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To replace kidney function, peritoneal dialysis (PD) utilizes hyperosmotic PD fluids with specific physico-chemical properties. Their composition induces progressive damage of the peritoneum, leading to vasculopathies, decline of membrane function, and PD technique failure. Clinically used PD fluids differ in their composition but still remain bioincompatible. We mapped the molecular pathomechanisms in human endothelial cells induced by the different characteristics of widely used PD fluids by proteomics. Of 7894 identified proteins, 3871 were regulated at least by 1 and 49 by all tested PD fluids. The latter subset was enriched for cell junction-associated proteins. The different PD fluids individually perturbed proteins commonly related to cell stress, survival, and immune function pathways. Modeling two major bioincompatibility factors of PD fluids, acidosis, and glucose degradation products (GDPs) revealed distinct effects on endothelial cell function and regulation of cellular stress responses. Proteins and pathways most strongly affected were members of the oxidative stress response. Addition of the antioxidant and cytoprotective additive, alanyl-glutamine (AlaGln), to PD fluids led to upregulation of thioredoxin reductase-1, an antioxidant protein, potentially explaining the cytoprotective effect of AlaGln. In conclusion, we mapped out the molecular response of endothelial cells to PD fluids, and provided new evidence for their specific pathomechanisms, crucial for improvement of PD therapies.

Rationale: Patients with chronic kidney disease (CKD) have an exceedingly high cardiovascular risk; which further increases in patients on peritoneal dialysis (PD). The pathophysiological role of reactive metabolites accumulating in CKD such as glucose degradation products (GDP) is uncertain. Objective: Delineating the impact of GDP present in PD fluids in accelerated vasculopathy development in patients with CKD. Methods and Results: Omental and parietal peritoneal tissues were obtained from 107 children with CKD before dialysis and 90 children on chronic PD with PD fluids containing very low or high concentrations of GDP. Omental arterioles, protected from local PD fluid exposure by surrounding fat, were microdissected for multiomics analyses. High-GDP exposed omental arterioles exhibited 3-fold higher advanced glycation endproduct concentrations and upregulated genes involved in cell death/apoptosis and suppressed genes related to cell viability/survival, cytoskeleton organization, and immune response biofunctions. Vasculopathy-associated canonical pathways concordantly regulated on gene and protein level with high-GDP exposure included cell death/proliferation, apoptosis, cytoskeleton organization, metabolism and detoxification, cell junction signaling, and immune response. Parietal peritoneal arterioles of patients exposed to high-GDP fluids exhibited lumen narrowing compared to patients with CKD stage 5 (end-stage kidney disease) and patients on low-GDP PD, intima thickness was increased. Protein quantification verified increased proapoptotic activity and cytoskeleton disintegration, single-molecule-localization microscopy demonstrated arteriolar endothelial ZO-1 (zonula occludens-1) disruption. Absolute and per endoluminal surface length, arteriolar endothelial cell counts inversely correlated with GDP exposure, caspase-3, TGF (transforming growth factor)-β–induced pSMAD2/3 (phosphorylated SMAD2/3), interleukin-6, ZO-1 abundance, and lumen narrowing. In vitro, 3,4-dideoxyglucosone-3-ene reduced lamin-A/C and membrane ZO-1 assembly, increased pSMAD2/3, and ionic and 4 and 10 kDa permeability of arterial endothelial cells. Conclusions: Our findings indicate a fundamental role of GDP in PD-associated vasculopathy, exerted by endothelial cell junction and cytoskeleton disruption, and induction of apoptosis. They should redirect the focus of research and intervention on targeting reactive metabolite overload in CKD and PD. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01893710.

