Human peritoneal mesothelial cells isolated from spent dialysate fluid maintain contaminating macrophages via production of macrophage colony stimulating factor

BACKGROUND: Human peritoneal mesothelial cells (HPMC) are useful for the analysis of peritoneal reactions to various insults and to peritoneal dialysate. HPMC can be readily obtained from spent dialysis fluid, but leucocyte contamination is a major problem when using these cells for in vitro experiments. Therefore, we examined the persistence of leucocyte contamination in HPMC cultures obtained from spent dialysate. METHODS: Cells were obtained from spent patient dialysate bags by centrifugation and analysed for specific cell phenotypes by flow cytometry at the initial collection and during sequential passages in cell culture. Cell proliferation was assessed by either bromodeoxyuridine incorporation or a dehydrogenase assay. Cytokine secretion was analysed by enzyme-linked immunosorbent assay. RESULTS: Spent dialysate bags contained two major cell populations: CD45+ leucocytes and cytokeratin-8/18+ cells. Initially, most collected cells were CD45+, but their numbers decreased rapidly during the first week of culture. However, a persistent contamination of CD45+ leucocytes, approximately 20% of cells, was evident during the next three passages. This persistent CD45+ contamination was identified as CD68+ macrophages and contained bromodeoxyuridine + proliferating cells. These macrophages could be removed by fluorescence-activated cell sorting using anti-CD45 antibody, resulting in highly purified HPMC which expressed cytokeratin-8/18 and calretinin. Supernatant obtained from these purified HPMC contained macrophage colony stimulating factor and induced proliferation of bone marrow-derived macrophages. CONCLUSION: Spent dialysate contains macrophages which persist in culture and are associated with HPMC secretion of macrophage colony stimulating factor and macrophage proliferation. Therefore, contaminating macrophages should be specifically removed from HPMC preparations before performing in vitro studies.     

The association between soluble intercellular adhesion molecule-1 levels in drained dialysate and peritoneal injury in peritoneal dialysis

Background: Chronic inflammation of the peritoneum causes peritoneal injury in patients on peritoneal dialysis. Intercellular adhesion molecule-1 and its circulating form, soluble intercellular adhesion molecule-1, play pivotal roles in inflammation. However, their role in peritoneal injury is unclear.

Methods: We measured changes in intercellular adhesion molecule-1 expression in the peritoneum of a peritoneal injury model in rats. The associations between soluble intercellular adhesion molecule-1 levels in drained dialysate and the solute transport rate (D/P-Cr and D/D0-glucose) determined by the peritoneal equilibration test, and matrix metalloproteinase-2 levels in drained dialysate were investigated in 94 peritoneal drained dialysate samples.

Results: Intercellular adhesion molecule-1 expression was increased in the peritoneum of rats with peritoneal injury. Soluble intercellular adhesion molecule-1 levels in drained dialysate were significantly positively correlated with D/P-Cr (r?=?.51, p?r?=??.44, p?r?=?.86, p?Conclusions: Intercellular adhesion molecule-1expression is increased in the peritoneum of a peritoneal injury model in the rat, and soluble intercellular adhesion molecule-1 levels in drained dialysate are associated with peritoneal injury in patients on peritoneal dialysis. These results suggest that soluble intercellular adhesion molecule-1 could be a novel biomarker of peritoneal injury in patients on peritoneal dialysis.     

The effectiveness of oral essential aminoacids and aminoacids containing dialysate in peritoneal dialysis

Background/Aim: Oral essential amino acids (AAs) containing supplements (EAS) and AA containing dialysate (ACD) are frequently used in peritoneal dialysis (PD) patients with malnutrition. The present study was conducted to investigate two strategies and compare their effects on the malnutrition status of PD patients. Materials and Methods: A total of 31 EAS, 14 ACD patients were enrolled in this study. Serum albumin levels were lower than 3.5?g/dL in all subjects. EAS group patients took five pills containing AAs three times a day with meals. In the other, 2.000?cc of 1.1% ACD was given to patients daily during the study. Demographic and laboratory parameters were analyzed and compared at baseline and 6th month. Results: Significant increases in BMI, albumin, and protein in both groups. Mean albumin levels increased significantly by 0.54?g/dL in ACD group (p?0.005) and 0.49?g/dL in EAS group (p?0.001) following 6 months. Mean albumin and delta albumin levels did not differ between two groups. Conclusion: These strategies may play an important role in increasing albumin levels and improving the nutritional status of PD patients.     

