Dialysis

Effect of Glucose Degradation Products on Human Peritoneal Mesothelial Cell Function

JANUSZ WITOWSKI, KATARZYNA KORYBALSKA, JUSTYNA WISNIEWSKA, ANDRZEJ BREBOROWICZ, GERHARD M. GAHL, ULRICH FREI, JUTTA PASSLICK-DEETJEN and ACHIM JÖRRES
JASN April 2000, 11 (4) 729-739;

Abstract

Abstract. Bioincompatibility of conventional glucose-based peritoneal dialysis fluids (PDF) has been partially attributed to the presence of glucose degradation products (GDP) generated during heat sterilization of PDF. Most previous studies on GDP toxicity were performed on animal and/or transformed cell lines, and the impact of GDP on peritoneal cells remains obscure. The short-term effects of six identified GDP on human peritoneal mesothelial cell (HPMC) functions were examined in comparison to murine L929 fibroblasts. Exposure of HPMC to acetaldehyde, formaldehyde, glyoxal, methylglyoxal, furaldehyde, but not to 5-hydroxymethyl-furfural, resulted in dose-dependent inhibition of cell growth, viability, and interleukin-1 β (IL-1 β)-stimulated IL-6 release; for several GDP, this suppression was significantly greater compared with L929 cells. Although the addition of GDP to culture medium at concentrations found in PDF had no major impact on HPMC function, the exposure of HPMC to filter-sterilized PDF led to a significantly smaller suppression of HPMC proliferation compared to that induced by heat-sterilized PDF. The growth inhibition mediated by filter-sterilized PDF could be increased after the addition of clinically relevant doses of GDP. These effects were equally evident in L929 cells. In conclusion, GDP reveal a significant cytotoxic potential toward HPMC that may be underestimated in test systems using L929 cells. GDP-related toxicity appears to be particularly evident in experimental systems using proliferating cells and the milieu of dialysis fluids. Thus, these observations may bear biologic relevance in vivo where HPMC are repeatedly exposed to GDP-containing PDF for extended periods of time.

Over the past two decades, peritoneal dialysis has developed into an effective renal replacement therapy for treating patients with kidney failure. However, increasing use of chronic peritoneal dialysis regimens has raised concern about the long-term preservation of the peritoneum as a dialyzing membrane. It has soon become apparent that the currently used peritoneal dialysis fluids (PDF) are limited in terms of biocompatibility. Many studies have convincingly demonstrated the adverse effects of PDF toward peritoneal membrane and peritoneal host defense (1,2,3,4,5,6,7). One of the aspects of PDF that has been viewed as bioincompatible is the presence of glucose, which is added in high concentrations to most PDF as an effective osmotic agent. Both the development of diabetiform alterations in peritoneal ultrastructure in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) (8,9,10) and the impaired function of cells exposed to glucose-based PDF (5, 11) have been linked either directly or indirectly to the use of glucose. These effects may be related to the metabolic action of glucose per se, the corresponding rise in osmolality, the accumulation of glycated proteins, and/or the formation of glucose degradation products (GDP) (12).

In 1986, Henderson et al. suggested that abdominal pain experienced by patients receiving CAPD might be caused by irritant products of glucose degradation (13). The report has prompted research on the stability of glucose-based PDF and the potential toxicity of glucose derivatives. It has been demonstrated that breakdown of glucose contained in PDF gives rise to a number of products, several of which have been identified as low molecular weight aldehydes, including 5-hydroxymethyl-furfural, furaldehyde, acetaldehyde, formaldehyde, glyoxal, and methylglyoxal (14,15,16). The process occurs primarily during heat sterilization of PDF and to a lesser extent during their prolonged storage (17,18,19). It has been well documented that concentrations of GDP in heat-sterilized solutions are much higher than those detected in filter-sterilized fluids (20,21,22). The difference in biologic effects between heat- and filter-sterilized PDF has therefore been related—indirectly—to the presence of GDP. Indeed, in various experimental in vitro systems, heat-sterilized solutions have been shown to impair cell function to a greater extent than fluids sterilized by filtration (16, 20,21,22,23,24,25). The parameters that have been investigated included cell proliferation rate (15, 16, 20, 24, 25), cytokine synthesis (21, 22, 24, 26), phagocytosis (21), and superoxide generation (16, 21, 24). Using a different approach, Wieslander et al. have also demonstrated that several glucose-derived aldehydes were capable of directly inhibiting cell growth in culture (16). Most GDP studies to date have been performed with animal and/or transformed cells including mouse L929 fibroblasts (16, 20, 23, 25), RAW 264.7 macrophages (24), and human neuroblastoma SH-SY5Y cell line (24). Several studies have also used peripheral leukocyte subpopulations (21, 22, 24). However, very little is known about how exposure to GDP affects the function of peritoneal cells. The mesothelium forms the largest resident cell population in the peritoneal cavity. It is now well recognized that mesothelial cells not only form a barrier for the transport of solutes during peritoneal dialysis, but also play a crucial role in controlling the intraperitoneal inflammatory responses (27, 28). Thus, the potential detrimental effects of GDP toward mesothelial cells may impair intraperitoneal homeostasis in patients undergoing CAPD. In the present study, we have sought to examine whether the short-term exposure to GDP affects mesothelial cell functions, assessed as viability, proliferative capacity, and cytokine release.

