Mechanisms of epithelial-mesenchymal transition of peritoneal mesothelial cells during peritoneal dialysis

A growing body of evidence indicates that epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMC) may play an important role in the development and progression of peritoneal fibrosis during long-term peritoneal dialysis (PD) leading to failure of peritoneal membrane function. Here, we review our own observations and those of others on the mechanisms of EMT of HPMC and suggest potential therapeutic strategies to prevent EMT and peritoneal fibrosis during long-term PD. We found that high glucose and H2O2 as well as transforming growth factor-beta1 (TGF-beta1) induced EMT in HPMC and that high glucoseinduced EMT was blocked not only by inhibition of TGF-beta1 but also by antioxidants or inhibitors of mitogen-activated protein kinases (MAPK). Since MAPKs are downstream target molecules of reactive oxygen species (ROS), these data suggest that high glucose-induced generation of ROS and subsequent MAPK activation mediate high glucose-induced EMT in HPMC. We and others also observed that bone morphogenetic protein-7 (BMP-7) prevented EMT in HPMC. Glucose degradation products (GDP) were shown to play a role in inducing EMT. Involvement of a mammalian target of rapamycin (mTOR) in TGF-beta1-induced EMT has also been proposed in cultured HPMC. A better understanding of the precise mechanisms involved in EMT of HPMC may provide new therapeutic strategies for inhibiting peritoneal fibrosis in long-term PD patients.     

Cytologic features of atypical mesothelial cells in peritoneal dialysis fluid

From February 1988 through October 1988, 23 samples of peritoneal dialysis fluid from 20 patients with end-stage renal disease were cytologically analyzed in an attempt to determine the effect of the dialysate on the mesothelial cells lining the peritoneal cavity. The patients, five female and 15 male, ranging in age from 26 to 75 yr, had been on continuous ambulatory peritoneal dialysis (CAPD) from 1 mo to 6 yr, 4 mo. The patients had no history of cirrhosis, neoplastic disease, radiation and/or chemotherapy, or current findings of infection. Smears and cytosieve filter preparations were made. Smear analysis included the mesothelial cell pattern, the degree of mesothelial cell atypia, and the presence of atypical multinucleated cells and mitoses. In the majority of the fluid samples, reactive mesothelial cells were arranged singly and in sheets. Moderately and severely atypical mesothelial cells were glandular and papillary in configuration. All samples contained at least a few reactive mesothelial cells; in six, the highest degree of cellular atypia was moderate; in 17, it was severe. The development of severe cellular atypia did not appear to be time dependent (a finding noted in samples from patients on dialysis for 6 mo up to 6 yr). When present, multinucleated mesothelial cells showed moderate to severe atypia. In four cases mitotic figures were present. On the basis of these findings, it is proposed that peritoneal dialysis plays a role in the development of mesothelial cell atypia.     

腹透液增强腹膜间皮细胞CD40表达及其意义

目的:研究腹膜透析液对大鼠腹膜间皮细胞CD40表达的影响及CD40活化后与细胞间粘附分子-1(ICAM-1)分泌的相关性, 以揭示腹膜透析时腹腔局部炎症的发生机制。方法: 分离、培养大鼠腹膜间皮细胞, 分别用IFN-γ、4.25%腹透液、4.25%腹透液+IFN-γ作为刺激因子, 通过逆转录-多聚酶链反应(RT-PCR)及流式细胞仪(FACS)检测间皮细胞CD40表达;并通过CD40单克隆抗体(CD40mAb)活化高表达的间皮细胞CD40, 用FACS检测间皮细胞ICAM-1的表达。结果:腹膜间皮细胞结构性表达低水平CD40mRNA及蛋白。用4.25%腹透液刺激后, 间皮细胞CD40mRNA及蛋白的表达显著高于对照组。用4.25%腹透液+IFN-γ刺激后, 间皮细胞表面CD40mRNA及蛋白的表达进一步增加, 且明显高于单纯腹透液刺激组。活化CD40受体可显著增强间皮细胞ICAM-1的表达。结论:腹膜间皮细胞可表达CD40, 4.25%透析液及IFN-γ均能显著增加腹膜间皮细胞CD40的表达。间皮细胞CD40的上调, 可能是腹透过程中腹腔局部炎症启动、放大的重要机制之一。     

