Hepcidin overexpression caused iron-limited erythropoiesis. (A) Photograph of gross appearance and Wright-Giemsa–stained peripheral blood smear of representative C57BL/6 mice 4 months after viral transduction (AAV8) with AAV-mHepc1 (1.3 × 10¹³ particles/mouse) or AAV control (empty-vector: 8 × 10¹² particles/mouse). Overexpression of mHepc1 resulted in runted growth, hair loss, and microcytic, hypochromic anemia. Gross appearance of mice was captured using a standard digital camera and brightness adjusted using Microsoft PowerPoint. Peripheral blood smears were visualized using a Nikon ECLIPSE E600 microscope with a Nikon Plan Fluor 100×/1.30 NA oil objective. Images were captured using a Nikon Digital Camera DXM 1200f (Nikon) and processed using Nikon's ACT-1 Version 2.62 software. (B) AAV-hHepc expression in mice (AAV5; n = 4/group) caused an increase in serum levels of hHepc and a dose-dependent decrease in serum iron 2 weeks after viral transduction. H indicates AAV-hHepc; C, AAV-control (AAV-GFP). Infectious units administered via tail vein injection are shown in parentheses. (C) Control animals (AAV-GFP) responded to suprapharmacologic ESA treatment, whereas animals overexpressing hHepc did not show a significant Hb response (ESA administered 4 weeks after virus treatment as in panel B and Hb measured 1 week later; n = 4-5/group). Horizontal bars indicate groups compared for statistical analysis: ***P < .001; NS, no significance. Results in all figures are shown as mean ± SEM.