Pharmacologic inhibition of hepcidin expression reverses anemia of chronic inflammation in rats

Abstract

Anemia of chronic inflammation (ACI) is the most frequent anemia in hospitalized patients and is associated with significant morbidity. A major underlying mechanism of ACI is the retention of iron within cells of the reticuloendothelial system (RES), thus making the metal unavailable for efficient erythropoiesis. This reticuloendothelial iron sequestration is primarily mediated by excess levels of the iron regulatory peptide hepcidin down-regulating the functional expression of the only known cellular iron export protein ferroportin resulting in blockade of iron egress from these cells. Using a well-established rat model of ACI, we herein provide novel evidence for effective treatment of ACI by blocking endogenous hepcidin production using the small molecule dorsomorphin derivative LDN-193189 or the protein soluble hemojuvelin-Fc (HJV.Fc) to inhibit bone morphogenetic protein-Smad mediated signaling required for effective hepcidin transcription. Pharmacologic inhibition of hepcidin expression results in mobilization of iron from the RES, stimulation of erythropoiesis and correction of anemia. Thus, hepcidin lowering agents are a promising new class of pharmacologic drugs to effectively combat ACI.

Figures

Figure 1

LDN-193189 and soluble hemojuvelin protein (HJV.Fc) block Smad1/5/8 signaling and inhibit Hamp mRNA expression in primary rat hepatocytes. Primary rat hepatocytes were isolated from female Lewis rats and stimulated with BMP6 (25 ng/mL; 0.69nM) for 12 hours in the presence/absence of LDN-193189 (500nM) or HJV.Fc (25 μg/mL; 166nM). (A) Quantitative RT-PCR for Hamp mRNA expression relative to the housekeeping transcript β glucuronidase (Gusb) was then carried out. (B) In parallel, Western blots investigating Smad1 levels and Smad1/5/8 phosphorylation (pSmad1/5/8) as well as Stat3 levels and phosphorylation (pStat3) were carried out. 1TBP18 was used as nuclear loading control. (A) Results are reported as means ± SEM for 3 independent experiments with n = 6 per group, and the P values are shown as determined by ANOVA with Bonferroni correction for multiple tests. (B) One representative blot of 3 independent experiments is shown.

Figure 2

LDN-193189 and HJV.Fc both inhibit hepatic Hamp mRNA induction in vivo in a rodent model of anemia of chronic inflammation. Anemia of chronic inflammation (ACI) was induced in female Lewis rats on a single intraperitoneal injection of PG-APS as detailed in “Animals” and followed up for 3 weeks. (A-C) Hamp mRNA expression relative to the housekeeping gene β glucuronidase (Gusb), (D-F) SMAD protein expression and phosphorylation (pSMAD 1/5/8) and (G-I) Stat3 expression and phosphorylation (pStat3) (G-I) were determined in livers of control and ACI rats which were treated with either a single injection of vehicle, (B,E,H) LDN-193189 [3mg/kg] or (C,F,I) HJV.Fc (20 mg/kg), at 6 hours or 24 hours before sacrifice, respectively. (D-I) 1TBP18 was used as nuclear loading control. (D-I) One representative Western blot is shown. Original Western blots used for densitometric quantification of protein expression (D-I) are shown in supplemental Figure 2. (A-I) Results are reported as means ± SEM (n = 5 in control rats, n = 6 in all other groups). Calculations for statistical differences between the various groups were carried out by Student t test and P values are shown.

Figure 3

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.

Figure 4

Long term treatment with HJV.Fc reverses anemia in a rodent model of ACI by modulating the hepcidin-ferroportin axis and by mobilizing iron. ACI in female Lewis rats was induced by intraperitoneal administration of PG-APS and animals were followed up for 3 weeks. Then, ACI rats were treated with either HJV.Fc protein (20 mg/kg; ACI/HJV.Fc) or vehicle alone (ACI) by intravenous administration twice weekly over 28 days. (A) Hamp mRNA relative to Gusb mRNA expression in the liver, (B) hepatic Smad1 levels and Smad1/5/8 phosphorylation (pSmad1/5/8) as well as (C) Stat3 levels and Stat3 phosphorylation (pStat3), (D) serum iron levels and (E) the protein expression of ferroportin (FP-1) and ferritin in the spleen are shown after the termination of the experiment as detailed in the legend to Figure 3. (B-C) 1TBP18 was used as nuclear loading control and (E) β-actin as cytoplasmatic loading control. (F) Hemoglobin levels were determined in ACI rats once weekly starting with the initiation of HJV.Fc protein ♦ (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 mean ± SEM, n = 10 for vehicle treated ACI rats and n = 10 for HJV.Fc treated ACI rats. Calculations for statistical differences between the various groups were carried out by Student t test. Exact P values are shown. (B,D) One representative Western blot is shown. Western blots used for densitometric quantification (B-C) are shown in supplemental Figure 3B.