Regulation of complement C3 and C4 synthesis in human peritoneal mesothelial cells by peritoneal dialysis fluid

Although complement is activated in the peritoneal cavity during chronic peritoneal dialysis (PD), little is known about its role in peritoneal defence and injury related to long-term PD. We examined the impact of glucose and commercial peritoneal dialysis solutions on complement expression in HPMCs obtained by primary culture from omental tissues of consented patients undergoing elective abdominal surgery. Constitutive expression of C3 and C4 mRNA in HPMCs was up-regulated upon exposure to 75 mm glucose in a time-dependent manner. C3 and C4 protein was secreted in both apical and basolateral directions. Glucose doses beyond 100 mm markedly down-regulated C3 and C4 expression, and stimulated LDH release dose-dependently. Such cytotoxic effects were attenuated using equivalent doses of mannitol instead of glucose. Treatment with conventional lactate-buffered dialysis solution gave rise to down-regulation of C3 and C4 expression, and heightened LDH release in HPMCs. These effects correlated with the glucose strength of the solution, persisted despite replacement with a bicarbonate-buffered solution, aggravated by glycated albumin, and were partially abrogated by supplementation with 10% fetal bovine serum in the culture system. Our findings suggest that the artificial conditions imposed by PD lead to alterations in local complement synthesis that have implications for the role of the peritoneal mesothelium in both inflammation and defence.     

Comparison of icodextrin- and glucose-based peritoneal dialysis fluids in their acute and chronic effects on human peritoneal mesothelial cells

BACKGROUND: Icodextrin-based peritoneal dialysis fluids (PDFs) display several features that may potentially improve their biocompatibility compared to conventional glucose-containing solutions. So far, however, the studies assessing the biocompatibility profile of icodextrin toward human peritoneal mesothelial cells (HPMC) has produced mixed results. The present study was performed to examine the acute and chronic impact of icodextrin on HPMC in vitro in comparison with standard glucose-based PDF. METHODS: Omentum-derived HPMC were either acutely pre-exposed to or incubated chronically (for up to 10 days) in the presence of icodextrin-PDF. Parallel cultures were treated with conventional PDFs containing either 1.5% or 4.25% glucose. All fluids were tested at neutral pH. HPMC were assessed for viability, proliferation, IL-6 secretion and generation of reactive oxygen species (ROS). RESULTS: Incubation in the presence of icodextrin-PDF significantly reduced HPMC proliferation in a manner similar to that of 1.5% glucose-PDF. In addition, exposure to icodextrin-PDF impaired viability and IL-6 release from HPMC. This effect occurred both after the short pre-treatment with neat icodextrin-PDF for 1-4 hours and after prolonged incubation (up to 10 days) in media supplemented with icodextrin-PDF (1:1). The dysfunction of icodextrin-treated HPMC was of the magnitude that was between the effects exerted by 1.5%- and 4.25%-glucose PDF. Furthermore, exposure of HPMC to icodextrin-PDF induced a dose-dependent increase in ROS generation which was comparable to that produced by 1.5%-glucose PDF. CONCLUSION: Exposure to icodextrin-PDF may impair viability and function of HPMC. The detrimental effects of icodextrin-PDF are at least as serious as those produced by conventional heat-sterilized low glucose-based PDF.     

Feasibility of mesothelial transplantation during experimental peritoneal dialysis and peritonitis

The mesothelial cell layer lining the peritoneum orchestrates peritoneal homeostasis. Continuous exposure to peritoneal dialysis fluids and episodes of peritonitis may damage the monolayer irreversibly, eventually leading to adhesion formation and fibrosis/sclerosis of the peritoneum. Autologous mesothelial cell transplantation is thought to be one of the options to reduce dysfunction of the peritoneal membrane. In this article we will review the mesothelial cell transplantation experiments performed in the field of peritoneal dialysis and peritonitis. In addition we will focus on the trouble shooting using cultured autologous mesothelial cells for transplantation.     

