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NDT Advance Access originally published online on August 17, 2007
Nephrology Dialysis Transplantation 2008 23(1):126-135; doi:10.1093/ndt/gfm540
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


The TGF-β-induced gene product, βig-h3: its biological implications in peritoneal dialysis

Sun-Hee Park1, Soon-Youn Choi1, Mi-Hyung Kim1, Eun-Joo Oh1, Hye Myung Ryu1, Chan-Duck Kim1, In-San Kim2 and Yong-Lim Kim1

1Division of Nephrology and Department of Internal Medicine, 2Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, Kyungpook National University School of Medicine, Daegu, South Korea

Correspondence to: Yong-Lim Kim, MD, Division of Nephrology and Department of Internal Medicine, Kyungpook National University Hospital, 50 Samduk-dong, Jung-gu, Daegu 700-721, South Korea. Email: ylkim{at}knu.ac.kr



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. TGF-β is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-β induces βig-h3 in several cell lines, and βig-h3 may be a marker for biologically active TGF-β. However, no study has reported induction of βig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes.

Methods. We used cultured HPMCs to investigate the biological roles of βig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in βig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model.

Results. In vitro, TGF-β1 induced βig-h3 in cultured HPMCs, and βig-h3-mediated mesothelial cell adhesion occurred via {alpha}vβ3 integrin. βig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, βig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and βig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that βig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. βig-h3 protein expression was higher in the dialysis group.

Conclusion. In vitro, βig-h3 induced by TGF-β1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, βig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.

Keywords: βig-h3; fibrosis; high glucose; mesothelial cells; peritoneum; TGF-β1



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human peritoneal mesothelial cells (HPMCs) play an important role in the functional and morphological maintenance of the peritoneum. In peritoneal dialysis (PD), the repeated exposure of the peritoneum to hyperglycaemic, acidic and hyperosmotic dialysis solutions leads to a loss of peritoneal mesothelial cells and peritoneal fibrosis. Therefore, in patients undergoing PD, the peritoneal mesothelial cells are in a state of continuous repair [1] through continuous cell proliferation and migration. During long-term PD, the peritoneal membrane undergoes morphological changes such as interstitial fibrosis, the disappearance of mesothelial cells, vascular wall thickening, vasodilatation and increased angiogenesis [2,3]. These changes are linked to the functional deterioration of the peritoneal membrane and ultrafiltration failure.

It is well known that high glucose levels stimulate TGF-β1 in the HPMCs [4,5], and TGF-β is a key fibrogenic growth factor for mediating peritoneal fibrosis [5,6]. However, TGF-β is secreted in its latent form and undergoes extracellular modifications to become active [7,8]. Therefore, TGF protein and mRNA have some limitations as active biological markers of tissue fibrosis [9]. In contrast, the TGF-β-induced gene product βig-h3 has been suggested as a biological marker for active TGF-β [9].

Currently, there is no reported evidence that βig-h3 is induced in the peritoneal mesothelial cells and involved in peritoneal membrane changes during long-term PD. However, βig-h3 is known to function as a cell adhesion substrate by interacting with several integrins, meaning that it has wound-healing activity [10]. Therefore, we hypothesized that βig-h3 mediates peritoneal mesothelial cell adhesion and migration, thus maintaining the integrity of the peritoneal mesothelial cells. We also hypothesized that sustained injuries and repair processes trigger fibrosis cascades in the peritoneal membrane and that βig-h3 along with TGF-β has a central role in the peritoneal membrane fibrosis that develops during long-term PD. To test these hypotheses, we investigated in vitro the biological roles of βig-h3 in mesothelial cell injury and repair during PD and evaluated the expression of βig-h3 after exposures to high glucose levels in a chronic infusion animal model of PD.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
The in vitro study
Cell culture and induction of βig-h3
The HPMCs were collected from omental tissue that had been obtained with informed consent from patients undergoing abdominal surgery. The cells were isolated by enzymatic disaggregation with trypsin–EDTA as previously described [11], and a second passage of cells was employed in this study. The HPMCs were characterized by their morphology and biochemical characteristics using immunofluorescence with monoclonal antibodies to human cytokeratin 8/18 (Zymed, South San Francisco, CA), vimentin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), desmin (Santa Cruz Biotechnology, Inc.) and von Willebrand factor (Chemicon, Temecula, CA).

