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NDT Advance Access published online on November 25, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn645
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Glutathione depletion as a mechanism of 3,4-dideoxyglucosone-3-ene-induced cytotoxicity in human peritoneal mesothelial cells: role in biocompatibility of peritoneal dialysis fluids

Takashi Yamamoto1,2, Tadashi Tomo3, Eiji Okabe3, Shinji Namoto1, Koji Suzuki1 and Yoshihiko Hirao2

1 Research & Development, JMS Co. Ltd, Hiroshima 2 Department of Urology, Nara medical University, Kashihara 3 Second Department of Internal Medicine, Faculty of Medicine Oita University, Oita, Japan

Correspondence and offprint requests to: Takashi Yamamoto, Research & Development, JMS Co. Ltd, 12-17 Kako-machi, Naka-ku, Hiroshima, Japan. Tel: +81-82-243-5980; Fax: +81-82-243-5955; E-mail: t-yamamoto{at}jms.cc



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Result
 Discussion
 Conclusion
 References
 
Background. The potential detrimental effects of glucose degradation products (GDPs) contained in peritoneal dialysis fluids (PDFs) on peritoneal mesothelial cells (PMCs) may impair intraperitoneal homeostasis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A recent study showed that 3,4-dideoxyglucosone-3-ene (3,4-DGE) was the most strongly cytotoxic among all identified GDPs in PDFs. The present study examined the effects of clinically relevant concentrations of 3,4-DGE on the proliferative capacity of PMCs and oxidative injury to them.

Method. The concentrations of eight GDPs in commercially available PDFs were determined by HPLC. The effect of cell growth media spiked with GDPs on the proliferation capacity of PMCs was evaluated. As a marker of the cellular redox status, total cellular glutathione (tGSH) was determined in PMCs incubated with GDPs. The reaction of 3,4-DGE with GSH under nonenzymatic conditions was analysed by liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS).

Result. The concentrations of 3,4-DGE in a heat-sterilized single-compartment standard-type PDF (S-PDF) and in a heat-sterilized dual-chamber-type PDF (N-PDF) were 16 µM and 1.7 µM, respectively. The most cytotoxic GDP was 3,4-DGE, and the concentration at which it causes 50% inhibition of cell growth was 35 µM. A significant decrease in the cellular tGSH levels was observed in the cells treated with 10 µM 3,4-DGE. 3,4-DGE disappeared on incubation with GSH under nonenzymatic conditions for 1 h, and the 3,4-DGE-GSH conjugate was confirmed by accurate mass measurement using LC-ESI-MS. These data demonstrated that the change in the cellular redox status by GSH depletion might be a contributory factor in 3,4-DGE-induced cytotoxicity.

Conclusion. 3,4-DGE is a highly reactive GDP and is responsible for the depletion of the total intracellular glutathione. 3,4-DGE has an intense impact on PMC growth at concentrations found in standard PDFs. It is desired that the amount of 3,4-DGE in PDFs should be minimized.

Keywords: CAPD; 3,4-DGE; GDP; glutathione; peritoneal mesothelial cell



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Result
 Discussion
 Conclusion
 References
 
Peritoneal dialysis has developed into an effective renal replacement therapy for treating patients with kidney failure. However, the increasing use of chronic peritoneal dialysis regimens has raised concerns regarding the long-term preservation of the peritoneum as a dialyzing membrane. The mesothelium forms the largest resident cell population in the peritoneal cavity. It is now well recognized that peritoneal mesothelial cells (PMC) not only form a barrier for the transport of solutes during peritoneal dialysis but also play a crucial role in controlling the intraperitoneal inflammatory response. Many studies have convincingly demonstrated the adverse effects of peritoneal dialysis fluids (PDFs) on PMCs.

One of the aspects of PDFs is the presence of glucose that is added in high concentrations to most PDFs as an effective osmotic agent. PDFs are autoclaved for sterilization, and this process degrades the glucose to several low molecular weight aldehydes, including 5-hydroxymethyl-furfural (5-HMF), 2-furaldehyde (FUR), acetaldehyde (AA), formaldehyde (FA), 3-deoxyglucosone (3-DG), methylglyoxal (MGO), glyoxal (GO) and 3,4-dideoxyglucosone-3-ene (3,4-DGE). It has been well documented that the concentrations of these glucose degradation products (GDPs) in heat-sterilized fluids are much higher than those detected in filter-sterilized fluids [1]. In various in vitro experimental systems, heat-sterilized fluids have been shown to impair the cell function to a greater extent than filter-sterilized fluids. Wieslander et al. also demonstrated that several GDPs were capable of directly inhibiting cell growth in culture [1]. Thus, the potential detrimental effects of GDPs on mesothelial cells may impair intraperitoneal homeostasis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD).

