Investigating Dynamic Cellular Crosstalk and Pathomechanisms in Experimental Peritoneal Dialysis through Secretomics Analysis

Peritoneal Dialysis (PD) is a life-saving renal replacement therapy. Yet, the used of PD-fluids induce adverse effects leading to detrimental changes in the peritoneal membrane (PM), reducing the therapy efficacy. Cross-talk among different peritoneal cell types modulates PD-associated deterioration. Currently, there is no model available to study the interactions of these cells in close proximity. Here, we aimed to develop a co-culture model to explore cell-to-cell communication by analyzing the cellular proteome and secretome.

For modelling the PM, mesothelial and endothelial cells were co-cultured in transwell plates under optimized conditions for simultaneous culturing under non-starving conditions (5% FCS). Cells were exposed to PD-fluids in either co- or single-culture conditions. To overcome current limitation on secretomics analysis in non-starvation conditions, an equalizer approach was used to deplete high abundant proteins in combination with SILAC for tracing the cell origin of secreted proteins. For quantitative analysis of cellular and secreted protein abundances, LC-MS (TMT-18plex) was performed.

Co-cultured cells yielded differently regulated pathways following PD-fluid exposure compared to individual cultures. Analysis of the secretome revealed cell-specific pattern of secretion and low correlation with intra-cellular abundances. Combined proteome and secretome analysis revealed different ligand-receptor pairs expressed uniquely in co-culture. Furthermore, the secretome was able to capture different types of secretion. Using protein-protein interaction analysis, the identified cell-secreted proteins (~1900) formed 11 functional clusters, interacting with different receptors presented by the cells. The resulting interactome between cells through the secreted proteins in combination with differentially expressed cellular and secreted proteins revealed modulation of the cross-talk by PD-fluids. Identified secreted proteins had a high overlap with clinical samples from PD-patients and largely correlated with their clinical parameters. The combination of the modulation of the cross-talk by PD-fluids and clinical information revealed 3 novel candidates regulating pathways related to angiogenesis, EMT and NOTCH, which were validated in a chronic mouse model and patient samples.

This study shows that harmful effects of PD-fluid exposure on mesothelial also affect endothelial cells. Co-cultures revealed different responses compared to individual cultures, highlighting the importance of models that allow interactions between multiple cell types. Secretomics analysis showed to be essential to understand cell-to-cell communication, and showed a more complex modulation than classical ligand-receptor interaction. We further identified novel potential signaling axes between the cell types explaining pathophysiological changes of the PM during PD that may allow identifying therapeutic targets to reduce current limitations of PD.