Exposure to Recombinant Human Erythropoietin Increases Survival of Red Blood Cells in Wistar Rats.
Authors: Krzyzanski Wojciech , Gebreegziabher Asfiha G , Kawczak Piotr 
Published: October 2019

Abstract
Abstract 5087

Purpose
The primary goal of this study was to determine if exposure to recombinant human erythropoietin (rHuEPO) increases survival of RBCs in rats. A secondary objective was to test if rHuEPO prolongs survival of RBC by interaction with erythroid progenitor cells or by affecting cells of the reticuloendothelial system.

Methods
Normal male Wistar rats (337 ± 8 g) were divided into four groups (n=2). Animals in groups 0 and 1 were not treated whereas animals groups 2 and 3 received multiple doses of rHuEPO (Epogen, Amgen, CA) 450 IU/kg three times a week for two weeks via intravenous injections in the tail vein. Subsequently, blood samples (∼ 1-2 mL) were collected from all animals and labeled with water-soluble biotin as a tag for RBC. The biotinylated RBCs were injected back to animals according to the following pattern. Group 1 received blood from Group 2 and Group 2 from Group 1. Animals from Groups 0 and 3 were injected back with their own blood. Two days were allowed for a system equilibration. Next, blood samples (∼ 100 mL) were drawn weekly from each animal until the signal reached the limit of quantification. The biotinylated cells were detected by streptavidin conjugated to phycoerythin and analyzed by flow cytometry (FACSCalibur, Becton-Dickinson). The RBC absolute count was determined by a hematology analyzer (BC-2800 Vet, Mindray, China). The survival data were represented as the fraction of surviving cells. For quantification of RBC survival, the times necessary to reduce the size of the labeled sample by 25, 50, and 75% (T25, T50, and T75) were used.

Results
The mean survival curves for each group were compared to the control rats (Group 0). The survival curve for animals in Group 3 was distinctly higher that the survival curve for the control which was reflected by the T50 values of 31.5±1.6 and 13.6±0.1 days, respectively. This implies that the survival of RBCs is prolonged by the treatment with rHuEPO. If the effect of erythropoietin on RBC survival is mediated by its interaction with the erythroid progenitor cells, then an increase in the RBC survival in Group 1 is expected. This was somewhat confirmed by the survival curve for Group 1 that was above the control survival curve, however not to the extent of Group 3. Contrary, the survival curve for Group 2 overlapped with the control, supporting the hypothesis that the effect of rHuEPO on RBC survival is not mediated by its interaction with the reticuloendothelial system. The T50 values for Group 1 and 2 were 24.3±7.4 and 15.6±3.0 days, respectively.

Conclusions
The survival patterns of RBCs following two week long exposure to rHuEPO imply that their lifespan is prolonged when compared to control rats. Lack of an increase of RBC survival for Group 1 and a moderate increase of RBC survival for Group 2 support the endowment hypothesis that rHuEPO prolongs survival of RBCs possible by enhancing viability of the erythroid precursors.

Disclosures
No relevant conflicts of interest to declare.