FC 103PROTEOME WIDE OXIDATIVE STRESS PROFILING IN MESOTHELIAL CELLS INDUCED BY PERITONEAL DIALYSIS FLUID
Authors: Herzog Rebecca , Wagner Anja , Kratochwill Klaus 
Published: May 2021

Abstract

Background and Aims
Reactive oxygen species (ROS) in the peritoneal cavity may result both from CKD and the specific composition of peritoneal dialysis fluids (PDF). Elevated cellular oxidative stress is defined as a cellular oxidant/antioxidant imbalance which impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. So far only single targets or mediators of oxidative stress were investigated in mesothelial cells exposed to PD fluids. Here, we aim to analyze the broad impact and also identify individual targets of ROS during PD. Using the developed technique the anti-oxidative effect of alanyl-glutamine (AlaGln) supplementation of PDF was characterized on the proteome level.

Method
To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (100 µm hydrogen peroxide (H2O2)) and PD-fluid induced stress. Levels of oxidative stress were first evaluated by intracellular ROS levels and superoxide dismutase activity. Oxidative stress levels induced by PDF were titrated to comparable levels of H2O2 treatment to be able to characterize redox modifications and the effect of addition of 8 mM AlaGln. To detect alterations of the redox proteome we adapted and refined an approach combining redox-sensitive isobaric mass tags and high-performance liquid chromatography coupled to mass spectrometry (LC/MS). We used a sequential combination of direct and indirect labeling of redox-sensitive cysteine residues.

Results
Exposure to PDF increased intracellular ROS production and accumulation as well as cell damage assessed by LDH-release compared to control cells. Cells exposed to AlaGln supplemented PDF showed less cell damage compared to PDF alone. Addition of AlaGln not only reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but also led to different proteins being affected by redox modifications. The carefully optimized highly sensitive LC/MS-based redox proteomics workflow allowed identification of 5537 proteins of which 2614 contained a labeled cysteine. H2O2 treatment resulted in a shift of median oxidation from 11% under control conditions to 36%. While PDF alone increased the oxidation level to 31%, AlaGln supplemented PDF only led to 15% oxidation. Pathway analysis of proteins that changed their oxidation level &gt;50% following the treatment were subjected to molecular pathway analysis revealing distinct differences. PDF exposure leads to regulation of general cell processes like regulation of glucokinase, RNA-binding and SUMOylation, addition of AlaGln regulated more specific signaling pathways for example fibrosis related pathways like TGF-ß and SMAD signaling.

Conclusion
Redox proteomics of peritoneal cells could represent a novel tool for the identification of mediators of uraemia and PD-induced pathomechanisms, and also to evaluate anti-oxidant pharmacological interventions to improve PD outcomes.