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TRANSLATIONAL PHYSIOLOGY
1Department of Pediatrics, Univ. Kinderklinik Wien, 2Division of Biomedical Research, and 3Department of Pathology, Vienna Medical School, Vienna, Austria
Submitted 15 December 2005 ; accepted in final form 26 June 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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heat shock proteins; cytoskeletal repair; stress response; cyto-protection
Recent research demonstrated that PDF exposure not only results in cellular injury but also activates an endogenous machinery found in every cell, the so-called stress response (2, 5, 34). This stress response includes cellular mechanisms that facilitate cellular repair and survival after acute injury (8, 27, 29, 31). Main effectors of this stress response are the heat-shock proteins (HSP). Induction of HSP was described in mesothelial cells exposed to PDF in in vitro, ex vivo, and in vivo models of peritoneal dialysis (1, 2, 5, 34). Overexpression of HSP confers cytoprotection in mesothelial cells, improving cellular viability on PDF exposure (9).
Disruption of the cytoskeletal architecture is regarded as a classic marker of sublethal epithelial cell injury (21, 25, 38). Interestingly, marked morphological alterations have been described in mesothelial cells in the dialyzed peritoneal cavity, which can be related to such cytoskeletal disruption. Ultrastructurally, the number of microvilli was substantially reduced, intercellular gaps widened, and considerable detachment of mesothelial cells from the basement membrane on exposure to PDF was found (17–20). These observations are of potential clinical relevance as they likely represent the morphological correlate of impaired ultrafiltration, inefficient solute clearance, and significant protein loss.
We and others have recently described clear evidence for HSP-mediated repair during cytoskeletal restoration in the model of renal ischemia (3, 4, 6, 10, 11). No such data exist for peritoneal dialysis. In this study, we aimed to investigate the role of HSP in cytoskeletal stabilization in mesothelial cells following acute exposure to PDF in in vitro and in vivo models of peritoneal dialysis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture. Immortalized human mesothelial cells (Met5A, ATCC CRL-9444) were cultured in M199/MCDB 105 medium (1:1) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. Cultures were kept in 75-cm2 tissue culture flasks (Falcon, Becton Dickinson, Oxnard, CA) at 37°C in 5% CO2 and passaged by regular trypsinization. The medium was changed every 2–3 days. Confluence was reached on average after 6–7 days.
PDF exposure. Confluent cultures were exposed to a standard glucose monomer and acidic lactate-based PDF (Fresenius 2, Bad Homburg, Germany) containing 1.5% anhydrous dextrose at pH 5.5. Exposure times ranged from 30 to 120 min. Depending on the experimental setup, cells were then allowed to recover in regular growth medium for 0–24 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 for the times indicated. In selected experiments, cells were exposed to a more biocompatible peritoneal dialysis solution with almost neutral pH and low-glucose degradation products (PD-Bio 1.5%, Gambro, Lund, Sweden) for 4 and 8 h. At the end of each protocol, detergent extractability of cytoskeletal marker proteins, induction of HSP, and cell viability were assessed in parallel cultures as described below. Cells for use in immunofluorescence staining were grown on microscope slides (culture slides, Falcon, BD Bioscience, Franklin Lakes, NJ), using the same medium and atmosphere as mentioned previously.
In vitro conditioning protocol. Confluent mesothelial cells were pretreated for 60 min by standard PDF incubation (classic conditioning) and then allowed to recover in regular culture medium. Twenty-four hours after the pretreatment, cells were subjected to repeat standard PDF exposure and subsequent recovery studies.
Cell viability assessments. Cellular ATP levels of harvested extracts were measured by luciferase assay using a bioluminescent somatic cell assay kit (FLASC, Sigma, St. Louis, MO). Viability of cells was investigated after the indicated exposure times to PDF and 24-h recovery. Dead cells were identified by incapability of standard trypan blue dye exclusion. Cells were stained with trypan blue for 15 min and evaluated in a hemocytometric chamber. Cells capable of dye exclusion (viable) and cells stained with trypan blue (nonviable) were counted, and percent viability was calculated. For lactate dehydrogenase (LDH) analyses, supernatants were removed after the described experimental setup and kept at 4°C until analyzed within 48 h. Measurements were performed in duplicate with a Sigma TOX-7 LDH Kit according to the manufacturer's instructions. LDH efflux is calculated as the percentage of LDH values measured in each negative control experiment.
