The triggering of human peritoneal mesothelial cell apoptosis and oncosis by glucose and glycoxydation products
Eric Boulanger1,3,
Marie-Paule Wautier1,2,
Pierre Gane2,
Christophe Mariette4,
Olivier Devuyst5 and
Jean-Luc Wautier1
1 Laboratoire de Biologie Vasculaire et Cellulaire and 2 INSERM Unité 76, Institut National de la Transfusion Sanguine, Paris, France, 3 Clinique Néphrologique and 4 Clinique Chirurgicale, CHRU, Lille, France and 5 Division de Néphrologie, Université Catholique de Louvain Medical School, Brussels, Belgium
Correspondence and offprint requests to: Dr Eric Boulanger, Laboratoire de Biologie Vasculaire et Cellulaire, INTS, 6 Rue A. Cabanel, 75739 Paris Cedex 15, France. Email: eboulanger{at}ints.fr
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Abstract
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Background. Peritoneal dialysis fluids (PDFs) have been shown to alter mesothelial cell functions. To further determine the mechanisms involved, we investigated the effects of glucose, glucose degradation products (GDPs) and advanced glycation end products (AGEs) on the inhibition of human peritoneal mesothelial cell (HPMC) proliferation and the induction of apoptosis and oncosis.
Methods. Four PDF solutions, heat-sterilized dextrose-lactate, filtered dextrose-lactate and heat-sterilized dextrose-bicarbonate-lactate, each containing 15 or 45 g/l glucose, and heat-sterilized icodextrin-lactate, containing 75 g/l icodextrin, were tested. In addition, we analysed the independent and synergistic effects of two glucose compounds, i.e. 3-deoxyglucosone (3-DG), a major GDP, and N
-(carboxymethyl)-lysine (CML), a high-affinity AGE receptor (RAGE) ligand on HPMC viability. Cell proliferation was measured by methyl-[3H]thymidine incorporation. Oncosis was quantified by nuclear propidium iodide (PI) DNA-intercalating capability, and apoptosis by the decrease in mitochondrial transmembrane potential (
m).
Results. It was found that heat-sterilized dextrose-lactate inhibited HPMC proliferation to a greater extent than filtered dextrose-lactate, heat-sterilized dextrose-bicarbonate-lactate, or heat-sterilized icodextrin-lactate (P<0.001). Compared to filtered dextrose-lactate, heat-sterilized dextrose-lactate induced a significantly greater degree of apoptosis (P<0.05) and oncosis (P<0.01). Glucose-induced cell death and antiproliferative activity were significantly potentiated by the action of 3-DG or CML-albumin. By blocking the AGERAGE interaction recombinant soluble-RAGE reduced the PDF-induced inhibitory effect on cell proliferation (P<0.001) and apoptosis (P<0.05).
Conclusion. Heat-sterilized PDFs that contain high glucose concentrations and GDPs, which are AGE precursors, reduce cell proliferation, induce mesothelial cell apoptosis and oncosis, and may be involved in peritoneal damage. PDFs containing lower glucose derivative products are more biocompatible.
Keywords: apoptosis; biocompatibility; glycoxidation; peritoneal dialysis
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Introduction
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Treatment of end-stage renal failure by peritoneal dialysis (PD) is limited by the reduced efficacy of the therapeutic approach after a period of time, which is linked to the loss of peritoneal membrane integrity leading to ultrafiltration failure. Alterations in the peritoneal barrier are the result of the cumulative effects of peritoneal infections and repeated exposure to non-biocompatible peritoneal dialysis fluids (PDFs) [1]. To further investigate the mechanisms responsible for the degradative changes related to prolonged exposure to PD solutions, the biocompatibility of PDFs has been tested on various cells such as leucocytes, fibroblasts and mesothelial cells. Different factors, including the composition of the buffer, glucose and glucose degradation products (GDPs) formed during heat sterilization, have been shown to be critical for biocompatibility [2]. GDPs such as glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG) and 3,4-dideoxyglucosone-3-ene (3,4-DGE) are extremely reactive, and could form advanced glycation end products (AGEs) [3]. Moreover, GDPs are known to have both peritoneal and systemic effects. For standard PD treatment, the average annual dose of 3-DG per patient corresponds to more than 100 g/year. In the AGE molecule group, N
-(carboxymethyl)-lysine (CML), which can be generated from GDPs, is a high-affinity ligand for the AGE receptor (RAGE) [4]. We have previously demonstrated that CML-albumin binds to mesothelial cells via RAGE, and stimulates vascular cell adhesion molecule-1 (VCAM-1) expression and leucocyte adhesion, thus suggesting that this mechanism may be involved in human peritoneal mesothelial cell (HPMC) degradation [5].
