Erythropoietin-induced erythroid precursor pool depletion causes erythropoietin hyporesponsiveness

Abstract

Purpose: The purpose of this study is to demonstrate that the erythroid precursor depletion in bone marrow induced by recombinant human erythropoietin (rHuEPO) treatment may be another contributing factor to erythropoietin hyporesponsiveness.

Methods: Healthy Wistar rats were given single dose (SD) or multiple doses (MD) of rHuEPO (100 IU/kg). In MD study, animals were challenged with thrice-weekly over two weeks. Blood, bone marrow and spleen (for SD only) were collected. The erythropoietic responses in bone marrow and spleen were quantified using a flow cytometric immunophenotyping technique. A mathematical approach involving measuring reticulocyte age distribution was developed to evaluate the reticulocyte loss due to neocytolysis.

Results: A reduced level of erythropoietic responses below the baseline was observed for both MD and SD studies. In SD study, the reticulocyte decreased below the baseline after day 6. A depletion of the bone marrow erythroid precursor cells was observed. However, neocytolysis of reticulocyte only occurs from day 3-5 after rHuEPO injection.

Conclusions: The findings demonstrate that EPO-induced erythroid precursor depletion in bone marrow is responsible for reduced reticulocyte response and may contribute to erythropoietin hyporesponsiveness. Therefore, this study provides further justification for reducing the doses of erythropoietin-stimulating agents in anemic patients demonstrating hyporesponsiveness.

Fig 1

Flow cytometry analysis of bone marrow and spleen erythroblasts. (A) Flow cytometry analysis of fresh isolated bone marrow (upper two panels) and spleen (lower two panels) stained with antibodies against rat erythroid cells and CD71. The left panels show all cells. The gated population in the left panel was further analyzed in terms of the forward scatter (FSC) parameter shown in the right panel. Three cell populations P1, P2 and P3 are gated based on their FSC and CD71 level. (B) P1, P2 and P3 cells were sorted and stained with May Grunwald/Giemsa solution.

Fig 2

Reticulocyte flow cytometry analysis. (A) A distribution of the green fluorescent signal (FL1). The shaded region represents reticulocytes. (B) Reticulocyte age density distribution obtained from the FL1 distribution. The RET age is described by the time that a RET needs to become a mature RBC (RT). The density at RT=0 is the mature RBC production rate (k_RBC). The peak RT density approximates the RET production rate (k_RET).

Fig 3

Pharmacokinetics and pharmacodynamics of rHuEPO in multiple dosing regimens. (A) rHuEPO serum concentration vs time profile during multiple dosing regimens of rHuEPO. Solid grey circles represent individual observations. Solid black line represents mean profile with standard deviation (SD) error bars. Arrows represent the dosing events. Dashed line represents endogenous EPO level. Pharmacodynamic measurements include (B) reticulocytes, (C) red blood cells and (D) hemoglobin. Solid circles represent individual observations. Solid lines represent mean data with SD error bars. Black color is used for the treatment group and grey color is used for the control group. Arrows represent the dosing events.

Fig 4

Pharmacokinetic profile and spleen weight changes after a single IV injection of rHuEPO. (A) rHuEPO plasma concentration vs time profile after a single dose of rHuEPO. Solid grey circles represent individual observations. Solid black line represents mean profile with SD error bars. Arrow represents the dosing event. Dashed line represents endogenous EPO level. (B) Spleen weight vs time since rHuEPO administration profile for control and treatment groups. Solid circles represent individual observations. Solid lines represent mean data with SD error bars. Black color is used for the treatment group and grey color is used for the control group. Arrow represents the dosing event. * P < .05.

Fig 5

Erythropoietic response in bone marrow and spleen after single IV injection of rHuEPO. Panels show time courses of P1, P2 and P3 cell populations in bone marrow and spleen. Solid circles represent individual observations. Solid lines represent mean profile with SD error bars. Grey color denotes the control group and black color denotes the treatment group. Arrow represents the dosing event. * P < .05, ** P < .001, *** P < .001.

Fig 6

Pharmacodynamics of rHuEPO in peripheral blood after a single IV injection. (A) RET, (B) mature RBC, (C) RBC, and (D) hemoglobin concentration vs time since dose profiles from control and treatment groups are shown. Solid lines represent mean data with SD error bars. Solid circles represent observations. Black color is used for the treatment group and grey color is used for the control group. Arrow represents the dosing event. * P < .05, ** P < .001, *** P < .001.

Fig 7

Loss of reticulocytes. (A) Cell production rate vs time since rHuEPO administration profiles for control and treatment groups. Solid circles represent individual observations. Solid lines represent mean data with SD error bars. k_RET and k_RBC represent RET and mature RBC production rates, respectively. (B) Accumulated RET loss vs time since rHuEPO administration profiles, which were calculated based on the mean data. Black color is used for the treatment group and grey color is used for the control group. Arrow represents dosing event. * P < .05, ** P < .001, *** P < .001.