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*
Department of Internal Medicine II, Hiroshima University School of
Medicine, Hiroshima, Japan.
Department of Anatomy II, Hiroshima University School of Medicine,
Hiroshima, Japan.
Correspondence to Dr. Noriaki Yorioka, Department of Internal Medicine II, Hiroshima University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Phone: +81-82-257-5196, Fax: +81-82-255-7360; E-mail: nyorioka{at}mcai.med.hiroshima-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mesothelial cells are reported to possess several different types of intercellular junctions, including tight junctions, adherens junctions, desmosomes, and gap junctions (10,11,12). Tight junctions selectively regulate the passage of molecules through the paracellular pathway (gate function) and also separate molecules in the apical and basolateral plasma membrane domains (fence function) (13). Adherens junctions form a continuous adhesion belt that connects the cytoskeletal elements of one mesothelial cell to those of another.
Mesothelial cells have the capacity to produce a variety of matrix macromolecules. Synthesis of the extracellular matrix is regulated by several growth factors, among which transforming growth factor-ß (TGF-ß) is the critical one (14). TGF-ß has been detected in CAPD effluent (15), peritoneal mesothelial cells are known to synthesize this growth factor (16), and a high glucose concentration enhances TGF-ß gene expression by cultured human peritoneal mesothelial cells (HPMC) (17,18,19,20,21). Moreover, it has been reported that TGF-ß inhibits mesothelial cell growth and significantly increases the permeability of mesothelial cell monolayers for FITC-labeled albumin (22). Both of these effects of TGF-ß are completely blocked by an anti—TGF-ß neutralizing antibody (17,22).
Little is known about the effects of high glucose levels and TGF-ß in peritoneal dialysis fluid on the intercellular junctions of mesothelial cells. Accordingly, this study investigated the effect of a high glucose concentration on the intercellular junctions and on TGF-ß production by HPMC, as well as the effect of TGF-ß and an anti—TGF-ß neutralizing antibody.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Harvesting of the omentum was permitted by the Medical Ethics Committee of Hiroshima University School of Medicine, and informed consent was obtained from each patient.
Identification of Mesothelial Cells
The cultured cells were examined under an inverted phase contrast
microscope. In addition, immunostaining was performed with monoclonal
antibodies for human cytokeratin (Dako, Kyoto, Japan), vimentin (Dako), desmin
(Dako), and human Factor VIII (Dako). Visualization was done with a Texas
red-labeled secondary antibody (Jackson Immuno Research Laboratories, West
Grove, PA).
Culture Media
Fifteen different culture media were tested. Standard M199 medium
containing 1.0% FCS (vol/vol), 100 U/ml penicillin, 100 µg/ml streptomycin,
and 2 mM L-glutamine was used as the control (glucose concentration, 5.6 mM).
In the experimental cultures, M199 medium was supplemented with D-glucose
(Kanto Chemical Co., Inc., Tokyo, Japan), D-mannitol (Sigma Chemical Co., St.
Louis, MO) at various concentrations (75 mM [1.35%], 140 mM [2.5%], and 222 mM
[4.0%]), or with recombinant TGF-ß1 (R&D Systems Inc., Minneapolis,
MN) at various concentrations (100, 500, and 1000 pg/ml). In some experiments,
the 222 mM D-glucose or 500 pg/ml TGF-ß1 media also contained various
concentrations of an anti—TGF-ß neutralizing antibody (1, 4, and 10
µg/ml; R&D Systems). To assess the effect of blocking TGF- ß
activity, media containing TGF-ß1 were preincubated with this
neutralizing antibody for 2 h at room temperature. All media were sterilized
by filtration (0.22 µm, Millex-GV, Millipore Corp., Bedford, MA) and then
stored at 4°C for use within 3 d.
Assay of Cell Proliferation
Cells were grown to confluent monolayers in culture flasks, harvested with
0.25% (wt/vol) trypsin-0.02% (wt/vol) ethylenediaminetetraacetate in PBS, and
resuspended in fresh M199 medium containing 10% FCS. Then the cells (2 x
104/100 µl per well) were seeded into 96-well tissue culture
plates coated with rat type I collagen (Becton Dickinson). After the cultures
were confirmed to be subconfluent by microscopy, cells were washed twice with
PBS and the medium was replaced with 100 µl of fresh standard medium. The
cells were then cultured for an additional 48 h to allow them to become
confluent and quiescent, washed twice with PBS, and used in the following
experiments. Cell viability was assessed using a cell counting kit (Dojindo,
Kumamoto, Japan) that used a colorimetric assay based on cleavage of the
water-soluble tetrazolium salt WST-1
[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt] to a formazan dye by mitochondrial dehydrogenase in viable
cells
(24,25).