Materials and Methods

Materials

All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich Chemie (Deisenhofen, Germany). Tissue culture flasks and multi-well plates were from Falcon (Oxnard, CA), Becton-Dickinson (Heidelberg, Germany), and Sarstedt, Inc. (Newton, NC). Glucose degradation products studied included acetaldehyde (AcA), formaldehyde (FoA), 2-furaldehyde (FurA), glyoxal (Glx), methylglyoxal (M-Glx), and 5-hydroxymethyl-2-furaldehyde (5-HMF). GDP were studied at doses ranging in order of magnitude from the concentrations detected in commercial glucose-based peritoneal dialysis fluids to those shown to produce 50% growth inhibition in L929 fibroblasts (16). Working concentrations of GDP were prepared directly before the experiments. During exposure to GDP, the culture plates were sealed with the Parafilm M® (American Can Co., Greenwich, CT) to minimize the potential evaporation of volatile aldehydes (16).

Cell Cultures

Human peritoneal mesothelial cells (HPMC) were isolated from the specimens of omentum obtained from consenting nonuremic patients undergoing elective abdominal surgery. Cells were isolated and characterized as described in detail elsewhere (29). HPMC were propagated in the M199 culture medium supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), hydrocortisone (0.4 μg/ml), and 10% vol/vol fetal calf serum (FCS) (Life Technologies, Eggenstein, Germany). Cell cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. All experiments were performed using cells derived from at least five separate donors, and between the first and third passage.

Mouse L929 fibroblast cell line was kindly provided by Dr P. Scholz (Schering, Berlin, Germany). Cells were maintained and propagated in the same medium as used for HPMC cultures.

Proliferation Studies

Cell proliferation was measured by [3H]-thymidine incorporation assay. Cells were plated onto 48-well clusters at a density of 2.5 × 104/cm2 for HPMC or 1.0 × 104/cm2 for L929 fibroblasts, and cultured for 24 h. Preliminary experiments had determined the optimal seeding density for each cell type, which ensured that both populations were studied during the exponential phase of growth. The subconfluent cell cultures were then exposed to GDP in 10% FCS-containing medium and pulsed with [3H]-thymidine (as methyl-[3H]-thymidine; 1 μCi/ml; Institute of Radioisotopes, Prague, Czech Republic) for 24 h at 37°C. At the end of the exposure, the labeling fluids were removed and the cells were detached with trypsin:ethylenediaminetetra-acetic acid (0.05% wt/vol:0.02% wt/vol) solution and precipitated with 10% (wt/vol) TCA. The precipitate was washed again with 10% TCA and finally dissolved in 0.1N NaOH. The released radioactivity was measured in a beta liquid scintillation counter (LKB Wallac, Turku, Finland).

