#5930 ENDOTHELIAL CELL PROTEOME AND SECRETOME ALTERATION UNDER CHRONIC UREMIC EXPOSURE
Authors: Sacnun Juan Manuel , Kratochwill Klaus , Herzog Rebecca 
Published: June 2023

Abstract

Background and Aims
Dialysis therapies including peritoneal dialysis (PD) are described to lead to premature vascular aging, atherosclerosis and associated cardiomyopathies, which are associated with high cardiovascular morbidity and mortality. Chronic inflammation, incomplete clearance of uremic toxins, and dyslipidaemia, are factors affecting the vasculature system. However, how these factors affect the endothelium, the fist cell layer in contact with these molecules, and the molecular mechanisms triggered by uremic toxins remain poorly understood. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PD fluids reduce peritoneal damage in mesothelial and endothelial (EC) cells. Their potential systemic effects were not studied so far. Here, we demonstrate, in a newly developed model system, the changes induced by chronic exposure to serum of PD patients in the proteome and secretome of endothelial cells, and the effects of cytoprotective additives.

Method
For modelling systemic conditions of PD patients, primary EC were cultured for 5 passages in medium containing 10% serum collected from PD patients (n = 26) during regular PET tests, or from healthy donors (n = 12). Cytoprotective additives were added in parallel experiments. Cells were stably labelled (SILAC) to differentiate cell and donor serum proteins and the cellular proteome and secretome profiles were analysed by quantitative mass spectrometry. Prior to analysis of the secretome, equalizer beads, to enrich low abundant and deplete high abundant proteins, were used. ECIS (Electric Cell-substrate Impedance Sensing), was used to measure barrier function, growth rate, and permeability.

Results
Proteome analysis revealed perturbation of major cellular processes by serum of PD patients including inflammatory related processes such TLR regulation and complement activation, as well membrane related processes such as extracellular matrix interactions and junctions, and plasma lipoprotein remodelling. Addition of LiCl counteracted cell-adhesion related proteins. ECIS analysis showed that in uremic conditions the EC monolayer has a decreased barrier function compared to healthy conditions, and LiCl partially restores the tightness of the membrane. Secretome analysis showed differentially regulated proteins related to oxidative stress, senescence-associated secretory phenotype (SASP), and apoptosis. Interestingly, in the secretome, LiCl addition counter regulated INHBA and tissue factor pathway inhibitor 2, both proteins related to vascular calcification.

Conclusion
Surprisingly few studies have analysed uremic effects on EC using proteomics approaches, and no reports of chronic settings, modelling the patient situation, are available. Our data demonstrates that EC react to serum factors of PD patients with increased inflammation, permeability and a secretory profile. Interestingly, our model reflects many of the known effects on the vasculature, but unravels the molecular mechanism that may induce those processes. We have also identified potential mechanisms by which the addition of cytoprotective additives may counteract some of the uremic effects systemically. Secretomics data identified several proteins secreted by EC that are regulated in uremia with potential for cellular crosstalk with other cells of the vasculature, showing the potential to identify therapeutic targets to reduce the cardiovascular risk of PD patients and current limitations of the therapy.