1 Department of Pathophysiology, Poznan Medical School, Poland and 2 Division of Nephrology, University of Toronto, Canada
Correspondence and offprint requests to: Professor Andrzej Brborowicz, Department of Pathophysiology, Poznan Medical School, Ul. Swiecickiego 6, 60781 Poznan, Poland. Email: abreb{at}am.poznan.pl
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Abstract |
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Methods. Experiments were performed on primary mesothelial cell cultures. Free radical generation in these cells after exposure to acetaldehyde (ACT), glyoxal (GLYO) or methylglyoxal (M-GLYO) was detected with a fluorescent probe. Cell viability measurements were based on release of LDH from cell cytosol, and synthesis of IL-6 and proliferation after exposure to GDP. Effects of individual GDPs and of dialysis fluid free of GDP (GDP-free PDF) or containing GDP (GDP-high PDF) on cell viability were also studied in the presence of OTZ (1 mmol/l).
Results. All of the GDPs as well as the autoclaved dialysis fluid caused increased free radical generation. ACT increased LDH release from the cells by 374% (P<0.001), and this effect was abolished by OTZ. All of the GDPs inhibited cell growth (ACT, 47%, P<0.01; GLYO, 52%, P<0.01; M-GLYO, 26%, P<0.05) and this effect was reversed in presence of OTZ. ACT inhibited Il-6 synthesis in mesothelial cells by 74% P<0.01 and this effect was prevented by OTZ. GDP-high PDF but not GDP-free PDF reduced synthesis of IL-6 in mesothelial cells by 40% (P<0.01) an effect that was reversed by OTZ. Mesothelial cell growth was more strongly inhibited by GDP-high PDF (76%, P<0.01) than by GDP-free PDF (31%, P<0.05). OTZ improved growth of mesothelial cells in the presence of GDP-high PDF (+150%, P<0.01) and in presence of GDP-low PDF (+38%, P<0.05). OTZ prevented the cytotoxic effect of GDP-high PDF on mesothelial cells.
Conclusions. The GDP-induced stimulation of free radicals in mesothelial cells in the present study may provide a possible mechanism of GDP cytotoxicity. Because OTZ reduced the toxic effects of GDP on mesothelial cells, this compound may improve biocompatibility of peritoneal dialysis fluids.
Keywords: glucose degradation products; L-2-othiazolidine-4-carboxylic acid; mesothelium
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Introduction |
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In uraemic patients treated with peritoneal dialysis, glycation and lipooxidation of the peritoneal membrane is due not only to presence of reactive carbonyl compounds in plasma but also to their presence in commercially available heat-sterilized dialysis fluids [3]. Heat sterilization of the peritoneal dialysis fluids results in formation of various glucose degradation products (GDPs), such as GLYO and M-GLYO, which in turn induce formation of AGE [4]. These substances directly react with mesothelial cells to cause toxic effects [5]. They also stimulate the production of vascular endothelial growth factor in peritoneal cells, which over the long term may lead to formation of the hyperpermeable peritoneum [6]. In addition, GDPs present in the dialysis fluid are absorbed from the peritoneal cavity into the bloodstream to cause increases in plasma AGE concentrations [7]. Recently, several approaches have been proposed to reduce local and systemic toxicity of carbonyl compounds originating from the dialysis fluid. For example, GDP concentrations in the dialysis fluid can be decreased by autoclaving glucose solutions at very low pH in two chamber bags, by absorption of these compounds onto the surface of affinity beads to reduce their dialysis fluid levels [8], or by their enzymatic degradation with use of glyoxalase [9]. Although new generations of peritoneal dialysis fluids contain low levels of GDPs, we have previously found that low concentrations of these compounds during prolonged exposure exert toxic effects on mesothelial cells [10]. We therefore believe that the use of substances to protect against toxic effect of GDPs is of clinical relevance.
In the present in vitro experiments on human peritoneal mesothelial cells, we tested the cytotoxicity of individual GDPs in cells having increased intracellular glutathione concentrations caused by pretreatment with L-2-oxothiazolidine-4-carboxylic acid (OTZ). We found that enhancement of cell resistance to oxidative stress rendered the cells more resistant to injury by GDPs.