In order to reduce the formation of GDPs during the manufacture of PDFs, a dual-chamber system has been developed, which separates a highly concentrated glucose solution with a low pH from an alkaline lactate solution. The contents of both compartments are mixed together prior to use, and as a result, solutions with a low GDP content and neutral pH are currently being prescribed. Such solutions are associated with an improved proliferative response of PMCs as has already been confirmed by several in vitro and in vivo studies [2,3].

A recent study showed that 3,4-DGE is the most strongly cytotoxic among all the identified GDPs in PDFs, and that it retards mesothelial cell regeneration [4,5] and accelerates leukocyte apoptosis [6]. We have also observed that 3,4-DGE-induced cytotoxicity is enhanced by a synergistic effect of a combination of the acidic properties of PDFs and the presence of lactate [7]. It has been suggested that 3,4-DGE might play an important role in the biocompatibility of PDF. On the other hand, Witowski et al. demonstrated that exposure of PMCs to PDFs decreased cell viability and the total glutathione level in them, and pretreatment of PDFs with glutathione markedly reduced the inhibitory effects of high GDP-containing PDFs on PMCs [8]. Walker and colleagues demonstrated that the formaldehyde in PDFs can be scavenged by the addition of reduced thiol compounds, and they also suggested that the other aldehydes present in PDFs might also be scavenged by addition of thiol compounds such as L-cysteine [9]. These reports indicate that the interaction between GDPs and thiol compounds might play a major role in the biocompatibility of PDFs; therefore, the detailed mechanism of this interaction needs to be elucidated.

In the present study, we evaluated the effects of clinically relevant concentrations of 3,4-DGE on the proliferative capacity of mesothelial cells and oxidative injury. In particular, in order to understand the cytotoxic mechanism of PDFs, the interaction between 3,4-DGE and the –SH group that may lead to the decrease of cellular glutathione levels was investigated in detail.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Result
 Discussion
 Conclusion
 References
 
Chemical reagents
Acetaldehyde, formaldehyde, glyoxal, 2-furfural, 2,4-dinitrophenyl-hydrazine (2,4-DNPH), glutathione (GSH), glutathione oxidative form (GSSG) and N-acetyl-L-cysteine (NAC) were obtained from Wako Pure Chemical Industries (Osaka, Japan); 3-deoxyglucosone (Dojindo Laboratories, Kumamoto, Japan), methylglyoxal (Sigma-Aldrich, St Louis, USA), 5-hydroxymethyl-furfural (Merck-Schuchardt, Hohenbrunn, Germany) and Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Merck-KGaA, Darmstadt, Germany) were commercially available. All the above-mentioned reagents were used without further purification. 3,4-DGE was isolated from pyrolyzed glucose as described below.

Preparation of 3,4-DGE
3,4-DGE was extracted from the autoclaved glucose solution according to the method of Kato et al. [10] with slight modifications. The chemical structure of the isolated substance was determined by proton nuclear magnetic resonance spectroscopy (1H-NMR), infrared spectroscopy, ultraviolet spectroscopy and mass spectroscopy (MS). The substance showing a distinct absorption band at 228 nm was identified as 3,4-DGE. The 1H-NMR spectrum of this substance was then compared with the reference 1H-NMR spectrum of Anet [11] and was confirmed to be the cis-form of 3,4-DGE. The purity of the isolated 3,4-DGE was >95%, according to 1H-NMR spectroscopy and HPLC.

Analysis of GDPs
The amounts of 5-HMF, FUR and 3,4-DGE were directly determined by reverse-phase HPLC [1,4]. MGO, GO, 3-DG, AA and FA were analysed by reverse-phase HPLC with gradient elution after derivatization with 2,4-DNPH, according to a previously reported method [12] with slight modifications. PDF samples were obtained from a heat-sterilized lactate-buffered 2.25% glucose-containing PDF; Perisate®400 (JMS Co., Japan) was used as a single-compartment standard-type PDF (S-PDF) and Perisate®400N, as a dual-chambered neutral-pH-type PDF (N-PDF). It is well known that PD solutions that contain a high concentration of glucose have a large amount of GDPs [4]. In this study, we selected the 2.25% glucose solution that is widely used in patients.

