KY1070 effectively suppresses hepcidin levels in healthy mice and rats and leads to an improved erythroid output in combination with Darbepoetin alfa over time. (A-D) Healthy C57BL/6N mice were treated with a single dose of either PBS (n = 5), IgG4 isotype control (3 mg/kg; n = 5), KY1070 (3 mg/kg; n = 5), Darbepoetin alfa (10 µg/kg; n = 5), Darbepoetin alfa and IgG4 isotype control (n = 5), or KY1070 and EPO (n = 5). Liver Hamp mRNA levels (A), plasma iron (B), Tf-Sat, hepatic tissue iron levels (C), and reticulocyte counts (D) (shown as %Thiazol Orange^pos) were determined 3 days after indicated treatments. (E-G) Healthy Lewis rats were treated with IgG4 isotype control (3 mg/kg; n = 6), KY1070 (3 mg/kg; n = 7), Darbepoetin alfa (10 µg/kg; n = 7), or KY1070 and EPO (n = 7). Liver Hamp mRNA levels (E), hemoglobin levels (Hgb) (F), MCV, and hepatic tissue iron levels (C), and reticulocyte counts (G) (shown as %Thiazol Orange^pos) were determined at day 21 after a single KY1070 treatment ± weekly EPO application. (H) Flow cytometric analysis of rat bone marrow erythropoiesis at the end of treatment in rats of all different treatment groups. Representative dot plots for Lineage^neg cells with percentages of the different erythropoietic populations (I to IV) per femur are given; basophilic cells (I), polychromatic cells (II), orthochromatic cells and reticulocytes (III), and mature RBCs (IV); for gating strategy see supplemental Figure 3F. Two-way analysis of variance (ANOVA) with Tukey corrected post hoc Student t test for multiple comparisons between all groups was applied for panels A-G. Results are shown as means ± SEM. Significant levels are indicated for the Isotype-treated control group against all treatment groups and EPO vs KY1070 + EPO–treated rodents. *P < .05; **P < .01; ***P < .001. FSC-A, forward scatter area.