Background and Aims Patients with chronic kidney disease patients (CKD) have an exceedingly high cardiovascular risk. While vasculopathy is further accelerated during peritoneal dialysis (PD), the pathophysiological role of reactive metabolites such as glucose degradation products (GDP) is uncertain. Method Omental and parietal peritoneal tissues from 100 non-CKD individuals, 107 children with CKD5, 60 children treated with neutral pH, low GDP, and 30 children treated with acidic pH, high GDP PD fluids underwent standardized digital histomorphometry. Omental arterioles localized within the fat tissue, protected from direct PD fluid exposure were microdissected for multi-omics analysis. Key regulated pathways were validated by quantitative immunostaining, with localization microscopy in peritoneal tissues of matched cohorts and in vitro in human umbilical vein endothelial cells. Results Arterioles from children with CKD5 exhibited reduced lumen to vessel ratio (L/V) and reduced endothelial telomere length compared to non-CKD individuals; gene ontology analysis identified enrichment of arteriolar genes associated with nuclear telomere cap complex and focal adhesion. Pathway analysis of arteriolar cross-omics identified top canonical pathways including telomere extension by telomerase, actin cytoskeleton, integrin and tight junction signalling. Peritoneal vasculopathy progressed with PD vintage and was more pronounced with high versus low GDP exposure (p Quantitative validation in PD cohorts with similar PD vintage, dialytic glucose exposure and age (n=15 / group) verified increased proapoptotic activity and cytoskeleton disintegration with high-GDP exposure; single-molecule-localization microscopy demonstrated arteriolar endothelial zonula occludens-1 (ZO-1) disruption. Absolute and relative to endoluminal surface length, arteriolar endothelial cell counts were inversely correlated with GDP exposure, with apoptosis marker caspase-3, TGF-ß induced pSMAD2/3, interleukin-6, ZO-1 protein abundance and the degree of vasculopathy. In vitro, exposure to GDP 3,4-dideoxyglucosone-3-ene dose-dependently reduced nuclear endothelial lamin-A/C and membrane ZO-1 assembly. Transendothelial electrical resistance was decreased. ZO-1 and sealing tight junction claudin-5 protein abundance were decreased in cells after incubation with high GDP compared to low GDP PD fluid and culture media. On nanoscale level GDP reduced junction cluster formation in the membrane area. Conclusion Multi-omics analysis of omental arterioles from children without pre-existing vasculopathy and life-style related confounders identified key mechanisms of vascular aging in CKD5 and the major contribution of GDP to accelerated vasculopathy during PD, i.e. disruption of endothelial cell junctions and cytoskeleton and induction of apoptosis.

Background and Aims Infectious complications occur in a significant proportion of PD patients, limiting long-term applicability. Reduced peritoneal immune-competence, caused by the continuous exposure to PD-fluids, has been described as a therapy-related pathomechanisms, prompting the need for a tool to assess the functional peritoneal immune status. We established an ex-vivo stimulation assay to test host defence mechanisms in only 9ml of PD-effluent. The aim of this study was to analyse basal inflammation and immune-competence in the general PD population at routine conditions to evaluate the assay as surrogate parameter of immune competence and linking it to PD vintage and clinical outcome parameters. Method 147 of 284 (51.8%) adult and paediatric PD patients treated between April 2013 and September 2020 at the local Department of Nephrology were included in the analysis. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki. Patients were exclusively treated with neutral pH/multi-chamber PD fluids during the glucose dwells. The majority of the 558 included PD-effluent samples were obtained during standard 4-hours peritoneal equilibration tests (PET) with 3.86% glucose containing PDF. Samples from the pre-PET dwell and at PET time points 1-hour and 4-hours were collected and immediately processed. Additional effluent samples were obtained during unscheduled hospitalization and in the event of an acute peritonitis. Effluent samples were collected directly from the drainage bags into standard 9 ml additive-free sample tubes. For ex-vivo stimulation, 100 ng/ml toll-like receptor (TLR) 4 agonist LPS and TLR2 agonist Pam3Cys were added to the effluent in the 9 ml collection tubes in duplicates and incubated at 37°C for 24h. Unstimulated samples kept in parallel were used as controls. IL-6 and TNF-α concentrations were measured with ELISA in the supernatants. Results Ex-vivo stimulation of peritoneal cells significantly increased the IL-6 and TNF-α release compared to unstimulated controls and resulted in a dwell-time dependent increase, with a significant lower cytokine released at the 1h PET time point. To assess local inflammation IL-6 levels of crude effluent were determined. IL-6 concentrations remained stable over time on PD. Interestingly, we were able to show higher IL-6 levels in CAPD patients in comparison to APD. As chronic exposure to PD-fluids has been shown to dampen the peritoneal immune competence, consecutive peritoneal effluent bags, obtained from patients were analysed. In this subcohort of 183 4h-PET effluents we found a decline in cytokine secretion with time on PD (IL-6 r=-0.27, p=0.00015, TNFa r=-0.25, p=0.00071). In a subgroup the ex-vivo cytokine release of effluent samples from patients with an acute peritonitis was assessed. IL-6 levels of acute peritonitis effluent samples did not differ from the stimulated IL-6 levels of effluent samples without acute peritonitis (2.45 pg/mL vs 2.31 pg/mL, p=0.85, t-test) suggesting that the assay seemingly represents the in-vivo host-defence cytokine release accurately. Conclusion The study provides evidence of a correlation of declining local host defence and duration of PD-therapy. It supports the hypothesis of PD duration-dependent progressive impairment of the ability of the peritoneal immune cells to secrete cytokines in response to a pathogenic stimulus and thereby dampening the global peritoneal immuno-competence. This suggests the utility of this clinically feasible ex-vivo induced cytokine-release assay in peritoneal effluent as a surrogate of the functional peritoneal immune competence. Future analyses need to evaluate the assay as a tool to predict common clinical outcomes and define reference values to facilitate stratification of patient populations, clinical staging and to guide novel therapeutic interventions.

