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NDT Advance Access originally published online on June 24, 2008
Nephrology Dialysis Transplantation 2008 23(11):3494-3500; doi:10.1093/ndt/gfn353
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Effects of epithelial-to-mesenchymal transition on acute stress response in human peritoneal mesothelial cells

Regina Vargha, Thorsten O. Bender*, Andrea Riesenhuber, Michaela Endemann, Klaus Kratochwill and Christoph Aufricht

Kinderdialyse, Department of Pediatrics, AKH Vienna, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria

Correspondence and offprint requests to: Christoph Aufricht, Kinderdialyse, Department of Pediatrics, AKH Wien, Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel: +43-1-40400-3232; Fax: +43-1-40400-3238; E-mail: christoph.aufricht{at}meduniwien.ac.at



   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. During peritoneal dialysis, mesothelial cells undergo epithelial-to-mesenchymal transition (EMT), resulting in markedly altered protein expression. This potentially includes heat-shock proteins (HSP), the main effectors of cellular repair. Thus, chronic cellular processes, such as EMT, may influence acute stress responses and thus survival of mesothelial cells following non-lethal injury upon exposure to peritoneal dialysis fluid (PDF).

Methods. In this study, we investigated the effects of EMT on acute stress responses and cytoresistance in human peritoneal mesothelial cells. In vivo EMT was defined as a fibroblast-like growth pattern in mesothelial cells grown from peritoneal effluents, and in vitro EMT was induced by TGF-β1 in mesothelial cells grown from omental tissue. Morphologic EMT was validated by western blot analysis of EMT marker proteins (ezrin, alpha-SMA). Expression of HSP and cellular survival was evaluated in a simple in vitro PDF exposure model.

Results. In vivo and in vitro EMT resulted in marked effects on phenotypes of mesothelial cells, associated with differential HSP expression. In vivo ‘chronic’ EMT resulted in lower expression of HSP-27 and HSP-72, whereas in vitro ‘acute’ EMT was associated with increased HSP-27 and decreased HSP-72 expression. Following PDF exposure, there were no effects of in vivo EMT on the stress induction of HSP, and survival of epithelial versus fibroblast-like phenotypes was comparable. The non-stressful induction of HSP-27 following TGF-β1 pretreatment resulted in the attenuated stress induction of HSP, and in improved survival in following PDF exposure.

Conclusions. Taken together, this study confirms that mesothelial cells are not ‘unchanged’ or ‘static targets’ during the clinical course of PD treatment. The cellular processes during EMT play a complex role in acute cellular stress response and cytoresistance of mesothelial cells. Sequential analysis at different stages of EMT will be essential to provide more insights on cytoprotective cellular processes in in vitro and in vivo models of PD.

Keywords: epithelial-to-mesenchymal transition; heat-shock proteins; mesothelial cells; peritoneal dialysis; stress response



   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Peritoneal dialysis (PD) is a frequently used mode of renal replacement therapy in end-stage renal disease. Studies on the clinical outcome of PD, however, reported a risk of technical failure of up to 30% [1]. Pathomorphologic studies confirmed progressive peritoneal damage in patients after long-term PD [2,3]. Toxic physicochemical properties of PD fluids (PDF), such as acidic pH, high lactate, high glucose and glucose degradation products, are currently regarded as major culprits for peritoneal injury [4,5].

Recently, we and others have demonstrated that these stressors not only disrupt mesothelial cell homeostasis and cause injury but also induce a specific stress response that counteracts injury [6–9]. Overexpression of heat-shock proteins (HSP), the major effectors of the cellular stress response, upon pretreatment or transient transfection resulted in significant protection of mesothelial cell cultures against PDF exposure [10].

