Hepcidin suppression by shRNA mobilized iron and corrected anemia in mice treated with BA. (A) Experimental scheme detailing administration of shRNA (control = antiluciferase, H10 or H6 = antihepcidin: 2 × 10¹² particles/mouse via the hepatic portal vein) relative to BA (5 × 10⁸ particles/mouse, intraperitoneally) or saline control and ESA (100 μg/kg darbepoetin alfa, subcutaneously) or saline control (no BA). Serum iron, serum hepcidin, liver hepcidin mRNA, and red cell parameters were measured at sample collection (n = 4-5/group). (B) Liver hepcidin mRNA (■, measured in RLU/10 μg total RNA) and serum hepcidin (▩, measured relative to hHepc internal standard in relative units [RU]). BA treatment increased serum hepcidin. Antihepcidin shRNA (H6 or H10) decreased both liver hepcidin mRNA and serum hepcidin. (C) Serum iron was increased by antihepcidin shRNAs H10 and H6. (D) Treatment with BA induced an anemia that was reversed with antihepcidin shRNA. The combination of hepcidin suppression and ESA treatment was more effective than hepcidin suppression alone. All statistical differences against control without inflammation (▩) and against BA-control (■) are indicated. Each shRNA subset was also compared with and without ESA administration and any significant differences shown (■ vs □ for each shRNA tested). (E-F) MCV and MCH, respectively, were increased by hepcidin suppression. All statistical differences compared with control group with no inflammation (▩) are shown. (G) Reticulocyte count in BA-treated animals was not increased by either hepcidin suppression or ESA treatment. Combination treatment showed a synergistic increase in reticulocytes. All statistical differences compared with control group with no inflammation (▩) are shown. No significant differences were seen between shRNA groups treated with BA alone (■). Significant differences between shRNA treatments caused by ESA administration (□) are shown. For statistical comparisons, *P < .05; **P < .01; *** P < .001.