HPMC Interleukin-6 Release

Induction of Interleukin-6 Production. HPMC were grown to confluence in 24-well plates and rendered quiescent by serum deprivation. Briefly, HPMC monolayers were transferred to the culture medium containing 0.1% FCS (rest medium) for 48 h before stimulation. Previous experiments had demonstrated that these conditions did not impair HPMC viability (as assessed by lactate dehydrogenase [LDH] release and intracellular ATP levels) (30). HPMC in the rest medium were then exposed to GDP in the presence of recombinant human interleukin-1β (rhIL-1β) (100 pg/ml; R&D Systems, Wiesbaden, Germany) for 24 h at 37°C. After the incubation, the supernates were removed, centrifuged at 12,000 × g to remove any cellular debris, and stored at -70°C until assayed. Cell monolayers were washed with Hanks' balanced salt solution (HBSS) and solubilized with 0.1N NaOH. Total cellular protein was then analyzed with BCA protein assay (Pierce, Rockford, IL), using bovine serum albumin as the standard. Repeated cell counts revealed that 1 μg of HPMC protein corresponded to (mean ± SD) 2.1 ± 1.0 × 103 cells (n = 16). All data for interleukin-6 (IL-6) production were expressed as pg/μg cellular protein.

IL-6 Measurements. IL-6 concentrations in HPMC supernates were measured with a specific “sandwich-type” immunoassay using an enzyme-linked immunosorbent assay-matched antibody pair (R&D Systems). The assay was designed and performed according to the manufacturer's instructions. Sensitivity of the system, determined by adding 2 SD to the mean optical density of the zero standard (n = 16), was 2 pg/ml.

Viability Studies

Release of LDH. After incubation under various experimental conditions, the cell culture supernates were collected and assayed immediately for LDH using a commercially available LDH kit (Analco-GBG, Warsaw, Poland). Total intracellular LDH content was determined following the lysis of representative cell monolayers with 0.1% (vol/vol) Triton X-100 in HBSS.

MTT Assay. The MTT assay is conventionally used for measuring cell proliferation. However, because the metabolic conversion of the MTT salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolinum bromide) is mediated by active mitochondrial dehydrogenases of living cells, the test can also be used for assessing cell viability (31). Briefly, after exposure to GDP, the cells were treated with MTT (1.25 mg/ml in culture medium) for 4 h at 37°C. The formazan product generated was solubilized by the addition of acidic solution of 20% (wt/vol) sodium dodecyl sulfate and 50% (vol/vol) N,N-dimethylformamide. Absorbance of the converted dye was recorded at 595 nm with a reference wavelength of 690 nm.

Effect of PDF

Test Solutions. To characterize the effect of GDP in the milieu of PDF, the cells were exposed either to heat-sterilized PDF (H-PDF), filter-sterilized PDF (F-PDF), or filter-sterilized PDF supplemented with a known concentration of GDP (F-PDF + GDP). The fluids were prepared in the laboratory according to the following formula (g/L): NaCl - 5.786, CaCl2 × 2H2O - 0.257, MgCl2 × 6H2O - 0.102, sodium DL-lactate - 3.925, and anhydrous D-glucose - 15.0 or 42.5. The solutions from the same stock were then sterilized either by heat (121°C, 0.2 MPa, 20 min) or filtration through 0.2-μm pore size filter (Nalgene®, Nalge Nunc International, Rochester, NY). The extent of glucose degradation was estimated by measuring the absorbance at 284 nm, which is thought to reflect primarily the level of 5-HMF (Figure 1) (22). The pH of all fluids tested was adjusted to 7.3 with 0.1 M Na2CO3. The level of endotoxin in all PDF preparations was <0.01 IU/ml as determined by Limulus amoebocyte lysate assay using QCL-1000® Test kit (BioWhittaker, Walkersville, MD). The doses of GDP applied corresponded to the highest concentrations detected in PDF (14): AcA 420 μM, FoA 15 μM, FurA 2 μM, Glx 14 μM, M-Glx 23 μM, and 5-HMF 30 μM.

Figure 1.
Figure 1.

Spectra of ultraviolet absorbance of heat- and filtered-sterilized peritoneal dialysis fluids (PDF) containing either 1.5 or 4.25% glucose. Arrows indicate maxima at 284 nm.

Exposure to PDF. Confluent cell monolayers of HPMC and L929 fibroblasts were pretreated with different types of PDF for specified times up to 3 h. Afterward, the test solutions were removed and replaced by fresh culture medium (with 0.1% FCS) in which cells were incubated for the next 18 h. After this recovery phase, the viability of cells was determined by LDH release and MTT test as described above. In parallel experiments, HPMC were incubated in the presence of IL-1β (100 pg/ml) during recovery phase; supernates from these cultures were assayed for IL-6 while cell monolayers were solubilized and measured for protein contents.

