1Department of Pathophysiology and 2Department of Nephrology, University Medical School, Pozna, Poland, 3Department of Nephrology and Medical Intensive Care, Universitätsklinikum Charité, Berlin, 4Fresenius Medical Care, Bad Homburg, Germany, 5Department of Nephrology, University Medical School, Lublin, 6Department of Nephrology, University Medical School, Gda
sk and 7Department of Nephrology, Collegium Medicum, Jagiellonian University, Kraków, Poland
Correspondence and offprint requests to: Dr Janusz Witowski, Department of Pathophysiology, University Medical School, ul. wiecickiego 6, 60781 Pozna
, Poland. Email: jwitow{at}amp.edu.pl
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Abstract |
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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.
Keywords: biocompatibility; glucose degradation products; peritoneal dialysis; peritoneal mesothelial cells
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Introduction |
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The cytotoxic potential of GDPs present in PDFs has been demonstrated in several studies. We have recently observed significantly better preservation of viability and function in peritoneal mesothelial cells treated in vitro with new dual-chamber bag PDFs compared with conventional solutions of equal pH [2]. Indeed, by measuring heat shock protein expression, it has been confirmed that new generations of PDFs impose considerably less stress to mesothelial cells in culture [3].
Whilst these studies have shown differences in the response of mesothelial cells treated with unused PDF, it is not clear if such differences are still observable in vivo, where PDF is continuously being equilibrated. Following intraperitoneal instillation, the composition and properties of PDF change rapidly due to mixing with residual volume from preceding dwell and ultrafiltration from systemic circulation. It is well known that the low initial pH of PDF increases quickly, while concentrations of lactate and glucose decline with time. Moreover, it has been demonstrated that a substantial fraction of several GDPs present in PDF (glyoxal, methylglyoxal, 3-deoxyglucosone) is absorbed from the peritoneal cavity within a few hours [4]. As a result, the potential advantages of new PDFs, i.e. neutral pH and low GDP content, which are clearly seen in vitro, may in fact bear limited significance in the clinical setting.
In the present investigation, we have therefore assessed the function of peritoneal mesothelial cells treated with equilibrated dialysate effluent rather than by unused solutions. The samples of dialysate were obtained in the course of the Euro Balance Trial (EBT), a randomized, cross-over clinical study, during which patients were undergoing CAPD with the use of either conventional PDFs or a new pH-neutral low-GDP solution.
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Subjects and methods |
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Sample collection
At the end of treatment phases I and II, samples of peritoneal effluent were collected after a standardized overnight dwell of 10 h. The dwell was performed with a solution containing 2.3% glucose. Approximately 200 ml of effluent were withdrawn from the bag, filtered through a 0.2 µm pore size filter (Nalgene®; Nalge Nunc International, Rochester, NY, USA) to remove any cellular debris and aliquoted into polypropylene tubes (Sarstedt, Nümbrecht, Germany), centrifuged at 3000 g for 30 min and frozen at 70°C. After each treatment phase, all samples collected in participating centres were shipped in dry ice to the Department of Pathophysiology at Poznan Medical School for storage and analysis.
For initial experiments, samples of peritoneal effluent were obtained and pooled from eight stable CAPD patients not participating in the EBT. These samples were collected at specified times (15, 30 min, 1, 2, 4 h) after intraperitoneal infusion of conventional S-PDF containing 1.5% glucose.
Reagents
All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich GmbH, Deisenhofen, Germany. All tissue culture plastics were obtained from Nunc, Roskilde, Denmark.
Human peritoneal mesothelial cells (HPMCs)
Cells were isolated, characterized and maintained in culture as described in detail elsewhere [5]. The standard culture medium was M199 supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), hydrocortisone (0.4 µg/ml) and 10% (v/v) fetal calf serum (GibcoTM; Invitrogen, Karlsruhe, Germany).
Exposure of HPMCs to peritoneal effluent
Cells were rendered quiescent by serum deprivation for 24 h. Samples of effluent were mixed with an equal volume (1:1) of standard serum-free medium M199 and then added to cell cultures. Following a defined exposure period, cells were tested for viability, proliferation, growth factor release and cytokine secretion, as described below.
Cell viability
Cell viability was measured with the MTT assay. Briefly, after exposure to effluent, cells were incubated in medium containing 1.25 mg/ml of the MTT salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolinum bromide) for 4 h at 37°C. The formazan product generated was solubilized by the addition of acidic solution of 20% (w/v) sodium dodecyl sulphate and 50% (v/v) N,N-dimethylformamide. Absorbance of the converted dye was recorded at 595 nm with a reference wavelength of 690 nm.