Triton extraction. Cells were either lysed in buffer A [Na-K-ATPase; containing 0.1% Triton X-100 and (in mM) 60 PIPES, 2 CDTA, 1 EDTA, 1 EGTA, 100 NaC1, and 0.5 PMSF, as well as 0.75 mg/l leupeptin and 0.1 M DTT] or buffer B (all other marker proteins; containing 0.1% Triton X-100 and 300 mM sucrose, 500 mM Tris·HCl, 2 mM EGTA, 200 µM PMSF, and 10 µM leupeptin) (adapted from Ref. 14). The homogenate was centrifuged within 10 min at 35,000 g for 15 min at 4°C to separate the Triton-soluble protein fraction from the insoluble cytoskeletal pellet. Protein fractions were saved at –80°C until further Western blot analysis.
In vitro repair assay. This method was adapted from an assay originally designed for postischemic rat renal cortex (10, 11). In brief, Triton-insoluble protein aliquots (=cytoskeletal pellets) from mesothelial cells were isolated immediately after exposure to PDF ("injured") or after 24 h of recovery in culture medium ("recovered"). For assessment of in vitro repair, aliquots of injured cytoskeletal pellets were incubated in buffer with added recombinant HSP-72 at a concentration of 10 µg/100 µl (Stress Gen Biotechnologies, Victoria, BC) under conditions of blocked (2 mM CDTA) or enhanced ATP hydrolysis (5 mM MgATP). In addition, aliquots of recovered cytoskeletal pellets were incubated in parallel either in pure buffer or in buffer with added anti HSP-72 antibodies at a concentration of 50 µg/100 µl. After coincubations, differential centrifugation was repeated with 35,000 g for 15 min at 4°C. After repelleting, the supernatant represents nontranslocated HSP-72 (or anti-HSP-72 antibody, respectively) plus dissociated cytoskeletal proteins.
In Vivo Model of Peritoneal Dialysis
Acute rat model. The studies were carried out in adult male inbred Sprague-Dawley rats (average weight 310 g). After introduction of anesthesia (100 mg/kg ketamine and xylazine 5 mg/kg im), the animals were placed on a heated small-animal operating table. A sterile catheter was inserted into the peritoneal cavity through a small abdominal midline incision and, according to the protocol, 35 ml of prewarmed (37°C) test fluid (Medium 199 or PDF, CAPD3, FMC) were slowly infused in 45–60 s. In selected experiments, nonprewarmed PDF (room temperature, 20°C) or PDF warmed to 41.5°C were used. The animal was gently moved, a small volume of peritoneal fluid was aspirated, the catheter was withdrawn, and the abdomen was sutured. Animals awoke within 20 min after the procedure and had free access to food and tap water. At appropriate times (4 or 24 h) after the intraperitoneal injection, animals were again anesthetized, the peritoneal cavity was opened, and the peritoneal effluent was collected. In selected animals, material for morphological analysis was obtained.
In vivo conditioning protocols. For heat conditioning, transient hyperthermia of 41.5°C core temperature was induced for 15 min in anesthetized rats by the heated animal table and an infrared warming lamp, followed by 20 h of recovery.
All animals received humane care in compliance with the principles of laboratory animal care formulated by the Institute of Laboratory Animal Resources and the Guide For The Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.
Mesothelial cell detachment. For quantification, the peritoneal effluent was collected by gentle aspiration of 2 ml at baseline and after 4 h. Thereafter, animals were killed by cardial puncture and exsanguination, the abdomen was opened by a midline incision, and the complete intraperitoneal fluid was gently collected. The volumes of the collected fluids were recorded, and the total cell count and differential counts were assessed by hand count after giemsa staining and by machine count by a Coulter counter. The total number of detached mesothelial cells was then computed for each rat. This method was adopted from recent studies in human patients (7) and based on observations of mesothelial cell loss within 10 min following acute oxidative stress in rats (22).