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Material and methods |
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Mesothelial cells were grown to confluency in 75 cm2 culture flasks, were harvested with trypsin 0.05%EDTA 0.02% Hanks solution, and were resuspended in fresh medium and seeded into 24 well clusters. Experiments were performed either on mesothelial monolayers or on proliferating cells in 24 well clusters. We studied the effects of GDPs on the intracellular generation of free radicals, viability, the growth rate of the cells, and on interleukin-6 (IL-6) synthesis stimulated by interleukin-1ß. We selected individual GDPs and specific concentrations from our previous study in order to moderately inhibit the growth of mesothelial cells (5075% of growth rate in control) [5]. Acetaldehyde (ACT) (200 µM), GLYO (150 µM) and M-GLYO (150 µM) were tested. In other experiments, we tested the cytotoxicity of standard dialysis fluid prepared in the laboratory and sterilized either by filtration or by autoclaving on mesothelial cells. The cytotoxicity of these substances or fluids was additionally studied in presence of OTZ (1 mM), which significantly increases glutathione concentrations in mesothelial cells at these concentrations [12]. In these experimental groups, cells were pretreated with OTZ 4 h prior to exposure to the individual GDPs or dialysis fluid and OTZ was maintained in the solution during the study period. In preliminary experiments, we found that 1 mM OTZ, in the presence of the individual GDPs increased glutathione concentration in mesothelial cells for 4 h of incubation (mean increase of 63%, range 3984%).
Free radical production by mesothelial cells
Intracellular generation of free radicals was measured in mesothelial monolayers cultured in 24 well plates which were exposed for 8 h to the following media:
At the end of incubation, cells were harvested from the wells with 0.02% EDTA solution in Hanks and washed in a culture medium. We then measured the intracellular generation of free radicals with a probe: 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). During 45 min of incubation in the presence of free radicals, non-fluorescent DCF-DA was converted to the fluorescent 2',7'-dichlorodihydrofluorescein (DCF). Fluorescence in the cell lysate was determined using a microplate fluorescent reader (Victor-2, Perkin Elmer Life Sciences, Finland), with excitation at 485 nm and emission at 530 m.Total protein concentration in the cell lysates was measured with a BCA Protein Assay kit (Pierce, Rockford, IL).
Using the same methodology, intracellular generation of free radicals was measured in mesothelial cells exposed for 8 h exposure to medium M199 + 10% FCS mixed (1:1 v/v) with dialysis fluid made in the laboratory containing 85 mmol/l glucose (complete composition of the fluid is listed below), which was either autoclaved or sterilized by filtration.
Viability of mesothelial cells
Experiments were performed on mesothelial monolayers in 24 well clusters, which had been cultured in medium with low serum concentration (0.1% FCS) for the preceding 24 h. The cells were then exposed for 18 h to the following media:
At the end of incubation, the supernatant was collected and the activity of lactate dehydrogenase (LDH) was measured with a colorimetric assay obtained from Sigma.
Proliferation of mesothelial cells
Proliferation of mesothelial cells was evaluated from the rate of incorporation of radiolabelled thymidine into the DNA of cells present during the exponential phase of growth. Cells were seeded into 24 well culture plates at a density of 5 x 104 cells/cm2. After 24 h, culture media was removed and replaced with the following solutions:
These solutions were supplemented with 3H-methyl-thymidine (Institute of Radioisotopes, Prague, Czech Republic) in a final concentration of 1 µCi/mL. After a 24 h culture, proliferating mesothelial cells were harvested with a trypsin 0.05%EDTA 0.02% solution and precipitated with 10% trichloroacetic acid (TCA). The precipitate was washed twice with TCA and then lysed with 1 N NaOH. Radioactivity of the lysate was measured in a ß-liquid scintillation counter (1209 Rackbeta LKB Wallac, Finland).
Synthesis of IL-6 by mesothelial cells
Mesothelial monolayers in 24 well clusters were cultured for 24 h in medium M199 with a low concentration of serum (0.1% FCS). The media in the wells was then replaced with the following solutions:
Effect of the dialysis fluid on viability proliferation and IL-6 synthesis by mesothelial cells
The dialysis fluid used in these experiments was prepared in the laboratory and its composition was similar to commercially available solutions [(in mmol/l) Na, 132; Ca, 1.75; Mg, 0.75; Cl, 102; lactate, 35; glucose, 85]. Prepared dialysis fluid was sterilized either by filtration or by autoclaving. Large amounts of GDPs are formed during heat sterilization of these types of glucose-containing fluids [4]. We tested the effects of autoclaved and filtered dialysis fluids on the viability and growth of mesothelial cells and on synthesis of these cells stimulated by IL-6. During all experiments, the pH of the dialysis solutions was adjusted to 7.4.
Viability study
Mesothelial monolayers in 24 well clusters were exposed for 24 h to the following solutions:
At the end of incubation, the integrity of mesothelial monolayers was monitored in the microscope and documented with photography.
Proliferation study
Mesothelial cells were seeded into 24 well clusters at a density of 5 x 104 cells/cm2. After 24 h, the culture medium was removed and replaced with mixture of culture medium and dialysis fluid in ratio 0.75/0.25 (v/v). All fluids were supplemented with 10% FCS and 3H-methyl-thymidine at a final concentration of 1 µCi/ml. The following experimental groups were studied:
After 24 h of culture, cells were harvested with trypsin, and incorporation of radiolabelled thymidine into their DNA was measured as described above.