The cells were plated confluently and allowed to rest in 1% fetal calf serum (FCS) containing M-199 media for 24 h for growth synchronization. They were then exposed to recombinant human TGF-β1 (R & D Systems, Minneapolis, MN) at concentrations of 0.5–5 ng/ml for 72 h. Supernatant samples were obtained at each concentration of TGF-β1. To measure βig-h3 by ELISA, additional HPMCs were exposed to 1 ng/ml of TGF-β1, and supernatant samples were obtained at 12, 24, 36, 48, 60 and 72 h following exposure to TGF-β1.

The function analysis of HPMCs
Cell adhesion and spreading assay
A cell adhesion assay was completed using previously described methods [12,13] in which 96-well micro culture plates (Coaster, Cambridge, CA) were coated with wild-type βig-h3; βig-h3 domain I, II, III, IV; purified human plasma fibronectin (pFN) as a positive control and BSA as a negative control. All the plates were diluted in PBS at 4°C overnight and then rinsed three times in PBS. Uncoated surfaces were blocked with PBS containing 2% heat-inactivated BSA for 2 h at room temperature. The plates were rinsed again, and 2.5 x 104 of HPMCs were added to each well in 100 µl of culture medium. After incubation for 1 h at 37°C, unattached cells were removed with two rinses in PBS. For quantification of the attached cells, a hexosaminidase assay was completed. These cells were incubated for 1 h at 37°C in a 50-mM citrate buffer (pH 5.0) containing 3.75 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma, St Louis, MO) and 0.25% Triton X-100. The reaction was stopped and colour was developed by the addition of 50 mM glycine buffer (pH 10.4) containing 5 mM EDTA. The absorbance was measured at 405 nm in a Bio-Rad model 550 microplate reader (Bio-Rad, Hercules, CA).

For the cell spreading assay, 3 x 104 cells were applied to the respective substrates (2% BSA, 10 µg/ml of fibronectin and 10 µg/ml of wild-type βig-h3) in 96-well culture plates. After 2 h, the attached cells were fixed with 8% glutaraldehyde (Sigma) and stained with 0.25% crystal violet (Sigma) in 20% methanol (w/v). The cells were then observed under Nikon TE300 light microscopy (Nikon, Japan).

The assessment of wound healing following a scratching and cell migration test
HPMCs were cultured in a 35-mm cell culture dish coated with the respective substrates (BSA, fibronectin, and βig-h3). After 24 h, the cultured cell monolayer was scratched with a sterile yellow pipette tip. After applying a PBS wash, we captured the cell migration and wound-healing process from the wound edge over 6 h using a Samsung CCD camera (Samsung, Korea) connected to a computer. For more quantitative data, we summed the length of movement of a cell that occurred in each culture dish over 6 h by a MetaMorph program (Universal Imaging CorporationTM, Carl Zeiss, Inc., Downingtown, PA).

Flow cytometry for detecting HPMC integrin profiles
For the flow cytometry analysis, cells at confluence were detached with a gentle treatment of 0.25% trypsin and 0.05% EDTA in PBS. They were washed and then incubated with the antibodies for 1 h at 4°C. The cells were then incubated again with 10 µg/ml of affinity-purified and fluorescein-labelled secondary antibodies for 30 min at 4°C and analysed on a flow cytometer FACSCalibur system (Becton Dickinson, San Jose, CA) equipped with a 5-W argon laser at 488 nm. The following anti-human integrin monoclonal antibodies were used for the FACS analysis: {alpha}1 (FB12), {alpha}2 (P1E6), {alpha}3 (ASC-1), {alpha}v (P3G8), β1 (12G10), β3 (B3A), {alpha}vβ3 (LM609), {alpha}vβ5 (P1F6) and {alpha}5β1 (HA5) from Chemicon.