Mesothelial cells represent no ‘unchanged’ or ‘static’ targets during PD treatment. They frequently undergo progressive transdifferentiation from an epithelial phenotype to a mesenchymal phenotype, resulting in the epithelial-to-mesenchymal transition (EMT) process [11,12]. Given the markedly altered gene expression during EMT, and the recently shown cytoprotective effects of HSP against PDF exposure, the interplay between EMT and stress response will likely be relevant for mesothelial cell damage during PD [13–15]. Dependent on the down- or upregulation of HSP expression upon EMT, transdifferentiated cells might be more or less susceptible to PDF exposure [10,16].

In this study, we hypothesized that PD-induced chronic cellular processes such as EMT might influence the acute stress responses of mesothelial cells to PDF exposure. Therefore, the effects of EMT on HSP expression and cellular viability were tested in mesothelial cells grown either from peritoneal effluents from patients on chronic PD or grown from omental tissue, using a simple in vitro PDF incubation and recovery model.



   Patients and methods
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 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
In vivo EMT of peritoneal-effluent-derived mesothelial cell cultures (Figure 1)
Mesothelial cell cultures were grown from randomly collected peritoneal effluents in children treated with peritoneal dialysis with commercial PD fluid at our institution. Children had to be clinically stable and for >3 months free of peritonitis at the time of effluent collection.


Figure 1
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  		Fig. 1 This scheme shows the design of the study. Effects of EMT were assessed in peritoneal-effluent-derived cell cultures (empty symbols to the left) and omental tissue-derived mesothelial cell cultures (grey symbols to the right). In vivo EMT was validated by western analysis in a total of 31 peritoneal-effluent-derived cell cultures (from nine children on chronic peritoneal dialysis). Twenty-three cultures (in seven children) demonstrated epithelial (squares) and eight cultures (in five children) demonstrated non-epithelial (diamond) phenotypes; all of these cultures were used for basal HSP analysis to assess effects of EMT. Three children were included in both groups, as their mesothelial cells underwent EMT during the follow-up. Ten epithelial and five non-epithelial cell cultures were also available for PDF exposure to assess stress responses and cytoresistance. In vitro EMT was induced by TGF-β1 incubation in six cultures (from six healthy donors) and resulted in a conversion from morphologically epithelial (squares) to non-epithelial (diamonds) phenotypes. All of these cultures were used for basal HSP analysis to assess effects of EMT, and also underwent PDF exposure to assess stress responses and cytoresistance. The numbers of patients (pat.) and donors (don.) are given in the symbols, and the number of cell cultures used for the experiments are given next to the respective arrows.

 
Peritoneal cells were concentrated by the centrifuga- tion of dialysis fluid effluent and then cultured in Earle's M199 medium, 10% fetal-calf serum, 50 U/ml penicillin and 50 µg/ml streptomycin. Non-adherent cells were removed 2 days later by two brief washes with a medium. When the primary mesothelial cultures reached confluence (after 8 to 31 days), they were split (in a ratio of 1:3) two to three times. In vivo EMT during clinical PD was assessed by characterizing the phenotypes of mesothelial cell cultures microscopically into cobblestone-like epithelial versus non-epithelial fibroblast-like phenotype (these features remained stable during two to three cell passages).

In vitro EMT of omental-derived mesothelial cell cultures (Figure 1)
Peritoneal mesothelial cells were isolated from fresh specimens of omentum from six healthy adult donors. Cultures were propagated in tissue culture flasks (Falcon, Becton Dickinson, Oxnard, CA, USA) at 37°C in a humified air containing a 5% CO2 incubator and passaged by regular trypsinization. The medium was changed every 2–3 days. Experiments in primary cell cultures were performed using cells from the first three passages since later subcultures contain an increasing number of senescent cells.

For TGF-β1 incubation, mesothelial cells at confluence were transferred to a medium containing less (0.3%) FCS, exposed for 1 week with 3 ng/ml recombinant human TGF-β1 (R&D Systems, Minneapolis, MN, USA), and then transferred to the TGF-β1 free medium containing 0.3% FCS prior to the experiments in order to keep cells viable in a non-proliferative status. Parallel cultures underwent sham treatment with the original medium as controls and were simultaneously harvested.

