Glucose degradation products downregulate ZO-1 expression in human peritoneal mesothelial cells: the role of VEGF
Joseph C. K. Leung1,
Loretta Y. Y. Chan1,
Felix F. K. Li1,
Sydney C. W. Tang1,
Kwok Wa Chan2,
Tak Mao Chan1,
Man Fai Lam1,
Anders Wieslander3 and
Kar Neng Lai1
1 Department of Medicine and 2 Department of Pathology, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong and 3 Gambro AB, Lund, Sweden
Correspondence and offprint requests to: Professor K. N. Lai, Department of Medicine, University of Hong Kong, Room 409, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. Email: knlai{at}hkucc.hku.hk
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Abstract
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Background. Glucose degradation products (GDPs) are formed during heat sterilization of peritoneal dialysis fluid and, to a lesser extent, during their prolonged storage. In vitro studies have demonstrated that GDPs impair functions of peritoneal mesothelial cells, including proliferation, viability and cytokine release. In the present study, we studied the acute effect of GDPs on the expression of tight junction-associated protein, zonula occludens protein 1 (ZO-1), in human peritoneal mesothelial cells (HPMC). The role of the vascular endothelial growth factor (VEGF) induced by GDPs in the expression of ZO-1 was also examined.
Methods. HPMC were cultured with GDPs, including 2-furaldehyde (FurA), methylglyoxal (M-Glx) and 3,4-dideoxyglucosone-3-ene (3,4-DGE). The expression of ZO-1 and the synthesis of VEGF were examined. To define the role of VEGF on the regulation of ZO-1 expression, HPMC were cultured with GDPs in the presence or absence of neutralizing antibody to VEGF. The signal pathways involved in VEGF synthesis induced by GDPs were also characterized.
Results. ZO-1 expression in HPMC was downregulated in a time- and dose-dependent manner following culture with subtoxic concentrations of GDPs (FurA, M-Glx and 3,4-DGE). All three GDPs increased VEGF synthesis in HPMC. Exogenous VEGF downregulated the expression of ZO-1 and neutralizing anti-VEGF antibody reversed the effect of GDPs on ZO-1 expression in HPMC, suggesting the action of GDPs on ZO-1 expression was mediated by VEGF. All three GDPs activated the p42/p44 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signal transduction pathways. The GDP-induced VEGF and transforming growth factor (TGF)-ß synthesis in HPMC was partially reduced by either the p42/p44 MAPK inhibitor (PD98059) or the PKC inhibitor (staurosporine). More importantly, the VEGF and TGF-ß synthesis induced by GDPs in HPMC was completely blocked by synergistic action of both inhibitors.
Conclusions. We have demonstrated that short-term exposure to GDPs downregulates ZO-1 expression in HPMC through the generation of VEGF. Our study provides evidence that GDPs can directly induce VEGF and TGF-ß production in HPMC through the activation of p42/44 MAPK and PKC signal transduction pathways.
Keywords: glucose degradation products; peritoneal dialysis; peritoneal mesothelial cells; vascular endothelial growth factor; zonula occludens protein 1
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Introduction
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Continuous ambulatory peritoneal dialysis (CAPD) is now established as a major treatment modality for end-stage renal failure. It has distinct advantages over haemodialysis, namely its lower cost and simplicity of technique. The ability to maintain the functional integrity of the peritoneal membrane to allow adequate removal of fluid and metabolic waste is essential for the success of CAPD. Unfortunately, the peritoneal membrane exhibits structural changes that correlate with the duration of dialysis. The peritoneal membrane consists of a monolayer of mesothelial cells (mesothelium) resting on a basement membrane with the underneath interstitium. The pathological changes in the peritoneum are due to the exposure to non-physiological peritoneal dialysis fluid (PDF) that has a low pH and high glucose content. PDF also contains toxic substances, including glucose degradation products (GDPs) and advanced glycation end-products (AGE) [1]. These toxic compounds cause irreversible damage to the mesothelium, leading to a decline or failure in ultrafiltration and a loss in dialysis efficacy.