Growth Inhibition Studies. In these experiments, the test solutions were prepared by mixing equal volumes of PDF and FCS-containing M199 medium labeled with [3H]-thymidine (final FCS concentration was 10%, and final [3H]-thymidine activity was 1 μCi/ml). In controls, the medium was mixed with HBSS to normalize for the dilution of culture medium components. HPMC or L929 fibroblasts in the exponential phase of growth were incubated in the presence of the PDF mixtures tested for 24 h, and the incorporated radioactivity was released as described above.

Statistical Analyses

All statistical analyses were performed using GraphPad Prism™ 2.00 software (GraphPad Software, San Diego, CA). Multiple comparisons of paired data were made with nonparametric repeated-measures ANOVA with Friedman modification. Data derived from mesothelial cells and L929 fibroblasts were analyzed using Mann-Whitney U test for unpaired data and two-way ANOVA. A P value <0.05 was considered significant. All data are presented as mean ± SEM.

Results

Cell Proliferation

Exposure of HPMC to either AcA, FoA, FurA, Glx, or M-Glx resulted in dose-dependent inhibition of cell proliferation as measured by [3H]-thymidine incorporation (Figure 2). The most profound reduction was detected in response to FoA and M-Glx. At the highest doses of FoA and M-Glx tested, the proliferative capacity of HPMC was almost completely abolished. In contrast, no significant effect was observed in HPMC incubated in the presence of 5-HMF. Proliferation of L929 fibroblasts in response to GDP exhibited a similar pattern, although at several GDP doses the degree of inhibition was significantly less pronounced (Figure 2). For example, AcA, FurA, and Glx, which in high concentrations produced considerable reduction in [3H]-thymidine incorporation into HPMC, had a limited impact on the growth of L929 fibroblasts. Indeed, two-way ANOVA revealed that there was a significant difference between HPMC and L929 cells in growth inhibition curves in response to AcA (F = 3.73, P < 0.05), FurA (F = 4.92, P < 0.01), and Glx (F = 4.85, P < 0.01). However, no such differences were detected in cells treated with FoA, M-Glx, and 5-HMF. To determine whether GDP synergize in their suppression of cell proliferation, HPMC were incubated in the presence of all GDP combined together either at the lowest concentrations tested or at the highest doses at which individual GDP did not impair HPMC proliferation (henceforth referred to as “subtoxic;” in μg/ml: AcA - 1, FoA - 1, FurA - 10, 5-HMF - 100, Glx - 1, M-Glx - 1) (Figure 2). Incorporation of [3H]-thymidine into HPMC exposed to a combination of low GDP concentrations did not differ from that in control cells (Figure 3A). Combination of GDP at subtoxic doses, however, caused a slight but significant inhibition of HPMC proliferation (P < 0.05). Neither of the GDP combinations had a significant effect on L929 fibroblasts (data not shown).

Figure 2.
Figure 2.

Dose effect of individual glucose degradation products (GDP) on the proliferation capacity of human peritoneal mesothelial cells (HPMC) and L929 fibroblasts. Fetal calf serum (FCS)-stimulated [3H]-thymidine incorporation was measured in HPMC (▪) and L929 cells (□) exposed to increasing doses of GDP for 24 h. Data are expressed as a percentage of [3H]-thymidine incorporation into control cells incubated in the absence of GDP. Data represent the mean ± SEM of five experiments (with HPMC isolated from different donors). *, statistically significant difference compared to the respective control; #, difference in relative growth inhibition between L929 cells and HPMC.

Figure 3.
Figure 3.

Effect of GDP combinations on HPMC proliferation, viability, and IL-6 release. HPMC were exposed for 24 h to GDP combined together either at the lowest concentrations tested (low) or at the highest doses that did not impair HPMC proliferation when applied separately (subtoxic). Data represent a mean ± SEM of five ([3H]-thymidine incorporation; Panel A), six (IL-6 release; Panel B), 10 (lactate dehydrogenase [LDH] release; Panel C), or nine (MTT assay; Panel C) experiments with cells prepared from separate omental specimens. *P < 0.05 compared to the control.