Antibiotic activity in peritoneal dialysate

There are few studies investigating whether antibiotics added to 30% glucose concentrate preserve their activity in the delivered dialysate. Using a Drake-Willock proportioning system, samples were obtained from the "to" patient path at ten minutes after starting and at four hours. Samples were tested for minimal inhibitory dilution (MID) against Escherichia coli and Staphylococcus aureus. Antibiotics evaluated included amikacin, tobramycin, gentamicin, cephalothin, cefamandole, moxalactam, ampicillin, penicillin, carbenicillin, and vancomycin. In all antibiotics studied, similar MIDs were obtained at the ten-minute and four-hour samples. Compared to saline, dialysate significantly impaired the antibiotic activity (a difference of two or more tube dilutions) of all antimicrobial agents except amikacin and vancomycin.     

Body composition measurements using bioimpedance analysis in peritoneal dialysis patients are affected by the presence of dialysate

The presence of peritoneal dialysate when performing bioimpedance analysis may affect body composition measurements. The aim of this study was to evaluate the impact of dialysate on body composition measurements in Asians. Forty‐one patients undergoing maintenance peritoneal dialysis in our hospital peritoneal dialysis unit were included in this study. Dialysate was drained from the abdomen prior to measurement, and bioimpedance analysis was performed using multi‐frequency bioimpedance analysis, with each subject in a standing position (D‐). Dialysate was then administered and the measurement was repeated (D+). The presence of peritoneal dialysate led to an increase in intracellular water (ICW), extracellular water (ECW), and total body water (D‐: 20.33 ± 3.72 L for ICW and 13.53 ± 2.54 L for ECW; D+: 20.96 ± 3.78 L for ICW and 14.10 ± 2.59 L for ECW; P < 0.001 for both variables). Total and trunk oedema indices were higher in the presence of peritoneal dialysate. In addition, the presence of peritoneal dialysate led to an overestimation of mineral content and free fat mass (FFM) for the total body; but led to an underestimation of body fat (D‐: 45.80 ± 8.26 kg for FFM and 19.30 ± 6.27 kg for body fat; D+: 47.51 ± 8.38 kg for FFM and 17.59 ± 6.47 kg for body fat; P < 0.001 for both variables). Our results demonstrate that the presence of peritoneal dialysate leads to an overestimation of FFM and an underestimation of fat mass. An empty abdomen is recommended when evaluating body composition using bioimpedance analysis.     

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.     

Hypertonic glucose-based peritoneal dialysate is associated with higher blood pressure and adverse haemodynamics as compared with icodextrin.

BACKGROUND: Little is known about the haemodynamic effects of continuous ambulatory peritoneal dialysis (CAPD) despite its widespread use in the management of end-stage renal failure. We undertook a study to delineate the haemodynamic effects of CAPD using glucose-containing fluids (1.36 and 3.86% glucose) and icodextrin. METHODS: Eight CAPD patients were recruited for a prospective crossover study. Patients attended for two investigatory days (in random order). CAPD was carried out using 1.36% followed by 3.86% glucose (buffered with lactate/bicarbonate, Physioneal) on one study day and 1.36% glucose followed by 7.5% icodextrin (Extraneal) on the other day. Dwell times were 150 min. Blood pressure (BP) and a full range of haemodynamic variables including pulse (HR), stroke volume (SV), cardiac output (CO) and total peripheral resistance (TPR) were measured non-invasively using continuous arterial pulse wave analysis. RESULTS: BP was significantly higher during 3.86% glucose dwells as compared with 1.36% glucose or icodextrin dwells (P<0.0001). TPR during all three dwells was similar; the higher blood pressure was due to an increased HR, SV and, therefore, CO during 3.86% glucose dwells. The higher blood pressure during the 3.86% glucose dwells was present despite the highest ultrafiltration volume and sodium removal. CONCLUSION: This study demonstrates large magnitude haemodynamic changes in response to CAPD. In addition to the well-recognized adverse effects on blood glucose and long-term peritoneal membrane viability, CAPD fluids containing high glucose concentrations may also exert undesirable effects on systemic haemodynamics, with potential long-term consequences for patient outcomes.     