Background and Aims Tight junction (TJ) proteins have been suggested as molecular correlates for peritoneal semi-permeability and dialytic transport function in patients on peritoneal dialysis. Junction abundance in healthy individuals, in those with CKD5 and in patients on PD has not been described yet, the relation with peritoneal solute transport is unknown. Method Junction and transporter expression was analysed in multi-omics data sets from microdissected omental arterioles in children with normal renal function, CKD5 and on PD with low and high glucose degradation product (GDP) content (n=6/group). Parietal peritoneal tight junction proteins CLDN-1,-2,-3,-4,-5,-15, the adapter protein of claudins to actin cytoskeleton protein, zonula occludens-1 (ZO-1), the tricellular junction protein tricellulin (TriC), and transcellular transporters for sodium (ENaC), glucose (SGLT-1) and phosphate (PIT-1) were quantified in 40 non-CKD individuals, 20 children with CKD5 and 20 and 15 children on low- and high-GDP PD by quantitative, digital immunohistochemistry. Findings were correlated to 2-hour peritoneal equilibration test data obtained within 6 months of biopsy sampling (n=23). Primary human umbilical vein endothelial cells (HUVEC) were used to study the effects of single PD compounds on transepithelial electrical resistance (TER) and molecular size-dependent paracellular transport capacity. Co-stained monolayers were visualized by confocal microscopy. Single junction molecule localization and clustering were analysed by super resolution microscopy. Results Transcriptome and proteome pathway enrichment analysis of arteriolar junction and membrane protein demonstrated regulation in CKD5 versus health, and differential regulation by low- and high-GDP PD versus CKD5. In the parietal peritoneum all junctions and cellular transporters were expressed in endothelial and mesothelial cells. Pore forming CLDN-2, -4 and -15 were localized also in submesothelial immune cells. Parietal peritoneal junction abundance was age-dependent and also modified by CKD5 and PD. Mesothelial and endothelial abundance of the selective cation/water channel CLDN-2 increased in patients on low- and high-GDP PD fluids. Adaptor protein ZO-1 was upregulated in low GDP-PD versus CKD5, while sealing proteins CLDN -3 and -5 were downregulated. D/P creatinine, D/P phosphate, D/D0 glucose were similar in CKD5 and PD groups. D/P creatinine correlated with mesothelial CLDN-15, with arteriolar CLDN-2 and TriC and with endothelial ENaC. D/P phosphate correlated with endothelial CLDN-15, D/D0 glucose with mesothelial CLDN-4 and arteriolar CLDN-2. Capillary ZO-1 correlated with 24-h ultrafiltration standardized to body surface area and dialytic glucose exposure. In vitro, TER was decreased by low pH, glucose and 0.5µM methylglyoxal after 5h. Alanyl-glutamine (AlaGln) dose-dependently increased TER, and reduced 10kDa and 70kDa solute at 24mM, increased the abundance of ZO-1 and CLDN5 at cell-cell contacts, and on nanoscale clustering of the pore-forming CLDN2 and CLDN5. Conclusion Abundance of parietal peritoneal sealing and pore forming junctions and transcellular solute transporters varies with cell type and age and is differentially regulated by PD and associated with dialytic transport function. Our preliminary analyses illustrate the role of junctions and cellular transporters for solute transport across the peritoneal mesothelial and endothelial cell barrier. In-depth understanding of specific molecular functions should provide targets for modulation to improve efficacy of PD.

