Age-dependent retinal iron accumulation and degeneration in hepcidin knockout mice

Abstract

Purpose: Iron dysregulation can cause retinal disease, yet retinal iron regulatory mechanisms are incompletely understood. The peptide hormone hepcidin (Hepc) limits iron uptake from the intestine by triggering degradation of the iron transporter ferroportin (Fpn). Given that Hepc is expressed in the retina and Fpn is expressed in cells constituting the blood-retinal barrier, the authors tested whether the retina may produce Hepc to limit retinal iron import.

Methods: Retinas of Hepc(-/-) mice were analyzed by histology, autofluorescence spectral analysis, atomic absorption spectrophotometry, Perls' iron stain, and immunofluorescence to assess iron-handling proteins. Retinal Hepc mRNA was evaluated through qPCR after intravitreal iron injection. Mechanisms of retinal Hepc upregulation were tested by Western blot analysis. A retinal capillary endothelial cell culture system was used to assess the effect of exogenous Hepc on Fpn.

Results: Hepc(-/-) mice experienced age-dependent increases in retinal iron followed by retinal degeneration with autofluorescent RPE, photoreceptor death, and subretinal neovascularization. Hepc(-/-) mice had increased Fpn immunoreactivity in vascular endothelial cells. Conversely, in cultured retinal capillary endothelial cells, exogenous Hepc decreased both Fpn levels and iron transport. The retina can sense increased iron levels, upregulating Hepc after phosphorylation of extracellular signal regulated kinases.

Conclusions: These findings indicate that Hepc is essential for retinal iron regulation. In the absence of Hepc, retinal degeneration occurs. Increases in Hepc mRNA levels after intravitreal iron injection combined with Hepc-mediated decreases in iron export from cultured retinal capillary endothelial cells suggest that the retina may use Hepc for its tissue-specific iron regulation.

Figure 1.

Hepc^−/− mice have retinal degeneration. Bright-field micrographs of plastic sections show that compared with 9-month-old Hepc^+/+ mice (A), the age-matched Hepc^−/− mice had focal areas of retinal pigment epithelial hyperplasia (B) and subretinal neovascularization (B, red asterisk). Compared with 18-month-old control (C), 18-month-old Hepc^−/− mice (D–F) had focal areas of massively hypertrophic retinal pigment epithelial cells (D, arrow), with degeneration of overlying photoreceptor inner and outer segments and thinning of the outer and inner nuclear layers. Photomicrographs of 18-month-old Hepc^−/− mice show strong RPE65 immunoreactivity within the hypertrophic retinal pigment epithelial cells (H, arrows) compared with no primary antibody (G) negative control. Scale bars: 25 μm (A–D, G, H); 50 μm (E, F). OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 2.

Lipofuscin-like material in Hepc1^−/− hypertrophic RPE. Fluorescence photomicrographs showing green autofluorescent lipofuscin-like material in 18-month-old Hepc^−/− RPE (B, arrows), whereas the age-matched controls had only minimal autofluorescence within photoreceptor outer segments and none detected in the RPE. Nuclei are stained with DAPI (blue). Spectral analysis of relative autofluorescence emission intensities (with 488 nm excitation) revealed several similar emission peaks (arrows) among hypertrophic retinal pigment epithelial cells from Hepc1^−/− and DKO mice compared with RPE from the post mortem retina of a patient with AMD (C). Scale bar, 25 μm. OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 3.

Quantification and histochemical detection of iron in Hepc^+/+ and age-matched Hepc^−/− mice. Graphs of iron in the retinas and RPE/choroid of 12.5-month-old Hepc^−/− and Hepc^+/+ (control) mice measured by atomic absorption spectrophotometry. Total iron in nanograms per neurosensory retina (A) and in nanograms per RPE/choroid (B) is shown for two different genotypes. *Significant difference (P < 0.05). Bright-field photomicrographs of Perls'-stained plastic-embedded Hepc^+/+ (C1, C2) and Hepc^−/− (C3, C4) retinas (C1, C3) and ciliary bodies (C2, C4) demonstrated iron accumulation in the RPE (C3, arrow) and choroid of 18-month-old Hepc^−/− mice. Iron also accumulated in the nonpigmented ciliary epithelium in 18-month-old Hepc^−/− (C4, arrows). Controls (Hepc^+/+) did not have Perls' signal in either the RPE or the ciliary body (C1, C2). Scale bar, 50 μm.