Culturing mouse peritoneal mesothelial cells

Obtaining normal cells has become increasingly important for use in comparative genetic analytical techniques to examine alterations in gene expression during transformation and progression into malignancy. Normal mesothelial cells are not currently available in cell banks and are essential for comparison of genetic expression analysis in current mouse mesothelioma models. The purpose of this investigation was to extract normal mouse peritoneal mesothelial cells using minimal culture techniques to obtain sufficient cells for gene expression analysis. Mesothelial cells were collected from the mouse peritoneal cavity in vivo with minimal contamination of lymphocytes and macrophages. The cells were cultured for approximately eight days until they were just confluent and retained normal mesothelial phenotype and cytokeratin immunoperoxidase staining.     

Capnocytophaga cynodegmi peritonitis in a peritoneal dialysis patient

The first reported case of peritonitis caused by Capnocytophaga cynodegmi is presented. The patient was treated with peritoneal dialysis and had contact with a cat. C. cynodegmi is part of the normal oral flora of dogs and cats but is very rarely isolated in clinical specimens from humans.     

灯盏花素对腹膜透析液诱导人腹膜间皮细胞转化生长因子β1的影响

背景：从不同层面了解灯盏花素阻止/延缓腹膜功能衰竭的作用及其机制,从而在临床上推广使用灯盏花素来阻止/延缓腹膜功能衰竭从而延长终末期肾脏病患者腹膜透析时间、提高透析质量、减少透析失败率,提高腹膜透析远期疗效具有广泛的应用前景。 目的：观察灯盏花素对腹膜透析液诱导的人腹膜间皮细胞转化生长因子β1分泌及其增殖活性的影响。 方法：体外培养人腹膜间皮细胞,分为5组：分别为对照组、腹膜透析液组、灯盏花素终浓度为5,10,20 µmol/L组。检测各组上清液中转化生长因子β1的水平以及间皮细胞的增殖活性。 结果与结论：腹膜间皮细胞在腹膜透析液诱导下,转化生长因子β1分泌显著增加、细胞增殖活性显著降低。灯盏花素5 µmol/L组转化生长因子β1分泌低于腹膜透析液组(P < 0.05),细胞增殖活性高于腹膜透析液组(P < 0.05);灯盏花素10,20 µmol/L组转化生长因子β1分泌显著低于腹膜透析液组(P < 0.01),细胞增殖活性显著高于腹膜透析液组(P < 0.01)。结果显示灯盏花素可以抑制腹膜间皮细胞转化生长因子β1分泌,拮抗腹膜透析液对腹膜间皮细胞增殖活性的抑制作用。     

State of the art on autologous mesothelial transplant in animals and humans

Sixteen years ago rabbit and human mesothelial cells were successfully cultured and autoimplanted. The aim of the study was merely to demonstrate that mesothelial implant was possible and interesting not only in peritoneal dialysis, but also in the vaster field of medicine and surgery concerning all the mesothelial districts of the body. The aim of this paper is to recollect the steps which have led to autologous mesothelial transplantation and verify if the technique has been validated and adopted by others. Review of the literature published in the last 15 years shows that intraperitoneal transplantation of mesothelial cells has been effective in reducing the formation of peritoneal adhesions, and in remodeling the area of mesothelial denudation. New studies on the mesothelial cell opened the way to construction of transplantable tissue-engineered artificial peritoneum, to the utilization of mesothelial progenitor cells and to find simple methods to collect autologous mesothelial cells. Finally mesothelial transplantation may represent a new neovascular therapy in the prevention and treatment of ischemic coronary heart disease.     