Effects of EMT on markers of the cellular stress response (Figure 1): for PDF incubation, mesothelial cell cultures were exposed for 60 min and 120 min (peritoneal effluent derived) or for 30 min and 60 min (omental derived) to commercially available glucose-monomer- and acidic-lactate-based PDF (Fresenius 2, Bad Homburg, Germany), containing 1.5% anhydrous dextrose at pH 5.5, and were then allowed to recover in a regular growth medium for 24 h. Control cultures were kept in regular culture media at 37°C and underwent the same ‘sham procedures’ of media changes, i.e. exposure to PDF was paralleled by exposure to control medium (without FCS). At the end of each protocol, cell viability and protein expression was assessed in parallel cultures.

Viability of cells was investigated by lactate dehydrogenase (LDH) release. For LDH analyses, 250 µl aliquots of supernatants were removed after the described experimental setup and kept on –20°C until analysed within 48 h. Measurements were performed in duplicates with Sigma TOX-7 LDH Kit according to the manufacturer instructions. LDH efflux is calculated as a percentage of LDH values measured in each negative control experiment.

For western blotting, protein content was determined by the Bradford assay (Biorad, Vienna, Austria) and equal amounts of protein samples (3 µg/lane) were separated by standard SDS-PAGE using a Pharmacia Multiphore II unit. Size-fractionated proteins were then transferred to PVDF membranes by semi-dry transfer in a Pharmacia Multiphore II Novablot unit. Membranes are blocked in 5% dry milk in TBS-Tween (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4). Membranes are incubated with the respective primary antibody for 16 h (HSP SPA 801, SPA810 and SPA815, Stressgen, B.C., Canada; SMA Ab-1, NeoMarkers, MS, USA; ezrin clone 3212, Sigma, MO, USA). Detection was accomplished by incubation with secondary, peroxidase-coupled antibodies (anti-mouse or anti-rabbit IgG, both Dako Cytomation, CA, USA) and enhanced chemiluminescence (ECL) using the ECL western blotting analysis system (Renaissance, NEN-Life Science Products, Boston, MA, USA).

For analysis of western blots, calibration curves were constructed to determine the linearity of the ECL detection system. Light emission was proportional to protein loading in the range of 2–8 µg of a given sample, indicating that fourfold changes were within the linear range. Differential expression of respective proteins were derived from the ratio of specific immunodensitometric signals at three different exposures in the linear range of the protein/signal intensity relationship, normalized to an internal standard and compared between parallel incubations in each experiment.

Analysis of variance or Wilcoxon signed-rank test, where appropriate, was used to compare data from five to eight experiments. Data are considered to be significantly different if P < 0.05. Changes are expressed as mean and 95% confidence intervals.



   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
From June 2004 to December 2006, 31 mesothelial cell cultures were grown from nine clinically stable children (five boys, four girls, aged 4.2 ± 3.6 years) undergoing peritoneal dialysis for 0–65 months. The phenotype of mesothelial cell cultures was characterized microscopically into cobblestone-like epithelial in 23 from seven children. Peritoneal dialysis treatment was associated in five children with morphologic features of in vivo EMT in eight peritoneal-effluent-derived mesothelial cell cultures, as reflected by a fibroblast-like growth pattern in contrast to the cobblestone-like appearance of mesothelium derived from omentum (see Figure 2). There were no differences in dialysis prescription (numbers of high glucose concentration bags) or number of peritonitis episodes in children with different EMT phenotypes. Mesothelial cells that grew in a fibroblast-like phenotype were harvested significantly later during PD therapy than mesothelial cells that grew in an epithelial-like phenotype (10.5 ± 4.3 versus 6.3 ± 3.8 months, P < 0.05).


