| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Bench |
Department of Internal Medicine,1 Daegu Fatima Hospital; Division of Nephrology and Department of Internal Medicine,2 Department of Surgery,3 Department of Biochemistry and Cell and Matrix Research Institute,4 Kyungpook National University School of Medicine, Daegu, Korea
Correspondence to: Y.L. Kim, Division of Nephrology and Department of Internal Medicine, Kyungpook National University Hospital, 50, Samduk-dong 2Ga, Jung-gu, Daegu 700-721, Korea. ylkim{at}knu.ac.kr
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Objective: Glucose degradation products (GDPs) are
formed during heat sterilization and storage of peritoneal dialysis (PD)
fluids. 3,4-dideoxyglucosone-3-ene (3,4-DGE) has been identified as the most
bioreactive GDP. 3,4-DGE induces apoptosis in leukocytes and renal tubular
epithelial cells. Our aim was to evaluate the apoptotic effects of 3,4-DGE on
human peritoneal mesothelial cells (HPMCs). Methods: Primary cultured HPMCs were treated with 25 or
50 µmol/L 3,4-DGE. MTT assay was used to determine cell viability.
Apoptosis was measured using TUNEL assay and flow cytometry. Expressions of
procaspase-3, Bax, and Bcl-2 were estimated by Western blot. Activity of
caspase-3 was measured and the effect of the caspase inhibitor zVAD-fmk
(Z-Val-Ala-DL-Asp-fluoromethylketone) was evaluated by TUNEL assay. Results: 3,4-DGE treatment accelerated cell death in
HPMCs in a dose- and time-dependent manner. Treatment with 3,4-DGE (25 and 50
µmol/L) significantly increased apoptosis compared to control (p
< 0.05 and p < 0.01 respectively) by TUNEL assay. Flow
cytometry showed treatment with 50 µmol/L 3,4-DGE significantly increased
apoptosis compared to control (p < 0.05). Decreased expression of
procaspase-3 and increased activity of caspase-3 were observed in the presence
of 50 µmol/L 3,4-DGE compared to control and 25 µmol/L 3,4-DGE
(p < 0.05). 3,4-DGE-induced HPMC apoptosis was decreased after
pretreatment with the pan-caspase inhibitor zVAD-fmk in the 50 µmol/L
3,4-DGE-treated group (p < 0.001). The ratio of Bcl-2 to Bax
expression was decreased in the 25 µmol/L and the 50 µmol/L
3,4-DGE-treated groups compared to control (p < 0.05). Conclusions: 3,4-DGE promotes apoptosis in HPMCs by a
caspase-related mechanism.
KEY WORDS: 3,4-DGE; peritoneal mesothelial cells; apoptosis; caspase.
Currently, most peritoneal dialysis (PD) fluids are glucose based. Heat sterilization and storage of PD fluids promote degradation of glucose into glucose degradation products (GDPs) (1). One of the biggest concerns associated with glucose-based PD fluids is bioincompatibility, and GDPs are the known culprits.
Because glucose promotes apoptosis in several cell types, including vascular endothelial cells, renal tubular cells, and the blastocyst, glucose was the suspected cause of the cytotoxicity associated with PD fluids (1–3). However, under in vitro conditions, heat-sterilized PD fluids have been shown to impair cell function to a greater extent than filter-sterilized PD fluids (4). As well, addition of glucose to commercial, lactate buffered, 1.5% glucose-containing PD solution to achieve a 4.25% glucose concentration failed to reproduce the increased neutrophil apoptosis rate observed in commercial, lactate buffered, 4.25% glucose PD solutions (5). These data imply that the cytotoxic effect of PD fluids can be attributed to GDPs rather than to glucose itself.
A large number of GDPs generated during heat sterilization of PD fluids have been identified (6) and the toxicity of GDPs has been demonstrated in vitro in different cell types (5), including mesothelial cells (7,8). GDPs such as acetaldehyde, 3-deoxyglucosone (3-DG), formaldehyde, 2-furaldehyde, glyoxal, 5-hydroxymethylfurfural (5-HMF), and methylglyoxal have shown proinflammatory and immunosuppressive effects in various cell types (9–11). However, the concentrations of individual GDPs used to induce cytotoxic effects in these assays were much higher than those found in conventional PD fluids. Although Witowski et al. (9) demonstrated that GDPs at concentrations relevant to PD fluids could impair viability and function in peritoneal mesothelial cells in an in vitro model, the effect required very long incubation times, up to 36 days.