Breakdown of glucose in PDF generates a number of low molecular weight aldehydes, including 5-hydroxymethyl-furfural, 2-furaldehyde (FurA), acetaldehyde, formaldehyde, glyoxal, methylglyoxal (M-Glx) and 3,4-dideoxyglucosone-3-ene (3,4-DGE). The process occurs primarily during heat sterilization of PDF and also during a prolonged storage [2]. While many studies have examined the detrimental effect of glucose or AGE on the biology and function of peritoneal mesothelial cells, the direct effect of GDPs on mesothelial biology is less well known. Recent reports have shown that distinct GDPs can exert different biological activities on human peritoneal mesothelial cells (HPMC), including viability, cell proliferation and cytokine synthesis [3]. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that has been implicated in many structural and functional alterations of the peritoneal membrane during CAPD. VEGF is found in peritoneal effluent and is partly derived from peritoneal mesothelial cells [4]. It has been shown that one of the GDPs, methylglyoxal, enhances the production of VEGF in murine peritoneal mesothelial cells, which leads to progressive deterioration of peritoneal function [5]. VEGF and its receptors are upregulated in experimental murine and human diabetes. Inhibition of VEGF exerts beneficial effects on diabetes-induced functional and structural alterations, suggesting a deleterious role for VEGF in the pathophysiology of diabetic nephropathy [6]. One of the possible pathogenetic roles is the increased renal cell permeability by local or exogenous VEGF in diabetic nephropathy. VEGF increases the permeability of the microvascular monolayer of endothelial cells in the brain through disrupting zonula occludens protein 1 (ZO-1) and occludin organization, which leads to tight junction disassembly [7].
In this study, we studied a tight junction-associated protein, ZO-1, to determine whether GDPs could directly affect the integrity of HPMC. We further explored the role played by VEGF in modulating the ZO-1 expression in HPMC. The signal transduction pathways involved in the production of VEGF induced by GDP in HPMC were also investigated.
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Subjects and methods
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Materials
Medium 199 and fetal bovine serum (FBS) were obtained from Life Technologies (Rockville, MD, USA). Consumables for electrophoresis were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Rabbit anti-ZO-1 antibody was obtained from Zymed Laboratories (San Francisco, CA, USA). Monoclonal mouse anti-actin was obtained from Lab Vision (Fremont, CA, USA). Monoclonal mouse anti-human mesothelial cell (clone HBME-1), vimentin, polyclonal anti-human factor VIII and secondary antibodies for immunofluorescence staining and immunoblotting were obtained from Dako (Carpinteria, CA, USA). Monoclonal anti-human cytokeratin 18 was obtained from ICN Biochemicals (Aurora, OH, USA). Antibodies to phospho-p42/p42 mitogen-activated protein kinase (MAPK) and pan protein kinase C (PKC) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-phosphotyrosine was obtained from Upstate Biotechnology Inc. (Charlottesville, VA, USA). The GDP preparations of M-Glx and 3,4-DGE were obtained from Gambro AB (Lund, Sweden). All other chemicals, including FurA, were obtained from Sigma (St Louis, MO, USA).
Culture of HPMC
HPMC were obtained from omental tissues of patients undergoing elective abdominal surgery. The cells were isolated and characterized using procedures described previously [8]. Mesothelial cells showed typical cobblestone appearance at confluence. All cells showed uniform positive staining by HBME-1 for cytokeratin and vimentin, but negative staining for factor VIII antigen. The cells were maintained in medium 199 supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), transferrin (5 µg/ml), insulin (5 µg/ml) and 10% vol/vol FBS. HPMC were incubated at 37°C in a humidified atmosphere with 5% CO2. Once the monolayer of HPMC reached confluence, the culture medium was removed and medium 199 containing 0.1% vol/vol FBS was added to the cells for 48 h prior to further culture experiments. Under these conditions, the HPMC remained in a non-proliferative viable condition for up to 120 h. Confluent cells were split at a ratio of 1:3 and all experiments performed with cells of second to third passage.
Quantification of proliferation and viability of HPMC cultured with GDPs
The optimal or subtoxic dose of GDPs was determined by cell viability measured by a commercial colorimetric kit (Chemicon International, Temecula, CA, USA) based on cleavage of the tetrazolium salt WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulpho-phenyl)-2H-tetrazolium, monosodium salt]. Briefly, growth-arrested HPMC were seeded into 96-well plates (0.25 x 105 cells per well) before being exposed to GDPs for 48 h. WST-1 reagent was then added and incubated at 37°C for a further 2 h. The absorbance was measured using 450 nm as the primary wavelength and 600 nm as the reference wavelength. Results were expressed as percentage changes in absorbance compared with that of the medium control. The cytotoxic effect of GDPs on HPMC was measured by lactate dehydrogenase (LDH) assay kit (Roche Diagnostic, Indianapolis, IN, USA) according to the manufacturer's protocol. Results were expressed as percentage changes in LDH release compared with that of the total intracellular LDH.