Human peritoneal mesothelial cell cultures
Human peritoneal mesothelial cells (HPMCs) were isolated from the omental tissues obtained from consenting patients undergoing elective abdominal surgery. HPMCs were propagated in the M199 culture medium supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL) and 20% v/v fetal calf serum (FCS; Gibco Invitrogen Corporation, Carlsbad, CA, USA), and the cell culture was maintained at 37°C under a 5% CO2 atmosphere. All experiments were performed using the second or third passage cells.

Proliferation studies for individual GDPs
Cell proliferation was determined by enumerating the cells using a Coulter counter Z1 (Beckman Coulter, Fullerton, CA, USA). HPMCs were plated onto collagen-I-coated 24-well culture dishes (IWAKI, Tokyo, Japan) at a density of 2.0 x 104 cells/well in a M199 medium supplemented with 10% FCS without any antibiotics. The cells were incubated for 24 h to allow attachment. The cell cultures were then exposed to GDPs in 10% FCS containing a M199 medium for 48 h without changing the medium during incubation. It has been demonstrated in previous reports that cell death occurs within 24 h after the start of exposure to GDPs in serum-containing media [13,14]. Consequently, we speculated that the survived cells could grow and multiply subsequently in the culture period. Hence, in order to facilitate the determination of the toxicity behaviour of the GDPs, an incubation period of 48 h was established as a method of augmenting the survived cells.

After incubation, the wells were aspirated and washed twice with phosphate-buffered saline, followed by addition of 100 µL of trypsin–EDTA, and the plate was placed in an incubator at 37°C for ~5 min until the cells lifted. The cell counts were obtained from quadruplicate wells using a Coulter counter. Growth inhibition was determined by comparing the number of cells under the GDP treatment conditions with that under the control conditions. Concentration–response curves were obtained by adding different concentrations of each GDP.

Proliferation studies for combinations of GDPs
To estimate the cytotoxicity of 3,4-DGE in the heat-sterilized single-compartment standard-type PDF (S-PDF) and in the heat-sterilized dual-chamber-type PDF (N-PDF), the cells were exposed to culture media spiked with several combinations of GDPs for 48 h. The GDP combinations comprised an assortment of eight GDPs including 3,4-DGE at the concentration found in S-PDF (+8 GDPs from S-PDF), seven GDPs except for 3,4-DGE at their respective concentrations found in S-PDF (+7 GDPs from S-PDF), +8 GDPs from N-PDF or +7 GDPs from N-PDF. The doses of GDPs applied corresponded to those detected in S-PDF (lactate-buffered 2.25% glucose solutions; Perisate®400; JMS Co.) and were as follows: 60 µM AA, 3.9 µM FA, 7.5 µM GO, 7.7 µM MGO, 4.8 µM 5-HMF, 0.8 µM Fur, 311 µM 3-DG and 15 µM 3,4-DGE. Likewise, the doses of GDPs in N-PDF (lactate-buffered 2.25% glucose solutions; Perisate®400N; JMS Co.) were as follows: 2.3 µM AA, 0.7 µM FA, 0.5 µM GO, 0.1 µM MGO, 16 µM 5-HMF, 0.3 µM Fur, 49 µM 3-DG and 1.4 µM 3,4-DGE.

Glutathione assay
Measurement of total intracellular glutathione (tGSH) (including the reduced and oxidized forms, GSH and GSSG, respectively; i.e. tGSH = GSH + GSSG) was performed by Tietze's enzymatic recycling method using glutathione reductase according to the instructions in a GSH assay kit (Cayman Chemical, Ann Arbor, MI, USA). In brief, the confluent HPMC cell monolayers in six-well culture dishes were treated with 3,4-DGE, MGO or 3-DG in a M199 medium for 1–4 h. Subsequently, the HPMCs were washed twice with PBS (pH 7.4), harvested using a rubber policeman and were homogenized by a freeze-thaw method with 250 µL of PBS (pH 7.4) containing 1 mM EDTA in order to extract tGSH. A small amount of the supernatant after centrifugation was obtained for the protein assay (BCA protein assay; Pierce, Rockfold, IL, USA). For deproteinization, an equal amount of 5% w/v of metaphosphoric acid was added to the residual supernatant. After centrifugation (at 10 000 xg for 15 min), the resulting supernatant (400 µL) was neutralized with 20 µL of 50% v/v of triethanolamine for measurement of the tGSH levels in the sample, and the tGSH concentration was then determined by the kinetic method according to the procedure mentioned in the assay kit and was expressed as nmol mg–1 protein.