Peritoneal dialysis (PD) offers specific advantages over hemodialysis, enabling increased autonomy of patients with end-stage renal disease, but PD-related complications need to be detected in a timely manner. Fourier transform infrared (FTIR) spectroscopy could provide rapid and essential insights into the patients&rsquo, risk profiles via molecular fingerprinting of PD effluent, an abundant waste material that is rich in biological information. In this study, we measured FTIR spectroscopic profiles in PD effluent from patients taking part in a randomized controlled trial of alanyl-glutamine addition to the PD-fluid. Principal component analysis of FTIR spectra enabled us to differentiate between effluent samples from patients immediately after completion of instillation of the PD-fluid into the patients&rsquo, cavity and 4 h later as well as between patients receiving PD-fluid supplemented with 8 mM alanyl-glutamine compared with control. Moreover, feasibility of FTIR spectroscopy coupled to supervised classification algorithms to predict patient-, PD-, as well as immune-associated parameters were investigated. PD modality (manual continuous ambulatory PD (CAPD) vs. cycler-assisted automated PD (APD)), residual urine output, ultrafiltration, transport parameters, and cytokine concentrations showed high predictive potential. This study provides proof-of-principle that molecular signatures determined by FTIR spectroscopy of PD effluent, combined with machine learning, are suitable for cost-effective, high-throughput diagnostic purposes in PD.

Background and Aims The composition of all currently available peritoneal dialysis (PD) fluids triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels, eventually leading to failure of the technique. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of these cytoprotective additives in PDF in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial and endothelial cells were co-cultured in transwell plates. Mesothelial cells were grown in the upper compartment and primary human umbilical vein endothelial cells (HUVEc) or primary microvascular cells were grown in the lower compartment. PDF with or without cytoprotective compounds, was added to the upper compartment to only expose mesothelial cells directly to different dilutions of the fluid. Effects on cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release and live-dead staining of cells. Proteome profiles were analysed for both cell-types separately and in combination using two-dimensional difference gel electrophoresis (2D-DiGE) and liquid chromatography coupled to mass spectrometry (LC-MS). In vitro findings were related to PD-induced arteriolar changes based on abundance profiles of micro-dissected omental arterioles of children treated with conventional PD-fluids and age-matched controls with normal renal function. Results Marked cellular injury of HUVEc after PD-fluid exposure was associated with a molecular landscape of the enriched biological process clusters ‘glucose catabolic process’, ‘cell redox homeostasis’, ‘RNA metabolic process’, ‘protein folding’, ‘regulation of cell death’, and ‘actin cytoskeleton reorganization’ that characterize PD-fluid cytotoxicity and counteracting cellular repair process respectively. PDF-induced cell damage was reduced by AlaGln and LiCl both in mesothelial and endothelial cells. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton reorganization. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. Comparison to human arterioles confirmed overlapping protein regulation between endothelial cells in vitro and in vivo, proving harmful effects of PD-fluids on endothelial cells leading to drastic changes of the cellular process landscape. Conclusion In summary, this study shows harmful effects of PD-fluids also effecting endothelial cells and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the peritoneal membrane for testing direct and indirect effects of cytoprotective additives in PDF. When cultured and stressed in close proximity cells may respond differently. Characterisation of PD-induced perturbations will allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