Test solutions (100 µl/well) were added to confluent HPMC and incubated for
6, 12, 24, or 48 h, with the WST-1 solution (5 mM WST-1, 20 mM HEPES, and 0.2
mM 1-methoxy-5-methylphenazinium methylsulfate) being added at 10 µl/well
for the last 4 h. The absorbance was determined using an enzyme-linked
immunosorbent assay reader (SLT Lab Instruments, Salzburg, Austria) at an
excitation wavelength of 420 nm and an emission wavelength of 630 nm.
Immunostaining of Intercellular Junction Proteins
Cells were cultured on 24 x 24-mm glass coverslips coated with
porcine type I collagen (Nitta Gelatin Co., Osaka, Japan) in 60-mm culture
dishes (Becton Dickinson). After cultures were shown to be subconfluent by
microscopy, cells were washed twice with PBS, the medium was replaced with a
fresh standard medium, and the cells were incubated for another 48 h to reach
confluence. After the cells were washed twice more with PBS, they were then
cultured for 48 h in the test media. Next, the cells were fixed in cold
acetone/methanol at -20°C for 5 min, rehydrated in PBS, and blocked for 1
h in PBS containing 20% Block Ace (Dainippon Seiyaku Co. Ltd., Tokyo, Japan).
This was followed by overnight incubation with a primary antibody at 4°C.
The primary antibodies used were a rabbit antibody for ZO-1 (a tight junction
protein; Zymed Laboratories Inc., San Francisco, CA) at a dilution of 1:100
and a mouse antibody for ß-catenin (an adherens junction—associated
protein; Zymed Laboratories Inc.) at 1:100. After incubation with the primary
antibody, cells were washed five times with PBS. Then an appropriate secondary
antibody (1:100) labeled with FITC (Dako) or Texas red (Jackson Immuno
Research Laboratories) was applied for 1 h at room temperature, and the cells
were again washed five times with PBS. Stained specimens were examined under a
confocal laser scanning microscope (TCS-NT, Leitz, Weitzlar, Germany).
Western Blot Analysis of Intercellular Junction Proteins
Cells were cultured in 100-mm dishes. After the cultures were confirmed to
be subconfluent by microscopy, the cells were washed twice with PBS, the
medium was replaced with a fresh standard medium, and the cells were incubated
for an additional 48 h to reach confluence. After the cells were washed twice
with PBS, they were cultured for 48 h in the test media. Next, cells were
again washed twice with PBS and were lysed in a culture dish by adding 900
µl of TRIZOL® Reagent (Life Technologies). Cellular protein was
isolated, and 20 µg of total protein, as measured with the BCA protein
assay (Pierce, Rockford, IL), was dissolved in sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 3
min. Samples were subjected to SDS-PAGE on 8% gels and then transferred to
PVDF membranes (Millipore Corp.). The membranes were blocked for 1 h at room
temperature in PBS containing 1% Tween 20 (Katayama Chemical, Osaka, Japan)
and 20% Block Ace (PBST-20% Block Ace) and were incubated for 1 h with a
primary antibody:rabbit anti-ZO-1 (1:5000), mouse anti—E-cadherin
(Transduction Laboratories, Lexington, KY; 1:5000), or mouse
anti—ß-catenin (1:10,000). Then the membranes were washed (once for
15 min and twice for 5 min) in PBST-20% Block Ace and incubated for 1 h at
room temperature with an appropriate secondary antibody (1:5000) labeled with
horseradish peroxidase (Amersham Pharmacia Biotech, Buckinghamshire, England).
After incubation, the membranes were washed again (once for 15 min and four
times for 5 min), and the reaction products were detected by chemiluminescence
(Amersham Pharmacia Biotech). The intensity of each band was estimated using
NIH image software (version 1.61).
Assay of TGF-ß1 Production by HPMC
Supernatants were collected after the cells were cultured for 48 h with
various glucose or mannitol concentrations in 12-well tissue culture plates
(Becton Dickinson). The TGF-ß1 level in culture supernatants was
determined with an enzyme-linked immunosorbent assay kit (R&D Systems
Inc.) and was normalized for the number of cells. All experiments were
performed in duplicate. We also determined the TGF-ß1 level in each test
medium without HPMC.