Figure 2
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  		Fig. 2 Epithelial-to-mesenchymal transition (EMT) in peritoneal-effluent-derived mesothelial cell cultures during the clinical course of peritoneal dialysis. In the upper panel, the representative photomicrograph to the left shows the epithelial phenotype with its cobblestone-like appearance, to the right the spindle fibroblast-like phenotype with a multilayered crisscross growth pattern (magnification: x100). The morphologic phenotype of mesothelial cell is paralleled by differential cellular abundance of the marker proteins ezrin and alpha-smooth muscle actin (SMA) (lower panel).

 
The differences in phenotypes were also validated by reduced expression of the epithelial marker protein ezrin and increased expression of the mesenchymal marker protein alpha-smooth muscle actin. In three patients, this loss of the epithelial phenotype occurred during the observation period and thus allowed to confirm the effects of in vivo EMT intra-individually in sequential mesothelial cell cultures.

In vivo EMT of mesothelial cells also had marked effects on HSP, as shown in Figure 3. Expression of HSP-27, HSP-72 and HSP-73 was consistently lower in 8 fibroblast-like cell cultures than in 23 cobblestone epithelial peritoneal-effluent-derived mesothelial cell cultures (all P < 0.05). In the three patients whose mesothelial cells underwent in vivo EMT during the follow-up, the loss of the epithelial phenotype was associated with reduction of HSP-72 and HSP-73 expression in all three patients; HSP-27 was reduced in two of them.


Figure 3
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  		Fig. 3 Western analysis and densitometric analysis of HSP expression profiles following in vivo epithelial-to-mesenchymal transition (EMT) in peritoneal-effluent-derived mesothelial cell cultures. The box (25th and 75th percentile) and whisker (10th and 90th percentile) plots demonstrate a significantly higher basal HSP expression in the epithelial versus the non-epithelial phenotype under control conditions.

 
Following PDF exposure, it became evident that there were no effects of in vivo EMT on cytoresistance. As shown in Figure 4, PDF exposure resulted in significant HSP upregulation (compared to medium exposure) in all cell cultures, regardless of the EMT phenotype. Under these stressful conditions, HSP-27 and HSP-72 expression in the fibroblast-like cultures were no more different from levels in the epithelial-like phenotype. As shown in Figure 5, LDH release following acute PDF exposure was also not different between epithelial and fibroblast-like peritoneal-effluent-derived mesothelial cell cultures.


Figure 4
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  		Fig. 4 Effects of in vivo epithelial-to-mesenchymal transition (EMT) on acute cellular stress responses of peritoneal-effluent-derived mesothelial cell cultures upon exposure to peritoneal dialysis fluid (PDF). The box (25th and 75th percentile) and whisker (10th and 90th percentile) plots demonstrate stress-induced HSP expression upon PDF exposure for 60 min or 120 min and 24 h of recovery. Following PDF exposure, levels of HSP expression were comparable between the epithelial and non-epithelial phenotype. The basal expression line corresponds to HSP expression during exposure to the culture medium.

 

Figure 5
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  		Fig. 5 Effects of in vivo epithelial-to-mesenchymal transition (EMT) on cytoresistance of peritoneal-effluent-derived mesothelial cell cultures upon in vitro exposure to peritoneal dialysis fluid (PDF). The box (25th and 75th percentile) and whisker (10th and 90th percentile) plots demonstrate LDH release upon PDF exposure for 60 min or 120 min and 24 h of recovery. Following PDF exposure, levels of LDH release were comparable between the epithelial and non-epithelial phenotype. One hundred percent control corresponds to LDH release during exposure to the culture medium.

 
In vitro EMT of omental tissue-derived mesothelial cell cultures with a cobblestone-like epithelial phenotype by continuous TGF-β1 incubation resulted in a morphologic conversion to the fibroblast-like phenotype, as demonstrated in Figure 6. In parallel to phenotypical characteristics, TGF-β1 treatment also decreased the expression of the typical epithelial marker ezrin and increased the expression of the typical fibromyoblast marker alpha-smooth muscle actin. As shown in Figure 7, in vitro EMT during TGF-β1 incubation resulted in higher expression of HSP-27 (P < 0.05) and lower expression of HSP-72 (P < 0.05) under control conditions with medium exposure.