Identification of the GSH-3,4-DGE conjugate
The GSH-3,4-DGE conjugate was prepared nonenzymatically by incubation of 0.5 mM GSH with an equimolar amount of 3,4-DGE in a 10 mM phosphate buffer (pH 7.4) at room temperature for 1 h. The reaction was monitored by reverse-phase HPLC using an Atlantis C18 column (Waters, Milford, USA) with 0.1% formic acid as the mobile phase. The identity of the GSH-3,4-DGE conjugate was confirmed by a liquid chromatography–electrospray ionization–TOF mass spectrometry (LC-ESI-MS) analysis carried out on a QSTAR XL system (Applied Biosystems, Foster City, CA, USA).

Statistical methods
Data are expressed as the mean ± SD. Multiple comparisons of the data in Figure 3 were performed by the Bonferroni/Dunn method using parametric measures. The other statistical analyses were performed using Student's t-test. A P-value of <0.05 was considered statistically significant. Statistical analyses were performed using StatView for Windows (SAS Institute Inc.) and Microsoft Excel.


Figure 3
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  		Fig. 3 Time course of cellular tGSH in HPMCs incubated with 3,4-DGE. (A) HPMCs were exposed to either 30 µM 3,4-DGE or 30 µM MGO, and intracellular tGSH levels were measured. (B) The effect of antioxidant on 3,4-DGE-induced tGSH depletion. HPMCs were pretreated with 500 µM Trolox® or NAC for 1 h, and the cells were then treated with 30 µM of 3,4-DGE. The values represent the mean ± SD of triplicate determinations. *P < 0.05 compared to the control, **P < 0.05 compared to 3,4-DGE as assessed at the same time points.

 


   Result
Top
 Abstract
 Introduction
 Subjects and methods
 Result
Discussion
 Conclusion
 References
 
Proliferation study
Initial experiments have reconfirmed that exposure of HPMC to either 3,4-DGE, 3-DG, GO, MGO, FA, AA, 5-HMF or FUR results in a dose-dependent inhibition of cell proliferation. The concentration at which 50% inhibition of cell growth occurs (ICG50) of these aldehydes and the concentrations found in several 2.5%-glucose containing PDFs are shown in Table 1. The inhibitory effects of these GDPs, which could be represented in terms of ICG50, were practically equivalent to the values that had previously been reported by Wieslander and colleagues [1,4]. The most cytotoxic GDP was 3,4-DGE since its ICG50 was observed to be 35 µM. In the case of 3-DG, GO, MGO, FA, AA, 5-HMF and FUR, the gap between the ICG50 and their respective concentrations in the PDF was very wide; however, in the case of 3,4-DGE, this gap was small.


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  		Table 1 Concentration of GDP in commercially available PD fluids and the concentration that brings ~50% cell growth inhibition (ICG50)

 
We further investigated the influence of the identified GDPs at concentrations measured in the PDFs on mesothelial cell proliferative capacity. Figure 1 shows the cell growth inhibition from a combination of seven (without 3,4-DGE) or eight types (including 3,4-DGE) of GDPs at concentrations detected in either S-PDF or N-PDF. The impairment of cell growth by the eight types of GDPs from S-PDF was significantly greater than that by the seven types of GDPs. The other GDP combinations from N-PDF did not affect cell growth. 3,4-DGE at its concentration in S-PDF had a serious impact on HPMC growth.


Figure 1
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  		Fig. 1 Effect of different GDP combinations on HPMC proliferation. HPMCs were exposed for 48 h to GDPs combined at the concentrations found in either S-PDF or N-PDF, in the absence and presence of 3,4-DGE, respectively. The dose of each GDP applied to the cell growth media has been described in the ‘Subjects and methods’ section. The data represent the mean ± SD of three different experiments.*P < 0.05 compared to the control.

