Flow cytometric analysis of mesothelial cells in peritoneal dialysis effluent

According to recent reports, deformity and size of mesothelial cells reflect mesothelial condition. We applied flow cytometry (FCM) to the analyzation of mesothelial cells in peritoneal dialysis effluent (PDE) and the relationship between the period of peritoneal dialysis (PD) and peritoneal function. Eighteen patients treated for two to 89 months by PD were selected. Their dialysate: plasma creatinine ratio (D/P creatinine)was 0.67 +/- 0.086 (0.53 to 0.87). Overnight PDE was drained and centrifuged. The cell population of peritoneal cells identified by anti-cytokeratin, CD14 and 45 antibodies was studied by FCM. Cytokeratin positive cells were identified as mesothelial cells, distinct from macrophages, granulocytes or lymphocytes. The forward scatter (FSC) of cytokeratin positive cells, fluorescence intensity of cytokeratin and percentage of cytokeratin-positive cells in PDE were 395.6 +/- 55.5 (298.31 to 527.72), 333.9 +/- 272.9 (67.55 to 1,071.95), and 6.75 +/- 6.1% (0.44 to 21.14), respectively. There was a positive correlation between D/P creatinine and FSC, and a negative correlation between D/P creatinine and cytokeratin fluorescence intensity or the percentage of cytokeratin positive cells. However, there was no correlation between the period of PD and FSC, cytokeratin fluorescence intensity or the percentage of cytokeratin-positive cells. It was suggested that the alteration of mesothelial cells is not necessarily influenced by the period of PD, but influences peritoneal function. It was found that the analysis of cell population by FCM reflects the morphological and functional changes in the peritoneum of patients on PD.     

Role of peritoneal mesothelial cells in peritonitis

BACKGROUND: Peritoneal mesothelial cells have a remarkable capacity to respond to peritoneal insults. They generate an intense biological response and play an important role in the formation of adhesions. This review describes these activities and comments on their relationship to surgical drainage, peritoneal lavage and laparostomy in the management of patients with peritonitis. METHODS AND RESULTS: Material was identified from previous review articles, references cited in original papers and a Medline search of the literature. The peritoneal mesothelium adapts to peritonitis by facilitating the clearance of contaminated fluid from the peritoneal cavity and inducing the formation of fibrinous adhesions that support the localization of contaminants. In addition, the fluid within the peritoneal cavity is a battleground in which effector mechanisms generated with the involvement of peritoneal mesothelial cells meet the contaminants. The result is a complex mix of cascading processes that have evolved to protect life in the absence of surgery. CONCLUSION: Future advances in the management of patients with severe peritonitis may depend upon molecular strategies that modify the activity of peritoneal mesothelial cells.     

Zymosan induces nitric oxide production by peritoneal mesothelial cells

Introduction: The production of nitric oxide is an important peritoneal defense mechanism. We have evaluated the effect of various putative stimulants on nitric oxide production by peritoneal mesothelial cells. Methods: Wistar rats were randomized to either a control group or a peritonitis group (5 mg zymosan intraperitoneally). Groups of five animals were sacrificed at 4, 18, 24, 48 and 96 h after the induction of peritonitis and their peritoneal fluid was harvested for assay. Cultures of peritoneal mesothelial cells were stimulated with lipopolysaccharide, myeloperoxidase, TNFα, zymosan, peritoneal fluid from a control animal and peritoneal fluid from a peritonitis animal. Supernatants were collected after incubation for 4, 24 and 48 h for assay. The assay for nitric oxide was based upon the nitrite content of the samples. Results: The intraperitoneal administration of zymosan was associated with an increased production of nitric oxide (NO) when compared with control animals (P < 0.01). In cultures of peritoneal mesothelial cells, zymosan, but not the other putative stimulants, was associated with a marked output of nitric oxide (P < 0.001). Conclusion: Zymosan has a direct effect on peritoneal mesothelial cells, which are able to generate nitric oxide in the absence of co‐stimulatory molecules. This suggests that it may be possible to use some form of external stimulation to up‐regulate the NO response by peritoneal mesothelial cells.     

