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NDT Advance Access originally published online on January 18, 2006
Nephrology Dialysis Transplantation 2006 21(5):1340-1347; doi:10.1093/ndt/gfk051
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Dialysis and Transplantation

Diltiazem suppresses collagen synthesis and IL-1ß-induced TGF-ß1 production on human peritoneal mesothelial cells

Cheng-Chung Fang1, Chung-Jen Yen2, Yung-Ming Chen2, Tzong-Shinn Chu2, Ming-Tsan Lin3, Ju-Yeh Yang2 and Tun-Jun Tsai2

Departments of 1 Emergency Medicine, 2 Internal Medicine and 3 Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

Correspondence and offprint requests to: Professor Tun-Jun Tsai, Department of Internal Medicine, National Taiwan University Hospital, No. 7 Chung Shan South Road, Taipei 100, Taiwan. Email: paul{at}ha.mc.ntu.edu.tw



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. After long-term treatment with continuous ambulatory peritoneal dialysis (CAPD), some patients may develop peritoneal fibrosis. Peritoneal mesothelial cells (PMCs) participate in the inflammatory reactions in the peritoneal cavity, and transforming growth factor-ß1 (TGF-ß1) and interleukin-1ß (IL-1ß) are involved in peritoneal fibrosis. Diltiazem is used frequently in patients with CAPD to treat hypertension. The objectives of this study were to examine the effects of diltiazem on collagen- and IL-1ß-induced TGF-ß1 production on human PMCs and the signalling pathway of diltiazem in this induction.

Methods. Human PMCs were cultured from the enzymatic disaggregation of human omentum. Collagen synthesis was measured by [3H]proline incorporation into pepsin-resistant, salt-precipitated collagen. The expression of collagen I and III, and TGF-ß1 mRNA was evaluated by northern blotting. The production of TGF-ß1 by human PMCs was measured by immunoassay. The changes of intracellular calcium level after adding Fura-2-AM were measured by fluorescence spectrophotometry. Western blotting was used to assess mitogen-activated protein kinase (MAPK) signalling proteins.

Results. We found that diltiazem (<0.2 mM) inhibited collagen I and III mRNA expression and collagen syntheses on a dose-dependent basis. Diltiazem (0.2 mM) suppressed IL-1ß- (5 ng/ml) induced TGF-ß1 production on human PMCs at both the protein and mRNA levels. Diltiazem (0.2 mM) also inhibited IL-1ß- (5 ng/ml) induced collagen I and III mRNA expression. Intracellular calcium levels did not change after the treatment with diltiazem, IL-1ß or both. The IL-1ß-treated human PMCs increased phospho-JNK (stress-activated c-Jun N-terminal kinase) and phospho-p38 MAPK expression, while diltiazem could suppress this phenomenon.

Conclusions. Diltiazem suppressed collagen synthesis of human PMCs and inhibited IL-1ß-induced TGF-ß1 production on human PMCs. This signalling transduction may be through p38 MAPK and JNK pathways instead of intracellular calcium. These results suggest diltiazem to be a potential therapeutic regimen in preventing peritoneal fibrosis and support further in vivo studies.

Keywords: diltiazem; IL-1ß; mitogen-activated protein kinase; peritoneal mesothelial cell; signalling pathway; TGF-ß1



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
After long-term treatment with continuous ambulatory peritoneal dialysis (CAPD), the peritoneum may be altered, which progressively increases extracellular matrix (ECM) deposition and neovasculization [1,2]. These changes, so-called peritoneal fibrosis, affect the peritoneum as a dialysis organ and result in ultrafiltration failure [2]. Peritoneal mesothelial cells (PMCs) line the peritoneum and can secrete ECM [3] and various cytokines, such as interleukin-1ß (IL-1ß) [4], transforming growth factor-ß (TGF-ß) [5] and vascular endothelial growth factor (VEGF) [6]. All these cytokines are involved in the pathogenesis of peritoneal fibrosis. These facts support the contention that PMCs play an active role in peritoneal fibrosis. Therefore, the PMC is a target cell type in CAPD-related peritoneal fibrosis.