Antibacterial peritoneal defence in automated peritoneal dialysis: Advantages of tidal over continuous cyclic peritoneal dialysis?

In tidal peritoneal dialysis (TPD) only a part of the infuseddialysate is drained with each exchange, leaving a residualvolume on top of which fresh fluid is cycled. As the persistentpresence of a buffered intraperitoneal reserve volume mightfavour peritoneal macrophage (PMO) function, PMO obtained fromeight patients during a 3-h continuous cyclic peritoneal dialysis(CCPD) or TPD session were studied in a randomized cross-overtrial. PMO were studied for uptake of E. coli (complement-dependent)and S. epidermidis (antibody-dependent), as well as for theirkilling capacity and peak chemiluminescence response. In addition,dialysate was sampled during both treatment sessions and studiedfor pH, osmolality, and effect on the viability of donor phagocytesand mesothelial cells. TPD-derived PMO were significantly better able to phagocytoseE. coli than CCPD-PMO (48 ±8 versus 33±6% uptake,P<0.05), whereas the other tested functional capacities revealedno significant difference between TPD- and CCPD-PMO. DuringTPD dialysate pH ranged from 6 to 7 as compared to a pH rangefrom 5 to 7 in CCPD. The presence of a residual dialysate volumeresulted in less wash-out of cells and opsonins early in thetreatment, and to some extent blunted the noxious effects offresh dialysis solutions. Overall, however, tidal PD appearedto have no advantage over CCPD regarding preservation of peritonealdefences.     

Role of transforming growth factor beta in peritoneal fibrosis

SUMMARY: Technique survival of peritoneal dialysis is seriously limited by the development of peritoneal fibrosis. the mesothelial cell layer lining the peritoneum is important in the pathogenesis of peritoneal fibrosis. Mesothelial cells are able to produce transforming growth factor beta (TGF-β), and respond to stimulation by this cytokine. In this review, we will detail the evidence available so far for the role of the complex interaction between TGF-β and mesothelial cells in the development of peritoneal fibrosis.     

Low vs standard calcium dialysate in peritoneal dialysis: differences in treatment, biochemistry and bone histomorphometry. A randomized multicentre study.

BACKGROUND: In patients undergoing peritoneal dialysis (PD), low-calcium dialysate (LCD) has been proposed as the first choice for a better control of renal osteodystrophy. Our aim was to compare the effects on bone metabolism of LCD (calcium: 1.25 mmol/l) with that of a standard calcium dialysate (SCD; calcium: 1.75 mmol/l). METHODS: Forty-four PD patients were randomized to receive LCD or continue on SCD for a period of 12 months. Bone biopsies were taken at baseline and at 12 months. Biochemical data and treatment were evaluated every 3 months. RESULTS: Twenty-four patients completed the study. In the SCD group (n = 10), nine out of the 10 patients were initially diagnosed with adynamic bone lesion (ABL). After 1 year, six continued having ABL and three patients moved to high-turnover bone lesion (HTBL). The other patient, initially diagnosed with HTBL, changed to ABL. In the LCD group (n = 14), 10 patients were initially diagnosed with ABL. At 1 year, six of them continued having ABL and four patients changed to HTBL. Four patients were initially diagnosed with HTBL and did not change. Comparison between LCD and SCD groups showed an increase in serum parathyroid hormone (PTH) levels starting at month 3 and a higher intake of calcium salts in the former group (P<0.01). Serum calcium, phosphate levels and bone histological outcome did not differ between the two groups. CONCLUSIONS: LCD use for 1 year was associated with an increase in PTH levels, but did not lead to histological changes different from those observed in SCD group. The LCD solution allowed a higher oral intake of calcium salts with a satisfactory control of the serum Calcium-Phosphorus product.     

腹膜透析液对腹膜间皮细胞凋亡与增殖的影响

目的　探讨PD 2腹膜透析液 (腹透液 )在体内对腹膜间皮细胞凋亡和增殖细胞核抗原(PCNA)表达的影响。方法　在正常二级SD大鼠上建立腹膜透析模型 ,用不同浓度的PD 2腹透液灌注腹腔 ,二次 /天 ,2 0ml/次 ,生理盐水作对照。透析第 8周后杀检 ,取腹膜用LSABkit和TUNELkit作免疫 凋亡双重染色。结果　腹透液显著诱导腹膜间皮细胞凋亡 ,以高渗糖腹透液尤为显著 ,不同浓度腹透液对PCNA影响则不同 ,透析液诱导的凋亡与PCNA表达之间无相关性 ,但与腹透液浓度正相关 ,PCNA表达则与腹透液浓度负相关。结论　腹透液可诱导腹膜间皮细胞凋亡 ,并影响PCNA表达 ,其作用与腹透液浓度不同而异。     

Different effects of amino acid-based and glucose-based dialysate from peritoneal dialysis patients on mesothelial cell ultrastructure and function.

