Uremia and Hypoxia Independently Induce Eryptosis and Erythrocyte Redox Imbalance.

Red blood cell (RBC) death could contribute to anemia in chronic kidney disease (CKD) patients. Recent observational research has suggested a relationship between RBC death (eryptosis) and hypoxemia in hemodialysis patients. Thus, we studied the isolated and joint effects of a uremic toxin (indoxyl sulfate; IS) and hypoxia on RBC biology.

We incubated RBC from healthy donors with IS at concentrations of 0.01mM, 0.09mM and 0.17mM under both normoxic (21% O₂) and hypoxic (5% O₂) conditions for 24 hours. Eryptosis was evaluated by RBC phosphatidylserine (PS) exposure, cell volume, and cytosolic calcium which were quantified by Annexin-V+, forward scatter, and Fluo-3AM+ binding, respectively. RBC redox balance was reported by reactive oxygen species (ROS) production and intracellular reduced glutathione (GSH). Analyses were performed by flow cytometry.

Hypoxia induced a 2-fold ROS production compared to normoxia. PS exposure and cytosolic calcium increased, while cell volume decreased by hypoxia and likewise by IS. IS increased ROS production in a dose-dependent manner under conditions of both normoxia and hypoxia. The same conditions promoted a GSH decrease with IS intensifying the hypoxia-induced effects.In summary, our results indicate that the concurrent presence of hypoxia and uremia augments RBC death and may therefore, contribute to the genesis of anemia in CKD.