 
Glutathione assay
To account for the 3,4-DGE toxicity mechanism, we examined the effect of 3,4-DGE on the change in the cellular redox status. Because glutathione normally acts as an antioxidant to protect cells against oxidative stress and the –SH group of glutathione is important from many aspects of cell function, the level of total glutathione (tGSH) in HPMC after exposure to several GDPs was determined.

The dose–effect relationship of 3,4-DGE, MGO or 3-DG on the decrease in cellular tGSH is shown in Figure 2. The data indicate that 3,4-DGE significantly decreased tGSH to only 10 µM, which is equivalent to its concentration in PDFs; on the other hand, MGO and 3-DG had no effect on cellular glutathione at levels of even 300 µM. Hence, this result suggested that glutathione depletion might play a specific role in 3,4-DGE-induced cytotoxicity. Consequently, we performed the following experiment by using 30 µM of 3,4-DGE because 3,4-DGE-induced cytotoxicity was clearly observed at this concentration, as shown in Table 1.


Figure 2
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  		Fig. 2 Effect of carbonyl compounds on total cellular glutathione. HPMCs were incubated with 10–300 µM of 3-DG, MGO or 3,4-DGE for 60 min. Intracellular tGSH including the reduced and oxidized forms of glutathione was analysed by Tietze's method. The data represent the mean ± SD of triplicate determinations.

 
We further investigated the time course of cellular tGSH and the concentration of 3,4-DGE in the supernatant after incubation of HPMCs with 30 µM 3,4-DGE. The changes in the concentration of 3,4-DGE in the culture media are presented in Table 2. When the cell layer was absent, the concentration of 3,4-DGE in the M199 medium decreased slightly from 30 µM to ~20 µM at least 15 min after being mixed together. It is assumed that the decrease in 3,4-DGE is caused by the reaction of 3,4-DGE with the components of M199. In contrast, on incubation with HPMC, 3,4-DGE in the supernatant decreased appreciably to 3.0 ± 0.3 µM at 15 min. As shown in Figure 3A, a significant decrease in cellular tGSH levels was observed in the cells treated with 30 µM of 3,4-DGE within 120 min, while 30 µM MGO, which is a weakly cytotoxic GDP, did not influence cellular tGSH levels. This decrease in cellular tGSH was slightly delayed from the point of disappearance of 3,4-DGE from the culture medium. Moreover, in order to determine the concentration of intracellular 3,4-DGE, the homogenates of exposed HPMCs were prepared and analysed by HPLC; however, 3,4-DGE in the homogenates could not be quantified because many inhibitory substances were presented in the homogenate/lysate (data not shown). These data suggest that 3,4-DGE is highly reactive with HPMC and/or cellular glutathione.


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  		Table 2 The time course of the concentration of 3,4-DGE in the culture medium during incubation with HPMC

 
Next, we evaluated the effect of antioxidant pretreatment on intracellular tGSH levels (Figure 3B). 3,4-DGE-induced tGSH decrease was not affected by Trolox®, which is a cell-permeable vitamin E derivative with radical-scavenging properties, but was significantly delayed by the thiol antioxidant, NAC. The thiol group might play a major role in the disappearance of cellular tGSH.

3,4-DGE conjugation with glutathione
The interaction between 3,4-DGE and GSH under nonenzymatic conditions was evaluated by HPLC. To determine the status of 3,4-DGE, the UV detector was set at 228 nm in order to detect the discriminative absorption by the unsaturated bond in 3,4-DGE. At 1 h after mixing 3,4-DGE and GSH, the 3.4-DGE peak at a retention time of 5.3 min disappeared (Figure 4A, B). On the other hand, the 3,4-DGE peak remained unchanged after treatment with GSSG (Figure 4C, D). These data indicate that the thiol group in GSH might have undergone an additional reaction with the unsaturated bond of 3,4-DGE, and the existence of a 3,4-DGE-GSH conjugate was estimated. The conjugate was confirmed by accurate mass measurement using LC-ESI-MS; the compound corresponding to the peak at a retention time of 3.6 min in Figure 4B yielded m/z values of 434.1286, 452.1344 and 470.1488 as shown in Figure 5. The mass value of 452.1344 confirmed that the chemical formula for C16H26N3O10S (theoretical mass, 452.1333) was consistent with that of GS-DGE, as shown in the estimated structure in Figure 6. The two other values, 434.1286 and 470.1488, were speculated to be of its dehydration and hydration products, respectively.


