Interleukin-1 receptor-mediated inflammation impairs the heat shock response of human mesothelial cells.

Bioincompatibility of peritoneal dialysis fluids (PDF) limits their use in renal replacement therapy. PDF exposure harms mesothelial cells but induces heat shock proteins (HSP), which are essential for repair and cytoprotection. We searched for cellular pathways that impair the heat shock response in mesothelial cells after PDF-exposure. In a dose-response experiment, increasing PDF-exposure times resulted in rapidly increasing mesothelial cell damage but decreasing HSP expression, confirming impaired heat shock response. Using proteomics and bioinformatics, simultaneously activated apoptosis-related and inflammation-related pathways were identified as candidate mechanisms. Testing the role of sterile inflammation, addition of necrotic cell material to mesothelial cells increased, whereas addition of the interleukin-1 receptor (IL-1R) antagonist anakinra to PDF decreased release of inflammatory cytokines. Addition of anakinra during PDF exposure resulted in cytoprotection and increased chaperone expression. Thus, activation of the IL-1R plays a pivotal role in impairment of the heat shock response of mesothelial cells to PDF. Danger signals from injured cells lead to an elevated level of cytokine release associated with sterile inflammation, which reduces expression of HSP and other cytoprotective chaperones and exacerbates PDF damage. Blocking the IL-1R pathway might be useful in limiting damage during peritoneal dialysis.

HSP-mediated cytoprotection of mesothelial cells in experimental acute peritoneal dialysis.

BACKGROUND: Low biocompatibility of peritoneal dialysis solution (PDS) injures mesothelial cells but also induces heat shock proteins (HSP), the main effectors of the cellular stress response. This study investigated whether overexpression of HSP upon pharmacologic induction results in cytoprotection of mesothelial cells in experimental PD. METHODS: Stress response of mesothelial cells upon exposure to PDS was pharmacologically manipulated using glutamine as a co-inducer. In vitro, HSP-mediated cytoprotection was assessed by simultaneous measurements of HSP expression using Western blot analysis and viability testing using release of lactic dehydrogenase in cultured human mesothelial cells. In vivo, detachment of mesothelial cells from their peritoneal monolayer was assessed following exposure to PDS with and without the addition of glutamine in the acute rat model of PD. RESULTS: In vitro, mesothelial cell viability following exposure to PDS was significantly improved upon pharmacologic co-induction of HSP expression by glutamine (226% +/- 29% vs 190% +/- 19%, p = 0.001). In vivo, mesothelial cell detachment during exposure to PDS was reduced upon pharmacologic induction of HSP expression by glutamine (93 +/- 39 vs 38 +/- 38 cells, p = 0.044), resulting in reduced peritoneal protein loss (75 +/- 7 vs 65 +/- 4 mg, p = 0.045). CONCLUSION: Our results represent the first study of pharmacologic manipulation of HSP expression for cytoprotection of mesothelial cells following acute in vitro and in vivo exposure to PDS. PDS with added glutamine might represent a promising therapeutic approach against low biocompatibility of PDS but needs validation in a chronic PD model.

Stress responses and conditioning effects in mesothelial cells exposed to peritoneal dialysis fluid.

Renal replacement therapy by peritoneal dialysis is frequently complicated by technical failure. Peritoneal dialysis fluids (PDF) cause injury to the peritoneal mesothelial cell layer due to their cytotoxicity. As only isolated elements of the involved cellular processes have been studied before, we aimed at a global assessment of the mesothelial stress response to PDF. Following single or repeated exposure to PDF or control medium, proteomics and bioinformatics techniques were combined to study effects in mesothelial cells (MeT-5A). Protein expression was assessed by two-dimensional gel electrophoresis, and significantly altered spots were identified by MALDI-TOF MS and MS2 techniques. The lists of experimentally derived candidate proteins were expanded by a next neighbor approach and analyzed for significantly enriched biological processes. To address the problem of an unknown portion of false positive spots in 2DGE, only proteins showing significant p-values on both levels were further interpreted. Single PDF exposure resulted in reduction of biological processes in favor of reparative responses, including protein metabolism, modification and folding, with chaperones as a major subgroup. The observed biological processes triggered by this acute PDF exposure mainly contained functionally interwoven multitasking proteins contributing as well to cytoskeletal reorganization and defense mechanisms. Repeated PDF exposure resulted in attenuated protein regulation, reflecting inhibition of stress responses by high levels of preinduced chaperones. The identified proteins were less attributable to acute cellular injury but rather to specialized functions with a reduced number of involved multitasking proteins. This finding agrees well with the concept of conditioning effects and cytoprotection. In conclusion, this study describes the reprogrammed proteome of mesothelial cells during recovery from PDF exposure and adaption to repetitive stress. A broad stress response with a number of highly overlapping processes and multitasking proteins shifts toward a more specific response of only few less overlapping processes.

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

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-beta1 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-beta1 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.

Biocompatibility of a bicarbonate-buffered amino-acid-based solution for peritoneal dialysis.

