Long term treatment with LDN-193189 reverses anemia in a rodent model of ACI by modulating the hepcidin-ferroportin axis and by mobilizing iron. ACI was induced by intraperitoneal administration of PG-APS into female Lewis rats and animals were followed up for 3 weeks. Then, ACI rats were treated with either LDN-193189 (3mg/kg, ACI/LDN) or vehicle alone (ACI) by intraperitoneal administration every second day over 28 days as detailed in “Animals.” Rats were then killed and analyzed for (A) relative expression of Hamp/Gusb mRNA in the liver as determined by quantitative real-time RT-PCR, (B) hepatic Smad1 levels and Smad1/5/8 phosphorylation (pSmad1/5/8) as well as (C) Stat3 levels and Stat3 phosphorylation (pStat3) as examined by Western blot, (D) serum iron levels, and (E) the protein expression of ferroportin (FP-1) and ferritin in the spleen as visualized by Western blots. (B, C) 1TBP18 was used as nuclear loading control and (E) ß-actin as cytoplasmatic loading control. (F) Hemoglobin levels were measured in ACI rats once weekly starting with the initiation of LDN-193189 ♦ (light gray) or vehicle ● (dark) administration (day 0; 21 days after PG-APS injection). Results in panels A, B, C, D, F are reported as means ± SEM (n = 6 per group). Calculations for statistical differences between the various groups were carried out by Student t test and P values are shown. (B,C,E) One representative Western blot is shown. Western blots used for densitometric quantification (B-C) are shown in supplemental Figure 3A.