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 CSR-mediated cytoprotection.

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.

In this paper CIEF combined with MALDI-MS is described using a sheath-liquid-assisted automatic sample deposition from the separation capillary onto a MALDI target. Sample/matrix preparation techniques on the target resembling the dried droplet and the thin layer methods were evaluated in the context of the automatic spotting. Volatile buffers were used as IEF catholyte solutions. Test samples consisting of tryptic peptides, glycopeptides, and phosphopeptides of well-known proteins showed that CIEF-MALDI-MS can be used as effective preseparation method prior to MS, allowing to obtain the amino acid sequence coverage of proteins similar to that achieved with CZE-MALDI-MS and CZE-ESI-MS. Particularly, completeness and reliability of glycopeptide analysis is much enhanced by the preseparation. The effect is less pronounced but still significantly found with phosphopeptides present in the test protein. Finally, a test sample of five standard proteins demonstrates the suitability of this technique also for the treatment of intact proteins. This technique has potential to emerge as a faster method analogous and complementary to 2-DE and to IPG-IEF-MALDI-MS demonstrated before by the group of Loo [1].

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.