Cell Viability

In HPMC treated with either FurA or 5-HMF, the release of LDH did not differ from that in control cells. After exposure to all other GDP, the release of LDH was augmented, but only when GDP were applied at the highest concentrations tested (Figure 4A). FoA and M-Glx appeared to be most toxic, inducing a 5.7- and 2.6-fold rise in LDH release, respectively (n = 10, P < 0.001 and P < 0.01). This effect was accompanied by a simultaneous decrease in the MTT conversion (Figure 4B). Although combinations of GDP did not alter the magnitude of LDH release from HPMC (Figure 3C), both low and subtoxic doses of GDP reduced the conversion of MTT by 15.3 ± 5.4 and 18.1 ± 4.5%, respectively (n = 9, P < 0.05 for both) (Figure 3D). In contrast, neither individual nor combined GDP impaired the viability of L929 fibroblasts as assessed by LDH release and MTT assay.

Figure 4.
Figure 4.

Effect of individual GDP on the viability of HPMC and L929 fibroblasts. After a 24-h exposure to the highest GDP concentrations tested, the release of LDH (A) and the conversion of MTT (B) were assessed in HPMC (▪) and L929 cells ([UNK]). The LDH data were obtained from 10 experiments with HPMC isolated from different donors and three experiments with the L929 cell line, and represent a mean ± SEM-fold increase in LDH release above controls. The MTT assay was performed in nine separate experiments for both cell systems; results are expressed as a percentage of MTT conversion in control cells. *P < 0.05 compared to the respective control; #P < 0.05 L929 versus HPMC.

HPMC IL-6 Synthesis

Incubation of HPMC in the presence of either AcA, FoA, Glx, or M-Glx decreased IL-1β-induced IL-6 production in a dose-dependent manner (Figure 5). The greatest inhibition was observed in cells treated with high concentrations of FoA and M-Glx, in which the release of IL-6 was reduced by 85.1 ± 8.1 and 86.3 ± 6.7%, respectively (n = 5, P < 0.05 for both). Under the same conditions, FurA and 5-HMF did not affect IL-6 release by HPMC. The levels of IL-6 recorded in HPMC cultures exposed to the mixtures of low and subtoxic GDP doses were lower than those detected in the controls, and for the subtoxic combination a reduction of 25.6 ± 18.0% reached statistical significance (n = 7, P < 0.01) (Figure 3B).

Figure 5.
Figure 5.

Effect of individual GDP on interleukin-1β (IL-1β)-stimulated IL-6 release by HPMC. Growth-arrested HPMC were exposed to increasing concentrations of GDP in the presence of IL-1β (100 pg/ml) for 24 h. Data represent the mean ± SEM of five experiments performed with HPMC isolated from five separate donors. *P < 0.05 compared to the control.

Heat- versus Filter-Sterilized PDF

Effect of Short-Term Preexposure to PDF. Pretreatment of HPMC with H-PDF at pH 7.3 and either at low (1.5%) or high (4.25%) glucose concentrations resulted in a time-dependent reduction in IL-1β-driven IL-6 synthesis during the subsequent recovery period. This inhibition was first evident in HPMC preexposed to 4.25%-H-PDF for 1 h, and after 2 h also in cells pretreated with 1.5%-H-PDF. After a 3-h preexposure, the release of IL-6 was reduced from 17.9 ± 3.5 pg/μg cell protein in controls to 6.8 ± 2.8 and 7.0 ± 2.8 pg/μg cell protein in HPMC incubated with 1.5%-H-PDF and 4.25%-H-PDF, respectively (n = 8, P < 0.05 and <0.001) (Figure 6). In contrast, in HPMC treated with F-PDF, regardless of glucose concentration, the secretion of IL-6 was not significantly diminished and remained above levels detected in H-PDF-treated cells. Interestingly, the release of IL-6 from HPMC preexposed to F-PDF supplemented with a defined mixture of GDP was not different from that observed in cells treated with F-PDF alone.

Figure 6.
Figure 6.

IL-1β-stimulated IL-6 release by HPMC preexposed to differently sterilized PDF and GDP. HPMC were preexposed to heat-sterilized PDF, filter-sterilized PDF, or filter-sterilized PDF supplemented with exogenous GDP for 3 h and then exposed to control medium in the presence of IL-1β (100 pg/ml) for 18 h. Data represent mean ± SEM IL-6 release from eight experiments with HPMC isolated from different donors. *P < 0.05 compared to the control; #P < 0.05 compared to heat-sterilized PDF of respective glucose concentration.