BACKGROUND: Peritoneal dialysis fluid (PDF) containing amino acids has been introduced recently aiming to improve the nutritional status of PD patients. Dextrose-based PDFs have been implicated in progressive functional and structural deterioration of the peritoneal membrane. Limited data are currently available regarding the effect of amino acid-based PDF on the function and ultrastructure of human peritoneal mesothelial cells (HPMCs), which play a critical role in peritoneal membrane pathophysiology. METHODS: We investigated the effects of two commercially available PDFs, which utilized dextrose (1.5% Dianeal) or amino acids (1.1% Nutrineal) as the osmotic agent, obtained from patients after a 4 h dwell, on HPMC proliferation (MTT assay and cell counting) and viability [lactate dehydrogenase (LDH)release], interleukin-6 (IL-6) secretion (commercial enzyme-linked immunosorbent assay) and ultrastructure (scanning and transmission electron microscopy). RESULTS: Exposure of HPMCs to 1.5% Dianeal reduced cell proliferation, total cellular protein synthesis, IL-6 secretion and cell attachment, but prolonged the cell doubling time on recovery, and increased LDH release (P<0.001, P<0.001, P<0.0001, P<0.0001, P<0.001 and P<0.001, respectively). The 1.1% Nutrineal reduced HPMC proliferation (P<0.001) and increased IL-6 secretion (P<0.0001), but did not affect cell attachment, LDH release, protein synthesis or cell doubling time. Ultrastructural studies of HPMCs exposed to Dianeal showed cell flattening, increased cell surface area, reduced microvilli, and intracellular organelles compatible with dysfunctional mitochondria. In contrast, the ultrastructural morphology of HPMCs was relatively preserved after incubation with Nutrineal. CONCLUSIONS: Our results showed that HPMC ultrastructure, viability and protein synthesis were better preserved with amino acid-based PDF, compared with conventional dextrose-based PDF. The significance of IL-6 induction by Nutrineal remains to be elucidated.     

Peritoneal dialysis with solutions low in glucose degradation products is associated with improved biocompatibility profile towards peritoneal mesothelial cells.

BACKGROUND: In vitro experiments point to a better biocompatibility profile of new pH-neutral peritoneal dialysis fluids (PDFs) containing low levels of glucose degradation products (GDPs). The present study examines the impact on human peritoneal mesothelial cells (HPMCs) of equilibrated dialysates obtained during dialysis with either conventional or new PDFs. METHODS: Peritoneal dialysate was collected from 17 patients participating in a randomized, controlled, cross-over trial comparing a pH-neutral low-GDP solution (Balance) to a conventional solution (S-PDF). All patients were treated sequentially for 3 months with both PDFs. At the end of each treatment phase, peritoneal effluent was drained after a timed 10 h dwell. Samples of dialysate were then mixed with standard culture medium and added to in vitro cultures of HPMCs from healthy donors. Cells were assessed for proliferation, viability and cytokine release. RESULTS: Proliferation and viability of HPMCs were better preserved in the presence of effluent obtained during dialysis with Balance (P<0.046 and P<0.035, respectively). The proliferative response of HPMCs correlated with the concentration of fibronectin in dialysates (P = 0.0024). Effluent drained following a 3 month dialysis with Balance contained significantly increased levels of fibronectin (P = 0.004) and CA125 antigen (P = 0.0004) compared with S-PDF. There was no significant difference in constitutive and stimulated cytokine (IL-6, MCP-1, VEGF) synthesis by HPMCs treated with either Balance- or S-PDF-derived effluents. CONCLUSIONS: These results suggest that therapy with new pH-neutral low-GDP solutions contribute to an intraperitoneal milieu that improves mesothelial cell proliferation and viability. It may positively impact on the preservation of the peritoneal membrane integrity during long-term dialysis.     

