Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs

Abstract

Hepcidin is the principal iron regulatory hormone and its overproduction contributes to anemia of inflammation (AI). In vitro, hepcidin binds to and induces the degradation of the exclusive iron exporter ferroportin. We explored the effects and distribution of synthetic hepcidin in the mouse. A single intraperitoneal injection of hepcidin caused a rapid fall of serum iron in a dose-dependent manner, with a 50-microg dose resulting in iron levels 80% lower than in control mice. The full effect was seen within only 1 hour, consistent with a blockade of iron export from tissue stores and from macrophages involved in iron recycling. Serum iron remained suppressed for more than 48 hours after injection. Using radiolabeled hepcidin, we demonstrated that the serum concentration of hepcidin at the 50-microg dose was 1.4 microM, consistent with the inhibitory concentration of 50% (IC50) of hepcidin measured in vitro. Radiolabeled hepcidin accumulated in the ferroportin-rich organs, liver, spleen, and proximal duodenum. Our study highlights the central role of the hepcidin-ferroportin interaction in iron homeostasis. The rapid and sustained action of a single dose of hepcidin makes it an appealing agent for the prevention of iron accumulation in hereditary hemochromatosis.

Figure 1.

Hepcidin causes a dose-dependent fall in serum iron. Mice received a single dose of hepcidin by intraperitoneal injection. Four hours later, serum iron was determined. There is a strong correlation between serum iron and the dose of hepcidin administered (R = -0.929, P < .001). Four mice were used for every dose. Horizontal lines with P values indicate comparisons by Student t test.

Figure 2.

Hypoferremia is induced by hHep-25 but not its truncated variants. Mice received a single 50 μg injection of hHep-25, diluent, hHep-22, or hHep-20. Only the full-length, 25-amino acid hHep induced hypoferremia at 2 hours (n = 4 mice for every treatment). Means and standard deviations are shown, and comparisons indicated by solid lines, and P values were tested by Student t test.

Figure 3.

Hepcidin causes a rapid, sustained fall in serum iron. Mice received a single 50 μg intraperitoneal injection of hHep. Between 30 and 60 minutes after injection, serum iron achieved its nadir value. This effect was sustained for at least 48 hours. Four to 5 mice were used for each time point.

Figure 4.

Hepcidin is cleared from the circulation within 24 hours. Mice received a single dose of radiolabeled hepcidin at time 0. represents blood hepcidin; ▪, the corresponding urinary hepcidin. The 0.5-hour and 1-hour time points are from the same group of 4 mice. The 2- and 24-hour time points were obtained from 2 independent groups of 4 mice. Means and standard deviations are shown. ^*P < .001 compared with 0.5-hour values, #P = .004 compared with 1-hour values, both by Student t test.

Figure 5.

Hepcidin is taken up by ferroportin-rich organs. Mice received a single dose of radiolabeled hepcidin. One hour later, mice were perfused with PBS to remove blood, and organs were collected. ^*P = .03 by rank-sum and #P = .007 by t test compared with blood; &P < .04 by t test compared with intestine, lung, muscle, or paw.

Figure 6.

Hepcidin concentration declines from the proximal to distal small intestine. Mice (n = 4, each interrupted line representing an individual mouse) received a single dose of radiolabeled hepcidin, were killed 2 hours later, and their organs were perfused with PBS to remove blood. Hepcidin concentration (average shown as a bold line) decreased from proximal to distal position in the intestine (Spearman R = -0.929, P < .001, n = 4).