神经酰胺在葡萄糖腹膜透析液诱导的腹膜间皮细胞凋亡中的作用

目的 观察葡萄糖腹膜透析液(PDS)对人腹膜间皮细胞(PMC)凋亡的影响和细胞内神经酰胺在PMC凋亡中的作用,初步探讨神经酰胺信号通路是否参与高浓度PDS诱导的PMC凋亡.方法 PMC分别在正常对照、1.5％ PDS、4.25％ PDS条件下培养,以4.25％甘露醇作为高渗对照.高压液相色谱串联质谱法(LC-MS-MS)检测细胞内神经酰胺的变化.Annexin - FITC- PI双染流式细胞术检测细胞凋亡.Western印迹法检测bax、p53、bcl-2蛋白表达.结果 (1)PDS可上调PMC细胞内神经酰胺表达,正常对照组、高渗对照组对细胞内神经酰胺均无明显影响.酸性鞘磷脂酶抑制剂地昔帕明可显著抑制高浓度PDS诱导的神经酰胺生成[(56.08±12.24)μg/L比(91.25±15.89) μg/L,P＜0.01].(2)与1.5％ PDS组相比,4.25％PDS可显著诱导PMC细胞凋亡[(26.65±6.21)％比(4.04±1.86)％,P＜0.01],上调bax、p53蛋白表达(P＜0.01),下调bcl-2蛋白表达(P＜0.05).地昔帕明可明显抑制高浓度PDS诱导的PMC细胞凋亡,bax、p53上调以及bcl-2下调(均P＜0.05),外源性神经酰胺则可明显逆转地昔帕明的此类作用(P＜0.05).正常对照组、高渗对照组对细胞内神经酰胺表达及细胞凋亡均无明显影响.结论 细胞内神经酰胺增加可能参与了高浓度PDS诱导的PMC凋亡.     

Leptin induces TGF-beta synthesis through functional leptin receptor expressed by human peritoneal mesothelial cell

Marked increase in leptin concentration in spent peritoneal dialysate has been reported following continuous ambulatory peritoneal dialysis treatment. The present study was designed to determine whether functional leptin receptor is expressed by human peritoneal mesothelial cells and if so, the possible implication in dialysis. Expression of leptin receptors in cultured mesothelial cells and omental tissue was examined. The effect of leptin on the production of transforming growth factor-beta (TGF-beta) by mesothelial cells in the presence or absence of high glucose was determined using in vitro culture model of human peritoneal mesothelial cells and adipocytes. The signaling mechanism involved in leptin-induced TGF-beta synthesis by mesothelial cells was studied. Both mRNA and protein of the full-length leptin receptor are constitutively expressed in mesothelial cells. The leptin receptor expression in mesothelial cells was upregulated by glucose but not leptin. In adipocytes, glucose increased the mRNA expression and synthesis of leptin. The Janus kinase-signal transducers and activation (JAK-STAT) signal transduction pathway in mesothelial cells was activated by either exogenous or adipocytes-derived leptin. Exogenous leptin induced the release of TGF-beta by mesothelial cells. The TGF-beta synthesis induced by leptin was amplified by glucose through increased leptin receptor expression. Our novel findings reveal that functional leptin receptor is present on human peritoneal mesothelial cells. The leptin-induced TGF-beta synthesis in mesothelial cells is associated with the expression of leptin receptor and the activation of the JAK-STAT signal transduction pathway.     

Correction of anemia in uremic mice by genetically modified peritoneal mesothelial cells

BACKGROUND: During peritoneal dialysis, mesothelial cells become detached from the peritoneum and accumulate in the dialysate. Our aim was to evaluate the potential of peritoneal effluent (PF)-derived human peritoneal mesothelial cells (HPMC) as target for gene therapy. We used erythropoietin (EPO) as our target gene. METHODS: Various extracellular matrixes (ECM) were tested for optimal adhesion and growth of HPMC. The EPO gene was introduced to mouse peritoneal mesothelial cells (MPMC) and HPMC by transfection or retroviral transduction. EPO secretion from PMC was measured by enzyme-linked immunosorbent assay (ELISA) and by the TF-1 cell proliferation assay. We performed intraperitoneal or intramuscular transplantations of the genetically modified cells into regular or 5/6 nephrectomized Balb/c mice and nude mice. Finally, we measured serum EPO and hematocrit levels. RESULTS: ECM-coated plates provided up to sixfold increase in the efficiency of PMC isolation from PF. Gelatin coated dishes (20 microg/cm2) were found optimal for isolation of PF-HPMC. RPR-120535 liposome was found to be best for PMC transduction. In vitro studies showed EPO secretion from modified HPMC over 6 months. Intraperitoneal transplantation aided with collagen matrix was the most effective. EPO, in MPMC transplanted mice, was detected up to 3 weeks (peak at 13 +/- 1 mIU/mL), and anemia of uremic mice was corrected (35.3 +/- 0.9 mIU/mL to 41.9 +/- 1.1 mIU/mL). CONCLUSION: PF-HPMC can be considered as an appropriate target for gene therapy since these cells can be efficiently isolated, modified, and transplanted. Nevertheless, implantation techniques in the peritoneum should be directed at obtaining longer duration of transgene expression in vivo, and means should be developed for enabling regulated expression of the gene.     