Staining Procedures
Rhodamine-phalloidin (F-actin) staining. Cells were grown on culture slides and treated as described above. After the experiments, the cells were fixed in 4% paraformaldehyde. For staining, cells were first reconstituted in PBS and exposed to rhodamine-phalloidin (R415, Molecular Probes, Eugene, OR) in a final concentration of 0.5 µM for 30 min in the dark. Cells were washed five times with PBS. Slides were mounted using 50% glycerol/50% PBS and finally sealed with nail polish. Evaluation and microphotographs were done using a Zeiss fluorescence microscope.
Immunofluorescence staining. Cells were grown on culture slides and treated as described above. After the experiments, cells were washed two times with PBS and fixed with 4% paraformaldehyde in PBS, pH 7.4, for 20 min at room temperature. Cells were then permeabilized for 20 min with either 0.1% Triton X-100 or 0.1% saponin in PBS (pH 7.4). Nonspecific binding sites were blocked for 45 min in 5% fetal calf serum and 1% bovine serum albumin in PBS (pH 7.4). Ezrin and ZO-1 were immunodetected by sequential incubation with the respective antibody and a fluorescently labeled second antibody.
For immunofluorescent double labeling of detached mesothelial cells, aliquots of peritoneal effluate were spun onto slides, air-dried, and fixed with fresh cold 4% paraformaldehyde for 20 min. The fixed cells were blocked with 10% goat serum and permeabilized with 0.1% saponin in PBS for 2 min and washed with PBS. After 30-min incubation with the primary antibodies against cytokeratin and HSP-72 at room temperature, slides were rinsed with PBS and bound IgG was visualized using Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes). As a third step, nuclei were stained with 4,6-diamidino-2-phenylindole.
For immunohistochemical studies of resident mesothelial cells lining the peritoneal cavity, median samples from parietal peritoneum were used to prepare tissue blocks after fixation for 24 h at 4°C in 4% paraformaldehyde in PBS. The fixed peritoneal slices were paraffin embedded, and 4-µm sections were cut. After blocking of unspecific binding sites, primary antibodies to cytokeratin or HSP-72 were applied in PBS, 0.5% BSA for a total of 1 h. After repeated washes, the sections were then exposed to the secondary antibody conjugated to horseradish peroxidase in an equivalent procedure to the primary antibody.
Detection, Densitometry, and Statistics
Western blotting. Protein content was determined by Bradford assay (Bio-Rad) and equal amounts of protein samples (5 µg/lane) were separated by standard SDS-PAGE using a Pharmacia Multiphore II unit. Size-fractionated proteins were transferred to PVDF membranes by semidry transfer in a Pharmacia Multiphore II Novablot unit. Membranes were blocked in 5% dry milk in TBS-Tween (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 8.0). Membranes were incubated with the respective primary antibody. Detection was accomplished by incubation with secondary, peroxidase-coupled antibodies (Sigma), and enhanced chemiluminescence (ECL) using ECL Western blotting analysis system and protocols (Renaissance, NEN-Life Science Products, Boston, MA).
Densitometry was performed with image-analysis software (Molecular Analyst software, Bio-Rad). Differential expression and cellular distribution of respective proteins were derived from the ratio of specific immunodensitometric signals at 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.
Antibodies. Na-K-ATPase (05–369, Upstate Biotechnology), cadherin (A-CAM, C-2667, Sigma), cytokeratin (ZO622, Dako Cytomation), ezrin (3212, Sigma), HSP-27 and HSP-72 (SPA 800, SPA 810, Stressgen), ZO-1 (33–9100, Zymed), and E-cadherin (33–4000, Zymed) were used.
Statistical analysis. Values for treatment conditions were compared with control values using a Wilcoxon signed rank test (SPSS 7.5 for Windows, SPSS, Chicago, IL). The data are expressed as means ± SD. Differences were considered to be significant given a P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first part of the in vitro experiments demonstrates cytoskeletal disruption in mesothelial cells exposed to standard PDF for 60 min with no apparent morphological alterations, negligible cellular detachment, and >90% viability. PDF exposure for 120 min caused marked cellular detachment and decreased viability (Fig. 1). PDF exposure for 60 min also resulted in <20% increase of LDH release (106 ± 12% of control) but markedly reduced ATP levels to 20 ± 9% of controls (P < 0.05) immediately after exposure with fully restored levels at 24-h recovery. Exposure to the more biocompatible peritoneal dialysis solution could be extended up to 8 h without significant increases in LDH release (118 ± 24% of control).