The pathogenesis of peritoneal fibrosis is not clearly understood, but recurrent bacterial peritonitis [7] and glucose-containing dialysate may be the two most important causes [2]. CAPD induces chronic inflammation in the peritoneum either with or without bacterial infections [8,9]. Proinflammatory cytokines, such as IL-1ß and tumour necrosis factor-{alpha} (TNF-{alpha}), are released initially from peritoneal macrophages and induce further release of other cytokines from PMCs [10]. In an adenovirus-mediated gene transfer study in rats, overexpression of IL-1ß, but not TNF-{alpha}, caused sustained peritoneal fibrosis by increasing VEGF and TGF-ß expression [11]. Based on these findings, the authors of the study concluded that IL-1ß inhibition may be a therapeutic goal in acute peritonitis to prevent peritoneal damage.

TGF-ß is an important growth factor involved in ECM modulation and can increase ECM protein syntheses and decrease their degradation [12]. TGF-ß is a growth factor vital to tissue repair, but it may be responsible for tissue fibrosis due to excessive action. In a study performed in rats, overexpression of TGF-ß1 after adenovirus-mediated gene transfer caused peritoneal fibrosis and neoangiogenesis by TGF-ß1-induced VEGF synthesis [13]. Therefore, TGF-ß is one of the key growth factors involved in peritoneal fibrosis [14], and it is imperative to design therapeutic strategies for preventing peritoneal fibrosis by suppressing TGF-ß [15].

Calcium channel blockers are used frequently to treat hypertension in patients with CAPD, and have been demonstrated to inhibit matrix production in various types of cells [16]. Diltiazem (DT) reduces IL-6 production in mixed lymphocyte culture [17] and downregulates production of IL-12 by human dendritic cells [18]. Human PMCs have been shown to synthesize TGF-ß1, TGF-ß2 and TGF-ß3, which can be upregulated by IL-1ß [5]. Therefore, we wanted to investigate the effects of DT on collagen synthesis and IL-1ß-induced TGF-ß1 expression in human PMC.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Trypsin-EDTA, RPMI-1640 medium and trypan blue were obtained from Invitrogen Life Technologies (New York, NY). Culture flasks and plates were purchased from Corning (NY) and pre-coated with Vigtrogen 100® (Celtrix Lab., Palo Alto, CA) before loading cells. Fetal bovine serum (FBS) was obtained from Biochrom KG (Berlin, Germany). Bovine serum albumin (BSA), penicillin, streptomycin, insulin and DT were purchased from Sigma (St Louis, MO). Mouse anti-human monoclonal antibodies to phospho-extracellular signal-regulated kinase (ERK), total and phospho-p38 mitogen-activated protein kinase (MAPK), and total and phospho-stress-activated c-Jun N-terminal kinase (JNK), and horseradish peroxidase-labelled goat anti-mouse and anti-rabbit IgG were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-human monoclonal antibody to total ERK was manufactured by Cell Signalling Technology (Beverly, MA). Fura-2AM, SB 203580, SP 600152 and PD 98059 were purchased from Calbiochem Corp. (San Diego, CA). IL-1ß, TGF-ß1 immunoassay kit and TGF-ß neutralizing antibody were bought from R&D Systems Inc. (Minneapolis, MN). Agents used for western blot analysis originated from Bio-Rad Laboratories Inc. (Hercules, CA) unless otherwise specified.

Human peritoneal mesothelial cell culture
Specimens of human omentum were obtained from patients undergoing gastrectomy due to early gastric carcinoma. The patients supplied written informed consent prior to starting the procedure. The method of enzymatic disaggregation of omentum was used as previously described [19]. Briefly, a piece of omentum was washed in sterile phosphate-buffered saline (PBS) three times and then incubated with 15 ml of trypsin-EDTA (0.125%) for 20 min at 37°C with continuous rotation. After incubation, the omentum and the suspension were centrifuged at 50 g for 5 min at 4°C. The cell pellet was washed once and cultured with RPMI-1640 medium containing 20% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml) and insulin (30 µg/ml). In 2–4 days, the cells became confluent and were subcultured with medium containing 10% FBS. The cells were initially bipolar or multipolar but became cobblestone-like in appearance upon confluence. Using the immunofluorescence method, human PMCs were identified by the presence of vimentin and cytokeratin and the absence of desmin and factor VIII-related antigen. All experiments listed below were performed in passage 1–3 cells and repeated at least three times using cells from different subjects.