Immunofluorescence staining of ZO-1
Expression of ZO-1 on the peritoneal mesothelial monolayer incubated with GDPs was determined by immunofluorescence staining. Briefly, HPMC (1 x 105 cells per well) were cultured in a chamber slide (Nalge Nunc International, Naperville, IL, USA) and were exposed to 1 µg/ml FurA or M-Glx or 10 µg/ml 3,4-DGE for 48 h. The slides were then washed three times with phosphate-buffered saline (PBS) and were fixed with ice-cold methanol for 15 min at 20°C. Slides were washed with PBS and cells were permeabilized with 0.02% Tween-20 in PBS for 10 min. Non-specific binding was blocked by incubating the slides for 20 min with blocking buffer (5% normal goat serum and 3% bovine serum albumin in PBS). The slides were then incubated with anti-ZO-1 (1:100 dilution) overnight. After washing with PBS, the slides were incubated with fluorescein isothiocyanate-labelled goat anti-rabbit immunoglobulins (Dako) for 1 h at room temperature. The slides were stained with nuclear counterstain DAPI (4',6-diamidino-2-phenylindole diacetate) before mounting. Expression of ZO-1 was examined and photographed under a fluorescence microscope. To ensure the specificity of the staining, the following labelling controls were performed: (i) primary antibodies were substituted with pre-immune rabbit immunoglobulins; and (ii) staining was carried out without either the primary antibodies or the secondary antibodies.
Ultrastructural examination of HPMC exposed to GDPs
HPMC were grown on transwell membrane (Nalge Nunc) and were exposed to GDPs for 48 h. The membrane was then washed briefly with PBS and fixed with 2.5% glutaraldehyde before processing for scanning and transmission electron microscopy examination.
Expression of ZO-1 on cultured HPMC exposed to different GDPs or recombinant VEGF
HPMC were grown to confluence in six-well culture plates (1x106 cells per well). The cells were growth arrested with medium 199 containing 0.1% vol/vol FBS for 48 h. After changing new medium, the cells were exposed to different GDPs, including FurA, M-Glx and 3,4-DGE, of increasing concentrations (0.1, 1, 10 or 100 µg/ml) for 8 h (for assay of mRNA expression) or 48 h (for assay of protein expression). To study the time-dependent modulation of ZO-1 expression in HPMC after exposure to different GDPs, similar experiments were performed with cells cultured with 1 µg/ml FurA or M-Glx or 10 µg/ml 3,4-DGE for defined time periods (0, 6, 12, 24 or 48 h). At the end of the experiments, culture supernatant was collected for the measurement of VEGF and cells from the dosecourse study were harvested for total RNA extraction. In parallel experiments, HPMC were cultured with different concentrations of recombinant VEGF (100, 400, 1600 or 6400 pg/ml) for 48 h or cultured with 1600 pg/ml VEGF for different time periods (0, 6, 12, 24 or 48 h). A preliminary experiment had shown that HPMC cultured with
6400 pg/ml recombinant VEGF for 48 h did not cause changes in cell proliferation or viability as determined by WST-1 and LDH assay (data not shown). For all experiments, expression of ZO-1 by the HPMC was determined using immunoblotting as described below.
Effect of anti-VEGF on expression of ZO-1 by HPMC exposed to GDPs
HPMC were grown to confluence in six-well culture plates (1x106 cells per well). The cells were growth arrested for 48 h. After changing new medium, the cells were exposed to 1 µg/ml FurA or M-Glx or 10 µg/ml 3,4-DGE for 48 h in the presence or absence of anti-VEGF (0.5 µg/ml; added 30 min before the addition of GDPs). The concentration of blocking antibody was selected based on the preliminary data that a concentration of 0.5 µg/ml neutralizing antibody is required to achieve maximal inhibition of
10 ng/ml human recombinant VEGF on the proliferation of cultured human umbilical vein endothelial cells. At the end of the experiment, expression of ZO-1 by the HPMC was determined using immunoblotting as described below.
VEGF mRNA expression by cultured HPME exposed to GDPs
The VEGF gene expression by the HPMC was determined by reverse transcriptionpolymerase chain reaction [8]. Briefly, total RNA was extracted from HPMC using a Qiagen RNeasy kit (Qiagen GmbH, Hilden, Germany). Five µg total RNA was reverse transcribed to cDNA with Superscript II reverse transcriptase (Life Technologies, Paisley, UK) in a 20 µl reaction mixture containing 160 ng oligo(dT)1218, 500 µM of each dNTP and 40 U RNase inhibitor for 10 min at 37°C, 60 min at 42°C and 5 min at 99°C. The cDNA was stored at 20°C until PCR.