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As a next step, we searched for induction and cellular distribution of HSP during cytoskeletal disruption and repair of mesothelial cells following in vitro exposure to PDF. Under control conditions, we found constitutive expression of HSP-27 and low amounts of HSP-72, both predominantly Triton soluble (Fig. 3B). With increasing PDF exposure, HSP-27 and HSP-72 were both induced and increasingly shifted into the cytoskeletal fraction. At 24 h of recovery, HSP-27 and HSP-72 showed potently elevated total cellular amounts with a predominant redistribution into the noncytoskeletal (=Triton-soluble) protein fraction.
Densitometric analysis, as given in Fig. 4 for ezrin, ZO-1, and HSP-72, demonstrates the statistical significance of the effects of PDF exposure on Triton extractability of these marker proteins. Moreover, it also becomes evident that their stabilization during recovery from PDF exposure was at least temporally and spatially associated with induction and redistribution of HSP. These findings support the hypothesis that HSP interact with disrupted elements of the cytoskeleton and participate in its restoration.
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To define an in vivo correlate to cytoskeletal damage in mesothelial cells following acute exposure to PDF, we quantified mesothelial cell detachment from their peritoneal monolayer in the rat model of peritoneal dialysis. When PDF was intraperitoneally injected, only few mesothelial cells (
1% of the total cells in the effluent) were identifiable at baseline. As shown in Fig. 7A, this number significantly increased within a 4-h dwell and then returned to control levels after 1 day. To discriminate for effects of PDF vs. the fluid injection per se, we treated rats either with a control solution or with standard PDF and compared the increase in mesothelial cell counts in effluents between both groups. As shown in Fig. 7B, PDF resulted in a significantly higher increased detachment of mesothelial cells. There was no significant effect of PDF temperature on detachment of mesothelial cells (20°C: 62 ± 24, 37°C: 94 ± 46, and 41.5°C: 124 ± 43 mesothelial cells, P = 0.43).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first part of this study, sublethal PDF exposure was evaluated for its ability to induce cytoskeletal disruption in mesothelial cells in vitro. Ezrin, cadherin, ZO-1, and Na-K-ATPase are essential for an intact cellular architecture (14, 15, 23, 24, 32). Based on the known effects of PDF exposure on ultrastructural morphology in mesothelial cells, such as reduced numbers of microvilli, widened intercellular gaps, cellular detachment, and loss of cellular polarity, these proteins were particularly attractive markers for PDF-induced cytoskeletal disruption. In addition to classic morphological assessment of cytoskeletal integrity, this study primarily used test systems that we and others had previously established as markers for acute nonlethal injury in renal tubule cells (6, 10, 11, 28, 33, 37, 40). The detergent Triton X-100 solubilizes cell membranes, and the cellular pool of proteins can be fractionated into an insoluble pellet (=cytoskeletal-associated fraction) and a soluble supernatant (=noncytoskeletal, dissociated fraction) by simple differential centrifugation. The distribution of such marker proteins between pellet and supernatant has been shown to accurately and reproducibly quantify cytoskeletal disruption on nonlethal cellular injury (28, 37).
Cytoskeletal findings in mesothelial cells exposed to PDF were comparable to observations in polarized renal tubular epithelia following sublethal injury. Prolonged exposure to standard PDF resulted in severe loss of cell-cell and cell-matrix contact of mesothelial cells and caused cellular detachment. Shorter exposures, or improved biocompatibility, resulted in disruption of the actin-based cytoskeleton and increased Triton extractability of the marker proteins with otherwise apparently unchanged morphology. Effects of acute nonlethal exposure to PDF can thus be quantified by dose-dependant, reversible disruption of the cytoskeletal anchorage of marker proteins in mesothelial cells. These results confirm that methods based on detergent extractability of membrane proteins may be transferred to the in vitro model of peritoneal dialysis.
In the following parts of this study, cytoskeletal restoration was investigated in the in vitro model of peritoneal dialysis, focusing on the concept of HSP-mediated repair. Both HSP-27 and HSP-72 showed distinct temporal patterns of postexposure cellular expression that can be readily related to the recycling of injured proteins. The finding of a shift of these HSP into the Triton-insoluble pellet also strongly suggests association with the disrupted cytoskeleton during the cellular reorganization process (3, 4, 6, 10, 11). The prevalence of such cellular recycling mechanisms in cellular repair of the peritoneal mesothelium is further supported by previous reports that overall de novo expression of proteins is reduced in these cells on exposure to PDF (13, 26). These results therefore suggest that HSP represent attractive candidates to interact with disrupted elements of the cytoskeleton and participate in its restoration in mesothelial cells during recovery from exposure to PDF. These observations, however, do not prove direct functional interactions and provide only indirect evidence for HSP-mediated repair.
As a next step, we therefore manipulated HSP expression in mesothelial cells and analyzed the effects of this intervention on cytoskeletal integrity on in vitro exposure to PDF. It is typical for HSP-mediated processes that pretreatment with a nonlethal dose of cellular stress results in survival of a subsequent otherwise lethal dose of the same or other type of injury (12). The finding of an increased resistance to repeat cellular injury is called cytoprotection, and the treatment resulting in this cytoprotection is termed "conditioning" (29, 31). Assuming functional interactions between cytoskeletal proteins and HSP in mesothelial cells during recovery from PDF exposure, increased HSP levels should improve cytoskeletal integrity (4, 10, 11). For these experiments, ezrin was chosen as the cytoskeletal marker protein, because ezrin was rapidly solubilized on sublethal PDF exposure and because loss of microvilli can be regarded as a hallmark of cytoskeletal architecture disruption following PDF exposure.
Our results demonstrated that conditioning by prior nonlethal PDF exposure indeed improved cytoskeletal stabilization as assessed by detergent extractability of ezrin following repeat PDF exposure in the in vitro model of peritoneal dialysis. Interestingly, the conditioning treatment did not affect Triton extractability of ezrin immediately after PDF exposure but resulted in a significantly more rapid stabilization during the subsequent recovery period. This finding suggests that the increasing abundance of cytoskeletal-associated HSP in the conditioned mesothelial cell (caused by both cellular redistribution and induction) is rather mediating enhanced postexposure repair than mere nonspecific resistance against cellular injury.
These results are in good agreement with the known chaperoning effects of HSP, but they still provide only indirect evidence for HSP-mediated repair of mesothelial cells on exposure to PDF. Incubation of denatured proteins such as luciferase or citrate synthase with purified HSP or with total cellular (reticulocyte) HSP-rich lysates has resulted in significant in vitro repair, readily quantifiable by gain of function (reviewed in Ref. 29). Therefore, we adapted a simple in vitro repair system analyzing functional interactions between HSP and disrupted elements of the cytoskeleton that were recently established in subcellular fractions of ischemic renal cortex. This assay is based on the ability of molecular chaperones to bind to hydrophobic sites of denatured proteins and subsequently undergo conformational changes, resulting in refolding, stabilization, and eventually release of reconfirmed proteins (4, 10, 11).
Using this functional assay, we found more direct evidence for the role of HSP-72 during repair of the cytoskeleton of mesothelial cells after nonlethal injury. Cellular distribution of ezrin was clearly affected by HSP-72 abundance and/or function. Coincubation with recombinant HSP-72 resulted in ATP-dependent stabilization of the cytoskeletal protein fractions from injured mesothelial cells, obtained immediately after PDF exposure. On the other hand, cytoskeletal redistribution of the membrane proteins during recovery could be abolished on coincubation with blocking antibodies against HSP. These results demonstrate that exogenously added as well as endogenously upregulated HSP interacted with substrates in the cytoskeletal fraction of mesothelial cells. These findings are consistent with HSP-mediated repair of disrupted proteins involved in the cytoskeletal organization of mesothelial cells (10, 11). Based on their known function, HSP-25 and/or HSP-90 might have been effective to convert the disrupted cytoskeletal elements to a "folding-competent" state, which can subsequently be refolded by HSP-72 (11).
Taken together, our results in the in vitro model of peritoneal dialysis suggest a novel and essential role of HSP in the repair of cytoskeletal integrity of mesothelial cells. First, markers of transient cytoskeletal disruption were defined in mesothelial cells on nonlethal cell injury following PDF exposure. Restoration of cytoskeletal integrity was then correlated with induction and cellular redistribution of HSP in that system. Finally, using conditioning protocols and novel repair assays, direct evidence for HSP-mediated cytoskeletal repair was delineated in mesothelial cells recovering from acute sublethal PDF exposure in vitro.
In the peritoneal cavity, however, rapid physicochemical equilibration of PDF and interactions between different peritoneal cell types will cause considerably increased complexity of both injury- and repair-related cellular processes (34, 36). In the next set of experiments, we therefore performed studies in intact animals to clarify the potential biological relevance of our in vitro findings.
Previously, we have described strong induction of HSP in the rat model of in vivo exposure to PDF (2). In that report, mesothelial cells were harvested by intraperitoneal trypsin digestion, precluding any differentiation between adherent and detached cell populations. In the present study, we separately investigated these mesothelial cell populations in PDF effluents and peritoneal tissue in the acute rat model. Weakening of cell-cell and cell-basal contact with subsequent cellular detachment is well accepted as a classic marker in several other models of sublethal cellular injury causing cytoskeletal disruption (21, 28, 37). In ischemic renal failure, this phenomenon is made responsible for renal tubular obstruction and urinary backleak (21). However, cellular detachment in the kidney cannot be directly evaluated, whereas our data suggest that simple assessment of mesothelial cell counts in PDF effluent might be used as a direct marker of sublethal mesothelial cell injury in the in vivo model of peritoneal dialysis.
When PDF was intraperitoneally injected, the number of detached mesothelial cells significantly increased within a 4-h dwell. Exposure to PDF resulted in a significantly higher count than exposure to control medium fluid. This is in good agreement with clinical observations, since the loss of mesothelial cells from the peritoneal surface is traditionally regarded as one of the hallmarks of PDF toxicity (17–20). Interestingly, our findings also provide strong evidence that the vast majority of the detached mesothelial cells are indeed viable but sublethally injured, as their protective cellular mechanisms were strongly induced (8, 35). Overexpression of HSP-72 was specifically found in those mesothelial cells that were detached following in vivo exposure to PDF but not in mesothelial cells that remained resident in the peritoneal monolayer until the end of the dwell. As a next step, we hypothesized that manipulation of HSP expression in the resident population before PDF injection might induce cytoprotection against in vivo exposure of the peritoneum to PDF.
Indeed, pretreatment by transient hyperthermia resulted not only in upregulation of HSP-72 but also in significantly reduced mesothelial cell detachment. These results are in good agreement with many studies that showed that organisms and tissues which overexpress HSP-72 are more resistant to cellular injury (12, 29, 31). Recently, transfection of mesothelial cells with HSP-72 resulted in protection of cell viability against subsequent PDF exposure in the cell culture model (9). In vivo, we found stabilization of the cytoskeletal integrity in the rat model of renal ischemia during repeat injury, following a classic conditioning protocol: at high cellular abundance of HSP-72, repeat renal ischemia resulted in neither increased Triton extractability nor altered cellular localization of cytoskeletal marker proteins in proximal tubular cells, in contrast to marked disruption after single ischemia at basal levels of HSP (4). Repeated exposure to PDF, as is the rule in the clinical setting, may therefore not only cause repeat injury to mesothelial cells but rather represents a kind of inadvertent conditioning treatment counteracting acute PDF toxicity. Future studies are needed to assess the effects of differential HSP expression on markers of more chronic peritoneal injury (39).
Taken together, our in vivo data clearly suggest the biological relevance of HSP-mediated cytoskeletal stabilization in mesothelial cells during peritoneal dialysis. First, mesothelial cell detachment could be related to PDF exposure. Next, HSP expression was found to be induced in detached cells but remain low in the resident mesothelial cell population. Finally, overexpression of HSP in that resident population resulted in significant reduction of mesothelial cell detachment following acute in vivo PDF exposure in the rat model.
Although HSP-mediated cytoprotection has long since been described as a potential therapeutic concept for acute cellular injury, its clinical application is seriously hampered because this concept strictly depends on planned "time course and dosages" of cellular insults (27, 29, 31). The majority of clinically relevant injuries, however, are complex and occur rather unpredictably with regard to both time course and dose. In peritoneal dialysis, with its repeated (and thoroughly predictably timed and dosed) exposure of mesothelial cells to PDF, therapeutic manipulations of HSP expression represents a unique opportunity for transferring cytoprotection from bench to bedside in future studies in more chronic models (30).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* M. Endemann and H. Bergmeister contributed equally to this study. 
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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