Recently, 3,4-dideoxyglucosone-3-ene (3,4-DGE), an intermediate in the degradation of glucose to 5-HMF, was identified as the most cytotoxic GDP in PD fluids (6). More recent studies have shown that 3,4-DGE induces apoptosis in renal tubular epithelial cells (7) and promotes leukocyte apoptosis (8). However, its direct effect on peritoneal mesothelial cells has not been addressed yet.
The aim of our study was to investigate the biological effects of 3,4-DGE in cultured human peritoneal mesothelial cells (HPMCs).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
HPMCs were isolated using the trypsin-EDTA method, and cells were cultured in M199 medium containing 20% fetal bovine serum (FBS). We used second- and third-passage cells in this study.
3,4-DGE TREATMENT IN HPMCs
Cultured HPMCs were plated at confluence and allowed to rest in M199 medium
containing 1% FBS for 24 hours for cell-cycle synchronization. Next, HPMCs
were treated with 25 µmol/L or 50 µmol/L 3,4-DGE. 3,4-DGE was generously
provided by Dr. Anders Wieslander (Gambro, Sweden). Cells not treated with
3,4-DGE were used as control.
ASSESSMENT OF CELL DEATH AND APOPTOSIS
Viability of HPMCs against 3,4-DGE was determined by MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St.
Louis, MO, USA]. In brief, cells (2 x 104 cells/well) were
seeded in 96-well plates (Nunc, Roskilde, Denmark) and then treated with
3,4-DGE for 24, 48, and 72 hours; cells were reacted with 500 µg/mL MTT for
4 hours. After melting formazan made by adding 200 µL dimethyl sulfoxide
(DMSO) solution (Amresco, Solon, OH, USA), absorbance was measured at a
wavelength of 570 nm using a microplate reader (Bio-Rad Model 550; Hercules,
CA, USA). Four independent experiments were done.
The terminal uridine nick 3' end-labeling (TUNEL) assay was performed as follows to identify apoptosis. After treatment with 3,4-DGE for 72 hours, cells were fixed for 1 hour at 15°C – 25°C with 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4), washed with PBS, incubated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 minutes at 4°C, washed with PBS two times, incubated with 150 µL TUNEL reaction mixture for 1 hour at 37°C, washed with PBS three times, stained with DAPI 1 µg/mL for 3 minutes, washed with PBS three times, and mounted with ProLong gold antifade reagent (Invitrogen, Eugene, OR, USA). Four independent experiments were done. The number of TUNEL-positive cells was counted in three sections per experiment. To be considered TUNEL positive, each green fluorescent signal (FITC) had to correspond to the nuclei location (DAPI). We evaluated the apoptotic cells as a percentage of the FITC-to-DAPI ratio.
Apoptosis was quantified by flow cytometry. HPMCs were trypsinized, fixed with 100% ethanol for 1 hour at 4°C, suspended with 1.12% sodium citrate buffer (with 50 µg/mL ribonuclease A) for 30 minutes at 37°C, and then incubated with 50 µg/mL propidium iodide for 20 minutes. Finally, samples were analyzed on the FACScan (Becton Dickinson, Franklin Lakes, NJ, USA). Three independent experiments were done.