Figure 4
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  		Fig. 4 Reverse-phase LC analysis of the nonenzymatically conjugated substance formed by the reaction of GSH with 3,4-DGE. Samples were obtained (A) immediately after mixing 0.5 mM GSH with 0.5 mM 3,4-DGE in a phosphate buffer (pH 7.4), (B) at 1 h after mixing GSH with 3,4-DGE, (C) immediately after mixing 0.5 mM GSSG with 0.5 mM 3,4-DGE in a phosphate buffer (pH 7.4) and (D) at 1 h after mixing GSSH with 3,4-DGE.

 

Figure 5
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  		Fig. 5 The TOF-MS spectrum of the compound corresponding to the peak at a retention time of 3.6 min in Figure 4B. The 3,4-DGE-GSH conjugate was identified by reverse-phase LC coupled with electrospray ionization-TOF mass spectrometry (LC-ESI-MS) with positive ion detection. The mass spectrum is an intensity versus mass-to-charge ratio (m/z) plot. The unit of the horizontal axis is the atomic mass unit (amu); amu is synonymous with Dalton. The conjugated product was observed as a protonated molecule [M+H]+ at m/z = 452.1344 and was identified as C16H26N3O10S.

 

Figure 6
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  		Fig. 6 The presumed reaction scheme of 3,4-DGE and GSH. 3,4-DGE reacts with GSH to form a thioether conjugate, arising from the addition of the nucleophilic sulfur of GSH to the electrophilic C-4 of 3,4-DGE.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Result
 Discussion
Conclusion
 References
 
It is well known that heat-sterilized PDFs include many types of GDPs. PDFs containing a high proportion of GDPs have been suggested to be the cause of peritonitis and ultrafiltration failure [3,15], inhibiting the respiratory burst of peritoneal macrophages and neutrophils in vivo [16]. In recent years, biocompatible PDFs in which the degradation of glucose can be prevented during the manufacturing process by using a dual-chambered bag have been widely used. Likewise, we observed that the concentration of GDPs in the PDF in the dual-chambered container (N-PDF) was significantly less than that in the PDF in the conventional single-compartment (S-PDF) (Table 1).

3,4-DGE is presently regarded to be the major GDP because it is the most strongly toxic among all the identified GDPs in PDFs; therefore, we first investigated the effect of 3,4-DGE on the HPMC proliferative response. The concentration of 3,4-DGE that brought about a 50% inhibition in cell growth was 35 µM, which is distinctly higher than that the ICG50 of the other GDPs. The concentration of 3,4-DGE in 2.25% glucose-containing S-PDF was ~15 µM, and this amount was third highest, next only to 3-DG and acetaldehyde. The concentration of 3,4-DGE that was capable of inducing a cytotoxic effect was found in S-PDF. In the case of 3-DG, GO, MGO, FA, AA, 5-HMF and FUR, the gap between the ICG50 and the concentration in PDF was very wide; it can be considered that there is no effect of these GDPs on the biocompatibility of PDFs.

As expected, the combination of eight types of GDPs including 3,4-DGE showed a strong cytotoxicity towards HPMCs as compared to the seven types of GDPs without 3,4-DGE, at the concentrations of GDPs found in S-PDF (Figure 1). These data suggest that 3,4-DGE plays a key role in the potential detrimental effects of PDFs. Correspondingly, N-PDF contained only 1.7 µM of 3,4-DGE. The generation of 3,4-DGE through heat sterilization could have been repressed under a low pH condition in the glucose solution (data not shown). It was anticipated that the improved biocompatibility of N-PDF is attributable to the decreased 3,4-DGE as well as to the effects of a neutral pH. The stability of 3,4-DGE in PDFs was previously reported: the concentration of 3,4-DGE in 1.5% PDF was drastically decreased from 125 to 25 µM after sterilization and storage for 30 days at 25°C, and the cytotoxicity of the PDF also decreased in parallel with the decrease in 3,4-DGE [17]. Moreover, it was also reported that higher concentrations of 3,4-DGE were found when PDF was stored at a temperature >25°C. Thus, coordinating the formulation of the glucose solution in the PDF, the storage conditions and the storage period after sterilization are considerable determinant factors for the biocompatibility of PDF.