Specific primers for VEGF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed from known GenBank accession numbers (VEGF: XM004512; GAPDH: AF261085). The sequences of each primer were as follows: VEGF, sense primer 5'-GGCAGAAT-CATCACGAAGTGGTG and anti-sense primer 5'-CTG-TAGGAAGCTCATCTCTCC; GAPDH, sense primer 5'-TGAAGGTCGGAGTCAACGGATTTGGT and anti-sense primer 5'-CATGTGGGCCATGAGGTCCA-CCAC. The PCR was carried out in the following profile: first cycle, 94°C for 3 min, 58°C for 1 min, 72°C for 1 min; second to thirtieth cycles, 95°C for 45 s, 58°C for 40 s, 72°C for 45 s. The final cycle was 94°C for 1 min and 72°C for 10 min. The PCR reaction from VEGF and control (GAPDH) amplicons were mixed and separated by 1.5% wt/vol agarose gels, stained with ethidium bromide and the gel image captured and analysed using the Gel Doc 1000 Gel Documentation System and Quantity One software (Bio-Rad Laboratories). The result of VEGF mRNA yield was expressed as a ratio of the VEGF amplicon to GAPDH amplicon. Precautions were taken to ensure the validity of the results as described previously [8].
Tyrosine phosphorylation of ZO-1 and activation of p42/p44 MAPK and PKC signal transduction pathways in HPMC by GDPs
HPMC were grown to confluence in six-well culture plates (1x106 cells per well). The cells were growth arrested with medium 199 containing 0.1% vol/vol FBS for 48 h. After changing new medium, the cells were exposed to 1 µg/ml FurA or M-Glx or 10 µg/ml 3,4-DGE for 30 min. In a parallel experiment, individual or a combination of inhibitors to p42/p44 MAPK (PD98059, 25 mM) and PKC (staurosporine, 20 nM) were added 30 min before the addition of GDPs. At the end of experiment, the expression of phospho-p42/p44 and phospho-PKC by HPMC was determined using immunoblotting as described below. Tyrosine phosphorylation of ZO-1 was determined by immunoprecipitation using anti-ZO-1 antibody and protein A Sepharose followed by phosphotyrosine immunoblotting as described below.
Western blot analysis
HPMC were lysed with lysis buffer containing protease inhibitor cocktails (Sigma). The cell extracts were pelleted at 150 000 g for 60 min to remove cell debris. The protein concentrations were measured by a modified Lowry method using bovine serum albumin as standard (DC Protein Assay Kit; BioRad). Ten micrograms of total protein from the extract or immunoprecipitated pellet from 106 cells were electrophoresed through a 15% SDSPAGE gel before transferring to a PVDF membrane. After blocking for 1 h at room temperature in blocking buffer (1% gelatin in PBS with 0.05% Tween-20), the membrane was incubated for 16 h with rabbit anti-ZO-1 antibody (1:1000), monoclonal anti-actin antibody (1:1000); rabbit anti-phospho p42/p44 MAPK (1:5000), rabbit anti-phospho pan PKC antibody (1:4000) or rabbit anti-phosphotyrosine antibody (1:1000) in PBSTween. The membrane was washed and incubated for 2 h at room temperature with a peroxidase-labelled goat anti-rabbit or anti-mouse immunoglobulin (Dako). After further washing, the membrane was detected with ECL chemiluminescence (Amersham Pharmacia Biotech, Arlington, IL, USA). For semi-quantitative determination of protein expression, western blotting images for some experiments were scanned on a flatbed scanner and the density of the bands was quantitated using ImageQuant software (Molecular Dynamic, Sunnyvale, CA, USA). Densitometry results were reported as percentages of medium control after normalization with the average arbitrary integrated values of the actin signal.
Measurement of VEGF and TGF-ß in supernatants of cultured HPMC
The supernatant concentration of VEGF and transforming growth factor (TGF)-ß1 was measured by an enzyme-linked immunosorbent assay (ELISA; R&D System, Minneapolis, MN, USA). The VEGF ELISA has a detection limit of 50 pg/ml and a coefficient of variation of 8.5%. This assay recognizes two soluble isoforms: VEGF121 and VEGF165. The TGF-ß1 ELISA has a detection limit of 30 pg/ml and a coefficient of variation of 6.8%.
Statistical analysis
All data were expressed as means±SD. Intergroup differences for variables were assessed by multivariate analysis of variance for repeated measures. Statistical analysis was performed using statistical software (Statview; SAS Intelligence, Cary, NC, USA). All P-values quoted are two-tailed and the significance is defined as P<0.05.
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Results
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Viability of HPMC cultured with GDPs
We demonstrated previously that amongst various GDPs tested, FurA, M-Glx and 3,4-DGE exerted maximal biological impact on HPMC in terms of induction of AGE production [8]. The present study focuses on examining the effects of these three GDPs on the expression of intercellular junction-associated protein, ZO-1, on HPMC. Figure 1 depicts the cell proliferation and viability following incubation with different GDPs for 48 h determined by the WST kit and LDH assay. HPMC remained viable when exposed to FurA or M-Glx for
1 µg/ml or to 3,4-DGE for
10 µg/ml (Figure 1A). There was a dose-dependent decrease in viability of HPMC beyond these concentrations. The cell viability decreased significantly when the concentration of GDPs was raised to 100 µg/ml, with maximum toxicity in M-Glx (P<0.05). There was no cytotoxicity when cells were exposed to all GDPs tested at <10 µg/ml (Figure 1B). For all subsequent experiments conducted with fixed doses of GDP, subtoxic concentrations of 10 µg/ml were selected for 3,4-DGE and 1 µg/ml for FurA and M-Glx.
Effect of GDPs on expression of ZO-1 by HPMC
Growth-arrested HPMC were cultured with optimal (subtoxic) concentrations of GDPs for different time periods (6, 12, 24 and 48 h). Expression of ZO-1 determined by western blot was noted to decrease when HPMC were exposed to the three GDPs for >12 h and reached statistical significance at 48 h (P<0.05) (Figure 2A). Expression of ZO-1 was decreased to 75%, 72% and 60% of the value from HPMC cultured with the medium alone for FurA, M-Glx and 3,4-DGE, respectively. There was a dose-dependent decrease of ZO-1 expression in HPMC exposed to GDPs (Figure 2B). Expression of ZO-1 in HPMC was significantly decreased following culture for 48 h with FurA or M-Glx at a concentration of
1 µg/ml and with 3,4-DGE at a concentration of
10 µg/ml.
Morphometric study and immunofluorescence staining of ZO-1 of HPMC exposed to GDPs
Growth-arrested HPMC grown on chamber slide were exposed to FurA, M-Glx or 3,4-DGE at optimal concentration for 48 h. The morphological changes under phase-contrast microscopy and staining patterns for tight junction-associated protein, ZO-1, are shown in Figures 3 and 4. Peritoneal mesothelial cells became more granular with vacuole formation after exposure to GDPs (Figure 3BD). Condensed regions were located along the intercellular boundaries of HPMC following incubation with GDPs. Ultrastructural examination using electron microscopy also demonstrated that in HPMC cultured with 3,4-DGE, the number of microvilli on the cell surface was decreased and the junctions between cells were dissociated (Figures 4B and 4D). In contrast, microvilli and cell junctions were better preserved when HPMC were exposed to control culture medium (Figures 4A and 4C). Immunofluorescence staining also shows that the GDPs altered the organization of the tight junction-associated protein, ZO-1 (Figure 5BD). The staining of ZO-1 became discontinued along the cell contour of HPMC following incubation with GDPs while intact and continuous ZO-1 expression was observed in HPMC cultured with medium alone (Figure 5A). The degrees of morphological changes and altered ZO-1 expression were most severe in HPMC incubated with 3,4-DGE while least effects were observed with FurA.

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Fig. 3. Representative microphotographs showing the morphology of HPMC exposed to (A) medium only, (B) medium with 1 µg/ml FurA, (C) medium with 1 µg/ml M-Glx and (D) medium with 10 µg/ml 3,4-DGE. HPMC became more granular and vacuole formation was observed after exposure to GDPs. Condensed regions were observed along the cell contour (arrowheads). Magnification: x200.
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Fig. 4. Representative microphotographs from scanning (A, B) and transmission (C, D) electron microscopy showing the ultrastructure of HPMC exposed to medium only (A, C) or medium with 10 µg/ml 3,4-DGE (B, D). In HPMC cultured with 3,4-DGE, the number of microvilli on the cell surface was decrease and the junction (arrowheads) between cells was dissociated (B, D). In HPMC exposed to control culture medium, microvilli were better preserved (A) and cellular junctions (short arrow) and desmosomes (long arrow) were intact (C).
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Fig. 5. Immunofluorescence staining of ZO-1 in HPMC exposed to (A) medium only, (B) medium with 1 µg/ml FurA, (C) medium with 1 µg/ml M-Glx and (D) medium with 10 µg/ml 3,4-DGE. Condensed regions were located along the intercellular boundaries when HPMC were cultured with GDPs (arrowheads). The expression of ZO-1 was discontinued along the cell contour in HPMC cultured with GDPs while there was continuous ZO-1 expression in HPMC cultured with medium. Cells were counterstained with DAPI. Magnification: x200.
