Cellular Physiology Biochemistry

Uremia and Inadequate Oxygen Supply Induce Eryptosis and Intracellular Hypoxia in Red Blood Cells

Gabriela Ferreira Dias, Sara Soares Tozoni, Gabriela Bohnen, Nadja Grobe, Silvia D. Rodrigues, Tassiana Meireles, Lia S. Nakao, Roberto Pecoits-Filho, Peter Kotanko, Andréa Novais Moreno-Amaral

Background/aims: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition.

Methods: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography.

Results: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis.

Conclusion: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.

About the Contributors

Nadja Grobe, MS, PhD

Supervisor, Laboratory Research

Nadja received her MS and PhD in biochemistry from the Martin Luther University Halle-Wittenberg, Germany. Prior to joining RRI in 2017, she gained more than 10 years of experience in guiding and implementing chemistry, biochemistry, and biomedical-focused research teams in nonprofit, academia, and government. Her previous research has been funded by the American Heart Association, the National Institutes of Health, and the American Society of Nephrology.

Dr. Peter Kotanko, MD

RRI Research Director

SVP, Corporate Research & Development

Peter Kotanko, MD, is Research Director at the Renal Research Institute (RRI), New York. Prior to joining RRI, from 1997 to 2007 he served as vice chair of a department of internal medicine at an academic teaching hospital in Graz, Austria. Prior to moving to Graz in 1989, he worked from 1982 to 1989 in the Department of Physiology and the University Clinic of Internal Medicine in Innsbruck, Austria. From 1995 to 1996 he trained in nephrology at the Hammersmith Hospital, London, United Kingdom.