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Lederhuber.euPediatr Nephrol (2010) 25:169–172DOI 10.1007/s00467-009-1284-9 Cellular stress-response modulators in the acute rat modelof peritoneal dialysis Michael Boehm & Helga Bergmeister &Klaus Kratochwill & Regina Vargha &Hans Lederhuber & Christoph Aufricht Received: 9 February 2009 / Revised: 25 May 2009 / Accepted: 15 June 2009 / Published online: 25 August 2009# IPNA 2009 Abstract Cytotoxicity of peritoneal dialysis fluids (PDF) Keywords Heat-shock proteins . HSP-72 .
not only results in cellular injury, but also induces heat- Peritoneal dialysis . Indomethacin . Quercetin shock proteins (HSP), the main effectors of the cellularstress response. This study investigated effects of modula-tion of mesothelial HSP expression on peritoneal membrane integrity during acute PDF exposure. In the acute in vivorat model of peritoneal dialysis (PD), either the HSP Peritoneal dialysis (PD) is a safe, cost-effective, and widely coinducer indomethacin or the HSP suppressor quercetin used form of renal replacement therapy in children with was added to standard PDF (CAPD 3, Fresenius, Ger- end-stage renal failure. However, PD fluids (PDF) are not many). HSP-72 expression, number of detached mesothelial biocompatible, and exposure of mesothelial cells to PDF cells, and peritoneal protein loss were evaluated at the end results in marked cytotoxicity . Recently, we and others of a 4-h dwell time. Compared with pure PDF exposure, have demonstrated that cytotoxicity of PDF not only results addition of indomethacin resulted in increased expression in cellular injury in experimental models of PD, but also of mesothelial HSP-72, reduced mesothelial cell exfolia- induces cytoprotective heat-shock proteins (HSP) tion, and reduced peritoneal protein loss. Addition of Induction of HSP-72, the main effector of the mammalian quercetin resulted in decreased expression of HSP-72, cellular stress response, was found in mesothelial cells in in increased mesothelial cell exfoliation, and higher peritoneal vitro, ex-vivo, and in vivo PDF exposure systems.
protein loss. Differences were statistically significant Pretreatment by heat or PDF and transfection of HSP-72 between indomethacin-treated and quercetin-treated rats.
results in cytoprotection of mesothelial cells against PDF Mesothelial HSP expression was related to markers of exposure, and heat pretreatment of rats prevented mesothe- peritoneal membrane integrity upon in vivo PDF exposure, lial cells from detachment from their peritoneal lining in the consistent with HSP-mediated cytoprotection. These data in vivo model of PD [, ]. Although these findings suggest clearly demonstrate the potential for clinically feasible that HSP-mediated cytoprotection might be an attractive pharmacologic interventions with the cellular stress re- novel approach to reduce PDF-mediated peritoneal injury, sponse as a novel therapeutic approach to improve PD neither of these pretreatments will ever be feasible in the clinical setting. In this study, we therefore investigated theeffects of pharmacologic manipulation of the mesothelialHSP expression on peritoneal membrane integrity duringacute in vivo PDF exposure.
M. Boehm : H. Bergmeister : K. Kratochwill : R. Vargha :H. Lederhuber : C. Aufricht (*)Department of Pediatric and Adolescent Medicine, Medical University of Vienna,AKH Wien, Waehringer Guertel 18-20, Standard chemicals were purchased from Sigma-Aldrich 1090 Vienna, Austriae-mail: [email protected] (St. Louis, MO, USA) if not specified otherwise. Animal Pediatr Nephrol (2010) 25:169–172 protocols were approved by the institutional committee on using the Mann-Whitney U test. Differences were consid- animal research. For the acute in vivo model of PD , ered to be significant given a p<0.05.
male Sprague Dawley rats (average weight 310 g) under-went anesthesia (ketamine 100 mg/kg, 5 mg/kg xylazine,intramuscularly) and were placed on a heated small-animal operating table. A sterile catheter was inserted into theperitoneal cavity through a small abdominal midline As shown in Fig. (upper panel), addition of cellular stress- incision. In three independent experiments on separate response modulators to PDF had significant effects on HSP days, a total of 18 rats (six per treatment group) were expression of mesothelial cells lining the peritoneal cavity in slowly infused with 35 ml of conventional, single-chamber the acute rat model of PD. Compared with pure PDF bag, acidic pH, L-lactate-buffered PDF with 4.25% exposure, addition of indomethacin resulted in a 38±26% (236 mmol/L) D-glucose, and an osmolarity of 511 increase of HSP-72 in isolated mesothelial cells, whereas mosm/L (CAPD3, Fresenius Medical Care, Germany) addition of quercetin resulted in a 42±29% reduction.