含糖透析液对慢性腹膜透析大鼠腹膜功能及间皮细胞形态的影响

目的:探讨含糖透析液对慢性大鼠腹膜透析模型腹膜功能和腹膜间皮细胞形态的影响及它们之间的关系。方法:40只SD大鼠随机分为4组,除对照组外,余3组每天分别腹腔内注入20mL4.25%透析液(HG)、1.5%透析液(LG)、林格氏液(RG)。8周后进行腹膜功能试验,并行细胞印片进行形态学分析。结果:HG组及LG组腹腔内透出液量和净出超量明显低于对照组和RG组(P<0.01),4h透析液与血浆尿素氮浓度之比(D/P_urea)显著高于对照组和RG组(P<0.05),尿素氮清除率(C_urea)显著低于对照组和RG组(P<0.01)。细胞印片上HG组及LG组间皮细胞的细胞密度显著少于对照组和RG组,表面积显著大于对照组和RG组(P<0.01)。但以上改变在HG组及LG组间无显著差异。使用含糖透析液8周所致的腹膜超滤功能下降和间皮细胞肥大呈负相关(r=-0.896,P<0.05)。结论:长期应用含糖透析液可使腹膜超滤功能下降,这一作用可能与腹膜间皮细胞肥大有关。     

Role of mesothelial cells in peritoneal antibacterial defence.

Whether phagocytosis of Staphylococcus aureus by polymorphonuclear neutrophils, monocytes, and peritoneal macrophages takes place when the staphylococci are adherent to monolayers of human mesothelial cells in the absence of opsonins was investigated. Adherence of S aureus to mesothelial monolayers increased significantly when the bacteria were opsonised with pooled human serum, but phagocytosis by polymorphonuclear neutrophils and monocytes occurred independently. Phagocytosis by peritoneal macrophages, however, was only marginal. Pretreatment of polymorphonuclear neutrophils with inhibitors of cellular metabolism and motility reduced their phagocytic capacity. These results indicate that the surface of mesothelial cells provides favourable conditions for the elimination of staphylococci in the peritoneal cavity. Phagocytic motility seems to be important in surface phagocytosis. The inability of peritoneal macrophages to phagocytise staphylococci adherent to the mesothelial cells suggests that they contribute little to the antibacterial defence of the peritoneal membrane of patients receiving peritoneal dialysis.     

Output of peritoneal cells during peritoneal dialysis.

Peritoneal dialysis provides a good source for the collection of macrophages. Six patients with chronic renal failure undergoing peritoneal dialysis for the first time were studied, and maximum cell egress, mostly macrophages, occurred at 24-48 hours and diminished after 48 hours.     

The effect of peritoneal rest in combination therapy of peritoneal dialysis and hemodialysis: using the cultured human peritoneal mesothelial cell model

The effects of peritoneal rest for 24 h during peritoneal dialysis and hemodialysis combination therapy were investigated using cultured human peritoneal mesothelial cell (HPMC) models. Cell activity was investigated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide (MTT) assay after exposing HPMCs to peritoneal dialysis fluids (PDFs) with different pH levels. The following PDFs (50 µl/well) were used for exposure durations of 30 or 240 min: acidic heat-sterilized PDFs (L-H PDF, pH 5.5) and neutral heat-sterilized PDFs (N-H PDF, pH 6.7). Control wells were exposed to M-199 Hanks medium containing 20% fetal bovine serum (FBS) for 30 or 240 min. Supernatants were then aspirated from each well and M-199 culture medium containing 20% FBS (50 µl) was added to each well to rest HPMCs for 24 h before investigation of MTT activity. The activity of HPMCs exposed to L-H PDF for 240 min decreased to ∼20% and 15% when compared with controls (glucose concentrations of 1.36% and 3.86%, respectively; P < 0.01 versus control, Tukey–Kramer test), and to ∼60% and 40% after exposure to N-H PDF for 240 min (glucose: 1.36% and 3.86%; P < 0.01). The activity of HPMCs exposed to L-H PDF for 240 min followed by rest was ∼20% and 4% when compared with controls (glucose: 1.36% and 3.86%; P < 0.01) and was 93% and 96% when compared with controls after exposure to N-H PDF for 240 min followed by rest (glucose: 1.36% and 3.86%). These findings suggest that rest for 24 h after exposure to N-H PDF improves the activity of HPMCs.