Background and Aims PD-fluids lead to generation of reactive oxygen species (ROS) in the peritoneal cavity. The caused oxidative stress, defined as a cellular oxidant-antioxidant imbalance impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. We aim to analyze the impact and specific targets of ROS during PD and the anti-oxidative mechanism of supplementation of PD-fluid with alanyl-glutamine (AlaGln) on a global proteome-wide level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (H2O2) and PD-fluid induced stress. Levels of oxidative stress were first validated by increased intracellular ROS and superoxide dismutase activity with PD-fluid and H2O2 treatment and a reduction of these parameters by the addition of AlaGln. To detect alterations of the redox proteome, cysteine residues were either directly or indirectly labeled with fluorescent dyes (redox-2D-DiGE) or isobaric tags (iodo-TMT). Results: The gel-based approach allowed global visualization of the reduced and oxidized cysteines and revealed redox profiles of 540 protein spots. Compared to control, we found an increase in oxidized and decrease in reduced cysteines in all PD treatments. The development of a highly sensitive LC/MS-based redox proteomics workflow allowed identification of ∼950 proteins affected by redox-stress in mesothelial cells and confirmed the quantitative levels seen on cysteine oxidation. The addition of AlaGln reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but further showed different proteins to be affected by redox modifications. Conclusion: Redox proteomics of peritoneal cells could represent a novel approach for the identification of mediators of PD-induced pathomechanisms, but also to evaluate effects of novel anti-oxidant therapeutical or pharmacological interventions.

Life-saving renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. However, the pathophysiological mechanisms involved are incompletely understood, limiting identification of therapeutic targets. We report that addition of lithium chloride (LiCl) to PDF is a translatable intervention to counteract PDF-induced mesothelial cell death, peritoneal membrane fibrosis, and angiogenesis. LiCl improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most consistently counter-regulated by LiCl. In vitro and in vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like up-regulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGF-β-independent activation of TGF-β-regulated targets. In contrast, αB-crystallin knockdown decreased VEGF expression and early mesothelial-to-mesenchymal transition. LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically up-regulated in response to PDF and increased in peritoneal mesothelial cells from biopsies from pediatric patients undergoing PD, correlating with markers of angiogenesis and fibrosis. LiCl-supplemented PDF promoted morphological preservation of mesothelial cells and the submesothelial zone in a mouse model of chronic PD. Thus, repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy.

Readily available peritoneal dialysis (PD) effluents from PD patients in the course of renal replacement therapy are a potentially rich source for molecular markers for predicting clinical outcome, monitoring the therapy, and therapeutic interventions. The complex clinical phenotype of PD patients might be reflected in the PD effluent metabolome. Metabolomic analysis of PD effluent might allow quantitative detection and assessment of candidate PD biomarkers for prognostication and therapeutic monitoring. We therefore subjected peritoneal equilibration test effluents from 20 stable PD patients, obtained in a randomized controlled trial (RCT) to evaluate cytoprotective effects of standard PD solution (3.86% glucose) supplemented with 8 mM alanyl-glutamine (AlaGln) to targeted metabolomics analysis. One hundred eighty eight pre-defined metabolites, including free amino acids, acylcarnitines, and glycerophospholipids, as well as custom metabolic indicators calculated from these metabolites were surveyed in a high-throughput assay requiring only 10 μl of PD effluent. Metabolite profiles of effluents from the cross-over trial were analyzed with respect to AlaGln status and clinical parameters such as duration of PD therapy and history of previous episodes of peritonitis. This targeted approach detected and quantified 184 small molecules in PD effluent, a larger number of detected metabolites than in all previous metabolomic studies in PD effluent combined. Metabolites were clustered within substance classes regarding concentrations after a 4-h dwell. PD effluent metabolic profiles were differentiated according to PD patient sub-populations, revealing novel changes in small molecule abundance during PD therapy. AlaGln supplementation of PD fluid altered levels of specific metabolites, including increases in alanine and glutamine but not glutamate, and reduced levels of small molecule indicators of oxidative stress, such as methionine sulfoxide. Our study represents the first application of targeted metabolomics to PD effluents. The observed metabolomic changes in PD effluent associated with AlaGln-supplementation during therapy suggested an anti-oxidant effect, and were consistent with the restoration of important stress and immune processes previously noted in the RCT. High-throughput detection of PD effluent metabolomic signatures and their alterations by therapeutic interventions offers new opportunities for metabolome-clinical correlation in PD and for prescription of personalized PD therapy.