Cell viability test
To exclude the toxic effect of DT on human PMCs, after incubating with various doses of DT for 1, 3 and 5 days in 24-well plates, cell viability was assessed by the trypan blue exclusion method. The number of dead cells from the supernatant of each well was counted and the proportions of dead cells among the cells adhering to the plates were also counted in separate wells by the addition of trypan blue directly. All samples were treated in triplicate.

Test of injury to human PMC plasmalemma
To exclude the toxicity of DT to the integrity of human PMC plasmalemma, the leakage of lactate dehydrogenase (LDH) from the cytosol of human PMCs was measured. Cells were plated down at a density of 5x104 cells/well in a 24-well plate and grown to a confluent monolayer. Then, the medium was replaced by 0.5 ml of RPMI-1640 medium with 0.5% FCS after washing twice with warm PBS. Various concentrations of DT were added and 0.015% Triton X-100 was used as a positive control. The plates were incubated for 1, 3 and 5 days and then the medium was removed and its LDH activity was measured by a Toshiba autoanalyser. All experiments were done in triplicate.

Collagen synthesis
Human PMCs were plated down at a density of 2.5x104 cells/well in a 96-well plate in 200 ml of medium supplemented with 10% FCS. After incubation for 48 h, the cells became confluent. Then the medium was replaced by 10% FCS-supplemented medium containing 50 mg/ml of ascorbic acid with or without different concentrations of DT. After an additional incubation for 48 h, cultures were labelled with 0.5 mCi of [3H]proline (100 Ci/mmol, ICN) and 50 mg/ml ß-aminopropionitrile for the final 24 h of incubation. The [3H]proline incorporation into pepsin-resistant, salt-precipitated collagen was determined as previously described [20]. After termination of incubation, [3H]proline-labelled collagen was extracted from each well by the addition of pepsin-containing acetic acid (final concentration of 1 mg/ml in 1 M acetic acid) and purified by successive salt precipitation at acid and neutral pH in the presence of carrier collagen. The final precipitates were dissolved in 0.5 M acetic acid, incorporated into a scintillation cocktail and counted by a liquid scintillation counter. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) uptake (absorbance at 570 nm) for human PMCs was found to vary linearly with cell numbers [19]. The MTT assay was used to count cell numbers in identically treated microplates and was used to factor the amount of collagen synthesized. All experiments were done in triplicate.

Northern blot analysis
Human PMCs were cultured in a 50 cm2 dish with 10% FCS-containing RPMI to a confluent monolayer. After growth arrest for 2 days with serum-free RPMI, the supernatants were replaced with 10% FCS-containing RPMI with various concentrations of DT to assay the mRNA expression of collagen I and III. For the experiments studying IL-1ß-induced collagen and TGF-ß1 gene expression, serum-free RPMI with agents of various concentrations were added. PD 98059 (30 µM, a specific inhibitor of the ERK pathway [21]), SB 203580 (10 µM, a specific inhibitor of the p38 MAPK pathway [22]), SP 600152 (30 µM, a specific inhibitor of the JNK pathway [23]) or DT (0.2 mM) was used as the pre-treatment for 1 h before adding 5 ng/ml of IL-1ß. The dosages of PD 98059, SB 203580 and SP 600152 were used according to the results of western blotting, demonstrating at those dosages that these agents could suppress the target MAPKs. After 24 h, the total RNA was isolated using the acid guanidinium thiocyanate–phenol–chloroform method. The amount of RNA was quantified using spectrophotometry with absorbance at 260 nm. A 10 µg aliquot of RNA was then electrophoresed on a 1% agarose gel containing 1.0 M formaldehyde in MOPS buffer (0.2 M morpholinopropanesulfonic acid, 0.05 M Na acetate, 0.01 M EDTA). Equivalency of sample loading and lack of degradation were verified by ethidium bromide staining of 28S and 18S rRNA bands. The RNA was transferred to a nylon membrane by overnight capillary action, followed by fixation in a UV cross-linker.

The RNA blots were pre-hybridized for 3 h at 65°C in a hybridization solution composed of 50% formamide, 5x SSC (1x SSC = 120 mM NaCl, 15 mM Na citrate, 13 mM KH2PO4 and 1 mM EDTA, pH 7.2), 0.1% Na lauryl sarcosine, 2% SDS and 5% blocking reagent, then hybridized overnight at 65°C with 20 ng/ml probe in the same solution, followed by washing in 2x SCC/0.1% SDS and 0.1x SSC/0.1% SDS at 65°C. The blots were developed using alkaline phosphatase-conjugated anti-digoxigenin antibody at Lumi-phos 530 according to the manufacturer's directions. The signal intensity recorded on X-ray film was quantified by computerized densitometry. The blots were also probed with a cRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to control for variation in RNA loading and transfer.