In the present work, we investigated whether PDFs could induce cell death. Cell death may occur in a disorganized and chaotic manner, associated with swelling of the cell, a process that is known as necrosis or, more specifically, oncosis [6]. In contrast, apoptosis is a highly regulated form of programmed cell death. Morphological alterations such as shrinkage and blebbing are observed. Apoptotic cells retain an intact cellular membrane and undergo removal by macrophages (Figure 1). Various means can be used to evaluate apoptosis, including an analysis of immunolocalization of active forms of effector proteins, assessment of mitochondrial alterations, DNA fragmentation, TUNEL assay and annexin V binding. To further understand the factors involved in PDF toxicity, we first studied the effect of four different PDFs, i.e. heat-sterilized dextrose-lactate, filtered dextrose-lactate, heat-sterilized dextrose-bicarbonate-lactate and heat-sterilized icodextrin-lactate (a glucose polymer) on HPMC proliferation, and the two pathways leading to cell death (i.e. oncosis and apoptosis). To evaluate the role of carbohydrates in this respect, we studied the independent and synergistic effects of glucose, 3-DG and CML-albumin on HPMC functions and AGERAGE interactions, and their possible implication in the cytotoxic effects of PDFs.

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Fig. 1. The two different ways of cell death. Oncosis is marked by cellular swelling followed by cell membrane rupture and apoptosis is marked by cellular shrinking, condensation and margination of the chromatin and ruffling of the plasma membrane with eventual breaking up of the cell in apoptotic bodies.
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Materials and methods
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Cell culture and characterization
HPMC isolation from freshly obtained omental tissue and the preparation of primary culture were carried out as previously described [5]. The study of HPMC from uraemic patients should be of major interest; however, for ethical reasons and feasibility, it could not be achieved. Cells were characterized by their morphological appearance and flow cytometry analysis. Immediately prior to and at confluence, cells adopted the polygonal cobblestone appearance characteristic of epithelial cells and formed a monolayer. HPMCs expressed the von Willebrand factor and the mesothelial marker cytokeratin-18, and failed to express the endothelial marker, PECAM-1 (CD31). RAGE expression on HPMC membrane has been previously described [5].
PDFs and glucose solutions
All manufactured PDFs were generously provided by Baxter S.A. (Maurepas, France and Nivelles, Belgium). Experiments with heat-sterilized icodextrin-lactate were carried out with the manufactured solution (pH 5.5; 40 mmol/l lactate, 75 g/l icodextrin). The formulation of heat-sterilized dextrose-lactate and heat-sterilized dextrose-bicarbonate-lactate PDFs has been described in Table 1. Heat-sterilized dextrose-bicarbonate-lactate solution was supplied in the form of a double-chamber bag system. To reduce GDP formation during heat sterilization, glucose (pH 3) was used in one of the chambers. In addition, some experiments were performed using dextrose-lactate solution sterilized by filtration (i.e. filtered dextrose-lactate).
To further explore the respective effects of glucose, GDPs and AGEs, we prepared culture solutions containing glucose at different concentrations (1, 15, 25 and 45 g/l) and compared the results of the proliferation assay, apoptosis and oncosis induction in the absence and after addition of 3-DG (100, 250 and 500 µmol/l) and/or CML (10, 20 and 40 nmol/ml). In PDF effluents, 3-DG was reported to range between 100 and 450 µmol/l [7]. CML concentrations tested are in the range of that found in uraemic patients [8].