PurposePeritoneal 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.MethodsPrimary 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.ResultsPDF 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.ConclusionsExposure 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.

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD. We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln). With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense. Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention.

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 effluent (PDE) represents a rich pool of potential biomarkers for monitoring disease and therapy. Until now, proteomic studies have been hindered by the plasma-like composition of the PDE. Beads covered with a peptide library are a promising approach to remove high abundant proteins and concentrate the sample in one step. In this study, a novel approach for proteomic biomarker identification in PDEs consisting of a depletion and concentration step followed by 2D gel based protein quantification was established. To prove this experimental concept a model system of artificial PDEs was established by spiking unused peritoneal dialysis (PD) fluids with cellular proteins reflecting control conditions or cell stress. Using this procedure, we were able to reduce the amount of high abundant plasma proteins and concentrate low abundant proteins while preserving changes in abundance of proteins with cellular origin. The alterations in abundance of the investigated marker for cell stress, the heat shock proteins, showed similar abundance profiles in the artificial PDE as in pure cell culture samples. Our results demonstrate the efficacy of this system in detecting subtle changes in cellular protein expression triggered by unphysiological stress stimuli typical in PD, which could serve as biomarkers. Further studies using patients' PDE will be necessary to prove the concept in clinical PD and to assess whether this technique is also informative regarding enriching low abundant plasma derived protein biomarker in the PDE.

Non-physiological components of peritoneal dialysis fluids (PDF) lead to the injury of peritoneal mesothelial cells resulting in the failure of peritoneal dialysis (PD) potentially via inadequate induction of the protective heat shock response (HSR). Glycogen synthase kinase-3β (GSK-3β) is a negative regulator of cell survival partly by suppression of the HSR and is influenced by stress stimuli also present in conventional PDF. The effects of PDF on GSK-3β activation and the impact of GSK-3β inhibition with lithium (LiCl) were investigated on cell survival with special regard to HSR, in particular to heat shock transcription factor 1 (HSF-1) activation and Hsp72 production in an in vitro model of PD using MeT-5A and primary mesothelial cells. Incubation of cells with the PDF Dianeal® (glucose-based, low pH, high glucose degradation products (GDP)) and Extraneal® (icodextrin-based, low pH, low GDP) caused activation of GSK-3β compared to the other tested PDF, i.e. Balance®, Physioneal® (normal pH, glucose-based, low GDP) and Nutrineal® (moderately acidic, amino acid-based). Inhibition of GSK-3β with LiCl in Dianeal® and Extraneal®-treated cells dose-dependently decreased cell damage and death rate and was paralleled by higher HSF-1 activation and Hsp72 expression. GSK-3β is activated by low pH GDP containing PDF with and without glucose as osmotic agent, indicating that GSK-3β is involved in mesothelial cell signalling in response to experimental PD. Inhibition of GSK-3β with LiCl ameliorated cell injury and improved HSR upon PDF exposure. Thus, GSK-3β inhibitors likely have therapeutic potential as cytoprotective additive for decreasing PDF toxicity.