TGF-ß1 assay
Two hundred thousand human PMCs per well were loaded into a 6-well plate and cultured by medium supplemented with 10% FCS. After growth arrest for 2 days with serum-free RPMI, the supernatants were then replaced with various agents. PD 98059 (30 µM), SB 203580 (10 µM), SP 600152 (30 µM) or DT (0.2 mM) was used as the pre-treatment for 1 h before adding 5 ng/ml of IL-1ß. Then, the cells were incubated for 48 h. The supernatants were collected and TGF-ß1 was assayed by immunoassay. The protein content of each well was determined by bicinchoninic acid assay. All experiments were done in triplicate.

Intracellular calcium
Human PMCs were suspended in PBS containing 2 mM EDTA by periodic shaking, washed in a Ca2+-containing solution (in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 11 glucose, 10 HEPES, pH 7.4, 0.1% BSA), and incubated in 4 µM Fura-2-AM with 0.04% Pluronic for 35 min. Cells were then washed and resuspended in the Ca2+-containing solution and maintained on ice until assay. Intracellular calcium ([Ca2+]i) was measured in cells suspended in the Ca2+-containing solution as the ratio of fluorescence with 340 and 380 nm excitation and 510 nm emission (Fluorolog 2; Spex Industries, Edison, NJ). The fluorescent ratio was calibrated by adding digitonin to a final concentration of 75 µg/ml, next adding 1 M EDTA in a 1:50 dilution, then adding 10 N NaOH at a 1 : 700 dilution to the solution. Calcium [Ca2+]i was calculated as described previously [24].

Western blotting
Human PMCs were cultured to subconfluence in a 50 cm2 dish with 10% FBS-containing RPMI. The medium then was changed to serum-free RPMI for 2 days and then different agents were added. PD 98059 (30 µM), SB 203580 (10 µM), SP 600152 (30 µM) or DT (0.2 mM) was used as the pre-treatment for 1 h before adding 5 ng/ml of IL-1ß. After a 30 min incubation, the cells were lysed by ice-cold lysing solution [65 mM Tris base, pH 8.0, containing 154 mM NaCl, 1 mM EDTA, 1% IGEPAL, 1 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (1 µg/ml), pepstatin (1 µg/ml), aprotinin (1 µg/ml) and 0.25% Na deoxycholate]. Samples were rotated for 15 min at 4°C, then centrifuged at 12 000 g for 5 min at 4°C. Supernatant was recovered, and the protein concentration was measured by the bicinchoninic acid assay (BioRad), with BSA as the standard. Samples were incubated for 5 min at 95°C in loading buffer (12 mM Tris–HCl, pH 6.8, with 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol and 0.1% bromophenol blue), and 50 µg of protein were loaded on SDS–polyacrylamide gels of different percentages corresponding to the molecular weight of the target proteins. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membrane by electroblotting. Membrane was blocked in 1% BSA/0.05% Tween/PBS solution overnight at 4°C. Mouse anti-human monoclonal antibodies to phospho-ERK, phospho-p38 MAPK and phospho-JNK were used as primary antibodies. A horseradish peroxidase-labelled goat anti-mouse IgG was used as a secondary antibody. Blots were developed by incubation in a chemiluminescence substrate and were exposed to X-ray films.

Statistical analysis
All data are expressed as means±SEMs. Statistical analyses comparing drug effects were performed by using the Student's t-test. The comparisons of the dose effect of agents were done by one-way analysis of variance (ANOVA) and post hoc tests. A P-value <0.05 was considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
To test the proper dose of DT on PMCs in vitro, we examined different concentrations of DT, from 0.2 to 0.05 mM, on PMC viability by the trypan blue test. We found that there were no differences in dead cell rate in both supernatant and adherent cells among control and DT-treated wells. The dead cells of each well were <1% of the total cells. The levels of LDH did not increase after 1, 3 and 5 day incubation periods in DT-treated wells, which excluded cell membrane damage by DT on the PMCs (data not shown). We found that DT, at 0.2 and 0.1 mM, downregulated collagen I and III mRNA expression on a dose-dependent basis. The results of densitometry showed that DT decreased serum-stimulated procollagen {alpha}1 (I) and procollagen {alpha}1 (III) mRNA expression, while GAPDH mRNA was not affected (Figure 1). At the protein level, DT also decreased collagen synthesis of PMCs (Figure 2).