either without additive or with quercetin (4 mg/kg) or with As shown in Fig. (middle panel), these effects on HSP indomethacin (50 μM) in 45–60 s. The animal was gently expression were associated with effects on mesothelial moved, a small volume of peritoneal fluid aspirated, the cellular outcome. Compared with pure PDF exposure, the catheter withdrawn, and the abdomen sutured. Animals extent of mesothelial cell exfoliation was reduced from awoke within 20 min after the procedure and had free median 92 to 42 cells (by 52±36%) with addition of access to food and tap water. At 4 h after the intraperitoneal indomethacin, whereas addition of quercetin resulted in a injection, animals were again anesthetized, sacrificed by 143±58% increase to 241 cells. Addition of cellular stress cardial puncture and exsanguination, the abdomen opened response modulators also had effects on peritoneal protein by a midline incision, and the complete intraperitoneal fluid loss as a functional parameter of peritoneal membrane collected. The volume was recorded and total cell count and integrity. Protein loss in the peritoneal effluent was also mesothelial cell counts assessed by hand count after reduced from median 52 to 47.5 mg by 4±3% with Giemsa staining and by machine count by a coulter counter.
indomethacin and increased by 10±7% to 55 mg with To assess peritoneal membrane integrity, amount of quercetin (Fig. lower panel). Differences in these ultrafiltration, total number of detached mesothelial cells parameters became statistically significant when compared and peritoneal protein loss were then computed for each rat between indomethacin-treated and quercetin-treated rats.
from dialysate samples obtained at the end of the dwell There were no differences in peritoneal ultrafiltration time. Mesothelial cells were isolated by a 20-min peritoneal between groups (indomethacin: 10.5±2.1 ml; quercetin: washout with 20 ml phosphate-buffered saline (PBS) 10.7±3.8 ml; PDF=100%, 10.6±3.2 ml).
containing 0.1% trypsin and 0.1% ethylenediaminetetraac-etate (EDTA) For Western blotting, equal amounts of protein samples from isolated mesothelial cells (5 μg/lane) were separatedby standard sodium dodecyl sulfate polyacrylamide gel The results of this study clearly confirm the link between electrophoresis (SDS-PAGE) using a Multiphor II unit (GE HSP expression and cellular outcome in mesothelial cells Healthcare, Uppsala, Sweden). Size-fractionated proteins during in vivo PDF exposure. Our data show potential for were transferred to polyvinylidene fluoride (PVDF) mem- pharmacologic interventions to induce HSP-mediated cyto- branes by semidry transfer in the Multiphor II Novablot protection against PDF-induced cellular injury in mesothe- unit (GE Healthcare). Membranes were blocked with 5% lial cells. Molecular chaperones, with HSP being the dry milk in Tris-buffered saline Tween-20 (TBST) buffer.
prototype, constitute a large family of soluble proteins that Membranes were incubated with the primary anti-HSP-72 are found throughout the mesothelial cell; the amount of all antibody (SPA 810, Assay Designs/Stressgen, Ann Arbor, HSP isoforms in some instances can constitute up to 5% of MI, USA). Detection was accomplished by incubation with total cellular proteins These proteins are known to secondary peroxidase-coupled antibodies (Sigma-Aldrich) cooperate in transport and folding of proteins, without and enhanced chemiluminescence (Western Lightning altering their own structure, by binding to hydrophobic, reagent, Perkin Elmer, Boston, MA, USA). Densitometry normally hidden domains of immature or denatured was performed with the image analysis software Quanti- proteins. They prevent disruption of cytoskeletal structures tyOne (Bio-Rad, Hercules, CA, USA). Differential expres- and might thus stabilize the mesothelial cell monolayer sion was derived from the ratio of specific signals in the lining the peritoneal cavity [, ].
linear range of the protein/signal-intensity relationship.
As detachment of mesothelial cells likely represents the Values for different treatment conditions were compared morphologic correlate of impaired peritoneal membrane
Pediatr Nephrol (2010) 25:169–172 Fig. 1 Effects of pharmacologic modulation of heat-shock protein-72 (HSP-72) expression on peritoneal membrane integrity in the acute ratmodel of peritoneal dialysis. Western analysis and densitometry of ratmesothelial cells harvested by trypsin peritoneal washout after a 4-hdwell showed increased HSP-72 expression with the HSP coinducerindomethacin (dark gray) and decreased HSP-72 expression with theHSP suppressor quercetin (light gray) compared with pure peritonealdialysis fluid (PDF) (white) (blot and upper panel). Increased HSP-72expression in mesothelial cells upon indomethacin addition to PDFwas associated with significantly lower detachment (middle panel) anddecreased protein loss into the dialysate effluent (lower panel). Dataare shown as box (giving the 25th and 75th percentile), whiskers(giving the 10th and 90th percentile), and median plots. Data wereobtained in six rats in each group in three independent experiments mesothelial cells indeed resulted in significant cytoskeletalstabilization of mesothelial cell against PDF exposurefollowing heat pretreatment by whole-body hyperthermiaAs heat pretreatment represents no practical therapeuticoption in the clinical setting of PD, our study introducednonstressful interventions with HSP expression during PDFexposure, using well-accepted modulators of the cellularstress response .