Cell proliferation
Proliferation of HPMCs was assessed by measuring incorporation of 3H-thymidine into cultures during the exponential phase of growth. For these experiments, cells were plated onto 48 well clusters at density of 2.5 x 104/cm2 and cultured for 24 h. The subconfluent HPMC cultures were then exposed to effluent being studied mixed with an equal volume of M199 serum-free culture medium labelled with 3H-thymidine (used as methyl-3H-thymidine at 1 µCi/ml; Institute of Radioisotopes, Prague, Czech Republic). After 24 h of incubation at 37°C, cells were detached with 0.05% trypsin/0.02% EDTA solution and precipitated with 10% (w/v) trichloroacetic acid. The precipitate was dissolved in 0.1 N NaOH and the released radioactivity was measured in a beta liquid scintillation counter (Wallac; Perkin Elmer, Warsaw, Poland).
Cytokine and growth factor synthesis
Quiescent HPMC monolayers were exposed to a mixture of effluent and medium and incubated for 24 h. In addition, selected HPMC cultures were stimulated with IL-1ß (100 pg/ml). The post-culture supernates were centrifuged to remove any cellular debris and measured for IL-6, MCP-1 and VEGF, as described below.
Immunoassays
All immunoassays were performed using multi-well MaxiSorp test modules (Nunc). Immunoassays for IL-6, MCP-1 and VEGF were designed using ELISA-matched antibody pairs and performed according to the manufacturers instructions. Antibodies against IL-6 and VEGF were purchased from R&D Systems Europe (Abingdon, UK), and those against MCP-1 from Pharmingen (BD Biosciences, Heidelberg, Germany). For determination of fibronectin in effluent anti-human fibronectin antibodies from Dako (Glostrup, Denmark) and Biogenesis (Quartett, Berlin, Germany) were used. The concentration of CA125 in effluent was measured using an electrochemiluminescence assay (ECLISA; Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. The sensitivity of the immunoassays was as follows: IL-6, 3 pg/ml; MCP-1, 5 pg/ml; VEGF, 10 pg/ml; fibronectin, 0.15 ng/ml; CA125, 0.6 U/ml. Since the concentrations of target molecules in samples often exceeded the range spanned by the standard curve, the samples were appropriately diluted with the assay diluent (150 mM NaCl, 20 mM Trizma base, 0.1% bovine serum albumin, 0.05% Tween-20, pH 7.3). Pilot experiments determined that at all dilutions used, the presence of effluent in samples being measured did not significantly interfere with the assays. Recovery of exogenous antigens added to diluted samples at concentrations within the range of standard curves were 96.7114.1%. The background levels of cytokines and growth factors in effluent-containing media were always assayed in parallel and subtracted from the final results to assess specific mesothelial cell-derived cytokine and growth factor release. All results were normalized per 1 µg of cell protein, which was found to correspond to 2.1±1.0 x 103 cells (mean±SD, n = 16). Protein concentrations in cell lysates and peritoneal effluent were estimated with the Bradford method using bovine serum albumin as a standard.
Statistical analysis
Each sample of peritoneal effluent was tested nine to 11 times with mesothelial cells isolated from separate, non-uraemic donors. A median value obtained from these experiments was taken for further analysis. Data from the two arms of the study were combined, so that comparisons were made between S-PDF and Balance irrespective of the order in which patients received these fluids. Since the run-in phase served mainly for standardizing the procedures and training, it was not included in the analysis, so that statistical comparisons were made only with the data obtained at the end of each treatment phase. The data were analysed with the Wilcoxon test for paired non-parametric data using GraphPad PrismTM 3.00 software (GraphPad Software Inc., San Diego, CA, USA). A P-value of <0.05 was considered significant.
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Results |
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Discussion |
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We have attempted to combine all these approaches by allowing the peritoneal dialysis solutions tested to equilibrate in vivo, by in vitro exposing peritoneal mesothelial cells to drained dialysates, and by relating observed cell responses to the levels of certain mediators in effluent. Since using such a protocol can generate the data that are influenced not only by potential differences between the solutions examined, but also by the inter-patient variability, and diverse responses of HPMCs prepared from different donors, the following precautions have been introduced. To minimize differences originating from the patients population (i) the cross-over design of the underlying clinical trial, (ii) parallel in vitro testing of samples from the same patient and (iii) the analysis by paired statistics were employed. The differences stemming from HPMC cultures were reduced by testing a given effluent sample with all cell lines and then by taking a median rather than a mean from these measurements as an average value representing this sample.
Our experiments have shown that the intraperitoneal dwell led to a rapid change in the properties of unused PDF, so that the effluent drained from the peritoneal cavity maintained rather than inhibited HPMC growth in vitro. The maximal proliferation stimulating activity was observed after 2 h of intraperitoneal dwell but then declined with time. We have chosen to examine an effluent from the overnight dwell as we believed it would represent a balanced mixture containing not only growth-promoting stimuli but also inhibitory factors.
Interestingly, the effect of spent dialysate on HPMC proliferation appeared to be greater with Balance than with S-PDF effluent. Although we have been able to analyse samples from only 17 patients, the significance of these results is increased by the fact that, in contrast to other studies assessing ex vivo function of peritoneal cells, the EBT was designed as a cross-over study in which each patient served as their own control. In addition, the viability of HPMCs in vitro seemed to be better preserved following treatment with Balance. Since the stimulatory activity of dialysate towards HPMC proliferation could be attributed to the gradual diffusion of plasma-derived components and/or the local release of growth factors, we have hypothesized that the difference in dialysate levels of these growth-promoting stimuli could explain the difference between the effects of Balance and S-PDF. Indeed, we have found that effluent drained during dialysis with Balance contained significantly higher concentrations of fibronectin compared with those obtained during dialysis with S-PDF.
Fibronectin is an extracellular matrix protein that is known to stimulate mesothelial cell adhesion and proliferation [7]. The majority of fibronectin found in the peritoneal cavity is most probably of systemic origin [8]. It remains to be determined if Balance PDF may affect transperitoneal transport of fibronectin. On the other hand, although HPMCs do produce fibronectin, its fraction appears to be comparatively small. In fact we have been unable to accurately measure specific release of fibronectin from cells stimulated with effluent because of extremely high background (i.e. dialysate) levels of fibronectin compared with those secreted by HPMCs. However, in our previous study we have demonstrated that prolonged in vitro exposure of HPMCs to several identified GDPs resulted in a time-dependent loss of HPMC viability and, consequently, their ability to secrete fibronectin [9]. One may therefore imagine that dialysis with a low-GDP solution will help to maintain fibronectin synthesis in HPMCs and will enhance their proliferation in vivo. This view may be supported by changes in dialysate levels of CA125. Longitudinal changes in CA125 concentrations are believed to reflect mesothelial cell mass and viability [10]. We have found that intraperitoneal levels of CA125 were significantly increased when patients were receiving Balance compared to when they were treated with S-PDF. This observation concurs with previous reports of increased CA125 during dialysis with a new generation of dialysis fluids [11,12]. Although these solutions were based on different buffers (lactate or bicarbonate), they were all manufactured in multi-chamber systems and contained only trace amounts of GDPs. Interestingly, we have observed that increased levels of CA125 correlated significantly with higher concentrations of fibronectin but did not correlate directly with the in vitro growth of HPMCs in the presence of dialysate. It may indicate that while CA125 is a marker of mesothelial cell mass, it does not necessarily have to reflect cellular thymidine turnover measured over a short period of time.
The understanding of how peritoneal dialysis solutions affect HPMC growth in vivo appears to be of clinical significance. It has been demonstrated that the process of peritoneal dialysis induces continuous injury and regeneration in the peritoneal mesothelium [13]. Additional severe insult may be a consequence of peritonitis. Indeed, the Peritoneal Biopsy Registry® has documented loss of mesothelial lining in a significant number of patients undergoing CAPD [1]. Importantly, failure to re-mesothelialize is believed to be a prerequisite for subsequent development of peritoneal fibrosis and/or sclerosis that ultimately results in treatment failure [14].
The difference in HPMC growth in vitro in response to Balance and S-PSD effluents may appear small. However, the present study assessed proliferation of HPMCs only over a 24 h period. One may speculate that during long-term clinical CAPD with Balance these small differences may accumulate with time resulting in a peritoneal membrane covered with better preserved (or less injured) mesothelial cells. In this respect, recent data indicate that compared with conventional PDFs, new peritoneal dialysis solutions containing fewer GDPs improve mesothelial healing following in vitro wounding [15] and the process is associated with increased CA125 expression [16]. Furthermore, it is now known that GDPs accelerate the formation of advanced glycation end-products which are believed to contribute to peritoneal membrane dysfunction [4]. In this respect, it has been demonstrated that in vitro glycation of albumin in the presence of Balance PDF is significantly less pronounced compared with S-PDF [17]. More recently, reduced glycation in response to Balance PDF has been reported to also occur with collagen IV that forms the submesothelial basement membrane [18]. Interestingly, this Balance pre-exposed matrix has been found to better support subsequent growth of HPMCs in vitro compared with a template treated with S-PDF.
We have observed a similar release of cytokines from HPMCs incubated with test effluents. Additional stimulation with IL-1ß resulted in a synergistic increase in IL-6 secretion which appeared to be marginally greater in cells treated with Balance. Increased levels of IL-6 in dialysate have been recently shown to correlate significantly with dialysate VEGF, and both mediators were linked with increased peritoneal permeability [19]. While this association raises some concerns, we have found no evidence of increased VEGF release from cells exposed to Balance-derived effluent over the period of 24 h. On the other hand, recent data point to a crucial role of IL-6 in coordinating the inflammatory response [20]. In this respect, the readiness of cells to mount IL-6 may be viewed as promoting the prompt resolution of inflammation.
Taken together, our data suggest that dialysis with a new pH-neutral low-GDP solution is associated with improved proliferative response of peritoneal mesothelial cells, at least from non-uraemic patients. This impact may help to maintain integrity of the peritoneal membrane during long-term peritoneal dialysis.
Conflict of interest statement. None declared.
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References |
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