Figure 1
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  		Fig. 1. The mRNA expression of procollagen {alpha}1 (I) (A) and procollagen {alpha}1 (III) (B) on human peritoneal mesothelial cells. The cells were treated with different concentrations of diltiazem for 24 h. The activity ratios of collagen/GAPDH are expressed as mean±SEM, n = 3, *P<0.05 vs control.

 

Figure 2
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  		Fig. 2. Synthesis of collagen by human peritoneal mesothelial cells was measured after treatment with diltiazem. The results are expressed as a percentage of the control. Data are expressed as mean±SEM of three experiments conducted in triplicate. *P<0.05 vs control.

 
We further studied the effect of DT on IL-1ß-induced TGF-ß1 production. At 0.2 mM, DT also inhibited IL-1ß-induced TGF-ß1 mRNA expression (Figure 3) and TGF-ß1 production (Figure 4). To investigate the signalling pathway of DT, we studied the changes of intracellular calcium level after treatment with IL-1ß with and without DT. We found that the intracellular calcium concentrations did not change after IL-1ß treatment or after DT treatment (Figure 5). The data suggested that intracellular calcium changes may not be involved in the mechanism of IL-1ß-induced TGF-ß1 production. Therefore, we further studied the signalling pathway of MAPK. The IL-1ß-treated human PMCs activated ERK, JNK and p38 MAPK pathways. DT, unlike PD 98059 (a specific inhibitor of the ERK pathway), could not suppress the ERK pathway (Figure 6A). DT, like SB 203580 (a specific inhibitor of the p38 MAPK pathway) and SP 600152 (a specific inhibitor of the JNK pathway), could suppress both phospho-p38 MAPK (Figure 6B) and phospho-JNK (Figure 6C) expression. Both SB 203580 and SP 600152 could suppress TGF-ß1 mRNA expression in a manner similar to DT, whereas PD 98059 failed to do so (Figure 3). Similar effects were observed on IL-1ß-induced TGF-ß1 production (Figure 4). Therefore, we concluded that DT could inhibit IL-1ß-induced TGF-ß1 production on human PMCs. The signalling transduction may be through JNK and p38 MAPK pathways instead of intracellular calcium.


Figure 3
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  		Fig. 3. TGF-ß1 mRNA expression on human peritoneal mesothelial cells. The cells were pre-treated with different agents for 1 h, then 5 ng/ml of IL-1ß was added for an additional 24 h. SB 203580 and SP 600152 could downregulate the IL-1ß-induced TGF-ß mRNA expression in a similar manner to DT, whereas PD 98059 failed to do so. The activity ratios of TGF-ß1/28S tRNA are expressed as mean±SEM, n = 3, *P<0.05 vs control. DT, 0.2 mM diltiazem; PD, 30 µM PD 98059; SB, 10 µM SB 203580; SP, 30 µM SP 600152.

 

Figure 4
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  		Fig. 4. TGF-ß1 production by human peritoneal mesothelial cells. The cells were pre-treated with different agents for 1 h, then 5 ng/ml of IL-1ß was added for an additional 48 h. The production of TGF-ß1 was induced by IL-1ß. SB 203580, SP 600152 and DT could suppress the effect of IL-1ß, but PD 98059 failed to do so. Data are expressed as mean±SEM of three experiments conducted in triplicate. *P<0.05 vs control. DT, 0.2 mM diltiazem; PD, 30 µM PD 98059; SB, 10 µM SB 203580; SP, 30 µM SP 600152.

 

Figure 5
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  		Fig. 5. Peak intracellular calcium concentrations of human peritoneal mesothelial cells were measured after treatment with calcium ionophore (A23187, 5 µM), 0.2 mM diltiazem (DT), 5 ng/ml of IL-1ß, and both of the above. Data are expressed as mean±SEM of three experiments. *P<0.05 vs control.