WESTERN BLOT FOR PROCASPASE-3, Bax, AND Bcl-2
After treatment with 3,4-DGE for 72 hours, HPMCs were lysed in RIPA buffer
(50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1%
SDS, 10 µg/mL leupeptin, 2 mmol/L phenylmethylsulfonyl fluoride). The
lysates were centrifuged at 12000g for 15 minutes and protein
concentration was measured by Bradford's method. Total protein (20 µg) was
separated on 10% SDS-polyacrylamide gel and transferred to a polyvinylidene
difluoride membrane. The membrane was blocked with 10% nonfat dry milk in 10
mmol/L Tris-buffered saline with 0.1% Tween 20 (TBS-T), followed by overnight
incubation at 4°C with diluted primary antibodies in TBS-T. Primary
antibodies were procaspase-3 [1:5000; Cell Signaling Technology (CST),
Beverly, MA, USA], Bax (1:10 000; CST), Bcl-2 (1:10 000; CST), and GAPDH
(1:5000; Abcam, Cambridge, MA, USA). After washing three times in TBS-T, the
membrane was incubated with secondary antibody (HRP-conjugated polyclonal goat
anti-rabbit immunoglobulin; 1:10 000; Dako, Glostrup, Denmark) in TBS-T for 1
hour at room temperature and developed to detect specific protein bands using
advanced ECL reagents (Amersham Bioscience, Piscataway, NJ, USA). The levels
of expression were estimated using Scion image, an image-analysis program
(Scion, Frederick, MD, USA).
CASPASE-3 ACTIVITY ASSAY
After treatment with 3,4-DGE for 72 hours, a colorimetric caspase-3 assay
kit (Sigma) was used to measure caspase-3 activity. After preparation of cell
lysates from apoptotic cells, 20 µL cell lysate with assay buffer was
placed in the wells. Then the lysate was incubated with 10 µL caspase-3
substrate at 37°C for 90 minutes. Absorbance was measured at a wavelength
of 405 nm and calculated using p-nitroaniline. Three independent experiments
were done.
CASPASE INHIBITOR
Cultured HPMCs were plated and allowed to rest for 24 hours in M199 medium
containing 1% FBS. After treatment with 100 µmol/L zVAD-fmk, a pan-caspase
inhibitor, for 1 hour, cells were treated with 25 µmol/L or 50 µmol/L
3,4-DGE for 72 hours. Cell morphology was observed and then TUNEL assay was
done to quantify apoptosis. Three independent experiments were done.
STATISTICAL ANALYSIS
Results are expressed as mean ± SE. Statistical analysis was
performed using SigmaStat software (Jandel Scientific, San Rafael, CA, USA). A
t-test was used to assess significance between groups; p < 0.05
was considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
According to the TUNEL assay, 3,4-DGE was cytotoxic to HPMCs [Figure 1(a)] and the number of apoptotic cells increased in the 50 µmol/L 3,4-DGE-treated group (3,4-DGE-50) compared to the 25 µmol/L 3,4-DGE-treated group (3,4-DGE-25; p < 0.01) and the control group (p < 0.01) [Figure 1(b)]. There was also a significant increase in apoptotic cells in the 3,4-DGE-25 group compared to the control group (p < 0.05) [Figure 1(b)]. Flow cytometry analysis revealed that hypodiploid apoptotic cells increased in the 3,4-DGE-treated groups [Figure 2(a)]. The mean number of apoptotic cells was significantly higher in the 3,4-DGE-50 group than in the control group (p < 0.05) [Figure 2(b)] and the 3,4-DGE-25 group, although the difference was just shy of significant in the latter (p = 0.06). There was no significant difference in the mean number of apoptotic cells between the 3,4-DGE-25 group and the control group [Figure 2(b)].
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Recently, two studies demonstrated the induction of apoptosis by 3,4-DGE. Justo et al. (7) showed that 3,4-DGE induced apoptosis in renal tubular epithelial cells in a dose- and time-dependent manner. The lethal concentration range of 3,4-DGE was 25 – 50 µmol/L and the mechanism was Bax and caspase dependent; inhibiting either Bax or caspases prevented apoptosis. Catalan et al. (8) showed that 3,4-DGE promoted apoptosis in neutrophils and mononuclear cells; 3,4-DGE-induced apoptosis was caspase dependent and could be prevented by inhibiting caspase activity.
3,4-DGE is formed during the degradation of glucose and is an intermediate in the conversion of 3-DG to 5-HMF. 3,4-DGE is an unsaturated carbonyl compound similar to 3-DG but with an additional double bond in the carbon skeleton. The position of the double bond in relation to the carbonyl group increases the reactivity of this compound. No toxicity data on 3,4-DGE were available until 1994, when Kato et al. isolated the substance from seaweed and demonstrated its toxic and immunosuppressive effects on various cell systems (10).