Inhibition assay: a function-blocking assay using monoclonal antibodies to integrins
To identify the HPMC receptor for βig-h3, monoclonal antibodies to different types of integrins (Chemicon, Temecula, CA) were individually pre-incubated with HPMCs (2 x 105 cells/ml) in 0.05 ml of incubation solution at 37°C for 30 min. The pre-incubated cells were transferred onto plates pre-coated with βig-h3 proteins and then incubated further for 30 min at 37°C. The reaction was stopped, and colour was developed by the addition of 50-mM glycine buffer (pH 10.4) containing 5 mM EDTA. The absorbance was measured at 405 nm using an ELISA reader. The anti-human integrin monoclonal antibodies used for the function-blocking assay were {alpha}1 (FB12), {alpha}2 (P1E6), {alpha}3 (ASC-1), {alpha}5 (P1D6), {alpha}6 (GoH3), {alpha}v (P3G8), β1 (6S6), β3 (B3A), β4 (ASC-3), {alpha}vβ3 (LM609) and {alpha}vβ5 (P1F6) from Chemicon.

The anti-βig-h3 antibody and enzyme-linked immunosorbent assay (ELISA)
The anti-human βig-h3 antibody we used has been previously described [14]. The peritoneal fluid and cell culture supernatant were collected and centrifuged at 1500 r.p.m. for 5 min. The TGF-β1 levels in the peritoneal fluid were analysed using a human TGF-β ELISA kit (R & D Systems). The βig-h3 levels in the peritoneal fluid and cell culture supernatant were analysed by competitive ELISA. The 96-well plastic flat microtitre plates (Corning, NY) were coated with wild-type βig-h3 protein in 20-mM carbonate–bicarbonate buffer (pH 9.6) with 0.02% sodium azide and left overnight at 4°C. The plates were then rinsed three times in PBS–0.05% Tween-20 (PBST) and kept at 4°C. The peritoneal fluid and cell culture supernatant were pre-incubated with anti-βig-h3 antibodies (diluted to 1: 2000 in PBS-T) in 96-well plastic round microtitre plates for 90 min at 37°C. These pre-incubated samples were transferred to pre-coated plates and incubated for 30 min at room temperature. The plates were then rinsed three times in PBST and incubated for 90 min at room temperature with peroxidase-conjugated anti-rabbit IgG antibodies (Amersham, Santa Cruz Biotechnology, CA) diluted to 1: 2000 in PBST. The plates were again rinsed three times in PBST and then incubated in the dark for 60 min at room temperature with the substrate solution (after it had been dissolved in methanol at a concentration of o-phenylenediamine 10 mg/ml and diluted 1: 100 with deionized water). Next, 0.01 ml of 30% H2O2 was added per 100 ml. After the reaction was stopped with 8N H2SO4, the absorbance was read at 492 nm.

Animal studies
We used a chronic infusion model of animal PD, as previously described [15]. Briefly, 16 male Sprague–Dawley rats (250–300 g) were divided into two groups of eight animals each, and permanent PD catheters were inserted. Group C consisted of the control rats, which had catheters without infusion of the dialysis solution. Group T was infused twice daily with 25 ml of 4.25% glucose dialysis solution (Dianeal®, Baxter Healthcare Ltd, Singapore) for 8 weeks. We assessed the peritoneal transport rate by measuring the dialysate-to-plasma ratio (D/P) of solute at baseline and then at days 22 (3 weeks), 43 (6 weeks) and 57 (8 weeks) following the initiation of dialysis. During every test, the drained dialysate was collected to measure TGF-β1 and βig-h3. Each week, the dialysate effluent was cultured to assess for peritonitis. At the end of 8 weeks, all animals were sacrificed to obtain their peritoneal tissues. We then performed morphometric analysis to evaluate the structural changes of the peritoneal membrane and completed immunohistochemical staining and immunoblotting analyses to evaluate the expression of βig-h3.