Regulation of glucose transporters in human peritoneal mesothelial cells

BACKGROUND: Risk factors for peritoneal fibrosis and mesothelial cell (MsC) injury in CAPD are infections and bioincompatibility of the dialysate, including high glucose concentrations. To study a potential link between dialysate and glucose toxicity in MsC, we investigated the expression of facilitative glucose transporters (GLUT), which could contribute to glucose toxicity. METHODS: After induction of cell differentiation, MsC were incubated in regular medium or medium with 60 mM D-glucose, 30 mM glucose plus 30 mM mannitol, 60 mM mannitol, PD effluent, or with a cytokine mix. Expression of GLUT1, GLUT3, SGLT and GAPDH/L32 was studied by RNase protection assay. MsC were incubated under identical conditions with 14C-fluoro-deoxy-glucose for 30 minutes and glucose uptake was measured. To estimate Vmax and Km, 14C-fluoro-deoxy-glucose uptake rates were determined over a range of 0.6 to 10 mM unlabeled glucose. RESULTS: The cytokine mix significantly stimulated GLUT1 expression (3-fold) and GLUT3 (1.7-fold). There was a 1.4-fold increase in GLUT1 (p<0.05) and a 1.7-fold increase in GLUT3 (p<0.05) after incubation in high glucose but not in mannitol or PD-effluent controls. Glucose uptake studies confirmed this increase after incubation in 30 mM (p<0.05) and 60 mM glucose solutions. Kinetic studies showed the Km was approximately 3.7 mM for this transport. CONCLUSIONS: GLUT mRNA expression and glucose uptake are induced by high ambient glucose concentrations and cytokines. Unlike many other cells, MsC are not able to protect themselves from increased glucose concentrations by downregulation of GLUTs. The intracellular glucose concentration may therefore increase during CAPD, affecting growth factor expression and glycosylation, and contributing to glucose toxicity.     

Normal saline induces oxidative stress in peritoneal mesothelial cells

Background

Peritoneal adhesions are the most common complication of the abdominal surgery. Normal saline is frequently used to rinse the peritoneal cavity during abdominal surgery, although there is no well-established data describing effect of such procedure on the process of formation of peritoneal adhesions.

Methods

Effect of 0.9% NaCl solution on viability, oxidative stress, and fibrinolytic activity of human peritoneal mesothelial cells maintained in in vitro culture was evaluated.

Results

Exposure of mesothelial cells to 0.9% NaCl induces oxidative stress, derangement of their structure with subsequent increased release of tissue factor (+75%) and plasminogen activator inhibitor-1 (+19%), and simultaneous suppression of tissue plasminogen activator release (−39%). In effect, ration tissue plasminogen activator/plasminogen activator inhibitor-1 was reduced in 0.9% NaCl-treated cells by 50%. Pretreatment of cells with precursor of glutathione synthesis: l-2-oxothiazolidine-4-carboxylic acid prevented these changes.

Conclusions

Oxidative stress in the peritoneal mesothelium caused by 0.9% NaCl activates their procoagulant activity and impairs fibrinolytic properties of these cells. These effects disqualify 0.9% NaCl as rinsing solution during abdominal surgery.     

Direct activation of human peritoneal mesothelial cells by heat-killed microorganisms.

