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Long-term clinical outcome of peritoneal dialysis (PD) depends on adequate removal of small solutes and water. The peritoneal endothelium represents the key barrier and peritoneal transport dysfunction is associated with vascular changes. Alanyl-glutamine (AlaGln) has been shown to counteract PD-induced deteriorations but the effect on vascular changes has not yet been elucidated. Using multiplexed proteomic and bioinformatic analyses we investigated the molecular mechanisms of vascular pathology in-vitro (primary human umbilical vein endothelial cells, HUVEC) and ex-vivo (arterioles of patients undergoing PD) following exposure to PD-fluid. An overlap of 1813 proteins (40%) of over 3100 proteins was identified in both sample types. PD-fluid treatment significantly altered 378 in endothelial cells and 192 in arterioles. The HUVEC proteome resembles the arteriolar proteome with expected sample specific differences of mainly immune system processes only present in arterioles and extracellular region proteins primarily found in HUVEC. AlaGln-addition to PD-fluid revealed 359 differentially abundant proteins and restored the molecular process landscape altered by PD fluid. This study provides evidence on validity and inherent limitations of studying endothelial pathomechanisms in-vitro compared to vascular ex-vivo findings. AlaGln could reduce PD-associated vasculopathy by reducing endothelial cellular damage, restoring perturbed abundances of pathologically important proteins and enriching protective processes.

Peritonitis remains a major cause of morbidity and mortality during chronic peritoneal dialysis (PD). Glucose-based PD fluids reduce immunological defenses in the peritoneal cavity. Low concentrations of peritoneal extracellular glutamine during PD may contribute to this immune deficit. For these reasons we have developed a clinical assay to measure the function of the immune-competent cells in PD effluent from PD patients. We then applied this assay to test the impact on peritoneal immune-competence of PD fluid supplementation with alanyl-glutamine (AlaGln) in 6 patients in an open-label, randomized, crossover pilot trial (EudraCT 2012-004004-36), and related the functional results to transcriptome changes in PD effluent cells. Ex-vivo stimulation of PD effluent peritoneal cells increased release of interleukin (IL) 6 and tumor necrosis factor (TNF) α. Both IL-6 and TNF-α were lower at 1 h than at 4 h of the peritoneal equilibration test but the reductions in cytokine release were attenuated in AlaGln-supplemented samples. AlaGln-supplemented samples exhibited priming of IL-6-related pathways and downregulation of TNF-α upstream elements. Results from measurement of cytokine release and transcriptome analysis in this pilot clinical study support the conclusion that suppression of PD effluent cell immune function in human subjects by standard PD fluid is attenuated by AlaGln supplementation.

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.

Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P = .01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n = 3), glucocorticoid receptor NR3C1 (n = 4), and components of the mismatch repair pathways (n = 3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P = .02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.

Renal ischemia increases tubular immunogenicity predisposing to increased risk of kidney allograft rejection. Ischemia-reperfusion not only disrupts cellular homeostasis but also induces the cytoprotective heat shock response that also plays a major role in cellular immune and defense processes. This study therefore tested the hypothesis that upregulation of renal tubular immunogenicity is an integral part of the heat shock response after renal ischemia. Expressions of 70 kDa heat shock protein (Hsp70), major histocompatibility complex (MHC) class II, and intercellular adhesion molecule-1 (ICAM-1) were assessed in normal rat kidney (NRK) cells following ATP depletion (antimycin A for 3 h) and heat (42°C for 24 h). In vitro, transient Hsp70 transfection and heat shock factor-1 (HSF-1) transcription factor decoy treatment were performed. In vivo, ischemic renal cortex was investigated in Sprague-Dawley rats following unilateral renal artery clamping for 45 min and 24 h recovery. Upregulation of Hsp70 was closely and significantly correlated with upregulation of MHC class II and/or ICAM-1 following ATP depletion and heat injury. Bioinformatics analysis searching the TRANSFAC database predicted HSF-1 binding sites in these genes. HSF-1 decoy significantly reduced the expression of immunogenicity markers in stressed NRK cells. In the in vivo rat model of renal ischemia, concordant upregulation of MHC class II molecules and Hsp70 suggests biological relevance of this link. The results demonstrate that upregulation of renal tubular immunogenicity is an integral part of the heat shock response after renal ischemia. Bioinformatic analysis predicted a molecular link to tubular immunogenicity at the level of the transcription factor HSF-1 that was experimentally verified by HSF-1 decoy treatment. Future studies in HSF-1 knockout mice are needed.