Synthesis of IL-6
Mesothelial monolayers in 24 well clusters were maintained for 24 h in culture medium with low a serum concentration (0.1% FCS). The medium was then replaced in all wells with the following solutions:
After 6 h from the start of the incubation, the solutions in each well were supplemented with interleukin-1ß in an amount sufficient to achieve a final concentration of 100 pg/ml and incubation was continued for an additional next 18 h. Supernatants were then collected for measurement of IL-6 levels and synthesis of IL-6 was evaluated as described above.
Statistical analysis
Results are presented as mean±SEM. The data was analysed with Friedman tests, and post hoc analysis was performed using Dunns test. A P-value <0.05 was considered significant.
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Results |
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Autoclaved dialysis fluid significantly inhibited proliferation of mesothelial cells, as measured by the rate of 3H-methyl-thymidine incorporation into growing cells (3943±724 vs 16309±2396 in control cells, P<0.01). Although the growth of cells exposed to the filtered dialysis fluid was also inhibited, the effect was less pronounced (11321±1456, P<0.05). OTZ reduced the cytotoxic effects of the dialysis fluids and this effect was more pronounced with the autoclaved solution (Figure 4). Synthesis of IL-6 was significantly reduced in mesothelial cells exposed to the autoclaved dialysis fluid: 39.1±3.1 pg/µg cell protein as compared with controls (64.7±4.3 pg/µg cell protein, P<0.01). Filtered dialysis fluid did not suppress synthesis of IL-6 in these cells. The inhibition of IL-6 synthesis caused by the autoclaved solution was totally reversed in the presence of OTZ (Figure 5). We also demonstrated that OTZ prevented damage to mesothelial monolayers caused by incubation in culture medium mixed (1:1 v/v) with the autoclaved dialysis fluid (Figure 6).
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Discussion |
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The concentration of acetylaldehyde used in our experiments was within the range found in standard peritoneal dialysis fluids, whereas the concentrations of GLYO and M-GLYO were 10 times higher than that in dialysis fluids. However, we believe that the toxicity of these two substances at relatively high concentrations is of clinical significance, because even low concentrations of GDPs are not toxic in acute experiments, and become injurious to mesothelial cells after prolonged exposure [10]. The total elimination of GDPs from fluids containing high concentrations of glucose and that are sterilized by autoclaving is not possible. Therefore, development of methods that increase resistance of peritoneal membrane to the injurious effect of these compounds remains an important aspect of improved biocompatibility of dialysis fluids.
Increasing the intracellular glutathione concentration is a method that is used for enhancing resistance of cells against toxic effects of free radicals. OTZ is a precursor of cysteine, which is used for synthesis of glutathione. OTZ prevented organ damage in pathologies involving increased production of free radicals [16]. We have previously shown that acute in vitro exposure of mesothelial cells to a standard dialysis solution results in transient decreases in intracellular glutathione and that OTZ not only prevented this effect but even augmented the concentration of glutathione in these cells [17]. Yahyapour et al. [18] found that glutathione reduced the cytotoxic effect of heat-sterilized peritoneal dialysis fluid on neutrophils. However, it is possible that addition of reduced glutathione to the dialysis fluid may be impractical because of the rapid oxidation of this substance into its inactive oxidized form. We demonstrated in the present study that OTZ, which is a precursor of cysteine used for the intracellular synthesis of glutathione, prevented or reduced the cytotoxic action of free radicals on mesothelial cells. The growth rate and synthesis of IL-6 in mesothelial cells was improved when they were exposed to media containing both OTZ and GDPs compared with cells treated with GDPs alone. This effect was observed in mesothelial cells both when individual GDPs were used and when a heat-sterilized dialysis fluid containing high concentrations of a mixture of these substances was applied. In contrast, the filtered dialysis fluid, which is free of GDPs, exerted minimal in vitro cytotoxic effects (Figures 46). It was recently shown that GDPs retard the healing of the mesothelial monolayer after mechanical injury, and that this effect was due to inhibition of their growth rate [19]. We do not believe that the protective effect of OTZ against GDP-induced cytotoxicity was due to neutralization of these compounds. The addition of OTZ to a solution containing GDPs did not change the ultraviolet spectrum absorbance of the solution, indicating that these substances were not eliminated from the fluid (data not shown). Protective effects of OTZ may be even more visible in uraemic patients treated with peritoneal dialysis who have a deficient antioxidant system and are exposed to increased oxidative stress. We additionally showed that one of the GDPs, 3-deoxyglucosone, inactivates glutathione peroxidase, a key antioxidant enzyme. However, oral administration of OTZ to peritoneal dialysis patients caused significant increases in their whole-blood glutathione level [20].
In conclusion, GDPs stimulated generation of free radicals in mesothelial cells, and OTZ protected the cells against the cytotoxic actions of GDPs. An improved viability of mesothelial cells may enhance the long-term dialysis membrane functions of the peritoneum.
Conflict of interest statement. None declared.
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References |
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