Volume 133, Issue 4, Pages 1261-1271.e3 (October 2007) Lack of Haptoglobin Affects Iron Transport Across Duodenum by Modulating Ferroportin Expression  Samuele Marro, Donatella Barisani, Deborah Chiabrando, Sharmila Fagoonee, Martina U. Muckenthaler, Jens Stolte, Raffaella Meneveri, David Haile, Lorenzo Silengo, Fiorella Altruda, Emanuela Tolosano  Gastroenterology  Volume 133, Issue 4, Pages 1261-1271.e3 (October 2007) DOI: 10.1053/j.gastro.2007.07.004 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Iron overload in spleen macrophages of Hp-null mice. Spleen sections of a wild-type (A) and an Hp-null (B) mouse at 3 months of age stained with Perl’s reaction. Note iron loading in macrophages surrounding white pulp in Hp-deficient spleen (at high magnification in the inset). The result shown is representative of 10 mice analyzed. Bar, 250 μm. Gastroenterology 2007 133, 1261-1271.e3DOI: (10.1053/j.gastro.2007.07.004) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Analysis of the duodenum of Hp-null mice. (A) 55Fe mucosal uptake (MU) was measured in tied-off duodenal segments of wild-type mice fed a standard diet (SD) and, as positive controls, in wild-type mice fed an iron-free diet (IFD) and HFE-null mice on an SD. See text for details. Data represent mean ± SEM; n = 10. (B) 55Fe mucosal uptake (MU) and mucosal retention (MR) were measured in tied-off duodenal segments of wild-type and Hp-null mice. See text for details. Data represent mean ± SEM; n = 10. (C) Western blotting analysis of DMT1, Fpn1, and L-Ft expression in the duodenum of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to vinculin expression. Densitometry data represent mean ± SEM; n = 5 for each genotype. (D) Northern blotting analysis of Fpn1 mRNA levels in the duodenum of wild-type and Hp-null mice. Band intensities were measured by densitometry and normalized to β-actin expression. Densitometry data represent mean ± SEM; n = 4 for wild-type and n = 5 for Hp-null; *P < .05; **P < .01. Results shown in panels C and D are representative of 3 independent experiments; in each experiment, at least 4 mice per genotype were analyzed. Gastroenterology 2007 133, 1261-1271.e3DOI: (10.1053/j.gastro.2007.07.004) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Analysis of the liver of Hp-null mice. (A) qRT-PCR analysis of hepcidin expression in the liver of wild-type and Hp-null mice. Transcript abundance, normalized to 18S RNA expression, is expressed as a fold increase over a calibrator sample. (B) Western blotting analysis of TfR1, H-Ft, L-Ft, TfR2, and Fpn1 expression in the liver of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression. Densitometry data represent mean ± SEM; n = 5 for each genotype. Results shown are representative of at least 3 independent experiments; in each experiment, 5 mice per genotype were analyzed. Gastroenterology 2007 133, 1261-1271.e3DOI: (10.1053/j.gastro.2007.07.004) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Analysis of the spleen of Hp-null mice. (A) Western blotting analysis of L-Ft, H-Ft, TfR1, and Fpn1 expression in the spleen of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression levels. Densitometry data represent mean ± SEM; n = 5 for each genotype. *P < .05. Results shown are representative of at least 3 independent experiments; in each experiment, 5 mice per genotype were analyzed. (B) Three consecutive sections of the spleen of a wild-type mouse (a, c, e) and an Hp-null mouse (b, d, f) stained with antibodies to F4/80 (a and b), to L-Ft (c and d), and to Fpn1 (e and f), respectively. Note increased L-Ft expression in F4/80-positive cells of the marginal zone (indicated by arrows) in Hp-null mouse (more evident at higher magnification in the inset). Staining for Fpn1 is weak both in wild-type and Hp-null mice. No differences in Fpn1 expression are detected between wild-type and Hp-null mice. Bar, 500 μm. (C) Spleen/body-weight ratio in wild-type and Hp-null mice. Data represent mean ± SEM; n = 10 for each genotype. P < .01. (D) Percentage of F4/80+, CD11b+, CD19+, CD8+, and CD4+ cells within the spleen of Hp-null and wild-type mice. Data represent mean ± SEM; n = 4 for each genotype. (E) qRT-PCR analysis of Fpn1 and HO-1 expression on spleen macrophages isolated from wild-type and Hp-null mice. Transcript abundance, normalized to 18S RNA expression, is expressed as a fold increase over a calibrator sample. Data represent mean ± SEM; n = 3 for each genotype. **P < .01. Results shown are representative of 3 independent experiments; in each experiment, 3 mice per genotype were analyzed. Gastroenterology 2007 133, 1261-1271.e3DOI: (10.1053/j.gastro.2007.07.004) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Analysis of the kidney of Hp-null mice. (A) Western blotting analysis of Fpn1, L-Ft, and H-Ft, expression in the kidney of wild-type and Hp-null mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to vinculin expression levels. Data represent mean ± SEM; n = 5 for each genotype. *P < .05. Results shown are representative of at least 3 independent experiments; in each experiment, 5 mice per genotype were analyzed. (B) Kidney sections of a wild-type mouse (a) and a Hp-null mouse (b) stained with an antibody to L-Ft. Note the strong expression of L-Ft in proximal tubular cells of Hp-null mouse (more evident at high magnification in the inset). Bar, 500 μm. Gastroenterology 2007 133, 1261-1271.e3DOI: (10.1053/j.gastro.2007.07.004) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Fpn1 and HO-1 expression in RAW267.4 cells after exposure to hemoglobin or Hp. (A) qRT-PCR analysis of Fpn1 and HO-1 expression on RAW264.7 cells at different hemoglobin exposure times (0–24 hours). Transcript abundance, normalized to 18S RNA expression, is expressed as a fold increase over a calibrator sample. Data represent mean ± SEM, n = 3 for each experimental point. **P < .01, ***P < .001. Results shown are representative of 3 independent experiments. (B) Western blot analysis of Fpn1 expression on RAW264.7 cells at different hemoglobin exposure times (0–24 hours). Band intensities were measured by densitometry and normalized to vinculin expression. Densitometry data represent mean ± SEM; blot shown is representative of 3 independent experiments. *P < .05; **P < .01. (C) qRT-PCR analysis of Fpn1 and HO-1 expression on RAW264.7 cells after 6 hours of hemoglobin or Hp exposure. Transcript abundance, normalized for 18S RNA expression, is expressed as a fold increase over a calibrator sample. Data represent mean ± SEM, n = 3 for each experimental point. *P < .05, **P < .01. Results shown are representative of 3 independent experiments. Gastroenterology 2007 133, 1261-1271.e3DOI: (10.1053/j.gastro.2007.07.004) Copyright © 2007 AGA Institute Terms and Conditions