Peritoneal transport rate
To analyse the functional changes of the peritoneum, we assessed the peritoneal transport rate by measuring the dialysate-to-plasma ratio (D/P) of solute at 2 h after dialysate infusion using 25 ml of 4.25% glucose dialysis solution (Dianeal®, Baxter). The dialysate effluent samples that were obtained were centrifuged at 1500 r.p.m. for 5 min, and the supernatant was removed for analysis. The glucose concentration in the dialysate was assessed by the glucose oxidase method. Urea was measured using enzymatic methods via an automated analyzer (Hitachi 7600-110; Hitachi, Japan). The total protein amounts in the plasma and dialysate were determined by the Biuret method (Hitachi 7600-110; Hitachi, Japan). The rates at which the protein and urea nitrogen were transported were assessed by dividing the dialysate value by the plasma value (D/P) (the D/Pprotein values were expressed as D/P multiplied by 1000). The rate of glucose transport was measured by calculating D/D0, where D is the glucose concentration in the dialysate and D0 is the glucose concentration in the dialysis solution before its infusion into the peritoneal cavity.

Morphometry
The thickness of the parietal membrane, including the mesothelial cells and submesothelial interstitium was measured by sections from the parietal peritoneum using light microscopy employing 40x flat-field objectives. The thickness of the submesothelial matrix was measured using the computer program Optimas 6.0 (Optimas Corp., Bothell, WA).

Immunohistochemistry
The parietal peritoneum obtained at the end of week 8 was embedded in paraffin. For the immunoperoxidase labelling, endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal the antigens, sections were put in 1 mmol/l Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and were heated using a microwave oven for 10 min. Non-specific binding of the immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl for 30 min followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin and 0.2% gelatine. The sections were then incubated overnight at 4°C with polyclonal anti-mouse βig-h3 antibody that had been diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100 (1:2000). Labelling was visualized with horseradish peroxidase-conjugated secondary antibody (P448, 1:200, DAKO, Glostrup, Denmark), and immunolabelling controls were performed using rabbit IgG. Microscopy was carried out using a Zeiss light microscope (Axioplan2 Imaging, Carl Zeiss, Inc., Hallbergmoss, Germany).

Immunoblotting analyses
We performed immunoblotting analyses from all animals in group C (n = 6) and group T (n = 5). Using peritoneal tissue homogenate, we performed immunoblotting with polyclonal anti-human βig-h3 antibody (1: 5000, Regen Co., Ltd, Seoul, Korea) and anti-TGF-β antibody (1: 5000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein (10 µg) from each sample was solubilized at 100°C for 5 min and run on 10% polyacrylamide mini-gels (Bio-Rad Mini Protean II). The gels were then subjected to Coomassie staining to ensure identical loading. The sites of the antigen–antibody reaction were visualized using HRP-conjugated secondary antibodies (P447 or P448, diluted 1: 5000, DAKO, Glostrup, Denmark) with an enhanced chemiluminescence (ECL) system and by exposure to photographic film (Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech, Little Chalfont, UK). The band densities were quantified by computer scanning of films. We used Scion Image (Scion, Frederick, MD) for computer analysis of band pixel intensities.

Statistics
The Student's t-test was used where indicated for comparisons. When the data were not distributed normally, the Mann–Whitney rank sum test was used to compare the two groups. The serial changes of parameters were assessed with one-way repeated measures ANOVA tests. The Pearson's correlation coefficient was applied for correlation analysis. A P-value of less than 0.05 was considered to be significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
The in vitro study
βig-h3 expression is stimulated by TGF-β1 in HPMCs (Figure 1)
The cultured HPMCs were incubated with various concentrations of TGF-β1 and the βig-h3 levels were measured in the cell supernatant by ELISA. As shown in Figure 1A, TGF-β1 induced βig-h3 expression in the HPMCs in a dose-dependent manner. The maximal effect was observed at 2 ng/ml of TGF-β1. The βig-h3 levels also increased in a time-dependent manner (Figure 1B).


Figure 1
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  		Fig. 1. Induction of βig-h3 on human peritoneal mesothelial cells by the stimuli of TGF-β1. The βig-h3 protein levels of the cell supernatant were estimated by ELISA and expressed as nanogram per milligram of protein. βig-h3 protein was increased in dose (A) and time (B)-dependent manners via TGF-β1 stimuli. The data are expressed as mean ± SEM of at least three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs control.