Effect of PD fluid instillation on the peritonitis-induced influx and bacterial clearing capacity of peritoneal cells.

BACKGROUND: The commonly used peritoneal dialysis fluids contain glucose as the osmotic agent. Heat sterilization leads to the formation of glucose degradation products which contribute, together with glucose, to the formation of advanced glycation end-products (AGEs). AGEs have been shown to be present in the peritoneal cavity. Methods have been developed to minimize the amount of glucose degradation products in peritoneal dialysis fluids. In a rat peritoneal dialysis model, we compare the effect of a commonly used peritoneal dialysis fluid, Gambrosol, with a newly developed peritoneal dialysis fluid, PD-Bio, on the influx and functional capacity of the peritoneal cells after 2 weeks of peritoneal dialysis fluid instillation. METHODS: Three groups of animals were used: rats received daily infusion with 15 ml of either 4% Gambrosol (group 1) or 4% PD-Bio (group 2), and a control group of animals did not receive fluid (group 3). After 2 weeks of PD fluid instillation, all the animals were injected with a 0.5 ml suspension containing 3x10(8) colony-forming units of Staphylococcus aureus. The in vivo bacterial clearing capacity was determined after 15 h. RESULTS: A statistically significant higher leukocyte influx was found in the control group compared with both PD fluid-injected groups. No statistical differences in bacterial clearing were observed among the three groups, although the number of bacteria recovered from the PD-Bio group tended to be lower than that from the Gambrosol group. Moreover, in both PD fluid instillation groups, the bacteria tended to be cleared more slowly compared with the control group. The number of mesothelial cells in the PD fluid groups was significantly greater than in the control group. CONCLUSION: No differences were observed in bacterial clearing capacity, leukocyte influx and mesothelial cell number after a 2 week exposure of the peritoneal cavity to Gambrosol vs PD-Bio.     

糖化终末产物对人腹膜间皮细胞氧化应激的影响

目的研究不同浓度糖化终末产物（AGEs）对人腹膜间皮细胞（HPMC）氧化应激的影响。方法通过胰酶消化法从非。肾脏疾病腹腔手术患者的大网膜中得到HPMC,进行原代培养和传代,免疫组化法对培养的细胞进行鉴定。不同浓度的AGEs干预HPMC一定时间后,采用活性氧（ROS）捕获剂双氢-乙酰乙酸二氯荧光黄（DCFH-DA）孵育细胞,通过流式细胞仪检测细胞内的平均荧光强度测定细胞内ROS水平,观察不同浓度的AGEs对HPMC干预后细胞内ROS的变化。结果AGEs明显抑制HPMC活力（P〈0．05）,随着浓度和时间的增加,抑制率上升。AGEs干预HPMC后,细胞内的ROS水平明显升高。结论AGEs导致细胞发生氧化应激,其对细胞的损伤作用可能与氧自由基生成过多、ROS蓄积有关。     

Biocompatibility of a bicarbonate-buffered amino-acid-based solution for peritoneal dialysis

Amino-acid-based peritoneal dialysis (PD) fluids have been developed to improve the nutritional status of PD patients. As they may potentially exacerbate acidosis, an amino-acid-containing solution buffered with bicarbonate (Aminobic) has been proposed to effectively maintain acid-base balance. The aim of this study was to evaluate the mesothelial biocompatibility profile of this solution in comparison with a conventional low-glucose-based fluid. Omentum-derived human peritoneal mesothelial cells (HPMC) were preexposed to test PD solutions for up to 120 min, then allowed to recover in control medium for 24 h, and assessed for heat-shock response, viability, and basal and stimulated cytokine [interleukin (IL)-6] and prostaglandin (PGE(2)) release. Acute exposure of HPMC to conventional low-glucose-based PD solution resulted in a time-dependent increase in heat-shock protein (HSP-72) expression, impaired viability, and reduced ability to release IL-6 in response to stimulation. In contrast, in cells treated with Aminobic, the expression of HSP-72 was significantly lower, and viability and cytokine-producing capacity were preserved and did not differ from those seen in control cells. In addition, exposure to Aminobic increased basal release of IL-6 and PGE(2). These data point to a favorable biocompatibility profile of the amino-acid-based bicarbonate-buffered PD solution toward HPMC.