3-DG, glycated proteins, recombinant RAGE and RAGE expression
3-DG was purchased from Toronto Research Chemicals (Toronto, Canada) and tested in proliferation assays and cell death induction experiments. AGE-protein levels (non-CML and CML-albumin) were measured by ELISA in the PDF culture medium after 24 h [9]. CML-albumin was prepared as follows: ovalbumin and sodium cyanoborohydride were dissolved in phosphate-buffered saline (PBS). Glyoxylic acid was then added, and after a 16 h incubation the solution was dialysed against PBS. The extent of glycation determined by 2,4,6-trinitrobenzene sulphonic acid showed that 25% of the lysine present in the albumin molecule was glycated. Albumin was used as control, albumin solutions were passed through Detoxi-gel columns to remove possible endotoxin contamination (Pierce, Rockford, IL).
RAGE expression on HPMC was measured 30, 60 min, 2, 4, 8, 16 and 24 h after stimulation by a radiometric technique, as previously described [10].
For recombinant soluble-RAGE production, RAGE was cloned from a rat lung cDNA library, and recombinant rat-RAGE (rR-RAGE) was produced by baculovirus-transfected insect cells (Spodoptera frugiperda or Sf9). The resulting protein corresponded to the RAGE extracellular domain and acted by binding to AGEs, thereby blocking their interaction with endogenous RAGE. rR-RAGE at a concentration between 10 and 120 µg/ml had been tested previously, and an optimal blocking effect was achieved at 60 µg/ml. For RAGE antibody production, purified rat RAGE was used to immunize rabbits according to previously described methods. Anti-RAGE IgG were pre-incubated with HPMCs for 30 min at a concentration of 50150 µg/ml [5].
Cell proliferation assay
Cell proliferation was assayed by the incorporation of methyl-[3H]thymidine into acid-insoluble DNA of cultured HPMCs. Cells (15 x 103) were seeded into 96 well plates in culture medium for 24 h, then incubated for a further 24 h with PDF solutions mixed with culture medium, and finally pulsed with methyl-[3H]thymidine (Amersham Pharmacia Biotech, Buckinghamshire, UK). Results were expressed as counts per minute (c.p.m.). PDFs were mixed with culture medium at two ratios (PDF/medium: 1:1 and 3:1) since when added to HMPC in the absence of culture medium it resulted in rapid cell death. Cell proliferation was studied with cells exposed to four different PDFs: heat-sterilized dextrose-lactate, filtered dextrose-lactate, heat-sterilized dextrose-bicarbonate-lactate containing 15 or 45 g/l glucose, and heat-sterilized icodextrin-lactate containing 75 g/l icodextrin.
Apoptosis and oncosis
Para-umbilical biopsy samples of human parietal peritoneum were obtained in a patient that had been on PD for 4 years and a healthy subject at the time of laparotomy for benign surgery. The processing and fixation of these peritoneal biopsies was described in detail previously [11]. The use of these human biopsy samples was approved by the Ethical Review Board of the St-Luc Academic Hospital, Brussels, Belgium. Immunoperoxidase staining was performed on 6 µm paraffin sections, using a purified rabbit anti-human factor VIII IgG (Dako, Glostrup, Denmark), as described earlier in detail [11,12].
Early apoptosis-associated alterations were evaluated by flow cytometry analysis with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6-[3]), which accumulates in the mitochondria (Molecular Probes, Eugene, OR). The decrease in DiOC6-(3) staining indicates a disruption of the mitochondrial inner transmembrane potential (
m), a process which is associated with apoptosis [13]. For PDF-induced oncosis analysis, propidium iodide (PI) DNA-intercalating capability was measured. Apoptosis-inducing staurosporine was used as a positive control. After incubation, adherent cells were treated with trypsinEDTA for 4 min at 37°C and harvested. Cells were then washed and stained with DiOC6-(3) (80 nM) and PI (15 µg/ml) for 30 min at 37°C in the dark. DiOC6-(3) and PI fluorescence were measured by FACScan. Results were expressed in terms of the percentage of oncotic and apoptotic cells. Moreover, the independent and synergistic effects of glucose, GDP and AGE on the induction of cell death were also evaluated by adding 3-DG and/or CML-albumin to culture solutions at different glucose concentrations.