 
βig-h3: a new adhesion molecule in HPMCs (Figure 2)
βig-h3 supported the adhesion and spreading of HPMCs in a dose-dependent manner (data not shown), and its activities were comparable to those of fibronectin (Figure 2A). Each of the four fas-1 domains in βig-h3 was active in mediating HPMC adhesion except the first domain (Figure 2A). After incubation, the cells were rinsed with PBS, fixed in 8% glutaraldehyde, and stained with crystal violet. The cell adhesion and spreading observed with βig-h3 were comparable to that of fibronectin (Figure 2B).


Figure 2
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  		Fig. 2. Adhesion of HPMCs on each substrate. βig-h3 mediates adhesion and spreading of HPMCs. HPMCs were seeded onto surfaces coated with 2% BSA and each substrate. After incubation, attached cells were quantified by a hexosaminidase assay (A). Cells were fixed in 8% glutaraldehyde and stained with crystal violet (B). Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001 vs BSA. BSA, bovine serum albumin; FN, fibronectin; D1, first fas-1 domain; D2, second fas-1 domain; D3, third fas-1 domain; D4, fourth fas-1 domain.

 
βig-h3: a new migratory molecule in HPMCs (Figures 3 and 4)
The wound that we created after scratching the cell monolayer was closed by cell migration from the wound edge; within 6 h the wound in the fibronectin-coated cell culture dish was almost completely closed, whereas the wound in the BSA-coated dish was not. The wound closure in the βig-h3-coated dish was faster than in the BSA-coated dish, but it was slower than in the fibronectin-coated dish (Figure 3). The HPMCs were seeded to 2% BSA-, βig-h3- and fibronectin-coated culture dishes (Figures 4A–C). Using a MetaMorph program to gather more quantitative data, we summed the length of a cell's movement over 6 h (Figure 4D). The cell movement was highest in fibronectin (81.1 ± 7.3 pixels); βig-h3 (59.5 ± 3.2 pixels) also enhanced cell migration (P < 0.001) compared with BSA (0 pixels) (Figure 4D). Taking together the results from the scratching and cell migration tests, we infer that βig-h3 enhanced wound healing compared with the control.


Figure 3
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  		Fig. 3. The wound-healing process after scratching the cell monolayer. The wounds closed by cell migration from the wound edge. Within 6 h, the wound was almost closed in the fibronectin-coated cell culture dish but was not closed in the BSA-coated dish. The wound closure in the βig-h3-coated dish was faster than in the BSA-coated dish but slower than in the fibronectin-coated dish.

 

Figure 4
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  		Fig. 4. Migration of HPMCs on each substrate. HPMCs were seeded to 2% BSA- (A), βig-h3– (B), and fibronectin- (C) coated culture dishes, and the moving distance of a cell was measured using the MetaMorph program (D). The distance of cell migration was highest in fibronectin, followed by βig-h3. Compared to BSA, migration was significantly higher in both fibronectin and βig-h3. The data are expressed as mean ± SEM. ***P < 0.001 vs BSA.

 
{alpha}vβ3 integrin is a functional receptor for βig-h3 in HPMCs (Figures 5 and 6)
FACS analyses showed that the HPMCs expressed several integrins, including the {alpha}vβ3 integrin on their cell surfaces. There was strong expression of {alpha}3 and β1 integrins; moderate expression of {alpha}1, {alpha}2, {alpha}v, β3, {alpha}vβ3 and {alpha}5β1 integrins; and weak expression of {alpha}vβ5 integrin on HPMCs (Figure 5). The function-blocking assay using monoclonal antibodies to integrins revealed that the functional receptor of HPMCs for βig-h3 was the {alpha}vβ3 integrin (Figure 6).


Figure 5
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  		Fig. 5. Integrin profiles of HPMCs by flow cytometry analysis. There was high expression of {alpha}3 and β1 integrins; moderate expression of {alpha}1, {alpha}2, {alpha}v, β3, {alpha}vβ3 and {alpha}5β1 integrins; and weak expression of {alpha}vβ5 integrin on HPMCs.

