Uremia and Hypoxia Independently Induce Eryptosis and Erythrocyte Redox Imbalance

Abstract

Background/Aims: 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. Methods: We incubated RBC from healthy donors with IS at concentrations of 0.01mM, 0.09mM and 0.17mM under both normoxic (21% O 2 ) and hypoxic (5% O 2 ) 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. Results: 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. Conclusion: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.

Effect of hypoxia and uremia on oxidative stress on erythrocytes from hemodialysis patients

“…24 We have shown that hypoxia and IS promote eryptosis and alter the RBC redox system, increasing ROS formation and decreasing GSH content. 9 Here, we highlight the role of hypoxia in establishing oxidative stress and show for the first time that uremic serum potentiates these effects. This finding is particularly interesting for HD patients since arterial O 2 saturation can drop below 90% for more than 1/3 of treatment time in about 10% of patients, 7 increasing the RBC ROS production.…”

“…Nevertheless, IS reduced GSH in a dose-dependent fashion. 9 We also assessed the activity of important antioxidant enzymes in HD-RBC. SOD plays a critical role in redox homeostasis, converting superoxide into hydrogen peroxide, preventing oxidation of hemoglobin to methemoglobin, which cannot carry oxygen.…”

“…7,8 We previously showed that hypoxia induces ROS production and eryptosis in healthy RBC. 9,10 In the present study, we explore additional redox pathways that might be altered by hypoxia and uremic toxicity through enzymatic mechanisms such as xanthine oxidase (XO), elevated in the plasma of CKD patients 11 ; superoxide dismutase (SOD), that produces hydrogen peroxide, further converted to water by catalase and glutathione peroxidase 12 ; and heme oxygenase-1 (HO-1), induced by stress, catalyzes free heme degradation, releasing iron, CO, and bilirubin, products with antioxidant, anti-inflammatory, and antiapoptotic effects. 13 Besides the enzymatic defense, plasma and cells contain high levels of reduced glutathione (GSH) to protect cellular components from damage caused by ROS.…”

The Role of Eryptosis in the Pathogenesis of Renal Anemia: Insights From Basic Research and Mathematical Modeling

“…Importantly, about 10% of patients undergoing HD experience prolonged intradialytic hypoxemia (PIH), a clinical phenotype characterized by an arterial oxygen saturation below 90% for at least one third of the dialysis treatment time (Meyring-Wosten et al, 2016). Previously, we explored the effect of low oxygen partial pressure and the uremic toxin IS on eryptosis (Tozoni et al, 2019). Interestingly, we found that hypoxemia and IS independently increase eryptosis and ROS generation and decrease GSH levels, possibly contributing to the reduced RBC life span observed in CKD (Figures 1, 2).…”

“…Eryptosis can be triggered by a range of both endogenous and exogenous insults, including toxins, drugs, and acute and chronic diseases (Lang and Lang, 2015). Among the uremic solutes that accumulate in CKD, acrolein (Ahmed et al, 2013b), methylglyoxal (Nicolay et al, 2006), and indoxyl sulfate (IS) (Ahmed et al, 2013a;Dias et al, 2018;Tozoni et al, 2019) were shown to increase eryptosis. Moreover, stressors including osmotic shifts, oxidative stress, and energy depletion may also contribute to a shortened RBC survival (Lang et al, 2006).…”