Department of Nephrology, Leicester General Hospital, Leicester, UK
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Abstract |
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Methods. In order to investigate this in vitro, human peritoneal mesothelial cells (HPMC) were cultured in a 50:50 mix of dialysis solution and M199 for 12 h. The dialysate was laboratory manufactured and designed to be identical in composition to PD4 (LAB). The final glucose concentration ranged between 5 and 40 mmol/l. Experiments were conducted in the presence and absence of an anti-transforming growth factor-beta (TGF-ß) antibody. Cell viability was measured by lactate dehydrogenase (LDH) release. Fibronectin (FN) and TGF-ß protein were measured by ELISA, and FN gene expression was measured by Northern analysis. Separately, the effects of recombinant TGF-ß1 added to M199: dialysate at 5 mmol/l glucose were investigated.
Results. Forty millimoles per litre d-glucose LAB caused a decrease in cell viability, as evidenced by an increase in LDH release (6.0±1.3 vs 2.6±0.7%). This effect was dependent on osmolality. Forty millimoles per litre d-glucose LAB stimulated a 15.4±4.6% increase in FN, a 46.5±18.3% increase in TGF-ß protein (both P<0.05), and 1.4±0.09-fold increase in FN mRNA compared with 5 mmol/l d-glucose LAB. Exogenous TGF-ß 01 ng/ml induced a dose-dependent increase in FN protein (280±45% increase at TGF-ß 1 ng/ml, P<0.0001), and FN mRNA levels (10.0±1.8-fold at TGF-ß 1 ng/ml). The increase in FN in response to 40 mmol/l glucose was significantly reduced by anti-TGF-ß antibody to levels not different from control (93.8±6.6%, P<0.05 vs no Ab).
Conclusions. These data suggest that the pro-fibrotic effect of glucose dialysate on HPMC is mediated through stimulation of TGF-ß, which promotes FN gene expression and protein production.
Keywords: CAPD; dialysis solutions; extracellular matrix proteins; glucose; mesothelial cells; TGF-ß
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Introduction |
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The most commonly employed dialysate for CAPD is a hyperosmolar glucose lactate-buffered solution. This is inhibitory to many resident peritoneal cells, and has been shown in many studies to inhibit phagocytic activity by leukocytes and production of cytokines by mesothelial cells [8,9]. There is, however, little work specifically on the effect of glucose on mesothelial cell-derived extracellular matrix (ECM), although it has been suggested that it may increase fibronectin and decrease proliferation [10,11]. Although this would appear to be an appropriate response to aid peritoneal repair, it may also contribute to peritoneal fibrosis, and hence peritoneal membrane failure. Increasing glucose concentration has been shown to increase transforming growth-factor beta (TGF-ß) mRNA in peritoneal mesothelial cells [12], and in other cell types autocrine TGF-ß production has been implicated in the pathogenesis of ECM production in response to high glucose concentration [13]. In vivo, rats given exogenous intraperitoneal TGF-ß following surgical injury to their uterine horns have an increase in adhesion formation compared with untreated controls [14]. The aim of the current study therefore was to investigate the role of autocrine TGF-ß production in the control of glucose stimulated ECM protein production by peritoneal mesothelial cells.
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Subjects and methods |
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Cell preparation and characterization
Human peritoneal mesothelial cells (HPMC) were cultured using standard techniques from omentum obtained from uraemic patients at the time of first peritoneal dialysis catheter insertion [15]. The study was approved by the Leicestershire Ethics Committee and informed consent obtained. Omental tissue was washed three times in phosphate-buffered saline (PBS) and cut into pieces approximately 6 cm2. The removal of mesothelial cells was performed by incubating the specimen at 37°C for 20 min in 0.125% trypsin:0.05% EDTA with agitation. The cells were washed once before seeding at 1x104/ml in M199 medium with 10% fetal calf serum (FCS), insulin-transferrin-selenite (ITS), penicillin, streptomycin, hydrocortisone and L-glutamine. The cells were cultured in 5% CO2 at 37°C. All cell culture plastics were pre-coated with calf-skin collagen by incubating overnight with calf-skin collagen in 0.1% acetic acid, and then rinsed three times in PBS before use. Cells were cultured in 25-cm2 flasks with vented caps and experiments were performed on confluent cells from passages 24 on 12-well plates. Immunohistochemical analysis was performed using a horseradish peroxidase (HRP)-conjugated second-antibody technique. Confluent cells were rendered quiescent prior to experimentation by incubating for 48 h in M199 with 0.1% FCS, which has previously been shown to have no adverse effect on cell viability [16]. All experiments were performed in triplicate on HMPC derived from at least three separate patients.