 

Figure 6
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  		Fig. 6. Anti-integrin function-blocking antibodies for {alpha}v and β3 blocked the adhesion of HPMCs on βig-h3. Cells were pre-incubated with each antibody, and attached cells were quantified by a hexosaminidase assay. The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs none.

 
Animal studies
At the end of the study (8 weeks), analysis was performed in 11 of the 16 rats initially participating in the study (group C, n = 6; group T, n = 5). The study could not be completed for two group-C rats because of dialysate leakage (n = 1) and death (n = 1), or for three group-H rats because of death (n = 1) and peritonitis based on positive dialysate culture (n = 2).

Functional change of the peritoneal membrane with time on PD
We used a chronic infusion model of animal PD to assess the functional changes of the peritoneal membrane. At 8 weeks, the D/D0 glucose values were significantly lowered (P < 0.05) in Group T compared with Group C with 0.144 ± 0.014 and 0.303 ± 0.010, respectively. This difference was found to be statistically significant from 6 weeks. In addition, the ratio of dialysate protein to plasma protein (D/Ptotal protein, expressed as D/P x 1000) was significantly increased (P < 0.05) in Group T compared with Group C at 8 weeks (18.662 ± 1.014 and 8.209 ± 0.435, respectively). This difference was significant from 3 weeks. Also at 8 weeks, the ratio of the dialysate urea to the plasma urea (D/Purea) was significantly increased (P < 0.05) in Group T compared with Group C with 0.684 ± 0.038 and 0.526 ± 0.027, respectively.

Morphological changes of the parietal peritoneum
In the trichrome-stained peritoneal tissue, the thickness of the parietal peritoneum was significantly higher (P < 0.05) in group T compared with group C at 22.14 ± 4.26 µm and 9.95 ± 0.88 µm, respectively.

Levels of TGF-β1 and βig-h3 proteins in the dialysate effluent (Figure 7)
The dialysate TGF-β1 level increased (P < 0.05) in group T compared with group C at 8 weeks with 279.33 ± 59.64 pg/ml and 35.82 ± 9.20 pg/ml, respectively. This increase was statistically significant from 6 weeks (Figure 7A). In addition, the dialysate βig-h3 levels increased (P < 0.05) in group T compared with group C at 8 weeks with 190.09 ± 7.38 ng/ml and 117.42 ± 7.31 ng/ml, respectively. This increase was statistically significant from 3 weeks (Figure 7B), but plasma levels of TGF-β1 and βig-h3 did not differ between groups (data not shown). In a serial change, both the dialysate TGF-β1 and βig-h3 levels increased significantly until 3 weeks (P < 0.05) and remained at a plateau thereafter until 8 weeks.


Figure 7
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  		Fig. 7. Changes of TGF-β1 and βig-h3 in the drained dialysate. TGF-β1 (A) and βig-h3 (B) proteins in the dialysate were measured by ELISA. The black lines represent the parameters for Group C and the dashed lines for Group T. Dialysate TGF-β1 and βig-h3 were significantly higher in Group T compared with Group C after 8 weeks of dialysis solution infusion. The data are expressed as mean ± SEM. *P < 0.05 vs Group C.

 
The D/P ratio of βig-h3 in dialysis group was higher than the expected value based on the D/P ratio of albumin (mean D/P βig-h3, 0.250 in Group C and 0.505 in Group T; mean D/P albumin, 0.0016 in Group C and 0.0003 in Group T). These results indicate that βig-h3 levels in dialysate effluent are higher than those transported from the circulation to peritoneal cavity.

Correlation of the dialysate TGF-β1/βig-h3 and peritoneal transport (Figure 8)
Figure 8A and C shows that the levels of dialysate TGF-β1 and βig-h3 were negatively correlated with D/D0 glucose at 8 weeks (r = –0.84, P < 0.005 and r = –0.84, P < 0.005, respectively). Also, Figure 8B and D shows that the levels of dialysate TGF-β1 and βig-h3 were positively correlated with D/Ptotal protein at 8 weeks (r = 0.84, P < 0.005 and r = 0.92, P < 0.001, respectively).