Background. Exposure of mesothelial cells to peritoneal dialysis fluids (PDF) results in cytoprotective cellular stress responses (CSR) that counteract PDF-induced damage. In this study, we tested the hypothesis that the CSR may be inadequate in relevant models of peritoneal dialysis (PD) due to insufficient levels of glutamine, resulting in increased vulnerability against PDF cytotoxicity. We particularly investigated the role of alanyl–glutamine (Ala-Gln) dipeptide on the cytoprotective PDF stress proteome. Methods. Adequacy of CSR was investigated in two human in vitro models (immortalized cell line MeT-5A and mesothelial cells derived from peritoneal effluent of uraemic patients) following exposure to heat-sterilized glucose-based PDF (PD4-Dianeal, Baxter) diluted with medium and, in a comparative proteomics approach, at different levels of glutamine ranging from depletion (0 mM) via physiological (0.7 mM) to pharmacological levels (8 mM administered as Ala-Gln). Results. Despite severe cellular injury, expression of cytoprotective proteins was dampened upon PDF exposure at physiological glutamine levels, i ndicating an inadequate CSR. Depletion of glutamine aggravated cell injury and further reduced the CSR, whereas addition of Ala-Gln at pharmacological level restored an adequate CSR, decreasing cellular damage in both PDF exposure systems. Ala-Gln specifically stimulated chaperoning activity, and cytoprotective processes were markedly enhanced in the PDF stress proteome. Conclusions. Taken together, this study demonstrates an inadequate CSR of mesothelial cells following PDF exposure associated with low and physiological levels of glutamine, indicating a new and potentially relevant pathomechanism. Supplementation of PDF with pharmacological doses of Ala-Gln restored the cytoprotective stress proteome, resulting in improved resistance of mesothelial cells to exposure to PDF. Future work will study the clinical relevance of CSRmediated cytoprotection.

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.

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.

Introduction. Senescence of peritoneal mesothelial cells represents a biological program defined by arrested cell growth and altered cell secretory phenotype with potential impact in peritoneal dialysis. This study aims to characterize cellular senescence at the level of global protein expression profiles and modification of proteins withO-linked N-acetylglucosamine (O-GlcNAcylation).Methods. A comparative proteomics analysis between young and senescent human peritoneal mesothelial cells (HPMC) was performed using two-dimensional gel electrophoresis.O-GlcNAc status was assessed by Western blot under normal conditions and after modulation with 6-diazo-5-oxo-L-norleucine (DON) to decreaseO-GlcNAcylation orO-(2-acetamido-2-deoxy-D-glucopyranosylidene) aminoN-phenyl carbamate (PUGNAc) to increaseO-GlcNAcylation.Results. Comparison of protein pattern of senescent and young HPMC revealed 29 differentially abundant protein spots, 11 of which were identified to be actin (cytoplasmic 1 and 2), cytokeratin-7, cofilin-2, transgelin-2, Hsp60, Hsc70, proteasomeβ-subunits (type-2 and type-3), nucleoside diphosphate kinase A, and cytosolic 5′(3′)-deoxyribonucleotidase. Although the global level ofO-GlcNAcylation was comparable, senescent cells were not sensitive to modulation by PUGNAc.Discussion. This study identified changes of the proteome and altered dynamics ofO-GlcNAc regulation in senescent mesothelial cells. Whereas changes in cytoskeleton-associated proteins likely reflect altered cell morphology, changes in chaperoning and housekeeping proteins may have functional impact on cellular stress response in peritoneal dialysis.

Peritoneal dialysis effluent (PDE) represents a rich pool of potential biomarkers for monitoring disease and therapy. Until now, proteomic studies have been hindered by the plasma-like composition of the PDE. Beads covered with a peptide library are a promising approach to remove high abundant proteins and concentrate the sample in one step. In this study, a novel approach for proteomic biomarker identification in PDEs consisting of a depletion and concentration step followed by 2D gel based protein quantification was established. To prove this experimental concept a model system of artificial PDEs was established by spiking unused peritoneal dialysis (PD) fluids with cellular proteins reflecting control conditions or cell stress. Using this procedure, we were able to reduce the amount of high abundant plasma proteins and concentrate low abundant proteins while preserving changes in abundance of proteins with cellular origin. The alterations in abundance of the investigated marker for cell stress, the heat shock proteins, showed similar abundance profiles in the artificial PDE as in pure cell culture samples. Our results demonstrate the efficacy of this system in detecting subtle changes in cellular protein expression triggered by unphysiological stress stimuli typical in PD, which could serve as biomarkers. Further studies using patients' PDE will be necessary to prove the concept in clinical PD and to assess whether this technique is also informative regarding enriching low abundant plasma derived protein biomarker in the PDE.

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.