Experimental design
Quiescent HPMC on 12-well plates were exposed to a 50:50 mixture of dialysate and M199 with 0.1% FCS. The dialysate used was a laboratory-manufactured, filter-sterilized dialysate of identical electrolyte composition and pH to the widely used commercial dialysate PD4 (Baxter Healthcare Ltd). The use of this laboratory-manufactured dialysate enabled adjustment of the final test medium added to mesothelial cells to a D-glucose concentration of between 5 and 40 mmol/l. Mannitol at 40 mmol/l was used as an osmotic control. Experiments were also performed using commercial 1.36% glucose dialysate diluted 50:50 with medium to allow comparison with the laboratory-manufactured dialysate. A 1.36% glucose solution contains 76 mmol/l D-glucose, and hence the 40 mmol/l final glucose concentration in the laboratory-manufactured test medium corresponds approximately with a 50:50 dilution of this commercial solution with culture medium. Further experiments were performed using 3.86% commercial dialysate to investigate cell viability at higher glucose concentration. The majority of experiments were conducted at the 12-h time point to best mimic the overnight CAPD dwell, as longer dwells without replacement of dialysate do not occur in clinical practice. Additional experiments were conducted at 8, 12, 24 and 48 h time points to investigate any adaptation in the response to glucose over time.
Cell supernatants were harvested at 12 h and stored at -20°C for subsequent measurement of fibronectin and total TGF-ß. Cell monolayers were lysed by scraping the cells from the culture plate into 0.5 mol/l sodium hydroxide, and sonicating twice for 10 s. Total protein was measured using a modified Lowry technique (BioRad DC protein assay, BioRad, UK).
Assessment of cell viability
Cell viability was assessed by measurement of lactate dehydrogenase (LDH) activity in the cell-culture supernatant and cell monolayer dispersed in deionized water using a commercial assay (Sigma DG1340-K). Rate of change of absorbance (A) per minute was measured using a Cecil CE 2040 spectrophotometer, which is directly proportional to LDH activity.
Measurement of supernatant fibronectin and TGF-ß
Supernatants were assayed for fibronectin as previously described [17]. Briefly, Immunoplates (Nunc) were coated using a polyclonal rabbit anti-human fibronectin antibody diluted 1:1000 in coating buffer. Appropriately diluted samples were incubated overnight. A monoclonal mouse anti-human fibronectin antibody diluted 1:500 in wash buffer was added for 2 h, the plates washed and an HRP-conjugated anti-mouse-immunoglobulin (1:1000) added for 2 h. HRP was detected using o-phenylenediamine (OPD) as the peroxidase substrate. Total TGF-ß was measured using a commercial sandwich ELISA (Promega Ltd.).
Effect of exogenous TGF-ß on fibronectin production
Quiescent HPMC on 12-well plates were exposed for 12 h to a 50:50 mixture of laboratory dialysate and M199 with 0.1% FCS at a final glucose concentration of 5 mmol/l. Exogenous human platelet derived TGF-ß1 (R&D Systems Ltd, UK) was added at concentrations of between 0.1 and 5 ng/ml, and supernatant fibronectin levels measured by ELISA as described above.