Figure 8
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  		Fig. 8. Correlations of dialysate TGF-β1 and βig-h3 vs peritoneal transport characteristics (D/D0 glucose and D/Ptotal protein). The parameters were TGF-β1 vs D/D0 glucose (A), TGF-β1 vs D/P total protein (B), βig-h3 vs D/D0 glucose (C) and βig-h3 vs D/P total protein (D). There were significant negative correlations between dialysate TGF-β1 and D/D0 glucose and dialysate βig-h3 and D/D0 glucose, respectively, and there were significant positive correlations between dialysate TGF-β1 and D/Ptotal protein and dialysate βig-h3 and D/Ptotal protein, respectively.

 
Expression of βig-h3 in the peritoneal tissue (Figure 9)
Immunoperoxidase labelling showed that βig-h3 was expressed in the mesothelium and submesothelial matrix of the parietal peritoneum (Figure 9A) and in the endothelium of the capillaries and veins of the omentum (Figure 9B). The immunoblotting analyses showed that the βig-h3 expression was higher in Group T compared with Group C (P < 0.05) (Figure 9C).


Figure 9
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  		Fig. 9. The expression of βig-h3 in peritoneal tissue by immunoperoxidase labelling and immunoblotting analyses. βig-h3 was expressed in the mesothelium and the submesothelial matrix of the parietal peritoneum (A), as well as in the endothelium of the capillaries and veins in omentum (B). Expression of βig-h3 protein was higher in Group T compared with Group C (C). Immunoblotting analyses were performed from all animals in Group C (n = 6) and T (n = 5) (C). Magnificationx 630 (A), x100 (B). *P < 0.05 vs Group C.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
βig-h3 is a secretory protein composed of fasciculin I-like repeats containing sequences that allow for the binding of integrins and glycosaminoglycans in vivo. The expression of βig-h3 is highly induced by TGF-β in several cell types such as the proximal tubular epithelial cells, dermal fibroblasts and keratinocytes [12,16,17]. βig-h3 is also known to affect cell growth [18] and differentiation [19]. In a previous report, we showed that the expression of βig-h3 was up-regulated in the diabetic rat kidney and human proximal tubular epithelial cells when they were treated with high levels of glucose [14]. Also, in a clinical study, we reported that urinary levels of βig-h3 protein were elevated in patients with diabetic nephropathy [20].

The in vitro data presented in this report show that TGF-β1 induces βig-h3 in HPMCs and that βig-h3 plays a role in adhesion and migration of HPMCs and at least in part in enhancing wound healing. In our in vivo study using a chronic infusion model of animal PD, dialysate levels of βig-h3 significantly increased from the initiation of PD at 3 weeks and remained at a plateau until 8 weeks. In addition, dialysate βig-h3 levels and expression of βig-h3 in the peritoneal tissue were higher in the dialysis group than in the control group. These findings suggest that up-regulation of βig-h3 begins as early as 3 weeks in response to exposure to a high-glucose dialysis solution with catheter insertion and maintains increased levels thereafter.

From the initiation of PD, peritoneal mesothelial cells are injured from catheter insertion and subsequent exposures to high glucose dialysates. Our data suggest that βig-h3 plays an important role in repairing mesothelial cell injuries via cell adhesion and migration to denuded areas of the peritoneum. However, repeated injuries and repair processes are linked to fibrosis cascades in the peritoneal tissue, ultimately leading to peritoneal fibrosis and ultrafiltration failure. In our data, along with alterations in the peritoneal structure and functions during PD, the βig-h3 concentration in the dialysate effluent was positively correlated with the peritoneal solute transport. It seems that the correlations between transport and dialysate TGF-β1 and βig-h3 are related not only to the transport from circulation but also to local production in the dialysis group. Zweers et al. [21] reported that VEGF and TGF-β1 in dialysate can reflect the local production of these markers, using comparison of expected and measured dialysate-to-serum ratios of macromolecules and TGF-β1 or VEGF. The magnitude of locally produced TGF-β1 or βig-h3 could be estimated by comparison of their D/P ratios to those of macromolecules known to be transported across the peritoneal membrane without production.