Amino-acid-based peritoneal dialysis (PD) fluids have been developed to improve the nutritional status of PD patients. As they may potentially exacerbate acidosis, an amino-acid-containing solution buffered with bicarbonate (Aminobic) has been proposed to effectively maintain acid-base balance. The aim of this study was to evaluate the mesothelial biocompatibility profile of this solution in comparison with a conventional low-glucose-based fluid. Omentum-derived human peritoneal mesothelial cells (HPMC) were preexposed to test PD solutions for up to 120 min, then allowed to recover in control medium for 24 h, and assessed for heat-shock response, viability, and basal and stimulated cytokine [interleukin (IL)-6] and prostaglandin (PGE(2)) release. Acute exposure of HPMC to conventional low-glucose-based PD solution resulted in a time-dependent increase in heat-shock protein (HSP-72) expression, impaired viability, and reduced ability to release IL-6 in response to stimulation. In contrast, in cells treated with Aminobic, the expression of HSP-72 was significantly lower, and viability and cytokine-producing capacity were preserved and did not differ from those seen in control cells. In addition, exposure to Aminobic increased basal release of IL-6 and PGE(2). These data point to a favorable biocompatibility profile of the amino-acid-based bicarbonate-buffered PD solution toward HPMC.

Quercetin protects human mesothelial cells against exposure to peritoneal dialysis fluid.

During peritoneal dialysis, mesothelial cells have been shown to undergo severe damage due to continuous exposure to peritoneal dialysis fluid (PDF) with cytotoxic physicochemical properties. In this study, we investigated the cytoprotective role of the bioflavonoid Quercetin in the in vitro model of peritoneal dialysis. Immortalized human mesothelial cells (Met5A) were exposed either to regular growth medium or to standard acidic lactate-buffered PDF (Dianeal PD4) or to a more biocompatible lactate-bicarbonate-buffered PDF (Physioneal 40). Parallel cell cultures were supplemented with 200 microM Quercetin. Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by the release of cytoplasmic lactate dehydrogenase and fluorescence-activated cell sorting (FACS). PDF exposure with bioincompatible Dianeal PD4 resulted in severe disruption of cell cultures and in significantly increased lactate dehydrogenase (LDH) release (p=0.0007 vs. control). Addition of 200 microM Quercetin significantly decreased the LDH release (p=0.04 vs. "pure" Dianeal PD4 exposure), comparable to control exposure and to more biocompatible Physioneal 40 exposure (p=0.37) and resulted in marked preservation of cell culture monolayers and cellular viability as assessed by FACS. Introduction of cytoprotective agents such as Quercetin may represent an alternate approach to protect mesothelial cells from cytotoxicity of frequently used PDFs, comparably effective to the introduction of novel, more biocompatible, PDFs.

Evidence for HSP-mediated cytoskeletal stabilization in mesothelial cells during acute experimental peritoneal dialysis.

Low biocompatibility of peritoneal dialysis fluid (PDF) injures mesothelial cells and activates their stress response. In this study, we investigated the role of heat shock proteins (HSP), the main cytoprotective effectors of the stress response, in cytoskeletal stabilization of mesothelial cells in experimental peritoneal dialysis. In cultured human mesothelial cells, cytoskeletal integrity was assessed by detergent extractability of marker proteins following in vitro PDF exposure. Effects of HSP on stabilization of ezrin were evaluated by a conditioning protocol (PDF pretreatment) and repair assay, based on coincubation of cytoskeletal protein fractions with recombinant HSP-72 or HSP-72 antibodies. In the rat model, detachment of mesothelial cells from their peritoneal monolayer during in vivo PDF exposure was assessed with and without overexpression of HSP-72 (by heat conditioning). In vitro, cytoskeletal disruption on sublethal PDF exposure was demonstrated by significantly altered detergent extractability of ezrin and ZO-1. Restoration was associated with significant induction and cytoskeletal redistribution of HSP during recovery. Both the conditioning protocol and in vitro repair assay provided evidence for HSP-72-mediated cytoskeletal stabilization. In the rat model, overexpression of HSP-72 following heat conditioning resulted in significantly reduced detachment of mesothelial cells on in vivo exposure to PDF. Our results establish an essential role of HSP in repair and cytoprotection of cytoskeletal integrity in mesothelial cells following acute in vitro and in vivo exposure to PDF. Repeated exposure to PDF, as is the rule in the clinical setting, may not only cause repeat injury to mesothelial cells but rather represents a kind of inadvertent conditioning treatment.

Ex vivo reversal of in vivo transdifferentiation in mesothelial cells grown from peritoneal dialysate effluents.