To investigate the effects of RAGE activity on HPMCs, we used rR-RAGE to block AGE-protein binding to mesothelial RAGE.
DNA fragmentation was studied with a TiterTACS kit (TACS-Sapphire; R&D Systems, Abingdon, Berkshire, UK). HPMCs were cultured in a 96 well plate (15 x 103 cells per well) and the induction of apoptosis took place under the conditions described above for DiOC6-(3) staining. Apoptosis was analysed by absorbance at 450 nm [13]. Annexin V membrane binding (R&D Systems) was studied by flow cytometry analysis on cell suspensions according to the manufacturer's recommendations.
Statistical analysis
Results are presented as the mean ± standard error of the mean (SEM). Statistical significance was determined using a one-way analysis of variance (ANOVA) followed by the parametric Dunnett's test.
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Results
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PDF-induced inhibition of HPMC proliferation
Inhibition of methyl-[3H]thymidine incorporation was found to be dependent upon the ratio of PDF added to the medium. Compared to a PDF/medium ratio of 3:1 (v/v), the most marked inhibition of HPMC proliferation was observed at a PDF/medium ratio of 1:1. At the latter ratio, no methyl-[3H]thymidine incorporation inhibition was observed when these cells were exposed to heat-sterilized dextrose-bicarbonate-lactate containing 15 g/l glucose, whereas inhibition was maximal (98±1.1%) when cells were exposed to heat-sterilized dextrose-lactate containing 45 g/l glucose. Subsequently, the experiments on PDF-induced inhibition of HPMC proliferation were performed with this PDF/medium ratio.
HPMC proliferation was inhibited by glucose in a concentration-dependent manner (Figure 2). Heat-sterilized dextrose-lactate solution induced the greatest inhibition of thymidine incorporation, which was strongest at a concentration of 45 g/l glucose. Heat-sterilized dextrose-bicarbonate-lactate PDF (containing 15 g/l glucose) did not have a significant effect on HPMC proliferation. Filtered dextrose-lactate PDF (1545 g/l glucose) displayed a glucose-dependent inhibition of cell proliferation, but was significantly less pronounced than that observed with heat-sterilized dextrose-lactate (P<0.001). The effect of heat-sterilized icodextrin-lactate (75 g/l icodextrin) on methyl-[3H]thymidine incorporation in HMPCs was similar to that observed with heat-sterilized dextrose-bicarbonate-lactate PDF (at a concentration of 45 g/l glucose); however, heat-sterilized icodextrin-lactate PDF was less toxic than heat-sterilized dextrose-lactate PDF at the two concentrations tested (15 and 45 g/l glucose) (P<0.001).

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Fig. 2. PDFs containing a high glucose concentration, with heat sterilization and low pH alter HPMC cell proliferation. Methyl-[3H]thymidine incorporation was measured after 24 h exposure to heat-sterilized dextrose-lactate (L), dextrose-lactate sterilized by filtration (L. Filt), heat-sterilized dextrose-bicarbonate-lactate (B/L), each containing 15 or 45 g/l glucose; and heat-sterilized icodextrin-lactate (ICO) containing 75 g/l icodextrin. The results are the mean of four sets of experiments, each of which was performed in quintuplicate. Results are expressed in c.p.m. (***P<0.001).
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Cell count measured by video microscopy demonstrated that the extent of methyl-[3H]thymidine incorporation was correlated with cell number. Compared to the control medium, when cells were exposed to heat-sterilized dextrose-lactate PDF at a glucose concentration of 15 and 45 g/l, HPMC/mm2 levels were reduced by 30.2±2 and 66.6±2.4%, respectively (P<0.001). This effect was not reversible when cells were cultured for 48 hours in M199 containing 10% fetal calf serum.
PDF induction of apoptosis and oncosis
Examination of representative sections from the peritoneum of a control subject and a long-term PD patient (Figure 3) showed the characteristic changes associated with long-term PD, i.e. alteration of the mesothelial integrity, thickening of the submesothelial area and vascular proliferation evidenced by an increased density of the vascular structures stained for factor VIII. The cell death can be explained by two different mechanisms, oncosis (necrosis) and apoptosis followed by phagocytosis.

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Fig. 3. Biopsies of human peritoneal membrane. Structure of the parietal peritoneum of a control subject (A) and a long-term PD patient (B). Low-power micrographs of representative sections stained for factor VIII and counterstained with toluidine blue. The changes associated with long-term PD include loss of integrity of the mesothelium (m), thickening of the submesothelial area and vascular proliferation, as evidenced for the dark vascular staining for factor VIII. Original magnification x100.
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As for the cell proliferation assay, PDF/culture medium ratio tested was 1:1 for apoptosis and oncosis induction experiments. Only heat-sterilized dextrose-lactate PDF containing a high glucose concentration (45 g/l) induced a 2.4-fold increase in annexin V binding to HPMCs and a 2-fold increase in the number of apoptotic HPMCs when analysed for DNA fragmentation (TACS-Sapphire). Since DiOC6-(3) staining was found to be the most reproducible and reliable technique, most of the experiments were carried out using this marker. A typical flow cytometry profile of HPMCs stained with DiOC6-(3) and PI is shown in Figure 4. Apoptosis corresponded to a reduction in DiOC6-(3) fluorescence, while oncosis was associated with an increase in PI fluorescence. Compared to control medium, heat-sterilized dextrose-lactate PDF at a low glucose concentration (15 g/l) induced significant HPMC apoptosis (P<0.05) (Figure 5). After 24 h exposure to heat-sterilized dextrose-lactate PDF with a 45 g/l glucose concentration (high GPD levels), a large number of HPMCs were found to be apoptotic (40.8±12.2%) (P<0.05) or oncotic (41.6±9.7%)(P<0.01) compared to cells exposed to filtered dextrose-lactate or heat-sterilized dextrose-bicarbonate-lactate solutions. No significant increase in cell death was found to be induced by the other PDFs, i.e. heat-sterilized icodextrin-lactate (75 g/l icodextrin) or heat-sterilized dextrose-bicarbonate-lactate (15 and 45 g/l glucose).

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Fig. 4. Typical flow-cytometric profile of HPMCs incubated with high glucose concentration PDFs. Heat-sterilized dextrose-lactate (L), filtered sterilized dextrose-lactate (L. Filt) or dextrose-bicarbonate-lactate (B/L) buffered PDFs (each containing 45 g/l glucose) were tested. Oncosis was assessed by PI fluorescence on the vertical axis, and apoptosis was evaluated by DiOC6-(3) fluorescence on the horizontal axis. Apoptosis corresponded to a reduction in DiOC6-(3) fluorescence, while oncosis was associated with an increase in PI fluorescence. Viable cells can be seen in region R1, apoptotic cells in region R2, and oncotic cells in region R3. Results are expressed in arbitrary units of fluorescence.
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Fig. 5. PDF-induced HPMC apoptosis and oncosis. Heat-sterilized dextrose-lactate (L), dextrose-lactate sterilized by filtration (L. Filt), dextrose-bicarbonate-lactate (B/L) and heat-sterilized icodextrin-lactate (ICO) PDFs were tested. The results are the mean of five experiments, and are expressed as a percentage of fluorescent cells after 16 h exposure to PDF (*P<0.05, **P<0.01, ***P<0.001).
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The decrease of DiOC6-(3) labelling demonstrated the involvement of the mitochondria in the genesis of apoptosis. To further explore which pathways could be implicated, we added vascular endothelial growth factor (VEGF), which is known to prevent apoptosis via the Bcl-2 pathway. VEGF added at concentrations of 10, 50 and 100 pg/ml reduced apoptosis of 18, 32 and 34%, respectively.
Glycated protein generation with PDFs
When added to the culture medium, the heat-sterilized dextrose-lactate PDF containing 45 g/l glucose resulted in a higher AGE-protein level compared to that observed with filtered dextrose-lactate at a similar concentration (P<0.001) (Figure 6). Despite the separation of glucose during heat sterilization, heat-sterilized dextrose bicarbonate-lactate PDF induced more AGE-protein formation than filtered dextrose-lactate solution. Icodextrin incubated with culture medium resulted in a low level of AGE formation as previously reported [14].

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Fig. 6. AGE-protein formation in PDFs. AGE-protein concentration was measured by ELISA assay in PDF added to the culture medium. The following solutions were tested: heat-sterilized dextrose-lactate (L), dextrose-lactate sterilized by filtration (L. Filt), heat-sterilized dextrose-bicarbonate-lactate (B/L), each containing 15 or 45 g/l glucose; and heat-sterilized icodextrin-lactate (ICO) containing 75 g/l icodextrin. Results are the mean of two experiments performed in triplicate, and are expressed as µg/ml of AGE-protein (***P<0.001).
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Independent and synergistic effects of glucose, 3-DG and CML-albumin
The above results, which were obtained when HPMCs were exposed to PDFs, indicated that glucose and GDPs may have individual and synergistic effects. In order to investigate this possibility, glucose, 3-DG and/or CML-albumin were added to cell culture medium to evaluate the effect of each compound. Methyl-[3H]thymidine incorporation was inhibited in a glucose concentration-dependent manner (P<0.001) (Figure 7A). Addition of 3-DG (range 100500 µmol/l) to 1, 15 and 25 g/l glucose potentiated the inhibition of HPMC proliferation (P<0.001), which reached a maximum at 500 µmol/l of 3-DG. CML-albumin potentiated glucose antiproliferative activity in a CML-albumin concentration-dependent manner, with maximum activity observed when 40 nmol/ml of CML-albumin was added to 15 g/l glucose (P<0.05). The synergistic effect of CML-albumin was not detectable at 45 g/l glucose concentration, probably because proliferation inhibition reached a maximum level with glucose alone. The addition of 3-DG and CML-albumin together to the glucose solution did not increase the effect compared to that of each compound given alone. 3-DG and/or CML added to 1 or 15 g/l glucose solution did not induce detectable apoptosis or oncosis (data not shown). Glucose alone at a concentration of 45 g/l resulted in a higher extent of apoptosis and oncosis than that observed with 25 g/l glucose (P<0.05) (Figure 7B). CML-albumin addition enhanced the extent of apoptosis induced by glucose at concentrations of 25 and 45 g/l, and this enhancement was a maximum with 40 nmol/ml CML-albumin. Similarly, the addition of 3-DG (500 µmol/l) to a 45 g/l glucose solution increased the extent of apoptosis (P<0.01).

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Fig. 7. CML-albumin or 3-DG potentiate the effect of glucose on HPMC viability. CML (10, 20 or 40 nmol/ml) and/or 3-DG (100, 250 or 500 µmol/l) were added to the culture medium (glucose concentrations: 15, 25 or 45 g/l). (A) The results of the cell proliferation assay are the mean of two sets of experiments performed in quintuplicate, and are expressed in c.p.m. (B) The results of cell apoptosis and oncosis induction are the mean of two sets of experiments performed in duplicate (*P<0.05, **P<0.01, ***P<0.001).
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Enhancement of cell viability by blockade of AGERAGE interaction
RAGE expression was quantified using a radiometric technique and the results were expressed in c.p.m. RAGE expression on cells in culture in basal conditions varied between 1170±280 and 1800±200 c.p.m. per 104 cells. After stimulation by TNF
, RAGE expression was increased 1.31.4 fold. The maximum expression was observed after 4 h and returned to the basal values after 24 h. Similar results were obtained by others using either TNF
or AGE-albumin as stimulus [15].
Pre-incubation of heat-sterilized dextrose-lactate (15 g/l glucose) with rR-RAGE significantly prevented PDF-induced inhibition of cell proliferation (P<0.001) (Figure 8A). No effect of rR-RAGE was detected when cells were exposed to filtered dextrose-lactate (15 g/l glucose). HPMC apoptosis was significantly reduced when the AGERAGE interaction was blocked using rR-RAGE (60 µg/ml) (P<0.05; see Figure 8B). When rR-RAGE was added to the 45 g/l glucose solution with 3-DG no reduction of apoptosis was recorded.

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Fig. 8. Blockade of the AGERAGE interaction prevents the deleterious effect of heat-sterilized dextrose-lactate buffered PDFs on HPMC viability. (A) Heat-sterilized dextrose-lactate (L) and filtered dextrose-lactate (L. Filt) (15 g/l glucose) were pre-incubated with rR-RAGE (60 µg/ml). The results are the mean of three sets of experiments performed in quintuplicate and expressed in c.p.m. (B) rR-RAGE was added to solutions L and L. Filt at a glucose concentration of 45 g/l. The results are expressed as a percentage of fluorescent cells and are the means of two experiments performed in duplicate (*P<0.05, ***P<0.001).
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Some experiments were performed with anti-RAGE antibody. Anti-RAGE (100 µg/ml) significantly reduced the antiproliferative activity of heat-sterilized dextrose-lactate PDF at a concentration of 15 g/l, no significant effect was observed at higher glucose concentrations (i.e. 25 and 45 g/l). Similarly, the anti-RAGE antibody did not significantly modify apoptosis induced by heat-sterilized dextrose-lactate at a concentration of 45 g/l glucose. The AGERAGE interaction appeared to be more evident when the glucose concentration was lower; however, rR-RAGE decreased the extent of apoptosis induced by heat-sterilized dextrose-lactate containing 45 g/l glucose. This result suggests that rR-RAGE may have a higher affinity to AGEs, and as previously observed is more consistently efficient than the anti-RAGE antibody in preventing AGERAGE interaction [10].
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Discussion
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In this study, we demonstrated that glucose, 3-DG and CML-albumin have distinct deleterious effects on HPMC viability, and that GDP and AGE potentiate the toxic effect of glucose. Blockade of the AGERAGE interaction partially prevented the PDF-induced inhibition of cell proliferation and apoptosis, indicating that besides AGE other glucose-derived compounds such as 3-DG can alter HPMC functions via a RAGE-independent pathway.
Our results demonstrate that heat-sterilized dextrose-lactate PDF and a high glucose concentration are predominant factors in cell proliferation inhibition. The difference in results between heat-sterilized dextrose-lactate and filtered dextrose-lactate further indicates that agents such as GDPs may participate in the mechanism of cell proliferation inhibition. A direct effect of 3-DG on cell culture is consistent with this hypothesis, as well as with its potentiation of the deleterious effect of high glucose concentration. Addition of 3-DG to mesothelial cells stimulated VCAM-1 expression, IL-6 and IL-8 production but this effect was only observed at concentrations above the physiological levels found in standard PDFs. Two other GDPs, 3,4-dideoxyglucosone-3-ene (3,4-DGE) and methylglyoxal, are of particular interest. 3,4-DGE has been quantified as a novel reactive GDP, the concentrations of which increase after heat sterilization of PDF [16]. Compared to heat sterilization, less peritoneal injury developed in rats receiving a daily intraperitoneal infusion of lactate sterilized by filtration. This result is consistent with our in vitro study and indicates that besides pH level and glucose concentration, GDPs can also be involved in the genesis of mesothelial damage. In addition to the role of glucose toxicity in the development of diabetic nephropathy, methylglyoxal has recently been described to induce rat mesangial cell apoptosis [17].