 

Figure 6
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  		Fig. 6. The changes of mitogen-activated protein kinase (MAPK) pathways on human peritoneal mesothelial cells by western blots. The cells were pre-treated with different agents for 1 h, then 5 ng/ml of IL-1ß was added for an additional 30 min. The changes in extracellular signal-regulated kinase (ERK), p38 MAPK and stress-activated c-Jun N-terminal kinase (JNK) are displayed as (A), (B) and (C), respectively. IL-1ß could activate the ERK, JNK and p38 MAPK pathways. Diltiazem, unlike PD 98059, could not suppress the ERK pathway (A). Diltiazem, like SB 203580 and SP 600152, could suppress both phospho-p38 MAPK (B) and phospho-JNK (C) expression. Data are expressed as mean±SEM of three experiments. *P<0.05 vs control. DT, 0.2 mM diltiazem; PD, 30 µM PD 98059; SB, 10 µM SB 203580; SP, 30 µM SP 600152.

 

Figure 7
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  		Fig. 7. Collagen I and collagen III mRNA expression on human peritoneal mesothelial cells. After the cells were pre-treated with different agents for 1 h, 5 ng/ml of IL-1ß was added for an additional 24 h. DT, SB 203580 and SP 600152 could downregulate the basal and IL-1ß-induced collagen I mRNA expression, whereas PD 98059 failed to do so (A). DT and SB 203580 could downregulate the basal and IL-1ß-induced collagen III mRNA expression, whereas SP 600152 and PD 98059 failed to do so (B). TGF-ß neutralizing antibody did not demonstrate a significant inhibitory effect on IL-1ß-induced collagen mRNA expression (A and B). The activity ratios of collagen/GAPDH are expressed as mean±SEM, n = 3, *P<0.05 vs IL-1ß treated; #P<0.05 vs control. (C) Control, IL-1ß, 5 ng/ml of IL-1ß; DT, 0.2 mM diltiazem; PD, 30 µM PD 98059; SB, 10 µM SB 203580; SP, 30 µM of SP 600152; {alpha}TGF-ß, 30 µg/ml of TGF-ß neutralizing antibody.

 
We also studied the effect of IL-1ß on collagen I and III gene expression. We found that IL-1ß could induce collagen I and III gene expression and be suppressed by DT (Figure 7). Furthermore, both SB 203580 and SP 600152 could suppress IL-1ß-induced collagen I mRNA expression in a similar manner to DT, whereas PD 98059 failed to do so (Figure 7A). The inhibitory effect of SB 203580 was also noted on collagen III mRNA expression (Figure 7B). The basal collagen I and III mRNA expression was suppressed by DT and SB 203580, whereas, SP 600152 could only suppress collagen I mRNA expression (Figure 7). The TGF-ß neutralizing antibody could not suppress IL-1ß-induced collagen I and III mRNA expression (Figure 7). Therefore, the effect of DT on IL-1ß-induced collagen I and III mRNA expression is through inhibition of MAPKs, instead of the inhibition of IL-1ß-induced TGF-ß1 production.



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
It is unclear which type of cell is responsible for peritoneal fibrosis. Dobbie et al. proposed the hypothesis of the development of peritoneal fibrosis as excess cytokines stimulating peritoneal mesothelial stem cell proliferation and collagen deposition [25]. However, some researchers proposed that peritoneal fibroblast plays the main role in peritoneal fibrosis [26]. Recently, there have been numerous studies performed to demonstrate that cells may change their characters under various stimuli, so-called epithelial-to-mesenchymal transition (EMT). Yanez-Mo et al. demonstrated that PMCs may undergo EMT during peritoneal dialysis under the stimulation by IL-1ß and TGF-ß1 [27]. When investigating the causes of peritoneal fibrosis in patients with CAPD, IL-1ß and TGF-ß have been shown to be the key factors in matrix production and EMT [14]. Peritoneal inflammation can also occur in patients with CAPD even without bacterial peritonitis, due to bioincompatible dialysates [8,9]. In the present study, we demonstrated that DT could suppress not only the collagen synthesis but also the IL-1ß-induced TGF-ß1 production by PMCs. Therefore, it is reasonable to hypothesize that DT may be a potential therapy in preventing peritoneal fibrosis.