Figure 6
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  		Fig. 6 TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in omental tissue-derived mesothelial cell cultures. In the upper panel, the representative photomicrograph shows the conversion of a cobblestone-like epithelial phenotype (left) to a fibroblast-like morphology (right) following continuous in vitro incubation with TGF-β1 (3 ng/ml) for 7 days (magnification: x100). The western blot in the lower panel demonstrates concomitant effects of TGF-β1 on marker proteins ezrin and alpha-smooth muscle actin (SMA) (lower panel).

 

Figure 7
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  		Fig. 7 Western analysis and densitometric analysis of HSP expression profiles in in vitro epithelial-to-mesenchymal transition (EMT) of omental tissue-derived mesothelial cell cultures. Following TGF-β1 pretreatment, the box (25th and 75th percentile) and whisker (10th and 90th percentile) plots demonstrate significantly non-stressful induction of HSP-27 expression and decreased HSP-72 expression under control conditions.

 
Following PDF exposure, it became evident that in vitro EMT was also associated with improved cytoresistance. As shown in Figure 8, PDF exposure resulted in significant HSP upregulation (compared to medium exposure) primarily in the cell cultures without TGF-β1 pretreatment. Upon 30 min of PDF exposure, TGF-β1 pretreated cell cultures still showed significantly higher HSP-27 expression, and lower HSP-72 expression, than cells without TGF-β1 pretreatment. As shown in Figure 9, LDH release upon 30 min of PDF exposure was also significantly reduced following TGF-β1 pretreatment (P < 0.05). At more extendend PDF exposure (60 min), there were no more effects of in vitro EMT on HSP expression and cellular survival.


Figure 8
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  		Fig. 8 Effects of TGF-β1-induced in vitro epithelial-to-mesenchymal transition (EMT) on acute cellular stress responses of omental tissue-derived mesothelial cell cultures upon exposure to peritoneal dialysis fluid (PDF). The box (25th and 75th percentile) and whisker (10th and 90th percentile) plots demonstrate stress-induced HSP expression upon PDF exposure for 30 min or 60 min and 24 h of recovery. Following TGF-β1 pretreatment, stress induction of HSP expression was significantly attenuated. The basal expression line corresponds to HSP expression during exposure to the culture medium.

 

Figure 9
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  		Fig. 9 Effects of TGF-β1-induced in vitro epithelial-to-mesenchymal transition (EMT) on cytoresistance of omental tissue-derived mesothelial cell cultures upon exposure to peritoneal dialysis fluid (PDF). The box (25th and 75th percentile) and whisker (10th and 90th percentile) plots demonstrate LDH release upon PDF exposure for 30 min or 60 min and 24 h of recovery. Following TGF-β1 pretreatment, LDH release was significantly reduced (=improved cytoresistance) at the moderate PDF exposure. One hundred percent control corresponds to LDH release during exposure to the culture medium.

 


   Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
Peritoneal dialysis (PD) consists of repeated exposure of mesothelial cells to non-physiologic peritoneal dialysis fluid (PDF). In the clinical setting of PD, cellular insults mediated by PDF are dynamic, ranging during each intraperitoneal dwell from acute toxic effects in the early phase, towards more complex processes during later dwell [17,18]. For several years, research has mainly focused on early effects and resulted in delineation of low pH, high concentrations of glucose and unphysiological puffers (lactate) as major toxic agents [4]. Recently, the research focus shifted towards diabetiform cellular processes that will likely occur in the peritoneum upon more chronic exposure to PDF [19,20]. Exposure to high peritoneal glucose and its degradation products have been associated with profibrotic changes such as progressive EMT [11,15,21].

