Thorsten O. Bender

Human peritoneal fibroblasts are a potent source of neutrophil-targeting cytokines : a key role of IL-1β stimulation

Polymorphonuclear leukocyte (PMN) infiltration is a cardinal feature of peritonitis. CXC chemokine ligands 1 and 8 (CXCL1 and CXCL8), and the cytokine granulocyte colony-stimulating factor (G-CSF) are the key mediators of PMN accumulation. Increasing evidence points to an important role of human peritoneal fibroblasts (HPFB) in the response of the peritoneum to infection. We have examined the synthesis of PMN-targeting cytokines by HPFB exposed to intraperitoneal milieu as represented by peritoneal dialysate effluent (PDE) from patients undergoing peritoneal dialysis. PDE obtained during peritonitis, but not during infection-free periods, significantly increased production of CXCL1, CXCL8, and G-CSF by HPFB. The effect was largely blocked by antibodies to interleukin-1β (IL-1β), whereas neutralization of tumor necrosis factor-α (TNFα) had no major effect. Similar pattern of inhibition was observed when HPFB were exposed to conditioned media from endotoxin-stimulated peritoneal macrophages. Significance of IL-1β stimulation was further shown in experiments with recombinant cytokines. Compared with TNFα, exposure of HPFB to recombinant IL-1β resulted in a much higher release of PMN-targeting cytokines. The assessment of mRNA degradation revealed that the IL-1β-induced transcripts of CXCL1, CXCL8, and G-CSF were more stable compared with those induced by TNFα. These data indicate that HPFB can be a significant source of PMN-targeting cytokines when stimulated with IL-1β in the inflamed peritoneum.

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.

Dynamic O-Linked N-Acetylglucosamine Modification of Proteins Affects Stress Responses and Survival of Mesothelial Cells Exposed to Peritoneal Dialysis Fluids

The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD.

Peritoneal dialysis fluids can alter HSP expression in human peritoneal mesothelial cells

BACKGROUND Acute exposure of mesothelial cells to peritoneal dialysis fluid (PDF) has been shown not only to result in injury but also to induce cytoprotective heat shock proteins (HSP). The aim of the present study was to evaluate the expression of HSP in a more chronic in vitro PDF exposure system, searching for a role of glucose degradation products (GDP). METHODS Human peritoneal mesothelial cells (HPMC) were chronically incubated in filter- or heat-sterilized PDF (mixed 1:1 with cell culture medium), or in control cell culture medium. After incubation periods of 1, 3 and 10 days, cell extract was assessed for Ezrin, Hsp27 and Hsp72, and supernatant for IL-6 and IL-8. After 24-h exposure to the GDP 3.4-di-deoxyglucosone-3-ene (3.4-DGE), HPMC were assessed for expression of Hsp27 and Hsp72, and for release of LDH, IL-6 and IL-8. RESULTS In vitro PDF exposure for more than 1 day resulted in reduced cell mass, lower expression of the epithelial marker Ezrin and depressed cellular levels of both HSP, associated with increased IL-6 and IL-8 release. These effects occurred earlier and stronger with heat-sterilized than with filter-sterilized PDF. Exposure of HPMC to 3.4-DGE resulted in suppression of HSP, and increased release of LDH, IL-6 and IL-8. CONCLUSION Our data show that GDP (dys)regulate the mesothelial cell stress response. This was associated with reduced cell mass, loss of the epithelial phenotype and sterile cellular inflammation following extended exposure to heat-sterilized PDF. Toxic effects of PDF might thus be extended to reduced mesothelial cell stress responses.

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 (PGE2) 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 PGE2. These data point to a favorable biocompatibility profile of the amino-acid-based bicarbonate-buffered PD solution toward HPMC.

Regulation of Chemokine CCL5 Synthesis in Human Peritoneal Fibroblasts: A Key Role of IFN-γ

Peritonitis is characterized by a coordinated influx of various leukocyte subpopulations. The pattern of leukocyte recruitment is controlled by chemokines secreted primarily by peritoneal mesothelial cells and macrophages. We have previously demonstrated that some chemokines may be also produced by human peritoneal fibroblasts (HPFB). Aim of our study was to assess the potential of HPFB in culture to release CCL5, a potent chemoattractant for mononuclear leukocytes. Quiescent HPFB released constitutively no or trace amounts of CCL5. Stimulation of HPFB with IL-1β and TNF-α resulted in a time- (up to 96 h) and dose-dependent increase in CCL5 expression and release. IFN-γ alone did not induce CCL5 secretion over a wide range of concentrations (0.01–100 U/mL). However, it synergistically amplified the effects of TNF-α and IL-1β through upregulation of CCL5 mRNA. Moreover, pretreatment of cells with IFN-γ upregulated CD40 receptor, which enabled HPFB to respond to a recombinant ligand of CD40 (CD40L). Exposure of IFN-γ-treated HPFB, but not of control cells, to CD40L resulted in a dose-dependent induction of CCL5. These data demonstrate that HPFB synthesise CCL5 in response to inflammatory mediators present in the inflamed peritoneal cavity. HPFB-derived CCL5 may thus contribute to the intraperitoneal recruitment of mononuclear leukocytes during peritonitis.

Comparison of icodextrin- and glucose-based peritoneal dialysis fluids in their acute and chronic effects on human peritoneal mesothelial cells.