Effect of anti-TGF-ß antibody on fibronectin production
Confluent, quiescent mesothelial cells were exposed to a 50:50 mixture of dialysate and M199 with 5 mmol/l and 40 mmol/l glucose in the presence and absence of a pan-specific anti-TGF-ß antibody at concentrations between 1 and 10 µg/ml (R&D Systems Ltd). A pan-specific anti-PDGF antibody (R&D Systems Ltd) was used at 10 µg/ml concentration as a further negative control. The efficacy of antibody blocking was confirmed by assessing the effect of adding the anti-TGF-ß antibody (10 µg/ml) in the presence of increasing concentrations of exogenous TGF-ß.
Analysis of RNA by Northern blotting
Quiescent HPMC in 25-cm2 flasks were exposed for 8, 12, 24 and 48 h to a 50:50 mixture of M199 and dialysate as described above. In addition, cells were separately exposed for 12 h to a 50:50 mixture of laboratory dialysate at 5 mmol/l D-glucose and M199 in combination with 1 ng/ml exogenous TGF-ß. Total RNA was extracted using Trizol® reagent (Life Technologies, UK), a mono-phasic solution of phenol and guanidine isothiocyanate based on a method of Chomczynski and Sacchi [18], according to the manufacturer's instructions. Briefly, after removal of the test medium the cells were washed three times with PBS and the cells dispersed in 1.5 ml Trizol® reagent; 300 µl chloroform was added to each tube and vortexed for 15 s before standing at room temperature for 10 min. The sample was then centrifuged at 13 000 r.p.m. at 4°C for 15 min and the upper aqueous phase removed to a second tube. The RNA was precipitated by adding 750 µl propan-2-ol, vortexing, and standing for 10 min at room temperature before again centrifuging at 4°C, 10 000 r.p.m. for 10 min. The RNA pellet was washed by re-suspending in 70% ethanol before finally re-suspending in 20 µl of DEPC water. The RNA was stored at -70°C.
Aliquots (30 µg) of RNA were electrophoresed on a 1% agarose gel containing 1.9% formaldehyde in MOPS (3-(N-morpholino)propanesulphonic acid). The resolved RNA was transferred onto Hybond-N nylon membranes (Amersham Ltd) by capillary action using 20xSSC. Membranes were pre-hybridized for 4 h at 42°C with 200 µg/ml denatured salmon sperm DNA in 50% formamide, 1% SDS, 5xDenhardt's, and 5xSSPE (saline-sodium phosphate ethylenediaminetetra-acetic acid). The membranes were hybridized overnight with [32P]dCTP cDNA probe that had been Klenow DNA polymerase-labelled, using a random primer labelling system (Prime-a-Gene®, Promega Ltd) in fresh buffer. After hybridization the membranes were washed twice with 1% SDS, 2xSSPE at room temperature for 10 min, twice with 1% SDS, 0.2xSSPE at 65°C, then exposed to X-Omat LS film (Kodak) with intensifier screens at -70°C. Densitometric analysis of the transcripts was carried out on a BioRad GS 700 imaging scanner. RNA loading was normalized using a cDNA probe for cyclophilin. The human fibronectin cDNA probe was generously supplied by the UK HGMP Resource Centre, Cambridge, UK, and cyclophilin was a gift from SmithKline Beecham Pharmaceuticals.
Statistical analysis
To allow for variation in mesothelial cell fibronectin production between multiple experiments, the results are expressed as percentage increase over control (5 mmol/l glucose). Absolute levels of fibronectin and TGF-ß protein corrected for cell protein concentration are also given in the text where appropriate.
Data were expressed as means±SEM. For comparison of means between two groups, an unpaired t-test was used. For comparisons between multiple groups, ANOVA with a post-test for linear trend was applied. Statistical significance was defined as P<0.05.
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Results |
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Effect of increasing glucose concentration on LDH release (cell viability)
Increasing glucose concentration resulted in an incremental increase in LDH release by HPMC (Table 1). However, mannitol osmotic control caused a similar increase in LDH release suggesting that the effect was the result of increased osmolality of the test solution. The 50:50 mixture of commercial 3.86% PD4 resulted in significant cytotoxicity (10.6±1.71% LDH release vs 2.63±0.69% control), and for this reason was not used in subsequent experiments to investigate fibronectin and TGF-ß protein amounts.
