MO711: Evaluation of an in Vitro Co-Culture Model for Studying Modulation of Cross-Talk between Endothelial and Mesothelial Cells by Cytoprotective Additives in Peritoneal Dialysis Fluids

Abstract

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is a life-saving renal replacement therapy. The composition of all currently available peritoneal dialysis fluids (PDF) 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. 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 in mesothelial (MC) and endothelial (EC) cells. However, it is yet unclear if potential cross talk between cells present in the peritoneal membrane mediates the observed modulation of PD-associated changes. Currently, there is no model system available to study relevant processes in these cells when in close proximity. Here, we aimed to develop a co-culture model for investigating cell-to-cell communication in a PD-relevant setting using cellular proteome and secretome analyses and investigate the effects of cytoprotective additives. METHOD For modelling the peritoneal membrane in vitro, MCs and ECs were co-cultured in transwell plates. MCs were grown in the upper compartment and primary microvascular ECs were grown in the lower compartment. PDF with or without additives, was added to the upper compartment to expose MCs directly, while the ECs below were kept in medium. Cellular proteome and secretome profiles were analysed for both cell types cultured individually or together by a quantitative mass spectrometry approach. Prior to analysis of the secretome, a combinatorial peptide ligand library coupled to beads was employed to enrich low abundant proteins and deplete high abundant proteins. RESULTS Proteome analysis revealed perturbation of major cellular processes including cytoskeleton reorganization, stress response, and regulation of cell death that characterize PDF cytotoxicity. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to individual cultures. Several pathways relevant for PD, such as VEGF signaling in ECs or oxidative stress response in MCs were found only under co-culture conditions. Combined analysis of proteome and secretome showed specific ligand–receptor pairs expressed only under co-culture conditions regulating some of these pathways. Differentially regulated cellular and secreted proteins after PDF exposure were related to processes like angiogenesis, immune response, or and extracellular processes such as integrin signaling. Addition of 10 mM LiCl to PDF led to modulation of VEGF and oxidative stress response pathways in ECs as well as ligand–receptor pairs involved in these pathways, possibly explaining MC–EC crosstalk during PDF exposure of these cells and the cytoprotective effect of LiCl. CONCLUSION Harmful effects of PDF on MCs may also affect ECs, as demonstrated by our data. Interestingly, both cells type react differently when co-cultured compared to individual culture models, showing the importance of models that allow cell type interaction to mimic the in vivo conditions. We identified potential signaling axes between the cell types that could explain pathophysiological changes of the peritoneal membrane during PD treatment. We have also elucidated potential mechanisms by which the cytoprotective additive LiCl may modulate crosstalk between MCs and ECs to counteract adverse PDF effects in the local peritoneal environment. Linking local peritoneal damage with systemic vasculopathy through perturbations of PD-induced cellular crosstalk, may identify therapeutic targets to reduce the cardiovascular risk of PD patients and current limitations of the therapy.