None of the PDF examined increased the release of LDH from HPMC during the period of direct exposure (data not shown). During the overnight recovery phase, however, the LDH release became elevated in all cultures treated with PDF containing 4.25%, but not 1.5%, glucose (Figure 7A). This increase was significantly above background levels after a preexposure period of 3 h, with no differences observed between various types of 4.25%-PDF tested. This pattern of impaired HPMC viability was also recorded in the MTT assay (Figure 7B). In cells pretreated with H-PDF, F-PDF, or F-PDF + GDP (all with 4.25% glucose), the conversion of MTT was reduced to 71.4 ± 13.1% (P < 0.001), 79.6 ± 13.3% (P < 0.05), and 78.2 ± 11.5% (P < 0.05) of the control level, respectively (n = 10). In contrast to HPMC, the viability of L929 fibroblasts—assessed both in the LDH test and in the MTT assay—was not affected by the preexposure to PDF of any type.

Figure 7.
Figure 7.

Effect of preexposure to differently sterilized PDF and GDP on viability of HPMC and L929 fibroblasts. After a 3-h preexposure to various PDF types and subsequent 18-h recovery phase, the viability of cells was assessed by LDH release (A) or MTT assay (B). Data are expressed as a percentage of control values and represent means ± SEM of eight experiments with HPMC isolated from separate donors and of four experiments with L929 cell line. *P < 0.05 compared to the respective control; #P < 0.05 L929 versus HPMC.

Inhibition of Cell Growth in Response to PDF. The FCS-stimulated proliferation of HPMC was significantly reduced in the presence of autoclaved PDF, with solutions mixed with 4.25%-PDF being more inhibitory than those supplemented with 1.5%-PDF (Figure 8). The degree of inhibition exerted by these solutions was significantly greater than that of their filtered counterparts (66.6 ± 6.0% versus 31.9 ± 8.7% and 87.2 ± 3.2% versus 51.3 ± 7.8% of inhibition for 1.5%-and 4.25%-PDF, respectively; n = 10, P < 0.05 for both). However, the smaller inhibitory potential of F-PDF could be substantially augmented by the addition of a defined mixture of GDP (the differences between GDP-spiked F-PDF and F-PDF alone were not formally significant when compared by ANOVA, but the same test revealed no differences between F-PDF + GDP and H-PDF).

Figure 8.
Figure 8.

Growth of HPMC and L929 fibroblasts exposed to differently sterilized PDF and GDP. A 24-h [3H]-thymidine incorporation was measured in HPMC (▪) and L929 cells ([UNK]) exposed to a 1:1 mixture of culture medium and respective PDF. Data are expressed as a percentage of [3H]-thymidine incorporation into control cells. Data represent the mean (± SEM) from 10 experiments with HPMC isolated from separate donors and seven experiments with L929 cell line. *P < 0.05 compared to the respective control; ♦P < 0.05 compared to heat-sterilized PDF of respective glucose concentration; #P < 0.05 L929 versus HPMC.

The pattern of growth inhibition in L929 cells was very similar. Although the magnitude of inhibition in response to H-PDF did not differ from that observed in HPMC, the solutions sterilized by filtration (also with additional GDP) appeared to be less inhibitory in L929 fibroblasts than in HPMC.

Discussion

HPMC have been increasingly used for testing the biocompatibility profiles of dialysis fluids. The experiments have focused primarily on the evaluation of pH/buffering systems and osmotic agents (30, 32,33,34,35,36,37,38,39,40,41,42,43). In the present study, we have extended these HPMC applications to the assessment of biologic effects of GDP.