However, PD will be neither satisfyingly described by research of chronic diabetes-like models nor by acute toxicity models. The challenge, but also fascination, of studying PD therapy is the need to combine these models to an integrative approach [20]. For example, marked changes of the cellular protein expression pattern have been described during EMT, and will likely also include proteins that are involved in cellular processes counteracting acute toxic insults by PDF [13–15].

The first set of our experiments therefore investigated whether in vivo EMT of mesothelial cells (=occurring during PD) affected their expression of HSP, the major effectors of the acute cellular stress response. In a landmark paper, Yanez-Mo et al. showed that mesothelial cell cultures grown from peritoneal effluents of patients undergoing PD had markedly varied morphologic features, ranging from a cobblestone-like appearance similar to that of mesothelium derived from omentum to fibroblast-like cells [11]. In our study, in vivo EMT during the course of clinical PD therapy also resulted in a frequent fibroblast-like conversion of the cellular phenotype, associated with reduced expression of typical epithelial marker proteins and increased expression of mesenchymal marker proteins.

HSP represent a large family of extremely conserved soluble proteins that are found throughout the cell; the amount of all HSP isoforms in some instances can constitute up to 5% of total cellular proteins [6–8]. HSP are characterized by their induction when a cell undergoes various types of acute environmental stresses, and are categorized according to their molecular weight [22]. After acute cellular injury, they facilitate cellular repair and survival and induce cytoresistance against repeated injury. In previous studies we have described the marked upregulation of 27kD (HSP-27) and 70kD HSP (HSP-72) in mesothelial cells following exposure to PDF [6–9,17].

The comparison of HSP expression between the different EMT phenotypes of peritoneal-effluent-derived mesothelial cell cultures demonstrated consistently lower HSP expression in the fibroblast-like phenotype than in the epithelial phenotype. These findings are similar to those described in chronic epithelial wounding and, with regard to HSP-72, agree with reduced mRNA levels during the myofibro- blast conversion of mesothelial cells [23,24]. However, in a cross-sectional design, it is difficult to assess whether HSP expression was only indirectly related to in vivo EMT, as patients with low constitutive HSP might have been at increased risk for EMT during PD treatment. Fortunately, we were able to follow three patients whose mesothelial cells underwent in vivo EMT during the course of PD therapy. In these patients, the loss of their initial epithelial phenotype resulted in subsequent reduction of HSP expression. Thus, the association between EMT phenotype and HSP expression appears to indeed represent a direct effect of in vivo transdifferentiation processes.

In order to evaluate the effects of in vivo EMT on cellular stress responses and cytoresistance, we exposed different EMT phenotypes of peritoneal-effluent-derived mesothelial cells to PDF. Based on the findings of cytoprotection with HSP upregulation, one would expect that the reduced amount of HSP expression following in vivo EMT might correlate with reduced viability upon PDF exposure in the fibroblast-like phenotype [16,22]. However, the effects of acute PDF exposure did not differ between epithelial and fibroblast-like mesothelial cell cultures following in vivo EMT. Stressful upregulation resulted in comparably increased HSP expression in epithelial and fibroblast-like phenotypes following moderate and more extended PDF exposure. Exposure to PDF also resulted in comparable cellular viability in both EMT phenotypes. Cellular survival of peritoneal-derived mesothelial cells therefore correlated better with post-PDF-exposure levels of HSP than with pre-PDF-exposure levels. Thus, in vivo EMT decreased HSP expression only under control conditions, whereas HSP expression and cellular survival under the stress conditions of PDF exposure were not affected by the EMT phenotype.