Statistical Analyses
Results are expressed as the mean ± SD of three experiments, each
performed in duplicate. Data were examined using ANOVA (Fisher's protected
least significant difference test), and differences were considered
significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Standard Curve of WST-1 Colorimetric Assay
The absorbance showed a linear correlation with the number of cells (from
5.0 x 103 to 3.0 x 104 per well) in a
96-well culture plate (r = 0.966, P < 0.0001). Therefore,
the number of cells was calculated from the absorbance
(Figure 1).
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Proliferation of HPMC
There was a concentration-dependent decrease in cell proliferation after
culture for 48 h as the glucose concentration increased from 5.6 mM to 222 mM
(Figure 2). High glucose (222
mM) significantly depressed cell proliferation after 48 h when compared with
the lowest glucose concentration tested (5.6 mM). Similar results were also
seen after 6, 12, and 24 h of incubation. Mannitol also decreased cell
proliferation in a concentration-dependent manner, but the extent of
inhibition was less than that caused by glucose at the same osmolality. There
was a significant difference in cell proliferation after 48 h of incubation
with glucose or mannitol at 222 mM (P < 0.01).
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Immunostaining
In control cultures, the cell contour was clearly outlined by fluorescence
immunostaining for ZO-1 and ß-catenin. The contour immunofluorescence of
both ZO-1 and ß-catenin decreased and became discontinuous after
incubation with glucose, and this effect was concentration-dependent (Figures
3 and
4). In addition,
immunofluorescence for ß-catenin was markedly increased in the
perinuclear region. However, manitol caused only a slight decrease in the
expression of these proteins, and there was no discontinuity of staining along
the cell contour. All of these changes were more marked after 48 h of
culture.
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Western Blot Analysis
High glucose (222 mM) decreased the levels of ZO-1, E-cadherin, and
ß-catenin (Figure 5).
Mannitol also decreased ZO-1 and ß-catenin, but the extent of the change
was smaller than with glucose at the same osmolality. These changes all were
more marked after 48 h of culture.
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TGF-ß1 Production
In control cultures, confluent HPMC were shown to constitutively release
TGF-ß1, and the mean (± SD) TGF-ß1 level was 15.6 ±
0.4 pg/ml per 104 cells. The highest TGF-ß1 concentration
detected was 51.9 ± 2.6 pg/ml per 104 cells after culture
with high glucose (222 mM). However, the TGF-ß1 concentration was under
the detection limit of the assay in media without HPMC. Induction of
TGF-ß1 secretion by glucose or mannitol was concentration dependent and
was greater with glucose than with mannitol at the same osmolality (140 mM;
P < 0.01, 222 mM; P < 0.0001)
(Figure 6).
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Effect of Recombinant TGF-ß1
There was a concentration-dependent decrease of cell proliferation after
culture for 48 h with recombinant TGF-ß1 at concentrations from 100 to
1000 pg/ml (Figure 7). Contour
immunofluorescence for both ZO-1 and ß-catenin also decreased and became
irregular in a concentration-dependent manner
(Figure 8). Furthermore,
culture with recombinant TGF-ß1 decreased the levels of ZO-1, E-cadherin,
and ß-catenin, as shown by Western blotting
(Figure 9).
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Effect of the Anti—TGF-ß Neutralizing Antibody
Addition of the anti—TGF-ß neutralizing antibody to cultures
increased the proliferation of HPMC in the presence of 222 mM D-glucose or 500
pg/ml recombinant TGF-ß1 (Figure
10). We tested the neutralizing antibody at 1, 4, and 10 µg/ml
and found that it was most effective at 4 µg/ml. It also increased the
contour immunostaining for ZO-1 and ß-catenin in cells incubated with 222
mM D-glucose (Figure 11) or
500 pg/ml recombinant TGF-ß1 (Figure
12). Western blotting confirmed that the levels of ZO-1,
E-cadherin, and ß-catenin were increased by the neutralizing antibody
(Figures 13 and
14).