3,4-DGE is present at a concentration of 9 – 22 µmol/L in conventionally manufactured PD fluids (12), but may be as high as 125 µmol/L immediately after heat sterilization (6,11). According to Linden et al. (6), when increasing the temperature of PD fluids from room temperature to 37°C, the concentration of 3,4-DGE increases significantly; furthermore, after being removed by solid phase extraction, 3,4-DGE is regenerated during incubation. Therefore, the actual amount of 3,4-DGE in the incubation mixture may be at least twice as high as that suggested by the measured concentration. If so, the total amount of 3,4-DGE in conventional PD fluids is high enough to explain virtually all observed in vitro cytotoxicity. In addition, the concentration of 3,4-DGE depends on the balance between the reaction producing 3,4-DGE from the pool and the reverse reaction returning it to the pool (12). Increasing the temperature shifts the equilibrium in the direction of 3,4-DGE, while decreasing the temperature drives the equilibrium in the opposite direction. Therefore, the toxicity of PD fluids depends on the actual amount of 3,4-DGE and on the amount that may be recruited from the pool. In the present study we showed that both 25 µmol/L and 50 µmol/L 3,4-DGE promote apoptosis in HPMCs; these concentrations are similar to or slightly higher than that found in conventional PD fluids.
The cellular and molecular mechanisms of cell death induced by PD fluids had not been explored until recently (5). Apoptosis is an active mode of cell death that requires energy in the form of ATP to proceed (13), is under molecular control, and may be manipulated therapeutically (14,15). Apoptosis is precipitated by sequential activation of cysteine proteases of the caspase family in two distinct but converging pathways: the extrinsic pathway and the intrinsic, or mitochondrial, pathway. The intrinsic pathway is controlled by the Bcl-2 protein family. Bcl-2 itself inhibits apoptosis; in contrast, Bax promotes apoptosis (16).
The caspases behave as initiators and effectors of apoptosis. Caspases are activated by the sequential cleavage of procaspases. Initiator caspases proteolytically activate effectors, such as caspase-3, which in turn degrade other intracellular proteins, ultimately leading to cell death. Therefore, inhibiting caspase activity can prevent cell death. However, in some cases, caspase inhibition prevents apoptosis but not eventual cell death (17,18). In 3,4-DGE-induced apoptosis, inhibiting caspase activation does prevent apoptosis (7,8). In our study, we showed that exposure to 3,4-DGE decreased procaspase-3 expression and increased caspase-3 activity in HPMCs, which, combined with the observed increase in apoptosis caused by exposure to 3,4-DGE, suggests that the mechanism of apoptosis involves caspase-3 activation. This is supported by the results that pretreatment with the pan-caspase inhibitor zVAD-fmk decreased apoptosis induced by 3,4-DGE.
Many apoptotic stimuli regulate Bcl-2 and Bax levels. Usually, they decrease anti-apoptotic Bcl-2 expression and increase pro-apoptotic Bax expression, subsequently decreasing the Bcl-2/Bax ratio. Whether stressed cells live or die is largely determined by interplay between Bcl-2 and Bax. That is, the ratio of Bcl-2 to Bax may ultimately determine the fate of cells (19). In this study, we evaluated the expression of Bcl-2 and Bax by Western blot. We found that 3,4-DGE treatment upregulated Bax expression and downregulated Bcl-2 expression. As a result, the ratio of Bcl-2 to Bax expression decreased in 3,4-DGE-treated groups compared to control. This indicates that imbalance of Bcl-2 and Bax expression may be one mechanism of 3,4-DGE-induced HPMC apoptosis.
In summary, 3,4-DGE, a GDP that exists in conventional PD fluids, induces apoptosis in HPMCs and the mechanism is related to the caspase pathway. Decreasing the concentration of 3,4-DGE in PD fluids may prevent 3,4-DGE-induced apoptosis.
|
ACKNOWLEDGMENTS |
|---|
Received 23 July 2007; accepted 5 May 2008.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