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Effect of GDPs on mRNA expression and release of VEGF in HPMC
Figure 6 shows the dosecourse of VEGF mRNA expression in HPMC after culture with GDPs. VEGF mRNA expression was upregulated by the three GDPs at concentrations >1 µg/ml (P<0.05). Figures 7A and 7B show the dose- and time-course for VEGF synthesis in HPMC incubated with GDPs. VEGF synthesis in HPMC was readily upregulated by the three GDPs at concentrations >0.1 µg/ml (P<0.05). The GDPs induced the synthesis of VEGF in HPMC in a time-dependent manner, reaching a significant concentration at 24 and 48 h compared with baseline value (P<0.05).

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Fig. 6. Dose-course of VEGF mRNA expression in HPMC incubated with GDPs. All GDPs significantly upregulated VEGF mRNA expression by HPMC at a concentration >1 µg/ml. The results represent the mean±SD of five separate experiments. *P<0.05 vs medium control.
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Fig. 7. Dose- and time-course of VEGF synthesis in HPMC incubated with GDPs. (A) All GDPs significantly upregulated VEGF production by HPMC at a concentration >0.1 µg/ml. (B) There was a time-dependent release of VEGF by HPMC incubating with GDPs. The concentration of VEGF released by HPMC was significantly increased when cells were cultured with subtoxic concentrations of GDPs (10 µg/ml for 3,4-DGE and 1 µg/ml for FurA and M-Glx) for 24 and 48 h. The results represent the mean±SD of five separate experiments. *P<0.05 vs medium control.
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Direct modulatory effect of VEGF on expression of ZO-1 on cultured HPMC
The effect of VEGF on the expression of ZO-1 was studied by culturing HPMC with increasing concentrations of recombinant VEGF (0, 100, 400, 1600 and 6400 pg/ml) for 48 h. Recombinant VEGF at 1600 and 6400 pg/ml significantly downregulated the expression of ZO-1 (73% and 52% of the value from HPMC incubated with medium alone; P<0.05) (Figure 8A). Recombinant VEGF (1600 pg/ml) also downregulated ZO-1 expression by HPMC when incubation beyond 24 h (72% of value from HPMC incubated with medium alone; P<0.05) (Figure 8B). Most interestingly, the downregulatory effect of GDPs on the ZO-1 expression in HPMC was abolished with the addition of neutralizing anti-VEGF (50 µg/ml) (Figure 9).

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Fig. 8. Effect of recombinant VEGF on the expression of ZO-1 by HPMC. (A) HPMC were incubated with recombinant VEGF for 48 h. VEGF at concentrations of 1600 and 6400 pg/ml significantly downregulated the expression of ZO-1. (B) HPMC were incubated with recombinant VEGF (1600 pg/ml) for different time periods. The ZO-1 expression was downregulated when HPMC were cultured with VEGF for >24 h. The results represent the mean±SD of five separate experiments. *P<0.05 vs medium control.
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Fig. 9. The effect of anti-VEGF on the expression of ZO-1 by HPMC cultured with GDPs. Expression of ZO-1 was decreased when HPMC were exposed to 1 µg/ml FurA or M-Glx or 10 µg/ml 3,4-DGE for 48 h. Downregulation of ZO-1 expression was reversed by the addition of anti-VEGF (final concentration: 0.5 µg/ml). The results represent the mean±SD of five separate experiments. *P<0.05 vs medium alone; n.s., not significant.
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Signal transduction pathways involved in the activation of HPMC by GDPs
We then further explored the signal transduction pathways involved in the activation of HPMC by GDPs. Both phosphorylated p42 and p44 subunits of MAPK were upregulated in HPMC cultured with FurA, M-Glx or 3,4-DGE (Figure 10A). In addition, these GDPs also activated the PKC pathway in cultured HPMC (Figure 10A). The activation of these signal transduction pathways by GDPs was reduced by either PD98059 (inhibitor of the p42/p44 MAPK pathway) or staurosporine (inhibitor for the PKC pathway) alone and was readily abolished by the addition of both inhibitors. All three GDPs induced tyrosine phosphorylation of ZO-1 in HPMC and this ZO-1 phosphorylation was reduced by the presence of inhibitors to both the MAPK and PKC pathways (Figure 10B).

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Fig. 10. (A) Detection of phospho-p42 and phospho-p44 subunits of MAPK or phospho-PKC following activation by three different GDPs (10 µg/ml for 3,4-DGE and 1 µg/ml for FurA or M-Glx). Activation of the p42/p44 MAPK and PKC signal transduction pathway was significantly reduced in the presence of PD98059 (25 mM), staurosporine (20 nM) or both. (B) Detection of tyrosine phosphorylation of ZO-1 by GDPs (10 µg/ml for 3,4-DGE and 1 µg/ml for FurA or M-Glx). All GDPs tested induced tyrosine phosphorylation of ZO-1 and this ZO-1 phosphorylation was reduced in the presence of PD98059 (25 mM) plus staurosporine (20 nM).
