Mesothelial Stress Response and Cytoprotektion

Longterm peritoneal dialysis (PD) is frequently complicated by technical failure due to peritoneal membrane failure. Bio-incompatible PD fluids (PDF) and peritoneal inflammation are regarded as the major culprits. Exposure of human mesothelial cells (MC) to PDF results in marked cytotoxicity, ranging from minor dysfunction to cell death. Current research, mainly sponsored by pharmaceutical PDF manufacturers, aims to increase biocompatibility of PDF and thereby reduce MC damage. As an innovative alternative, our overall goal is to develop a novel therapeutic tool that enhances repair and/or prevent injury in MC upon PDF exposure. Our previous work demonstrated that non-lethal exposure to PDF indeed not only resulted in MC injury but also in specific cytoprotective responses. We recently showed in experimental PD that overexpression of heat-shock proteins (HSP) improved MC survival following PDF exposure. However, the stress response is known to induce complex cellular processes and HSP expression only represents a quite limited aspect. In the proposed program, we will apply a combined approach based on proteomics, fluorescence-assisted-cell- sorting and bio-informatic analysis to experimental PD. Protein profiles in MC exposed to isolated and combined physicochemical properties of PDF will be assessed in human cell culture systems and in the acute rat in-vivo model. Comparison between protein profiles will then characterize essential processes that are associated with cytoprotection against PDF exposure in MC. Finally, we will specifically manipulate the stress response by "cytoprotective small molecule" additives to PDF and search for the effects of these interventions on cytoprotection ("desired effects") and overall cellular protein expression/modifications patterns ("side effects?"). This research program will not only give relevant new insights on the stress response in MC upon PDF exposure, but also develop a novel "cytoprotective PDF" prototype. As the clinical setting of PD consists of repeated timed exposure of MC to acute and uniform cellular insults upon PDF exposure, PD represents an unique opportunity to transfer this approach from bench to bed-side, applying cytoprotective dialysates as an innovative therapeutic tool.

Peritoneal dialysis is often the only possible modality of renal replacement therapy for children or newborns with kidney failure and it is also a convenient alternative to hemodialysis. In peritoneal dialysis a fluid (PDF) is repeatedly filled in and drained from the peritoneal cavity. PDF removes water and toxic substances from the patient and thereby replaces kidney function. Unfortunately PDF are regarded as bio-incompatible because in the course of long-term treatment they harm the mesothelial cell layer, lining the peritoneal cavity, which leads to `technical` failure of the therapy. Our research group focuses on the molecular mechanisms leading to this damage of the peritoneal membrane and tries to selectively manipulate the stress response of mesothelial cells to render the cells more resistant to PDF. To elucidate the mechanisms responsible for this cell deterioration we applied a `proteomics` approach. With this method the components (proteins) of cells after PDF treatment can be separated in a gel based on their charge and their mass. By comparing the resulting gels, we could identify proteins of particular important functions after PDF stress. So called heat shock proteins (HSP) were identified as important cellular repair components after PDF exposure. These molecular paramedics are designated to stabilize other proteins - usually after exposure to heat - and thereby improve the cells` survival. Besides HSP mainly proteins involved in inflammatory processes, programmed cell death (apoptosis) and cytoskeletal assembly play an important role. However, our experiments showed that repeated exposure to PDF leads to a conditioning effect that prevents the cells from being protected again and again. In this project we analyzed not only the reaction of mesothelial cells to PDF but also to its constituents and we could identify so called glucose degradation products - originating from heat sterilization of the glucose-containing PDF - as possible inhibitors of a proper response of the cells to PDF stress. Following that, we tested various substances as possible PDF additives searching for potential drugs that can improve the mesothelial stress response. Above all, the natural occurring amino acid glutamine was evaluated in different models of experimental PD. Indeed we found a protective effect of glutamine in mesothelial cells after PDF exposure. This discovery can be used to enable the supply of more bio-compatible PDF in the future and to drive the development of cytoprotective PDF as novel therapeutic tools in PD.