, the alteration in the peritoneal membrane will further be aggravated by peritonitis, advanced glycation end-products and glucose degradation products. 30-70% dialysis patients are suffering from high TG, and PD patients are more vulnerable to be accompanied with high cholesterol and LDL-C. But within our limited understanding, there is no report on the role of lipids in the EMT and ECM accumulation of HPMC, the specific mechanisms are still unclear. The objective of this study was to observe the prevalence of dyslipidemia in peritoneal dialysis patients, to investigate the effect of LDL on epithelial-mesenchymal transition (EMT) and ECM accumulation in human peritoneal mesothelial cells(HPMCs), to study the role of the LDL receptor and PPARγsignaling pathway in these processes.METHODS AND RESULTSPart 1. Disorders of lipid metabolism in PD patientsAll 127 patients were withdrawn from peritoneal dialysis (PD) and were observed for the following 24 months. The control group include 95 MHD patients and 130 patients with chronic kidney disease (CKD 3-4). The clinical data was collected, blood biochemistry, dialysis adequacy, peritoneal transport characteristics of nitrogen balance and detect the full set of blood lipids(TG, TC, LDL-C, and HDL-C, Lp (a), apoA, apoB), hsCRP, dialysate levels of cancer antigen 125 (CA125) were measured regularly. The most important abnormalities are the increase in the serum level of triglyceride (elevated VLDL-remnants/IDL), small LDL particles and a low HDL cholesterol in PD patients. At 24 months, mortality was no significantly different in patients with or without LDL increased. But the CA125, the marker of mesothelial cell mass, had a good relationship with the TC and TG.Part 2. The effect and mechanism of LDL on epithelial-mesenchymal transition (EMT) and ECM accumulation in HPMCs2.1. Culture and identification of HPMCsHuman peritoneal mesothelial cell line (HMrSV5) were cultured with 10% fetal calf serum containing RPMI-1640 medium, the cultured cells were identified by morphology and immunohistochemistry. Results suggest that before cell confluent, HPMCs showed the appearance of circles, stars and polygons, confluent HPMCs looked like cobbleston. Immunohistochemical methods for cell identification, cultured HPMCs co-expressed cytokeratin and vimentin, but anti-human CD45 antigen andⅧfactor were negative.2.2. HPMCs uptake LDL into the cell by LDL receptorAfter co-cultured with the LDL for 24h, the LDL receptor was observed to expression on the cell membrane of HPMCs by immuno-fluorescence method. Oil red staining showed that LDL could be uptaken into the cells and abolished by LDL receptor blocker.2.3. The effects of LDL on EMT in HPMCsHPMCs were divided into four groups:0,25,50, 100μg/ml in accordance with LDL concentrations, stimulated for 24h, cell's morphological changes were observed under inverted microscope. With higher LDL concentrations, cells tended to be looser connection between them, and showed significant formation of fibroblast-like spindle morphology. The cytoplasm immuno-fluorescence intensity of a-SMA gradually increased with the increasing of LDL concentration, but was negative in control group. Then the cells were divided into 5 groups:control group,2.18%mannitol group (osmotic control), LG Group (glucose 30mmol/L), HG group (glucose 120mmol/L), HG+LDL group (100μg/ml). After co-cultured for 48h, compared with the control group, the expression of a-SMA mRNA and protein was significantly increased in HG +LDL group. At the same time, lower expression of E-cadherin mRNA was also observed in the same group.2.4. The effects of LDL on the synthesis and degradation of ECM in HPMCsIn supernatant of HPMCs co-cultured with HG+LDL, ELISA assay showed that the COL-I protein was significantly increased. The difference COL-I and FNmRNA expression was not observed with or without LDL by RT-PCR (realtime-PCR) method. On the other hand, the plasminogen inhibitor (PAI-1) mRNA and protein level of the cell culture supernatant of HG+LDL group were significantly increased than the control.2.5. The role of the PPARy signaling pathway in these processesPPARy mRNA and protein expression were detected by RT-PCR and Western blot. HG+LDL significantly increased the expression of PPARy mRNA. Different concentrations of GW9662 (5μg/ml, 10μg/ml), a kind of PPARy ligands blocker, were used to observe the abolished effect of EMT and ECM accumulation in above study. The improvement of morphological changes, trans-differentiation and up regulated PAI-1 was observed in HPMCs co-stimulated with HG+LDL.CONCLUSIONSUnder our experimental conditions:1. Peritoneal dialysis patients have more severe dyslipidemia than hemodialysis patients with higher TC, LDL-C and apoB.2. Human peritoneal mesothelial cells uptook LDL into cells via LDL receptor.3. LDL could induce HPMCs transdifferentiation in the context of high glucose, increase the secretion of COL-I protein and up-regulating the expression of PAI-1mRNA and protein, through which inhibited degradation of ECM.4. The PPARy signaling pathway may be play the role in these processes.