The second set of our experiments investigated effects of ‘experimental’ in vitro EMT (induced by TGF-β1) on HSP expression and cytoresistance against PDF in omental tissue-derived mesothelial cells [12]. In vitro EMT by TGF-β1 fully induced morphological conversion of epithelial mesothelial cell cultures into the fibroblast-like phenotype, and induced the typical changes of EMT marker proteins with the upregulation of alpha-SMA and down-regulation of ezrin–-identical to the findings with in vivo EMT [12,14,15]. Interestingly, however, effects of in vitro EMT on HSP expression were different to in vivo EMT. TGF-β1 incubation resulted in increased expression of HSP-27 and decreased expression of HSP-72. Such discordant regulation of HSP-27 and HSP-72 upon non-stressful pretreatment has been previously reported in other cell systems, and has been related to the activation of stress kinases by TGF-β1 [13,25–27]. HSP-27 has actin-binding (capping) properties, and demonstrates a high sequence homology to alpha crystalline that is essential for the organization of the actin-based cytoskeleton in the lens [22]. The concordant upregulation of alpha-SMA and HSP-27 upon TGF-β1 treatment likely reflects the marked alteration of the cytoskeletal apparatus during the myofibroblast conversion of mesothelial cells [27].

As a next step, the effects of in vitro EMT on cellular stress responses and cytoresistance were assessed in the omental tissue-derived mesothelial cell cultures. The non-stressful upregulation of HSP-27 by pretreatment with TGF-β1 resulted in sufficiently high HSP levels to attenuate stress-induced HSP expression and protect cellular viability upon moderate PDF exposure. More extended PDF exposure, however, apparently overrode effects of TGF-β1. In previous studies, similar cytoprotection was found following the upregulation of HSP expression upon pretreatment with PDF, heat or transient HSP-72 gene transfection [10].

Taken together, in vivo and in vitro EMT give similar results in respect to cell morphology and expression of marker proteins, but appear different in profile of HSP expression and cytoresistance to PDF exposure. We suggest that these findings might indicate different roles for HSP in different phases of mesothelial EMT. TGF-β1 is certainly the best-studied prototype mediator to initiate mesothelial EMT during aberrant peritoneal healing [15,28]. In clinical studies, Zweers et al. reported elevated TGF-β1 levels in patients on PD with and without peritonitis [29]. In the in vivo rat model of PD, Margetts et al. described clear evidence of mesothelial EMT induction by intraperitoneal transfection of TGF-β1 [30]. In vitro TGF-β1 incubation models to induce EMT have also been used by Yanez-Mo et al. and Yang et al. to describe the myofibroblast conversion of omental mesothelial cells [11,24]. Recently, we have transferred that system to peritoneal-effluent-derived mesothelial cell cultures [12]. In its initiation phase, early EMT—such as induced by TGF-β1—might cause/drive a positive selection bias within the mesothelial cell population by HSP-mediated cytoprotection in the hostile intra-peritoneal environment during ongoing PD therapy (=repetitive stress) [9,10,22]. However, maintaining chronic in vivo EMT is the result of the combined effects of chronic wounding, high exposure to glucose degradation products and smouldering (and/or acute) inflammation for several weeks to months and thus reflects a much more complex interplay of cellular regulatory pathways [14,15,28].

In conclusion, this study confirms that mesothelial cells are not ‘unchanged’ or ‘static targets’ during the clinical course of PD. In vivo EMT during PD therapy and in vitro EMT by TGF-β1 were associated with differential effects on cellular stress responses and cytoresistance. In vivo EMT resulted in decreased HSP expression under control conditions, whereas HSP expression and cellular survival were not affected during PDF exposure in peritoneal-derived mesothelial cell cultures. In contrast, in vitro EMT resulted in the non-stressful upregulation of HSP-27, with attenuated HSP expression and improved cellular survival during PDF exposure in omental-derived mesothelial cell cultures. Future studies are needed to further evaluate the complex cellular processes during chronic in vivo EMT and their role in acute cellular stress response and cytoresistance of mesothelial cells.



   Acknowledgments
 
Regina Vargha and Thorsten O. Bender contributed equally to this work. This work was supported by OeNB anniversary fund project 12028.

Conflict of interest statement. None declared.



   Notes
 
* Thorsten O. Bender is recipient of a long-term ERA-EDTA fellowship. Back



   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 20. 4.07
Accepted in revised form: 30. 5.08


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