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Low-dose erythropoietin induces erythroferrone
development of a validated assay for human ERFE led to the demonstration that ERFE is increased in patients with β-thalassemia and in response to blood donation or administration of high doses of rhEpo.8 Moreover, serum ERFE levels were found to be elevated in patients with chronic kidney disease treated with rhEpo9 and in children affected by iron deficiency anemia.10 However, another study did not find increased ERFE levels in patients with chronic kidney disease.11 In addition, ERFE did not change in patients with reduced erythropoiesis caused by andro- gen deprivation therapy12 or in athletes exposed to alti- tude13 and, unexpectedly, decreased in parallel with hep- cidin in subjects experiencing expansion of hemoglobin mass (Hbmass) due to exposure to altitude.14 The effect of accelerated erythropoiesis on ERFE in humans does, there- fore, remain uncertain. Here, we tested the hypothesis that, in healthy humans, ERFE is a physiological erythroid regulator, i.e. it responds to moderate erythropoietic stim- ulation, such as very low rhEpo doses or exposure to high altitude.
Methods
Study design
The pattern of variation of ERFE levels mirrored that of serum Epo (Figure 3A). Conversely, serum hepcidin decreased when Epo and ERFE rose, independently of the dose, and remained low as long as Epo and ERFE were above baseline values (Figure 1D). Both ERFE and hepcidin returned to placebo levels 1 week after the last injection. Interestingly, there was no cumulative effect of repeated rhEpo injections on ERFE levels (Figure 1C). Consistent with previous studies,3-5 the decrease in serum ferritin
A
B
Figure 3. Relationships between erythroferrone and erythropoietin with recombinant human erythropoietin and high altitude. (A, B) Relationship between individual serum concentrations (ln transformed) of erythroferrone (ERFE) and erythropoietin (Epo), before, during and after six injections of placebo, micro-dose or low-dose recombinant human erythropoietin (rhEpo) in healthy subjects (A), and at sea level and after 15 h of exposure to high alti- tude (3800 m) in healthy subjects (B). Regression equations, coefficients of determination and P values are reported in each panel. Relationships between hepcidin, Epo and ERFE during rhEpo injections are available in Online Supplementary Figure S2.
For the rhEpo study, Hbmass, indices of iron homeostasis and hematologic parameters were repeatedly measured in 24 healthy males given six injections (every second/third day) of saline (place- bo), rhEpo 20 IU/kg (micro-dose) or rhEpo 50 IU/kg (low dose) Written informed consent to participation in the study was pro- vided by all these subjects. The study (NCT03276910) was approved by the French ethics committee (CPP Est-III, EudraCT 2017-000375-82). For the high-altitude study, iron parameters were determined in 22 healthy subjects exposed to high altitude (3800 m) for 15 h. Written informed consent to participation in the study was provided by all these subjects. The study (NCT02778659) was approved by the French ethics committee (CPP Sud-Est-III, EudraCT 2015-004512-38).
Details of the methods are available in the Online Supplementary Material.
Results
Three days after the last rhEpo injection, Hbmass was high- er in subjects treated with low-dose rhEpo than in those given the placebo (Figure 1A). In contrast, treatment with micro-doses of rhEpo did not induce significantly higher Hbmass levels in comparison to those induced by placebo treatment. Hbmass was not significantly altered following placebo treatment but nonetheless tended to decrease, possibly because of frequent blood sampling. Short treat- ment duration may otherwise explain the marginal change of Hbmass following micro-doses of rhEpo. Unlike Hbmass, hemoglobin concentration and hematocrit progres- sively increased with both rhEpo doses (Figure 2A, B). As expected, circulating Epo levels increased after each injec- tion, but rapidly declined, in particular after micro-dose treatment (Figure 1B). ERFE levels showed a significant dose-related increase after each injection, remaining above placebo levels for up to 48 h (micro-dose) or 72 h (low dose) and decreasing thereafter (Figure 1C). Following low-dose rhEpo treatment, ERFE reached levels similar to those found in patients with anemia induced by bleeding (see the Online Supplementary Material, Analyses section).
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