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The effects of selective inhibition of these signalling pathways on VEGF and TGF-ß synthesis by HPMC incubated with different GDPs are depicted in Figures 11A and 11B. Whilst inhibiting MAPK or PKC pathways individually resulted in a significant reduction in VEGF and TGF-ß synthesis (P<0.05), the additive effect of PD98059 and staurosporine completely abolished the stimulatory effect of FurA, M-Glx or 3,4-DGE on the GDP-induced VEGF and TGF-ß synthesis in HPMC.

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Fig. 11. Supernatant concentration of (A) VEGF or (B) TGF-ß in cultured HPMC incubated with 10 µg/ml 3,4-DGE or 1 µg/ml FurA or M-Glx for 48 h. VEGF and TGF-ß synthesis were reduced when the cells were incubated in the presence of PD98059 (25 mM) or staurosporine (20 nM). The additive effect of PD98059 and staurosporine abolished the increased VEGF and TGF-ß synthesis induced by the GDPs. The results represent the mean±SD of five separate experiments. *P<0.05 vs medium alone; n.s., not significant.
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Discussion
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In CAPD, the peritoneal membrane is constantly exposed to non-physiological and hypertonic dialysate that exerts adverse effects on its structure and function. Heat sterilization and storage of PDF induce glucose oxidation and produce GDPs. Loss of ultrafiltration of the peritoneal membrane is an important cause of CAPD failure and is likely to occur due to insults from GDPs, high glucose levels, hypertonicity and/or AGE. We have found that amongst various identified GDPs, FurA, M-Glx and 3,4-DGE are most powerful promoters of AGE formation [8]. In the present research, we have focused on studying the direct short-term effect of these three GDPs on the organization of ZO-1, a tight junction-associated protein. In the mesothelial monolayer, different intercellular junctions hold mesothelial cells to each other. The intercellular junctions can dynamically alter their structural and functional properties under different conditions and can be modulated by various cellular and metabolic regulators [9]. Opening of intercellular junctions between mesothelial cells is one of the first manifestations of damage related to dialysis. Chronic exposure to dialysis fluid is associated with widening of the intercellular spaces between HPMC and the detachment of these cells from the peritoneum [10]. Modulation of the expression and organization of junction molecules by various components of PDF and growth factors released from peritoneal cells has been reported [11]. Using western blot and immunostaining, expression of intercellular junction-associated proteins, such as ZO-1 and ß-catenin, on HPMC is found to be decreased after culturing in a high glucose environment [11]. TGF-ß induced by high glucose concentration downregulates the expression of these junction proteins in HPMC [10], yet in another study, TGF-ß is reported to affect the integrity of cultured endothelial cells through the reorganization of the actin cytoskeleton but not the disassembly of adherent junctions [12].
In this study, we have demonstrated that purified GDPs are able to downregulate the ZO-1 expression in HPMC in a dose- and time-dependent manner. It is unlikely that the decreased ZO-1 expression resulted from a direct cytotoxicity of GDPs as there was no significant reduction in viability of HPMC after incubation with selected concentrations of GDPs for up to 96 h (data not shown). Our in vitro data have further confirmed that FurA, M-Glx and 3,4-DGE increase the production of VEGF. We have shown further that these GDPs increase the synthesis of TGF-ß. The peritoneal concentration of VEGF has been shown to be one of the key factors which correlates with peritoneal membrane function [4]. Culturing with VEGF is associated with disorganization of another junction protein, occludin, in endothelial cells [7]. Naturally, understanding the modulation and regulation of the intercellular junctions by mediators released from peritoneal cells following exposure to PDF and its components is of utmost importance in preventing peritoneal membrane dysfunction in CAPD. Our in vitro culture experiment has shown that recombinant VEGF downregulates the expression of ZO-1 in a dose- and time-dependent manner. More importantly, the downregulation of ZO-1 expression by GDPs can be abolished by a neutralizing antibody to VEGF. These findings are in accordance with the previous observation in cultured rat mesothelial cells that M-Glx stimulates the production of VEGF, which then enhances the vascular permeability and angiogenesis [5].
Peritoneal biopsies from patients on CAPD have revealed the loss of mesothelial lining and disruption of the interstitium with prolonged time of dialysis treatment. Vascular changes, including replication of the capillary basement membrane, result in considerable thickening of the subendothelial tissue that eventually leads to occlusion of the capillary lumina [13]. These peritoneal changes suggest that neoangiogenesis is responsible for some of the pathological changes in the peritoneum of patients on CAPD. It is logical to argue that AGE increases the expression of VEGF in the peritoneum, which, in turn, induces neoangiogenesis in the peritoneal microcirculation. Animal models of CAPD have provided supporting evidence for the role of neoangiogenesis in the development of ultrafiltration failure [14]. It is worth mentioning that other mediators in the peritoneal cavity, such as connective tissue growth factor, can bind VEGF, suggesting that the activity of VEGF may be regulated in vivo by other mediators [15].