We have found that none of the GDP studied impaired the viability and function of HPMC over a 24-h period if applied individually at low concentrations that approximated those seen in PDF. The addition of higher GDP doses, however, led to the significant suppression of HPMC functions with marked differences between various compounds examined. FoA, Glx, and M-Glx appeared to be most toxic, causing a profound dose-dependent reduction in both HPMC proliferation and IL-6 synthesis. These inhibitory effects could be partially attributed to direct HPMC injury because either FoA, Glx, or M-Glx at high doses caused a significant loss of HPMC viability as assessed by LDH release and MTT test. Adverse effects of AcA and FurA were detected only in [3H]-thymidine incorporation assay, while 5-HMF had no significant impact on any functional parameter studied. The level of 5-HMF in glucose-containing solutions is often regarded as a general indicator of glucose breakdown (14, 22, 44). The fact that 5-HMF does not seem to be acutely toxic to HPMC over a vast range of concentrations suggests that the assessment of PDF biocompatibility based solely on 5-HMF measurements may overlook the impact of other more toxic GDP. Another glucose derivative that is thought to better reflect the magnitude of glucose degradation is acetaldehyde (20, 21). In this respect, it was interesting to observe that AcA decreased HPMC proliferation already at a dose of 10 μg/ml, the level that could be reached in some H-PDF (16). The same dose, however, neither diminished IL-1β-stimulated IL-6 production nor damaged cell membrane integrity as judged by LDH release. These observations indicate that in assessing GDP-associated toxicity, it is necessary to examine more than one parameter because cell functions may—contrary to previous suggestions (22)—display different sensitivity to GDP action.

Although individual GDP at low doses did not exert significant effects on HPMC, one cannot rule out that, when combined, GDP may act in a synergistic manner. Indeed, a combination of low GDP doses appeared to interfere with the function of HPMC mitochondria as demonstrated by a slightly reduced metabolic conversion of MTT. This synergistic effect occurred more clearly at higher levels of concentrations. Exposure of HPMC to GDP combined together at maximal doses, which proved noninhibitory when tested separately, produced a detectable decrease in cell proliferation and IL-1 β-driven IL-6 synthesis. This subtoxic combination contained higher concentrations of Glx, 5-HMF, and FurA compared with the low-dose GDP mixture. Interestingly, we were unable to demonstrate any damage to cell membrane integrity under these conditions as judged by LDH release. In this respect, we have previously shown that short-term exposure of HPMC to dialysis solutions may impair the mitochondrial generation of ATP without increasing LDH release (30).

In general, our results are in agreement with those reported previously by Wieslander et al., who found that the same glucose-derived aldehydes at doses detected in PDF did not impair the proliferation of murine L929 fibroblasts (16). This lack of significant inhibition was evident even in cells exposed to GDP for as long as 72 h compared with 24 h in our system. To evaluate differences in HPMC and L929 fibroblast responses to GDP more precisely, we have analyzed their growth rates in the presence of GDP using the same type of culture medium, the same FCS batch, and identical exposure times. Preliminary experiments revealed that L929 fibroblasts could be easily maintained in the M199 HPMC-specific medium rather than in the MEM medium used in other studies (16, 20, 25). Growth responses of L929 cells recorded under these conditions confirmed the results of Wieslander's group in terms of either the character or magnitude of induced changes (16). However, as demonstrated by two-way ANOVA, AcA, Glx, and FurA suppressed the proliferation of L929 cells to a significantly smaller degree compared to HPMC. Lack of such differences in cells exposed to other aldehydes could be explained either by extreme toxicity occurring within a relatively narrow range of doses (FoA, M-Glx) or, conversely, by the absence of significant impact on cell proliferation (5-HMF). These results may suggest that HPMC are more susceptible to the insult from GDP. Indeed, we were unable to detect GDP-mediated injury to L929 cells, as measured by LDH release or MTT assay, even with the doses that were clearly cytotoxic for HPMC or any GDP combination.

