Heme oxygenase 1 is required for mammalian iron reutilization

Abstract

The majority of iron for essential mammalian biological activities such as erythropoiesis is thought to be reutilized from cellular hemoproteins. Here, we generated mice lacking functional heme oxygenase 1 (Hmox1; EC 1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron, to assess its participation in iron homeostasis. Hmox1-deficient adult mice developed an anemia associated with abnormally low serum iron levels, yet accumulated hepatic and renal iron that contributed to macromolecular oxidative damage, tissue injury, and chronic inflammation. Our results indicate that Hmox1 has an important recycling role by facilitating the release of iron from hepatic and renal cells, and describe a mouse model of human iron metabolic disorders.

Figure 1

Targeted disruption of the Hmox1 gene. (a) Hmox1 genomic locus and targeting vector. A 3.7-kb region including exons 3, 4, and a portion of 5 (e3–e5) was replaced with a pgk-neo cassette. The 5′ and 3′ probes used for screening ES cell clones and genotyping mice are shown. The 5′ probe hybridizes to an 11-kb KpnI fragment of the native Hmox1 gene and a 6.5-kb fragment from the disrupted gene. D, HindIII site; K, KpnI; X, XhoI; Xb, XbaI. (b) Southern blot analysis of KpnI-digested tail DNA from ES cell-derived mice. The blot was hybridized with the 5′ Hmox1 probe. Genotypes of one Hmox1 homozygous mutant mouse (−/−), two wild-type mice (+/+), and two heterozygous mice (+/−) are indicated. (c) Northern blot analysis of total splenic RNA from a wild-type mouse (+/+), a heterozygous mouse (+/−), and a homozygous mutant mouse (−/−). The blot was hybridized with a rat Hmox1 cDNA probe (13), which recognizes a major mRNA band of approximately 1.5 kb. The Hmox1 probe recognized an aberrantly sized mRNA from Hmox1^−/− mice that was barely detectable even in an overloaded lane.

Figure 2

Wasting, anemia, and serum iron deficiency in Hmox1^−/− mice. (a) Hmox1^−/− mice were consistently slightly smaller than Hmox1^+/− littermates, but weight loss between 20 and 40 weeks of age was clearly indicative of wasting. Data are shown mean ± SEM. One and 3 weeks, 9 Hmox1^−/− and 19 Hmox1^+/− males and females were analyzed; 6 weeks, 12 Hmox1^−/− and 24 Hmox1^+/− males only; 20 weeks, 9 Hmox1^−/− and 11 Hmox1^−/− males; and 40 weeks, 13 Hmox1^−/− and 15 Hmox1^+/− males. Significant differences (P < 0.05) were observed between Hmox1^−/− and Hmox1^+/− masses at all ages. In a–d, □, Hmox1^+/− mean values; ▪, Hmox1^−/− mean values. (b) Red blood cell counts (RBC), packed cell volume (hematocrit, Hct), blood hemoglobin concentration (Hb), and mean cell volume (MCV) analyzed in 7–11 male and female Hmox1^−/− and Hmox1^+/− littermates at 6, approximately 20, and approximately 50 weeks of age. Significant differences (P < 0.05) were observed for each parameter at all ages except for 6-week RBC and Hb values. (c) Serum iron (Fe), total iron-binding capacity (TIBC), and iron saturation analyzed in 5 Hmox1^+/− and 5 Hmox1^−/− male mice of each age group. Significant differences (P < 0.05) were observed in each parameter except for 6-week serum iron values. Iron saturation percentage is equal to 100(serum iron/TIBC). (d) Serum ferritin values analyzed in mice from c. Significant differences (P < 0.05) were observed at each age examined.