As DT is a calcium channel blocker, the signalling pathway of DT effects had been initially proposed to be through intracellular calcium. Crenesse et al. demonstrated that DT reduces JNK activation and consequently decreases hypoxia–reoxygenation-induced apoptosis in rat hepatocytes [28]. It was found that intracellular calcium levels were modulated during this experiment and it was concluded that DT's effect is through its property of being a calcium channel blocker. Cuschieri et al. found that both verapamil and DT can reduce lipopolysaccharide (LPS)-induced ERK activity and TNF-{alpha} expression in alveolar macrophages [29]. Although the authors postulated that DT may affect the flux of calcium from the endoplasmic reticulum that initiated the signalling events, their previous study failed to show a measurable change in intracellular calcium level after LPS stimulation [30]. Therefore, it is feasible to hypothesize that DT may possess directly inhibitory effects on MAPK. In the present study, we found that the intracellular calcium levels did not change with IL-1ß and/or DT treatment. Our data demonstrated that DT suppressed the JNK and p38 MAPK pathways on the PMCs after IL-1ß treatment, whereas the ERK pathway was not affected. We also showed that both SB 203580 and SP 600152 could suppress the IL-1ß-induced TGF-ß1 mRNA expression in the same manner as DT, whereas PD 98059 could not. Therefore, the effect of DT on PMCs may be conducted through JNK and p38 MAPK pathways without affecting intracellular calcium levels.

Calcium channel blockers have been shown to inhibit matrix production in various types of cells [16,31]. Therefore, we wanted to evaluate the effects of other calcium channel blockers on PMCs. We performed experiments on verapamil and nifedipine to measure the effects on collagen I mRNA expression and collagen synthesis. Our preliminary results demonstrated that verapamil could suppress the mRNA expression of collagen I and the collagen synthesis, whereas nifedipine could not. These findings warrant further studies on calcium channel blockers and PMCs.

The highest in vitro concentration of DT in this study was 0.2 mM (~90 mg/l). This concentration is high compared with therapeutic serum levels of 50–300 µg/l [32]. The effective concentration range of DT (0.05–0.2 mM) is narrow in this in vitro study. However, previous in vitro studies have used DT concentrations from 0.1 mM [17,18]. The study on alveolar macrophages demonstrated an effective concentration range from 0.1 to 0.5 mM DT [29]. The plasma level of DT may not reflect tissue levels of DT, and a higher concentration may be achieved in the peritoneal cavity through the intraperitoneal route in patients with CAPD. It has been shown that the blood pressure remains stable after giving 15 mg/kg of DT intraperitoneally in rats [33]. According to these data, the in vitro concentration of DT in our study is easily achieved in the dialysate. Nevertheless, the safety, proper dosage and effectiveness of DT in preventing peritoneal fibrosis require further study.

Another limitation of this study is that the donors of the PMCs were from patients with normal renal function. In a rat study, it was found that uraemia induces permeability and structural changes in the peritoneum [34]. Most of the in vitro studies were performed in PMCs from patients with normal renal function. One study compared the differences between the PMCs from non-uraemic patients and those from uraemic donors, and found that PMCs from uraemic patients more readily released IL-8 upon stimulation with IL-1ß [35]. Therefore, our results should be complemented by studies using cells from uraemic donors.

In conclusion, DT inhibited collagen synthesis and mRNA expression on human PMCs. DT also suppressed IL-1ß-induced TGF-ß1 production through inhibition of the JNK and p38 MAPK pathways on human PMCs. These results support the need for further in vivo studies on DT in preventing peritoneal fibrosis.



   Acknowledgments
 
The authors thank Professor Wan-Yu Chen and Ms Su-Li Hung for their kind assistance. This study was supported by grants from The National Science Council NSC 91-2314-B-002-349, Ta-Tung Kidney Foundation and Mrs Hsiu-Chin Lee Kidney Research Fund. This work was supported in part by the Department of Medical Research in the National Taiwan University Hospital. Part of the study has been presented in poster form at the World Congress of Nephrology, in Berlin, Germany, June 8–12, 2003.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 1. 3.05
Accepted in revised form: 13.12.05


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L. S. Aroeira, A. Aguilera, J. A. Sanchez-Tomero, M. A. Bajo, G. del Peso, J. A. Jimenez-Heffernan, R. Selgas, and M. Lopez-Cabrera
Epithelial to Mesenchymal Transition and Peritoneal Membrane Failure in Peritoneal Dialysis Patients: Pathologic Significance and Potential Therapeutic Interventions
J. Am. Soc. Nephrol., July 1, 2007; 18(7): 2004 - 2013.
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