OBJECTIVE: The aim of the study was to determine if human peritoneal mesothelial cells (HPMCs) can be activated directly by bacterial products contained in preparations of heat-killed Escherichia coli and staphylococci. SUMMARY BACKGROUND DATA: It has been shown recently that cytokine-activated HPMCs produce the inflammatory mediators, interleukin-1, interleukin-6, interleukin-8, and macrophage chemotactic protein-1. Studies concerning the effects of bacterial products on HPMCs are scarce and have not yielded conclusive results. METHODS: Growth-arrested HPMC monolayers were prepared from cell suspensions obtained by enzymatic disaggregation of small pieces of omentum. They were incubated for 24 hours with heat-killed E. coli (ATCC 25922), heat-killed staphylococci (ATCC 25933), or E. coli lipopolysaccharide, and the release of various cytokines in the culture media was measured by radioimmunoassays or enzyme-linked immunosorbent assays. Results were expressed as mean +/- standard error of the mean in picograms per milliliter of supernatant and analyzed with the Wilcoxon test; p values of less than 0.05 were considered significant. RESULTS: Baseline production of interleukin-6, interleukin-8, the chemokine "regulated upon activation, normal T cell expressed and secreted" (RANTES), and macrophage chemotactic protein-1 varied widely from one omental preparation to the other. E. coli increased the release of these mediators: from 1206 +/- 316 pg/mL to 8480 +/- 2189 pg/mL for interleukin-6, from 285 +/- 58 pg/mL to 3164 +/- 1053 pg/mL for interleukin-8, from 7 +/- 5 pg/mL to 684 +/- 264 pg/mL for RANTES, and from 2212 +/- 346 pg/mL to 7726 +/- 1473 pg/mL for macrophage chemotactic protein-1. Heat-killed staphylococci did not alter significantly the production of RANTES or macrophage chemotactic protein-1 but increased the production of the two other cytokines from 1325 +/- 389 pg/mL to 2206 +/- 523 pg/mL for interleukin-6 and from 318 +/- 70 pg/mL to 819 +/- 265 pg/mL for interleukin-8. CONCLUSIONS: The authors' results show that HPMCs are able to react to a direct stimulation with heat-killed microbes. They suggest that HPMCs, as well as resident macrophages, participate actively in the initiation and possibly in the modulation of intraperitonen inflammatory reactions.     

Antibiotics induce apoptosis of human peritoneal mesothelial cells

SUMMARY:   The peritoneal mesothelial cell is a critical component of the peritoneal membrane. The intraperitoneal use of several antibiotics to treat bacterial peritonitis is current clinical practice. Our previous study showed that cephalothin (CPL) and cefotaxime (CFT) have cytotoxic effects on human peritoneal mesothelial cells (HPMC), however, the exact mechanism of cytotoxicity has not been elucidated. In the present study, flow cytometry, TdT-mediated dUTP nick-end labelling (TUNEL) staining and electron microscopy were used to detect the apoptosis of HPMCs. Immunofluorescent staining was used to evaluate the cytochrome c distribution pattern. Western blotting was used to assess apoptotic signalling proteins. We found that CPL (0.5 mg/mL) and CFT (1 mg/mL) induced apoptosis of HPMCs, whereas cefazolin (0.5 mg/mL) and ceftriaxone (0.5 mg/mL) failed to induce apoptosis of HPMCs. While the DNA content of CFT- or CPL-treated cells was reduced, as determined by flow cytometry, cefazolin and ceftriaxone had no such effect. The CFT- or CPL-treated cells displayed the features of apoptosis both under the electron microscope and by using TUNEL staining. However, cefazolin and ceftriaxone produced the same result as the medium controls. Furthermore, CFT and CPL increased the expression of Bax and p53, and caused the translocation of cytochrome c from the mitochondria to the cytoplasm. The HPMC treated by CFT but not by CPL induced the cleavage of procaspase-3 to form active caspase-3. In conclusion, cefotaxime and cephalothin induce apoptosis of HPMCs in vitro . Signal transduction may be through the mitochondrial pathway.     

Remodeling of peritoneal-like structures by mesothelial cells: its role in peritoneal healing

BACKGROUND: Intraabdominal adhesions are a common complication following laparotomy. Since the exact mechanisms involved in this processes are unknown we have analyzed in vitro the role of mesothelial cells in peritoneal healing. MATERIAL AND METHODS: Human mesothelial cells from omental tissue were cultivated for 2 weeks in a three-dimensional culture either on or in a collagen type I matrix. The effects of blood and collagen matrix were analyzed by exposing mesothelial cells to an overlying blood clot, simulating intraperitoneal bleeding, or a second collagen layer. The production of collagen types III and IV, fibronectin, and laminin was analyzed with immunohistochemical methods. RESULTS: Mesothelial cells grown on a collagen matrix formed a monolayer of flat or cobblestone-like cells whereas those cultivated in a collagen matrix exhibited spindle-like morphology. Mesothelial cells failed to grow into an overlying collagen matrix, but did grow into a blood clot, emphasizing a potential role of blood clots in peritoneal adhesion formation. Independent of the culture systems mesothelial cells produced collagen type III, fibronectin, and laminin but not collagen type IV. CONCLUSIONS: Our experiments demonstrate remodeling of peritoneal-like structures by mesothelial cells in a three-dimensional culture reflecting their putative role in the reepithelialization after serosal defects, and also in the formation of peritoneal adhesions.     