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Although cell proliferation and the expression of intercellular junctional proteins both were increased in cultures with high glucose (222 mM), they did not reach control levels. Thus, the blocking effect of the neutralizing antibody on the actions of 222 mM glucose was weaker than that against 500 pg/ml of recombinant TGF-ß1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we assessed the effects on HPMC of culture with glucose at 75 mM (1.35%), 140 mM (2.5%), and 222 mM (4.0%), which were the same concentrations as those in commercial peritoneal dialysates. In the presence of high glucose, we confirmed that there was a concentration-dependent decrease in cell proliferation, as has also been demonstrated in previous studies (17,18,19,20,21,29). We also assessed the effect of mannitol on cultured HPMC as an osmotic control. Even though a concentration-dependent decrease in cell proliferation was induced by mannitol, its effect was significantly weaker than that of glucose.
It has been reported that chronic exposure to dialysis fluid induces widening of the intercellular spaces between HPMC and detachment of these cells from the peritoneum (2,3,4,5). In the present study, we investigated the effects of high glucose on the intercellular junctions of HPMC. Immunostaining for ZO-1 and ß-catenin was linear and continuous along the cell contour in control cultures but became weaker and discontinuous in a concentration-dependent manner after culture with high glucose. Conversely, mannitol caused only a slight decrease of staining, with no discontinuity. Western blotting confirmed that high glucose decreased the levels of ZO-1, E-cadherin, and ß-catenin. Mannitol also decreased ZO-1 and ß-catenin, but the extent of the change was smaller.
It is generally believed that tight junctions can dynamically alter their structural and functional properties under different conditions and are subject to modulation by a variety of cellular and metabolic regulators (13,30,31,32,33,34). It has also been shown that the cadherin/catenin complex plays an important role in cell adhesion and signal transduction, as well as in initiation and maintenance of the structural and functional organization of cells and tissues (35,36,37,38,39). In the absence of functional catenins, adherens junctions do not form (37). The findings of the present study suggest that high glucose peritoneal dialysis fluid decreases the intercellular junctions of mesothelial cells and impairs their function, thus causing peritoneal hyperpermeability. The results of our cell proliferation, immunostaining, and Western blotting experiments demonstrated that the inhibitory effect of high glucose was not solely related to hyperosmolality but also to some property of glucose itself or its metabolic effects. Mannitol was less toxic than glucose in the present study, but the effects of metabolites of mannitol in humans are not well understood. In contrast, glucose is known to be safe, effective, cheap, and readily metabolized, so glucose is used clinically in dialysis fluids (40).
TGF-ß is a classical cytokine that plays a central role in regulating tissue repair and remodeling after injury, along with platelet-derived growth factor and basic fibroblast growth factor. Production and deposition of the extracellular matrix is an essential part of wound repair. TGF-ß is unique among the cytokines in stimulating matrix synthesis, inhibiting matrix degradation by proteases, and modulating matrix receptors to increase cell adhesion (41). In the present study, to investigate the mechanism behind the differing effects on cell proliferation and intercellular junctions of glucose and mannitol, we measured TGF-ß1 levels in the culture supernatant. A concentration-dependent increase of TGF-ß1 was observed with exposure of HPMC to glucose, so we also investigated the effects of TGF-ß1 on HPMC. We showed that cell proliferation, immunofluorescence for ZO-1 and ß-catenin, and the expression of ZO-1, ß-catenin, and E-cadherin were decreased in a TGF-ß1 concentration-dependent manner. Moreover, the present study showed that an anti—TGF-ß neutralizing antibody could inhibit the effects of both high glucose and TGF-ß1 on HPMC, although its inhibition of the actions of high glucose was weaker than its blocking effect on TGF-ß1.
These findings suggest that TGF-ß induced by high glucose inhibits mesothelial cell proliferation and causes damage to intercellular junctions. The difference in cytotoxicity between glucose and mannitol may be largely related to TGF-ß1. The anti—TGF-ß neutralizing antibody reversed these effects only partially, suggesting that peritoneal dialysate causes damage not only as a result of the effects of glucose and glucose-induced TGF-ß but also as a result of hyperosmolality per se.
In conclusion, the present study first clarified that (1) high glucose and TGF-ß caused damage to the intercellular junctions of HPMC (2), an anti—TGF-ß neutralizing antibody increased the expression of intercellular junctions, and (3) the effects of high glucose were caused not only by glucose itself but also by hyperosmolality and by glucose-induced TGF-ß1. Damage to the intercellular junctions of peritoneal mesothelial cells by a high glucose concentration in the dialysate may induce peritoneal hyperpermeability, mesothelial cell loss, and a progressive reduction in dialysis efficacy.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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