Background Icodextrin-based peritoneal dialysis fluids (PDFs) display several features that may potentially improve their biocompatibility compared to conventional glucose-containing solutions. So far, however, the studies assessing the biocompatibility profile of icodextrin toward human peritoneal mesothelial cells (HPMC) has produced mixed results. The present study was performed to examine the acute and chronic impact of icodextrin on HPMC in vitro in comparison with standard glucose-based PDF. Methods Omentum-derived HPMC were either acutely pre-exposed to or incubated chronically (for up to 10 days) in the presence of icodextrin-PDF. Parallel cultures were treated with conventional PDFs containing either 1.5% or 4.25% glucose. All fluids were tested at neutral pH. HPMC were assessed for viability, proliferation, IL-6 secretion and generation of reactive oxygen species (ROS). Results Incubation in the presence of icodextrin-PDF significantly reduced HPMC proliferation in a manner similar to that of 1.5% glucose-PDF In addition, exposure to icodextrin-PDF impaired viability and IL-6 release from HPMC. This effect occurred both after the short pre-treatment with neat icodextrin-PDF for 1–4 hours and after prolonged incubation (up to 10 days) in media supplemented with icodextrin-PDF (1:1). The dysfunction of icodextrin-treated HPMC was of the magnitude that was between the effects exerted by 1.5%- and 4.25%-glucose PDF. Furthermore, exposure of HPMC to icodextrin-PDF induced a dose-dependent increase in ROS generation which was comparable to that produced by 1.5%-glucose PDF. Conclusion Exposure to icodextrin-PDF may impair viability and function of HPMC. The detrimental effects of icodextrin-PDF are at least as serious as those produced by conventional heat-sterilized low glucose-based PDF.

PERITONEAL DIALYSIS FLUID INDUCES p38-DEPENDENT INFLAMMATION IN HUMAN MESOTHELIAL CELLS

♦ Background: Noninfectious upregulation of proinflammatory pathways in mesothelial cells may represent an integral part of their stress response upon exposure to peritoneal dialysis fluids (PDF). ♦ Objective: The aim of this study was to evaluate the role of the stress-inducible mitogen-activated protein kinase (MAPK) p38 in regulation of inflammatory and stress responses in mesothelial cells following in vitro exposure to PDF. ♦ Materials and Methods: Human peritoneal mesothelial cells were exposed to Dianeal PD4 or Physioneal (Baxter AG, Vienna, Austria) containing 1.36% glucose and then allowed to recover. Phosphorylation of p38, induction of heat shock protein-70 (HSP70), release of lactate dehydrogenase (LDH), secretion of interleukin (IL)-8, gene transcription, and mRNA stability were assessed with and without the MAPK p38 inhibitor SB203580. ♦ Results: Exposure to Dianeal resulted in phosphorylation of p38 within 30 minutes (309% of control, p < 0.05) and increased IL-8 release (370% of control, p < 0.05), HSP70 expression (151% of control, p < 0.05), and LDH release (180% of control, p < 0.05). Exposure to Physioneal resulted in attenuated changes in IL-8, HSP70, and LDH. Addition of the p38 inhibitor SB203580 to Dianeal resulted in dampened IL-8 release (–55%; p < 0.05) and basal HSP70 expression, and unchanged LDH release. Effects of p38 on IL-8 were at transcriptional, posttranscriptional, and translational levels. ♦ Conclusion: These data confirm concordant p38-dependent upregulation of IL-8 and HSP70 following exposure to bioincompatible PDF. The MAPK p38 pathway therefore links proinflammatory processes and the cellular stress response in human peritoneal mesothelial cells.

Injury-induced inflammation and inadequate HSP expression in mesothelial cells upon repeat exposure to dual-chamber bag peritoneal dialysis fluids.

Purpose Peritoneal dialysis fluids (PDFs) may induce inadequate heat-shock protein (HSP) expression and injury-related inflammation in exposed mesothelial cells. The aim of this study was to relate cellular injury to these cellular responses in mesothelial cells following repeated exposure to 3 commercial PDFs with different biocompatibility profiles. Methods Primary cultures of human peritoneal mesothelial cells (HPMC) were exposed to a 1:2 mixture of cell culture medium and CAPD2 (single-chamber bag PDF; Fresenius, Bad Homburg, Germany), Physioneal (dual-chamber bag PDF; Baxter, Deerfield, IL, USA) or Balance (dual-chamber bag PDF, Fresenius) for up to 10 days exposure time (4 dwells). Supernatant was analyzed for LDH, IL-6, and IL-8, cells for HSP-72 expression, and protein content. Results PDF exposure resulted in a biphasic pattern of cell damage switching from an earlier phase with increased injury by single-chamber PDF to a delayed phase with increased susceptibility to dual-chamber PDF. Sterile inflammation was related to LDH release over time and could be reproduced by exposure to necrotic cellular material. PDF exposure resulted in low HSP-72 expression in all tested PDFs. Conclusions Exposure to single-chamber as well as to dual-chamber bag PDFs induce increased vulnerability of mesothelial cells to repeated exposure of the same solution. These effects were delayed with dual-chamber PDFs. Injury-induced inflammation and impaired HSP expression upon PDF exposure might initiate a vicious cycle with progredient mesothelial cell damage upon repeated PDF exposure. Certainly, interventional studies and translation of these results into the in vivo system is needed.

Cross-omics comparison of stress responses in mesothelial cells exposed to heat- versus filter-sterilized peritoneal dialysis fluids.

Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat-sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses.