P0942HYPOXEMIA AND UREMIA INDUCE OXIDATIVE IMBALANCE IN ENDOTHELIAL CELLS

Abstract Background and Aims Intradialytic hypoxemia is associated with oxidative stress, endothelial dysfunction, inflammation, higher erythropoietin requirements, and higher all-cause hospitalization and mortality in hemodialysis (HD) patients (Meyring-Wosten, et al., 2016). The aim of the present study was to further expand our insights into the hypoxia-uremia axis by investigating the oxidative balance in endothelial cells (EC) under hypoxic and uremic conditions. Method Human umbilical EC were incubated with DMEM medium supplemented with 10% of fetal bovine serum (FBS; control) or with sera obtained from healthy subjects (S-CON) or HD patients (S-HD, 1:10), respectively, for 40 minutes, 4 hours, and 24 hours under normoxic (21% O2) or hypoxic (5% O2) conditions (Culture Chamber ProOx, Biospherix). EC were analyzed by flow cytometer (BD Accuri™ C6 Plus) to assess a) intracellular production of reactive oxygen species (ROS, DCFH-DA probe, Abcam); b) reduced glutathione (GSH) content (ThiolTracker Violet probe, Thermo Fisher Scientific). Results S-HD increased intracellular ROS production at all time points compared to S-CON. Moreover, ROS production was higher under hypoxic conditions. ROS production declined after 24 hours. S-HD induced slightly higher GSH content when compared to S-CON in normoxia (Table 1). Conclusion In our in vitro experiments, hypoxia and uremia jointly favor EC oxidative imbalance. This effect is particularly pronounced after 4 hours. Translational studies are warranted to explore the clinical relevance of our findings. Table: Baseline (of 100%): EC incubated in DMEM medium supplemented with 10% FBS + antibiotics Normoxia (21% O2) Hypoxia (5% O2) ROS production [%] 28.9±2.4 52.3±7.2 GSH content [%] 8.4±1 8.1±0.8 Values after Incubation with S-CON or S-HD 40 minutes 4 hours 24 hours ROS production Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia S-CON [%] 102±5.2 121±6.32& 102.5±7.3 109.2±9.2 34.2±7.3 50.5±14.5 S-HD [%] 130.7±8.7* 146.2±8.3*& 150±13.9* 190.3±37.6* 71.8±11.4* 94.8±19.3*& GSH content Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia S-CON [%] 8.9±1.3& 6.5±2.3 7.6±1.2 7.4±1.6 7.9±1.5 8.1±1.2 S-HD [%] 12.7±1.8*& 6.3±0.9 9.3±1.6* 9.8±1.8* 10.4±1.3*& 7.5±0.8 * p