Ferroportin1-Deficient Zebrafish Treated with Iron Dextran Develop Anemia Despite Increased Macrophage Iron Stores.

Patients with type 4 hemochromatosis exhibit iron accumulation in the reticuloendothelial system and a tendency to develop anemia following venesection. These patients are heterozygous for mutations in the intestinal and macrophage iron exporter, ferroportin1 (fpn1), however it is not known whether these mutations cause a gain or loss of function. Of current animal models, only the Tp85c allele of the zebrafish anemia mutant weissherbst (weh) encodes a missense mutation (L167F) in the conserved region of fpn1, between transmembrane helix 3 and 4, where several missense mutations have been identified in individuals with type 4 hemochromatosis. Tp85c homozygote zebrafish die within 10–14 days post-fertilization, however administering iron dextran injections allows them to survive. To explore the effects of fpn1 deficiency on blood development and iron homeostasis in adult zebrafish, we raised Tp85c homozygotes along with control cohorts of injected and uninjected heterozygotes and wild types. Iron dextran injections were discontinued at 4 months of age in all cohorts. Although iron accumulation was observed in all iron-injected fish, only homozygotes exhibited iron accumulation in the intestinal epithelium on Perl’s staining, consistent with a block in enterocyte export. At 6 months of age, none of the cohorts were anemic, however at 12 months of age, only the homozygotes developed hypochromic anemia, characterized by reduced levels of hemoglobin per erythrocyte and impaired late erythroid maturation. Histologic evaluation of the anemic homozygotes revealed increased numbers of iron-loaded macrophages in the liver and in the kidney, the zebrafish bone marrow equivalent. Quantitative realtime rt-PCR, performed on liver and intestinal tissues at 1 year of age, revealed that homozygotes had increased intestinal expression of fpn1 compared to the other cohorts. Hepatic transcript levels of the secreted iron-regulator hepcidin were profoundly decreased in homozygotes, consistent with response to anemia. In contrast, hepatic transcript levels for transferrin, fpn1, and DMT1 were not significantly different among the cohorts. In summary, we provide evidence that Tp85c is a loss of function mutation affecting enterocytes and macrophages. After iron dextran injections were discontinued, Tp85c homozygotes developed anemia, despite increased macrophage iron stores. These studies support the hypothesis that fpn1-deficiency impairs iron cycling and suggest this as a possible mechanism in type 4 hemochromatosis.