Recent in vitro studies have revealed that HPMC cultured in conventional PDF have decreased cell viability, reduced ability to generate IL-6 and retarded remesothelialization compared with HPMC cultured in PDF with a lower GDP content [3,16]. In the present study, we have shown that even GDP alone can damage the tight junction on the mesothelial cell and, hence, alter the permeability barrier. This structural change is associated with increased release of VEGF. Of note is the concentration of GDPs used in our in vitro experiments. These are comparable to clinically relevant concentrations found in conventional PDF (223 µM for M-Glx, 0.052 µM for FurA and 922 µM for 3,4-DGE) [8,17]. We believe that our in vitro findings still contribute to the understanding of the effect of GDPs on ZO-1 expression, although our observations were made after brief exposure to a single GDP, while in maintenance peritoneal dialysis, HPMC are continuously exposed to multiple GDPs. Furthermore, in the actual clinical situation, carbonyl stress from uraemic circulation may also contribute to cellular changes in the peritoneal cavity [18]. Nevertheless, these in vitro studies by others and us strongly support the contributory role of GDPs in the detrimental effect of glucose-based PDF to HPMC. The use of a PDF of lower GDP content is attractive and less injurious to the peritoneal lining.
Information on the signal transduction pathway involved in the activation of HPMC by GDPs is, so far, little known. In the present study, we select p42/p44 MAPK and PKC pathways for further investigation based on two recent relevant experimental findings. First, it has been reported that high glucose upregulates TGF-ß1 and fibronectin synthesis by HPMC and that this high glucose-induced upregulation is mediated largely by PKC [19]. These results implicated by activation of PKC by conventional peritoneal dialysis solutions may constitute an important signal for activation of HPMC. Second, thermally generated products, including AGEs and non-AGEs, have been shown to activate the p42/p44 MAPK signal transduction pathway in inflammatory response and cellular proliferation [20]. We have demonstrated that p42/p44 MAP kinase and PKC are important signal transduction pathways involved in GDP-induced VEGF and TGF-ß synthesis in HPMC (with FurA, M-Glx and 3,4-DGE as prototype). The activation of these signal transduction pathways by GDPs was established further by blocking experiments using selective inhibitors of p42/p44 MAP kinase and PKC. Most intriguingly, the stimulatory action of these GDPs on VEGF and TGF-ß synthesis in HPMC is abolished completely by blockade of the two pathways using specific inhibitors. We have further demonstrated that GDPs induced tyrosine phosphorylation of ZO-1 in HPMC and that this phosphorylation was associated with activation of p42/p44 MAP kinase and PKC signal transduction pathways. Our findings suggest that the activation of both PKC and p42/p44 MAPK pathways is the dominant mechanism in the modulatory effect of VEGF and TGF-ß1 synthesis and ZO-1 phosphorylation by GDPs. A previous study in rat mesothelial cells has shown that M-Glx induces VEGF production through cross-linking of the mesothelial membrane [5]. The possibility that p42/p44 MAP kinase and PKC pathways are activated through membrane cross-linking by GDPs or through reactive carbonyl groups deserves further investigation. Our findings provide therapeutic potential not only for ameliorating the harmful effect of GDPs on HMPC, but also highlight that targeting VEGF synthesis could be a novel approach for microvascular injury related to glucotoxicity.
In summary, we have demonstrated that GDP-induced VEGF alters the expression of tight junction-associated protein, ZO-1, in HPMC. This upregulation of VEGF synthesis by GDPs provides further support for the role of VEGF in neoangiogenesis and remodelling of the mesothelial membrane in CAPD. Our findings have provided additional insight into the mechanisms and prevention of ultrafiltration failure in CAPD, although the exact pathophysiological event of long-term exposure of HPMC to GDPs remains to be elucidated.
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Acknowledgments
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The authors thank Drs K.M. Chu, K.W. Chu, S.M. Chu and J. Ho and members of the University Surgical Teams for the collection of omentum. The study was partly supported by the Research Grant Council, Hong Kong (HKU 7415/04M) and the CRGC, The University of Hong Kong. J.C.K.L. was supported by the L & T Charitable Foundation and INDOCÁFE.
Conflict of interest statement. None declared.
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References
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Received for publication: 14.10.04
Accepted in revised form: 8. 3.05