Icodextrin is a glucose polymer of an average molecular weight of 16 200 Da containing free carbonyl groups at lower concentrations than that found in conventional PDFs. This explains why icodextrin in the presence of protein may form AGEs as observed in vitro [14]. However, the dextrose concentration is low and may account for the less pro-apoptotic and oncotic effect of icodextrin. Physiological osmolarity of icodextrin may also improve HMPC viability.
CML-albumin potentiated the pro-apoptotic effect of glucose at intermediate concentrations, but this effect was not detectable at a higher glucose concentration. In diabetic rats, blockade of RAGE by anti-RAGE antibodies prevented the upregulation of transforming growth factor beta (TGFß) and the development of submesothelial fibrosis, but did not affect the upregulation of endothelial nitric oxide synthase expression [18]. These results are consistent with the partial inhibition of the deleterious effect of PDFs containing AGE precursors.
Glucose, GDPs and AGEs have a distinct toxicity, and can reduce HPMC proliferation and induce cell death. Apoptotic mesothelial cells have been found in the peritoneal effluent of stable dialysis patients. Apoptosis can be induced by several mechanisms, according to the stimulus, such as granzyme, TNF
, Fas-ligand, irradiation or chemical (Figure 9). Each of these inducers act through different intracellular pathways involving caspase cascades. The alternative pathway implicates Bax/Bcl-2 balance and alters mitochondria functions resulting in the release of cytochrome c. The different pathways lead to activation of caspase-9, DNA fragmentation and externalization of the phosphatidyl-ethanolamine/phosphatidyl-serine assymetry and a low annexin V binding. Our results demonstrated that mitochondria alterations occur, which is consistent with previous reports evidencing that both high glucose concentrations and AGEs alter mitochondria functions [19].

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Fig. 9. The two main apoptotic pathways. A diverse group of signals induce apoptosis via the death receptors, which activate the caspase cascade, or via the mitochondrial pathway and the release of cytochrome c. These two ways allow DNA cleavage and membrane assymetry changes.
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GDPs delay remesothelialization independently of glucose and our study is the first to demonstrate that GDPs and AGEs may potentiate the effects of glucose on alterations in cell function [7]. Apoptosis is greatly dependent upon mitochondrial activity and a high glucose concentration, or AGE and RAGE involvement stimulate the production of reactive oxygen intermediates in the mitochondria [20]. In bovine retinal pericytes, AGEs can induce apoptosis by stimulating the formation of reactive oxygen intermediates, and the activation of the diacylglycerol/ceramide pathway.
Three major biochemical mechanisms are implicated in the pathogenesis of cell damage resulting from high glucose concentrations: the hexosamine pathway, AGE formation and diacylglycerol-protein kinase activity [21]. Under our experimental conditions, we observed that at high glucose concentration, GDPs and AGEs are responsible for mesothelial cell toxicity, as evidenced by the inhibition of cell growth or the induction of cell death. PDF-induced mesothelial cell apoptosis and oncosis was only partially prevented by the AGERAGE blockade, indicating that significant toxicity could be related to the presence of GDPs and a high glucose concentration.
Glucose represents the major osmotic agent contained in most PDFs. However, it has deleterious effects on PDF biocompatibility and could be a source of potentially harmful derivative products. During heat sterilization, GDPs are formed, and these are precursors of AGE generation. These two glucose derivatives have independent and synergistic deleterious effects on HPMC when combined with glucose viability.
It therefore appears that the research which has been conducted to reduce AGE and GDP formation and to limit glucose concentrations in PDFs should improve their biocompatibility and help to reduce the side effects of PD.
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Acknowledgments
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The authors are grateful to Eric Daugas for his helpful advice, and to Philippe Dequiedt for his support and encouragement. We would also like to thank Pierre Ronco and Salim Mujais for their invaluable comments and suggestions.
Conflict of interest statement: None declared.
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Received for publication: 9.12.03
Accepted in revised form: 12. 3.04