Figure 4.

Hepc^−/− retinas and ciliary bodies have increased L-ferritin, and retinas have decreased transferrin receptor mRNA. Fluorescence photomicrographs of 3-, 9-, and 18-month-old Hepc^−/− retinas (B, D, F) showed stronger immunoreactivity (red), increasing with age, throughout the inner plexiform layer, outer plexiform layer, RPE, and choroid compared with the age-matched controls (Hepc^+/+; A, C, E). Immunoreactivity was quantified by measuring the mean pixel intensity within the RPE and neural retina of each photomicrograph (shown in the lower left corner). Fluorescence photomicrographs of 3-, 9-, and 18-month-old Hepc^−/− ciliary bodies (B′, D′, F′) showed strong immunoreactivity mostly within the nonpigmented ciliary epithelium, whereas age-matched controls (A′, C′, E′) had only weak signal. Scale bar, 50 μm. (G, H) Transferrin receptor mRNA levels in 4-month-old Hepc^−/− mice compared with the age-matched controls detected by qPCR in RPE/choroids (G) and in neural retina (H). *P < 0.05.

Figure 5.

Hepc^−/− mice have increased Fpn protein in the retina. Fluorescence photomicrographs of retinas from Hepc^−/− mice (4 months old) showing stronger Fpn immunoreactivity (B) compared with the age-matched Hepc^+/+ mice (A). Fpn immunoreactivity was prominent in the vascular endothelium (B, D, F, arrows) of Hepc^−/− mice but not in vessels from WT mice (C). Immunoreactivity was quantified by measuring the mean pixel intensity within the retinal pigment epithelial and neural retinas (G; P < 0.05). Scale bars: 25 μm (A, B); 10 μm (C); 12.5 μm (D–F). (C) Fpn, red; CD-31, green. (D) Fpn. (E) CD31. (F) Fpn, red; CD31, green. OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 6.

Exogenous Hepc peptide decreased Fpn protein levels in BRECs and also reduced iron release from BRECs. Western blot analysis for Fpn (A) in BRECs exposed to Hepc peptide for 1 hour and control BRECs not exposed to Hepc, quantified by densitometry and standardized to β-actin (B). BRECs exposed to Hepc peptide showed significant decreases (*P < 0.01) in the levels of Fpn protein compared with control BRECs. In a separate experiment, a monolayer of BRECs preloaded with 59Fe released significantly less (*P < 0.001) 59Fe into the basal chamber (as measured in gamma counts after 1 hour) when Hepc peptide was placed in the basal chamber compared with the no Hepc control condition (C).

Figure 7.

Hepc mRNA levels in mice with chronic (DKO) or acute (Holo-Tf injection) iron accumulation. Hepc mRNA levels measured by qPCR were significantly higher in Cp/Heph DKO mice than in age-matched controls (A). Needle injury significantly upregulated retinal IL6 (B) and Hepc (C) mRNA levels in WT but not in IL6^−/− (C) mice. Retinal Hepc mRNA levels were higher in Holo-Tf–injected IL6^−/− mice than in control IL6^−/− mice injected with Apo-Tf at 8 hours (D) and 24 hours (E) after injection. *P < 0.05.

Figure 8.

Western blot analysis of retinal p-ERK and p-SMAD after intravitreal injection of Holo-Tf. One hour after intravitreal injection of Holo-Tf, p-ERK levels increased compared with control Apo-Tf–injected contralateral eyes. Western blot analysis from four mice (eight retinas) is shown (A) and quantified by densitometry (B). p-ERK and ERK band intensities were standardized to α-tubulin. p-SMAD levels were unchanged in Holo-Tf compared with Apo-Tf–injected eyes. *P < 0.05.