Figure 3

Iron-loading in Hmox1^−/− tissues. (a) Kidney section from 50-week-old Hmox1^+/− mouse stained with Prussian blue for detection of loosely bound ferric iron, shown at low magnification. Stainable renal iron was rarely detectable in Hmox1^+/− or Hmox1^+/+ mice. (b) Kidney section from 50-week-old Hmox1^−/− mouse stained with Prussian blue. Note the intense blue positive staining in the proximal cortical tubules (arrows), which was consistently observed in Hmox1^−/− renal tissue. (c) Liver section from 50-week-old Hmox1^+/− mouse stained with Prussian blue and at low magnification. Mice containing functional Hmox1 displayed no hepatic iron deposits. (d) Liver section from 50-week-old Hmox1^−/− mouse stained with Prussian blue, demonstrating iron-loaded Kupffer cells (arrows), as well as diffusely staining hepatocytes. Note the faint-staining regenerative nodule indicative of injury (asterisk). All Hmox1^−/− mice displayed hepatic iron-loading by 50 weeks of age, varying in severity. (e) High-magnification view of liver section from 50-week-old Hmox1^+/− mouse stained with Prussian blue, displaying no detectable iron in hepatocytes. (f) High-magnification view of liver section from 50-week-old Hmox1^−/− mouse stained with Prussian blue, indicating iron-positive granules in hepatocytes (arrowheads), and intensely staining Kupffer cells (arrows). (All magnification bars = 100 μm.)

Figure 4

Markers of stress and inflammation in Hmox1^−/− mice. (a) Protein carbonyls were measured by the method utilizing 2,4-dinitrophenylhydrazine in supernatant harvested from Hmox1^+/− and Hmox1^−/− brain, liver, or kidneys. Estimates of lipid peroxidation were obtained by malondialdehyde (MDA) measurements of tissue homogenates. Data represent mean ± SEM from duplicate or triplicate determinations from each of 4 mice of each genotype for brain samples, and each of 6–8 mice of each genotype for liver and kidney samples. Open bars represent Hmox1^+/− values, while closed bars represent Hmox1^−/− values. ∗, Hmox1^−/− liver and kidneys, which consistently showed iron loading, had significantly greater oxidative damage than those from Hmox1^+/− animals (P < 0.05). Note that Hmox1^−/− brains, which had no iron deposition, showed no evidence of enhanced oxidative damage. (b) Average white blood cell counts (WBC), splenic mass, and splenic CD4⁺:CD8⁺ T-cell ratios analyzed from 7–11 mice of each age group for WBC, and 4–7 mice of each age group for splenic mass and T-cell ratios, at 6, approximately 20, and approximately 50 weeks of age. Significant differences (P < 0.05) were observed for each parameter at each age examined. □, Hmox1^+/− values; ▪, Hmox1^−/− values.

Figure 5

Hepatic and renal pathology of Hmox1^−/− animals. (a) Liver section from 50-week-old Hmox1^+/− mouse, stained with hematoxylin and eosin and shown at low magnification. (b) Liver section from 50-week-old Hmox1^−/− mouse, with typical pattern of lesions. Vascular lesions are indicated by arrows. (c) High-magnification view of typical periportal fibrosis and inflammation from a 50-week-old Hmox1^−/− mouse. This section was stained with Masson’s trichrome reagent, which reacts with collagen, an indicator of fibrosis, as bright blue. (d) High-magnification view of hepatic vascular lesion shown in b stained with hematoxylin and eosin. Note the proliferation of smooth muscle, the infiltration of neutrophils and lymphocytes into the vessel wall, and the monocytes adhered to the inner vessel wall (arrows). (e) High-magnification view of hepatic vascular lesion from a 50-week-old Hmox1^−/− mouse. This section was stained for ferric iron (blue). Note the iron-laden monocytes adhering to the vessel wall (arrows), as well as the iron-laden vascular and connective tissue (arrowheads). (f) High-magnification view of kidney section of 75-week-old Hmox1^+/− mouse, stained with hematoxylin and eosin, displaying normal glomerular morphology (arrows). (g) Kidney section of 75-week-old Hmox1^−/− mouse displaying severely damaged glomeruli with membranous proliferation, lobularity, crescent formation, and sclerosis (arrows). (All magnification bars = 100 μm.)