The nonsteroidal anti-inflammatory drug (NSAID) indo- methacin is known as coinducer of the stress response andthus to enhance cytoprotection [Albeit the detailedcellular mechanisms are still under investigation, indometh-acin is thought to potentiate trimerization and binding of theheat-shock transcription factor-1 (HSF-1) to DNA, therebyincreasing HSP transcription upon additional cellular stressIn contrast, the bioflavonoid quercetin is known assuppressor of the stress response and thus to inhibit HSP-mediated cytoprotection ]. Quercetin is thought toinhibit hyperphosphorylation and binding of HSF-1 toDNA, thereby decreasing HSP transcription upon addition-al cellular stress In previous studies, indomethacintreatment resulted in attenuation, whereas quercetin treat-ment resulted in aggravation of organ damage in experi-mental disease models, depending on their effects on HSPexpression ].
In our study in experimental PD, pharmacologic modu- lation of the mesothelial-cell stress response also resulted inconcordant effects on HSP-72 expression and peritonealmembrane integrity, assessed by mesothelial-cell detach-ment and peritoneal protein losses. As expected, addition ofthe coinducer indomethacin to PDF increased, whereasaddition of the suppressor quercetin decreased HSPexpression in mesothelial cells [–Consistent withthe concept of HSP-mediated cytoprotection, the higher integrity, such HSP-mediated cytoprotection might be an expression of HSP was clearly reflected in reduced attractive novel approach to reduce technical failure of PD mesothelial-cell detachment from its monolayer lining the . Based on this concept, we recently studied the peritoneal cavity during the in vivo PDF dwell [–In relationship between PDF-induced cytoskeletal disruption addition, we found evidence for an attenuated peritoneal and HSP-mediated cytoskeletal repair. In the acute rat barrier dysfunction, as demonstrated by a lower protein model of PD, overexpression of HSP in peritoneal content in the PD effluent of indomethacin-treated rats.
Pediatr Nephrol (2010) 25:169–172 Certainly, future studies in a chronic rat model of PD are fluids induce the stress response in human mesothelial cells. Perit needed to investigate chronic effects of modulation of Dial Int 21:85–88 4. Bender TO, Witowski J, Aufricht C, Endemann M, Frei U, mesothelial HSP expression during chronic PDF exposure.
Passlick-Deetjen J, Jorres A (2008) Biocompatibility of a With the resulting data, we could then assess the biological bicarbonate-buffered amino-acid-based solution for peritoneal relevance of our limited acute findings and extend the data dialysis. Pediatr Nephrol 23:1537–1543 with regard to peritoneal fibrosis, neoangiogenesis, and 5. Ruffingshofer D, Endemann M, Arbeiter K, Bidmon B, Mueller T, Herkner K, Aufricht C (2003) Induction of heat shock protein 72 ultrafiltration as important outcome variables. Taken to- in mesothelial cells exposed to peritoneal dialysate effluent. Perit gether, our acute experiments extend the previous findings Dial Int 23:74–77 of HSP-mediated cytoprotection of mesothelial cells fol- 6. Arbeiter K, Bidmon B, Endemann M, Bender TO, Eickelberg O, lowing heat pretreatment to a more feasible pharmacolog- Ruffingshofer D, Mueller T, Regele H, Herkner K, Aufricht C(2001) Peritoneal dialysate fluid composition determines heat ical intervention model. This study suggests potential for shock protein expression patterns in human mesothelial cells.
cytoprotective additives to PDF to optimize cellular Kidney Int 60:1930–1937 responses to pathophysiological stress upon PDF exposure.
7. Bidmon B, Endemann M, Arbeiter K, Ruffingshofer D, Regele H, Data such as ours represent a first step to innovative Herkner K, Eickelberg O, Aufricht C (2004) Overexpression ofHSP-72 confers cytoprotection in experimental peritoneal dialysis.
therapies to improve the long-term outcome of PD.
Kidney Int 66:2300–2307 8. Endemann M, Bergmeister H, Bidmon B, Boehm M, Csaicsich D, Acknowledgement This research work was funded by the Else- Malaga-Dieguez L, Arbeiter K, Regele H, Herkner K, Aufricht C Kröner-Fresenius Stiftung and by the FWF (Austrian Science Fund) (2007) Evidence for HSP-mediated cytoskeletal stabilization in Project P18130-B13 (both to CA). We are grateful to Michaela mesothelial cells during acute experimental peritoneal dialysis.
Endemann and Klaus Arbeiter for technical assistance and helpful Am J Physiol Renal Physiol 292:F47–F56 9. Aufricht C (2005) Heat-shock protein 70: molecular supertool? Pediatr Nephrol 20:707–713 10. Gotloib L, Waisbrut V, Shostak A, Kushnier R (1995) Acute and long-term changes observed in imprints of mouse mesotheliumexposed to glucose-enriched, lactated, buffered dialysis solutions.
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Regele H, Herkner K, Eickelberg O (2001) Peritoneal dialysis Kidney Int 59:1798–1802
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