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Effect of increasing glucose concentration on fibronectin protein production and gene expression
Basal fibronectin production (5 mmol/l glucose concentration) was 2.03±0.21 µg/mg cell protein. Increasing glucose concentration caused an increase in HPMC fibronectin production with a 15.4±4.6% increase at 40 mmol/l D-glucose (Table 2). Unlike the effect on cell viability, this effect was independent of the osmolality of the solution. Total cell protein did not vary between the test conditions. The time course of fibronectin protein production is shown in Figure 1
. The increase in fibronectin with increase in glucose concentration persists beyond 12 h, with a 135±11.0% increase at the 48 h time point (P=0.009). Northern analysis demonstrated an increase in fibronectin mRNA at 8 h which became statistically significant by 12 h (1.4±0.09-fold, P<0.05) in the 40 mmol/l glucose condition (Figure 2
). No difference in fibronectin mRNA was seen at either 24 or 48 h.
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Effect of increasing glucose concentration on TGF-ß protein production
Table 3 illustrates the increase in TGF-ß production by HPMC in response to increasing glucose concentration. There was an incremental rise in TGF-ß, reaching a 46.5±12.3% increase in the 40 mmol/l glucose condition. This represents a mean TGF-ß concentration in the culture supernatant of 150±24 pg/ml in the 40 mmol/l D-glucose condition. TGF-ß concentration with 40 mmol/l mannitol osmotic control was no different to that under control conditions.
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Effect of exogenous TGF-ß on fibronectin gene expression and protein production
Increasing concentrations of exogenous TGF-ß resulted in a dose-dependent increase in fibronectin protein levels over the concentration range 01 ng/ml (Figure 3), reaching a plateau of 280±45% increase at 1 ng/ml TGF-ß. At higher concentrations (up to 5 ng/ml) there was no further increase in fibronectin, suggesting that maximal stimulation of fibronectin production had been attained. Northern analysis comparing a 50:50 mixture of 5 mmol/l glucose dialysate:M199 with and without 1 ng/ml TGF-ß showed a significant (10±1.8-fold) increase in fibronectin gene expression (Figure 4
).
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Effect of anti TGF-ß antibody on fibronectin production in the presence of hyperosmolar glucose
Addition of the anti TGF-ß antibody at 10 µg/ml blocked the increase in fibronectin seen with the addition of exogenous TGF-ß over the pathophysiological concentration range of interest (01 ng/ml) (Figure 3). The effect of anti TGF-ß antibody on mesothelial-cell fibronectin production in response to glucose is shown in Figure 5
. As before 40 mmol/l D-glucose caused an increase (24.7±9.7%) in fibronectin production. Anti-TGF-ß antibody had no effect on fibronectin levels under control conditions (LAB at 5 mmol/l D-glucose). However, the increase in fibronectin in response to 40 mmol/l glucose was significantly reduced by increasing anti TGF-ß antibody concentrations to levels not different from control (93.8±6.6% at 10 µg/ml, P<0.05 vs no Ab). An irrelevant antibody (anti-PDGF) had no effect on the production of fibronectin by HPMC.
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Discussion |
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Increasing glucose concentration resulted in an increase in both fibronectin protein production and gene expression by HPMC. This effect was independent of the increase in osmolality of the dialysis solution. In contrast the increase in LDH release and hence decrease in cell viability of mesothelial cells appears to be purely the result of increasing osmolality of the solution, with no specific effect of high glucose concentration. The results show that above 40 mmol/l final glucose concentration, cell viability falls dramatically. In clinical practice these glucose concentrations are not sustained as there is a progressive decline in glucose concentration over the duration of a CAPD dwell, with a mean decrease to only 38% initial glucose concentration in 4 h during a PET [19]. For these reasons, further experiments were restricted to the pathophysiological concentration range of 540 mmol/l glucose final concentration. Results of fibronectin protein production were expressed corrected for total cell protein, and although cell viability decreased, there was no detectable change in total cell protein. The changes in fibronectin protein production cannot therefore be accounted for simply by changes in either cell viability or total cell protein.