The other biological effects of 3,4-DGE have been reported in the literature [10]. It has been reported that 3,4-DGE at micromolar concentrations suppressed the immune system, thymocyte proliferation, interleukin-1 production from lipopolysaccharide (LPS)-stimulated peritoneal macrophages and LPS-induced antibody production from spleen cells. Additionally, a recent study has revealed that GDPs including 3,4-DGE down-regulated the expression of tight junction-associated protein ZO-1 via the induction of VEGF synthesis in HPMCs [18]. The exposure of the peritoneum to PDFs containing a high concentration of 3,4-DGE may potentially injure the mesothelium and the peritoneal host defence system, resulting in peritoneal membrane dysfunction.

Exposure of HPMCs to pH-neutralized PDFs has been found to increase the intracellular H2O2 concentration [19]. Moreover, it is well known that MGO- and 3-DG-induced apoptosis is associated with the production of reactive oxygen species (ROS) [20,21]. For elucidation of the mechanisms of the strong biological activity of 3,4-DGE, we examined the effect of 3,4-DGE treatment on the changes in the cellular redox status. The redox status of the pool of cellular thiol plays a central role in the antioxidant defence against oxidative stress and in the regulation of a large number of signal transduction pathways. Because GSH represents the major thiol compound, the level of tGSH in HPMCs was determined. The results revealed that 3,4-DGE in the medium was immediately transferred to the HPMCs and significantly decreased cellular tGSH. This result suggested that increased oxidative stress due to tGSH depletion was the key mechanism in the cytotoxic effect of 3,4-DGE.

There are several possible ways by which 3,4-DGE may have reduced the intracellular tGSH levels; for example, 3,4-DGE may have directly reacted with GSH or 3,4-DGE may have inactivated the enzymes regulating the redox status such as glutathione reductase. Some mechanisms might underlie the observed tGSH decrease in the 3,4-DGE-treated HPMCs. Irrespective of the mechanisms, we first evaluated the effect of antioxidant pretreatment on intracellular tGSH levels (Figure 3B). The present observation is that 3,4-DGE-induced tGSH decrease was not affected by Trolox®, which is a non-thiol antioxidant, but was significantly delayed by the thiol-containing antioxidant, NAC. This result suggests that ROS generated by the exposure of HPMCs to 3,4-DGE should be a noncontributory factor and that the thiol group might play a major role in the disappearance of cellular tGSH. Consequently, the reaction of 3,4-DGE with GSH was analysed by liquid chromatography. We observed the disappearance of the 3,4-DGE peak at a 228 nm absorption within 1 h after being mixed together; hence, it was indicated that the {alpha},β-unsaturated carbonyl of 3,4-DGE may be the reactive site for this reaction. The thiol group in GSH might undergo an additional reaction with the unsaturated bond of 3,4-DGE and form the conjugate GSH&3,4-DGE. This conjugate product was elucidated by LC-ESI-MS analysis. One important cellular detoxification mechanism involves the binding of GSH to electrophilic chemicals and the export of the resulting GSH S-conjugates from the cell [22]. A similar detoxification reaction might take place for 3,4-DGE in HPMC.

Chronic renal insufficiency per se induces a state of oxidative stress; consequently, the progress of end-stage renal disease (ESRD) patients can be expected in terms of oxidative stress-related complications. The glutathione system closely contributes to ESRD-associated oxidative stress; the glutathione concentration in whole blood is dramatically decreased in dialysis patients [23]. Furthermore, the development of uraemic complications accompanied with the accumulation of reactive carbonyl compounds in uraemic plasma is observed as a strong possibility [24]. Thus, the loading of 3,4-DGE via PDF infusion may contribute to further deterioration of systemic uraemic complications. Further, clinical investigations from several approaches are desired.



   Conclusion
Top
 Abstract
 Introduction
 Subjects and methods
 Result
 Discussion
 Conclusion
References
 
The results obtained in this study indicate that 3,4-DGE at concentrations found in standard PDFs has a severe harmful effect on HPMCs as illustrated by the impaired proliferative cell response. 3,4-DGE can change the intracellular redox status accompanied by conjugation with cellular GSH. The influence of 3,4-DGE on the biocompatibility of PDFs is very important; it is desired that PDFs should be enclosed in a dual-chambered bag in order to decrease the concentration of 3,4-DGE.

Conflict of interest statement. Some author are employees of JMS Co. Ltd, which is a Japanese manufacturer of peritoneal dialysis fluids.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Result
 Discussion
 Conclusion
 References
 

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Received for publication: 31. 7.07
Accepted in revised form: 27.10.08


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