It has been assumed that high GDP concentration is responsible for the disadvantageous biocompatibility characteristics of H-PDF. We have therefore compared the function of cells preexposed to differently sterilized PDF. We have found that the inhibition in IL-6 release from HPMC preexposed to H-PDF was greater than that caused by F-PDF. However, HPMC viability was reduced to a similar degree regardless of the mode of sterilization and the presence of additional GDP. Importantly, none of the PDF studied impaired the viability of L929 fibroblasts in this system. Because it could be argued that the exposure period of 3 h was not long enough for GDP to exert a more specific impact on HPMC, in the next series of experiments we have exposed cells for up to 24 h to PDF diluted in pH neutral, FCS-supplemented medium, as described originally by Wieslander et al. (25). In this setting, and in line with several previous reports (21, 22, 24, 25), we recorded a significantly greater degree of inhibition in response to H-PDF compared with F-PDF. We have even been able to demonstrate a significant difference between proliferation rates of HPMC treated with H- and F-PDF of 1.5% glucose concentration, while in a recent report it has been suggested that in low glucose-based PDF, the levels of GDP might be too small to produce a distinction in biologic responses and that any significant difference between H- and F-PDF could only be detected in solutions with high (3.86%) glucose concentration (20). Although the difference in HPMC reaction to heat- and filter-sterilized fluids is in agreement with observations in other cell types, the extent of this response is somewhat surprising considering the fact that GDP at corresponding doses added to culture media had a very limited impact on HPMC proliferation. One may hypothesize that other factors present in PDF may influence the action of GDP. In the present study, we chose to examine GDP at neutral pH to exclude well-known inhibitory effects associated with acidic pH. However, it has recently been suggested that in the in vivo situation the combination of GDP and low pH may in fact accentuate the effects of GDP as demonstrated by changes in small solute transport during peritoneal dialysis in rats (45). To confirm the impact of GDP in the milieu of PDF, we exposed HPMC to F-PDF supplemented with the highest GDP concentrations detected in autoclaved PDF. The combination of F-PDF + GDP appeared to be more inhibitory toward HPMC proliferative capacity than F-PDF alone. This observation indicates that the six aldehydes tested in the present study may contribute, at least in part, to detrimental effects produced by PDF in vitro. In contrast, Wieslander et al. failed to demonstrate that aldehyde-spiked F-PDF differed in ensuing biologic effects from PDF without additional GDP (16). It is possible that the overall effects attributed to GDP are also mediated by the relative presence of different, not-yet-defined or quantified compounds. For example, other glucose derivatives that may be detected in PDF include formic acid, valeraldehyde (14), and 3-deoxyglucosone (46), and their impact on HPMC function remains to be elucidated.

The response of L929 fibroblasts to H-PDF was very similar to that of HPMC. In view of earlier results, this observation was rather unexpected and might indicate that proliferating L929 fibroblasts are much more vulnerable to GDP (and GDP-containing PDF) than the confluent L929 monolayers. On the other hand, greater inhibition of HPMC proliferation after exposure to F-PDF suggests that HPMC are more sensitive to PDF components other than GDP. Because the L929 cell line provides a well-characterized and reproducible cell system that is commonly used for cytotoxicity testing, these considerations should be taken in account when examining effects of GDP and/or PDF. Our data indicate that under certain experimental conditions, the exclusive use of L929 cells may underestimate the full extent of GDP-associated toxicity, and for this purpose it may be more appropriate to use primary cell cultures of peritoneal origin.

Taken together, our results indicate that GDP possess a significant cytotoxic potential toward HPMC. During the clinical setting of CAPD, mesothelial cells are continually exposed to GDP for extended periods of time. It is therefore possible that GDP exert cumulative effects on long-term cell function rather than produce direct acute injury to HPMC. The potential of GDP to evoke chronic HPMC dysfunction is currently being investigated. Mechanisms by which GDP may affect long-term HPMC functions are poorly defined. In recent years, it has become increasingly evident that GDP (M-Glx and 3-deoxyglucosone in particular) are potent promoters of advanced glycosylation end product (AGE) formation (47,48,49). It has been suggested that a considerable capacity of glucose-based PDF for AGE formation is related to the presence of GDP rather than glucose per se (50, 51). In this respect, Nakayama et al. have clearly documented the accumulation of AGE in the peritoneum of CAPD patients (52). It is also possible that the biologic impact of GDP is influenced by the uremic environment. Increased levels of 3-deoxyglucosone and glyoxal- and methylglyoxal-mediated protein cross-links have been reported in sera of uremic patients (53,54,55). It has also been demonstrated that methylglyoxal-modified proteins undergo a receptor-mediated endocytosis and stimulate production of inflammatory cytokines in mononuclear cells (56,57,58,59). Whether these mechanisms are involved in long-term effects of GDP on HPMC remains to be determined.

Acknowledgments

Acknowledgment

This work was supported by a grant from the Else Kröner-Fresenius Foundation (Bad Homburg, Germany).

Footnotes

  • American Society of Nephrology

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Effect of Glucose Degradation Products on Human Peritoneal Mesothelial Cell Function
JANUSZ WITOWSKI, KATARZYNA KORYBALSKA, JUSTYNA WISNIEWSKA, ANDRZEJ BREBOROWICZ, GERHARD M. GAHL, ULRICH FREI, JUTTA PASSLICK-DEETJEN, ACHIM JÖRRES
JASN Apr 2000, 11 (4) 729-739;
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