BACKGROUND: During peritoneal dialysis (PD), epithelial-mesenchymal transition (EMT) is likely involved in aberrant healing and progressive peritoneal fibrosis. Recently, EMT of the kidney was actively reversed into the opposite direction, into mesenchymal-epithelial transition (MET), by treatment with bone morphogenic protein-7 (BMP-7). In this study, the potential for ex vivo interconversion of in vivo transdifferentiation processes was investigated in mesothelial cells. METHODS: In vivo EMT was assessed in mesothelial cell cultures randomly grown from peritoneal effluents of seven patients on chronic PD. Then, ex vivo treatment with modulating factors was performed by incubating cobblestone-like cell cultures with transforming growth factor (TGF- beta1) and fibroblast-like cultures with BMP-7. Effects were assessed by morphological characterization, western analysis and reverse transcription-polymerase chain reaction of marker proteins ezrin and alpha-smooth muscle actin (alpha-SMA). RESULTS: PD caused progressive in vivo EMT with loss of the epithelial phenotype in the majority of mesothelial cell cultures over a 12-month period. EMT was reproducible by ex vivo treatment of cultured cells with TGF-beta1, converting the epithelial to the fibroblast-like phenotype. Ex vivo treatment with BMP-7 reversed in vivo and ex vivo EMT. During rhBMP-7 incubation the fibroblast-like growth pattern reversed into a more epithelial morphology, the expression of ezrin increased and alpha-SMA decreased. CONCLUSION: Our study shows that modulating factors of transdifferentiation, such as BMP-7, may be attractive tools in the balance between normal healing and aberrant profibrotic processes in mesothelial cells during peritoneal dialysis. Peritoneal-effluent-derived mesothelial cells are not mere biomarkers for in vivo EMT in the peritoneal cavity, but also represent an assay to test ex vivo interventions to reverse the profibrotic phenotype.

Overexpression of HSP-72 confers cytoprotection in experimental peritoneal dialysis.

BACKGROUND: Peritoneal dialysis is complicated by mesothelial cell injury due to low biocompatibility of peritoneal dialysis fluid (PDF). We have previously demonstrated that heat shock protein (HSP)-72 is potently up-regulated in response to PDF exposure of mesothelial cells in in vitro and in vivo models of peritoneal dialysis. The aim of this study was to evaluate potential cytoprotective effects of overexpression of HSP-72. METHODS: Cytoprotection was assessed by comparing cellular viability between pretreated versus nonpretreated human mesothelial cells (Met 5a; ATCC, Manassas, VA, USA, and primary cell cultures) subjected to extended, usually lethal PDF exposure times (120 min, CAPD2; Fresenius, Bad Homburg, Germany). Pretreatment was performed with exposure to PDF (60 min, CAPD2; Fresenius) or heat (15 min, 41.5 degrees C), and by transient transfection with HSP-72. RESULTS: When mesothelial cells were pretreated by nonlethal exposure to PDF or heat, HSP-72 was markedly up-regulated (>5-fold, P < 0.01). Pretreated human mesothelial cells were significantly protected against subsequent "lethal" exposures to PDF, as assessed by dye exclusion (>50% reduction, P < 0.05) and lactate dehydrogenase (LDH) release (>30% reduction, P < 0.05). Comparable cytoprotection (50% reduction by dye exclusion) was indicated by overexpression of HSP-72 in cultered human mesothelial cells (>5-fold) after transient transfection with HSP-72. This cytoprotection was confirmed at a cellular basis by double staining techniques with HSP-72 and ApopTag (apoptosis detection kit). CONCLUSION: Our study therefore shows that the mesothelial stress response confers cytoprotection in experimental peritoneal dialysis, mediated by the induction of HSP-72, and that the stimulus of the pretreatment does not have to be identical to the subsequent injury. These data offer the basis for an attractive novel therapeutic approach against PDF toxicity.

HSP-25 and HSP-90 stabilize Na,K-ATPase in cytoskeletal fractions of ischemic rat renal cortex.

BACKGROUND: We recently designed an in vitro system based on differential Triton-extractability of Na,K-ATPase from the cytoskeletal protein fraction isolated from rat renal cortex after renal ischemia. In the present study, we hypothesized that heat shock protein (HSP)-70, HSP-25 and HSP-90 work synergistically to stabilize the cytoskeletal anchorage of Na,K-ATPase. METHODS: Cellular proteins were fractionated by differential centrifugation into cytoskeletal pellets (I-PEL) obtained early (exhibiting abnormally high Triton extractability of Na,K-ATPase) and non-cytoskeletal supernatants (R-SUP) obtained late (exhibiting high abundance of HSP) after renal ischemia. For assessment of the role of HSP-70, HSP-25 and HSP-90 upon in vitro re-compartmentalization, I-PEL was either incubated in R-SUP with/without HSP antibodies, or in buffer with/without HSPs at different titers and combinations. Effects were evaluated by changes of Triton extractability of Na,K-ATPase after co-incubation. RESULTS: R-SUP was shown to contain significant amounts of HSP-70, HSP-25 and HSP-90. Incubation of I-PEL in R-SUP reduced Triton extractability of Na,K-ATPase. Addition of antibodies against each HSP significantly abolished these effects of R-SUP. Incubation of I-PEL with purified HSP-70, HSP-25 or HSP-90 each partly reproduced the effects of R-SUP, whereas the combination of all three HSP demonstrated a strong and more than additive effect on the cytoskeletal stabilization of Na,K-ATPase. CONCLUSIONS: The molecular mechanisms responsible for postischemic re-compartmentalization of Na,K-ATPase in rat renal cortex likely involves interactions between HSP-70, HSP-25 and HSP-90, stress proteins known to be induced in the ischemic kidney.