In the 12-h protocol a 15% increase in fibronectin protein production, and 1.4-fold increase in gene expression was observed. Although this represents a relatively small change, it must be remembered that patients on CAPD will be repeatedly replacing dialysis bags 45 times per day, and hence any small change in synthesis over 12 h potentially would result in considerable increase in ECM accumulation over many years of CAPD. Although no in-vitro cell culture protocol can truly mimic the condition in vivo, the 12-h time point was chosen to mimic the long overnight CAPD dwell, after which, in vivo, a patient would change the dialysate for a fresh solution. Longer dwells without replacement of dialysate do not occur in clinical practice. Experiments to investigate fibronectin mRNA levels at other time points showed that the effect was greatest at 12 h, and not apparent at 24 or 48 h. However, the same effects of high glucose concentration on fibronectin protein production are observed with longer time courses, indicating that there is little adaptation to the effect of high glucose even over longer dwells.
These experiments provide convincing evidence for the role of autocrine TGF-ß production by HPMC to regulate ECM production in response to glucose. Hyperosmolar glucose produced an increase in TGF-ß protein and fibronectin protein, and the latter could be inhibited by the addition of an anti TGF-ß antibody. This suggests that TGF-ß has a key regulatory role in promoting ECM production by HPMC. It is unclear whether the increase in fibronectin mRNA levels in response to TGF-ß results from increased gene expression or increased RNA stability. A high glucose concentration has been shown to promote an increase in ECM in other cell types [20,21]. Furthermore, exposing cultured mesangial cells to high glucose concentration results in an increase in collagen III deposition, which could be prevented by the addition of an anti TGF-ß antibody [13].
It is now recognized that the regulation of ECM turnover is the product of both alterations in protein synthesis, and protein degradation. The key elements in ECM protein degradation are the metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs). Fibronectin, investigated here, is a known substrate for MMP3 (stromelysin), and this is inhibited by TIMP I. The increase in fibronectin mRNA levels, coupled with the increase in protein levels seen in this study is evidence, at least in part, for an increase in ECM synthesis. However, a contribution from a decrease in degradation cannot be excluded. TGF-ß has previously been shown to increase production of biologically active MMP9 [22], PAI-1 [23], and the message for TIMP III [24] in HPMC. Conversely, hyperosmolality (either with D-glucose or mannitol) produced a decrease in MMP9 activity [22]. No studies have considered the role of MMP3 in HPMC ECM regulation. In the cell-culture system used in the experiments reported here, cells were exposed to a high glucose concentration (and hence osmolality), but also to a concurrent increase in autocrine TGF-ß levels. ECM accumulation is a fine balance between new matrix production, release of pre-formed matrix, and matrix degradation. It is clear from our results, however, that the balance of production and degradation in this model of CAPD favours an increase in ECM.
Hyperosmolar glucose is implicated in the pathogenesis of other important conditions, in particular diabetes mellitus. As a consequence, the mechanism by which hyperosmolar glucose influences cell functions has been widely studied. Two glycolytic pathways, the polyol and the hexosamine pathways have been investigated. Sorbitol (an intermediate of the polyol pathway) has been shown to accumulate in mesothelial cells exposed to high concentrations of glucose, and to cause cellular dysfunction [25]. In contrast, the hexosamine pathway has been explored in mesangial cells. There is evidence that both high glucose concentration, or the intermediate metabolite of the hexosamine pathway glucosamine, can stimulate mesangial cell TGF-ß production [26]. Either of these pathways could play a role in mediating the effect of glucose on ECM synthesis by HPMC through TGF-ß production, and is currently under further investigation.
In summary therefore, this study provides evidence that hyperosmolar glucose stimulates mesothelial cell ECM production, and that this effect is mediated through autocrine TGF-ß production. It also demonstrates that this effect can be prevented by using an anti TGF-ß-blocking strategy. These data raise the possibility that the use of local anti TGF-ß strategies could prolong CAPD technique survival by decreasing the tendency for peritoneal fibrosis.
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Acknowledgments |
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Notes |
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References |
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