In this report, we compared the D/P ratio of albumin and βig-h3 because both have similar molecular weights (albumin, 69 kD; βig-h3, 68–70 kD). The D/P ratio of βig-h3 in the dialysis group was higher than the expected value based on the D/P ratio of albumin. This finding suggests that βig-h3 levels in dialysate effluent are higher than those transported from the circulation to the peritoneal cavity, implying local production of βig-h3 in the peritoneum. However, Zweers et al. [21] reported that the TGF-β1 in the dialysate was not correlated with the peritoneal transport parameters in PD patients but that the dialysate VEGF was. They suggested that the lack of a relationship between the peritoneal transport parameters and the dialysate TGF-β1 may have been caused by a biologically inactive TGF-β1. In our results, both the dialysate TGF-β1 and βig-h3 were correlated with peritoneal transport. The correlation of βig-h3 was at least similar to or higher than that of TGF-β1.

In the immunohistochemical staining for βig-h3 in the peritoneal tissue, positive staining was identified in the mesothelium and submesothelial matrix of the parietal peritoneum, as well as in the vascular endothelium in the omentum of rats dialyzed with glucose-containing PD solutions. Based on this evidence and the increase of dialysate TGF-β1 and βig-h3 attributed to local production in the peritoneum, we concluded that dialysate βig-h3 levels were associated with the functional deterioration of the peritoneal membrane.

Our findings showed that βig-h3-mediated adhesion was blocked by antibodies against the {alpha}v and β3 integrin subunits. This result suggests that {alpha}vβ3 integrin is a functional receptor for βig-h3 in the mesothelium and that mesothelial cell attachment to βig-h3 is mediated by {alpha}vβ3.

The peritoneum is involved in the pathophysiology of many disease processes, such as cancer metastasis, endometriosis and chronic inflammatory states. In these states, the mesothelium may function not only as a barrier, but also as a substrate for ectopic cell attachment. The integrins play an essential role in cell-to-cell interaction, and their expression can be altered in these pathologic conditions. Tietze et al. [22] showed that mesothelial cells from human omentum majus (HOMC) strongly expressed β1, β3, {alpha}2, {alpha}3, {alpha}5 and {alpha}v. There was weak expression of {alpha}1 and {alpha}6 and no expression of {alpha}4. The data showed that HOMC attachment to fibronectin was mediated by {alpha}5β1, {alpha}vβ1 and {alpha}vβ3. In the current study, there was strong expression of {alpha}3 and β1 integrins; moderate expression of {alpha}1, {alpha}2, {alpha}v, β3, {alpha}vβ3 and {alpha}5β1 integrins; and weak expression of {alpha}vβ5 integrin on cultured HPMCs.

The role of mesothelial integrins is currently not well defined within the area of PD. It is not well known whether high glucose or TGF-β1 can modulate the expression of integrins in the peritoneal mesothelial cells, or if integrin profiles could be altered in detached peritoneal mesothelial cells during PD. Rout et al. [23] demonstrated that TGF-β1 modulates integrin expression in human peritoneal fibroblasts. They showed that TGF-β1 significantly up-regulated expression of the {alpha}5, {alpha}v and {alpha}6 integrin subunits and modulated their expression pattern; higher levels of these subunits were identified in the focal contacts of peritoneal fibroblasts.

In summary, βig-h3 may be associated not only with adhesion and migration of peritoneal mesothelial cells during the wound-healing process in vitro, but also with the functional deterioration of the peritoneal membrane in the chronic infusion model of PD. These results suggest that βig-h3 induced by TGF-β1 in HPMCs may play an important role in PD.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2004-002-E00079).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 5. 1.07
Accepted in revised form: 17. 7.07


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Perit. Dial. Int., February 1, 2009; 29(Supplement_2): S123 - S127.
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