Increased storage and secretion of phosphatidylcholines by senescent human peritoneal mesothelial cells

Background/aims

Human peritoneal mesothelial cells (HPMC) secrete phosphatidylcholines (PC) which form a lipid bilayer lining the peritoneum. They prevent frictions and adhesions and act as a barrier to the transport of water-soluble solutes while permitting water flux. PC may play an essential role in peritoneal integrity and function, the role of PD induced HPMC senescence on PC homeostasis, however, is unknown.

Methods

HPMC cell lines were isolated from four non-uremic patients. Expression of the three PC synthesis genes (rt-PCR), and cellular storage and secretion of PC (ESI-mass-spectrometry) were analyzed in young and senescent HPMC (>Hayflick-limit).

Results

Senescent cells displayed significantly altered morphology; flow cytometry demonstrated extensive staining for senescence-associated beta galactosidase. Nine different PC were detected in HPMC with palmitoyl-myristoyl phosphatidylcholine (PMPC) being most abundant. In senescent HPMC mRNA expression of the three key PC synthesis genes was 1.5-, 2.4- and 6-fold increased as compared to young HPMC, with the latter, phosphatidylcholine cytidylyltransferase, being rate limiting. Intracellular storage of the nine PC was 75–450 % higher in senescent vs. young HPMC, PC secretion rates were 100–300 % higher. Intracellular PC concentrations were not correlated with the PC secretion rates. Electron microscopy demonstrated lamellar bodies, the primary storage site of PC, in senescent but not in young cells.

Conclusion

Senescent HPMC store and secrete substantially more PC than young cells. Our findings indicate a novel protective mechanism, which should counteract peritoneal damage induced by chronic exposure to PD fluids.

     

Apoptosis of mesothelial cells caused by unphysiological characteristics of peritoneal dialysis fluids

There is an ongoing debate as to which peritoneal dialysis fluids (PDFs) provide the best preservation of peritoneal cells. To investigate this topic further, we measured apoptosis and necrosis of cultured mesothelial cells (MCs) after exposure to different single unphysiological features of PDFs and PDFs for whole. MCs were incubated in buffers containing plasticizers, high osmolarity by sodium chloride, low pH, and high glucose for 0.5, 4, and 24 h. The same procedure was repeated with different PDFs. Apoptosis and necrosis were measured by FACS-analysis (annexin-FITC and propidium iodide). We found that plasticizers were clearly able to induce apoptosis after 24 h (18 +/- 4%). The same result was observed with high osmolarity by sodium chloride (17 +/- 5%), but not for high glucose (9 +/- 8%). All fluids with low pH (5.2) caused severe and almost complete necrosis (after 4 and 24 h). Incubation in neutral, two-compartment PDFs (glucose 4.25%) without plasticizers for 4 h showed no significant necrosis (3%), but after 24 h apoptosis was detectable in 10 +/- 9% and necrosis in 29 +/- 8% of MCs. In conclusion, after improving PDFs and introducing neutral fluids, further attention should be drawn to inducers of apoptosis. Apoptosis can be detected quite early (24 h) and is caused by plasticizers and high osmolarity.     

Mononucleated phagocytes and mesothelial cells in serous peritoneal effusion fluid

Twenty one peritoneal effusions from patients with cirrhosis were studied in order to characterize mesothelial cells, monocytes and their vacuolated forms. Morphological investigations were performed after Papanicolaou, M.G.G. and Giemsa stains; cytochemical studies by P.A.S., Alcian Blue pH 2,5 and Sudan Black B techniques. Seven enzymatic activities were appreciated: Naphtyl Acetate Esterase, Acid Phosphatase pH 5, beta Glucuronidase, Lactico-dehydrogénase, Malico D, NADH.D and Peroxidase pH 7,6. The free mesothelial cells show a metabolic and functional active phase in which their hydrolasic equipment suggests an intense lysosomal activity only correlated to pinocytosis. Monocytes in transudates show a morphologically micro-vacuolated macrophagic transformation of pinocytic type (elicited macrophages ?); they only become cytophagic when an acute inflammation occurs (activated macrophages ?). This paper emphasizes analogy of behaviour of mesothelial cells and monocytes in transudates, but demonstrates differences in their energetic metabolisms and endocytic characteristics.