Hepatic and renal iron status in control (+/−) and mutant (<i>ms/−</i>) mice.

<p>Non-haem hepatic (<b>A</b>) and renal (<b>C</b>) iron content was measured as described in the Experimental section. Values are expressed as the means ± S.D. for both liver and kidney samples obtained from 15 mice of each genotype. L-ferritin levels in hepatic (<b>B</b>) and renal (<b>D</b>) cytosolic protein extracts (50 µg/lane) were assessed by western blot analysis <i>left-hand panels</i>. The blots were reprobed with polyclonal anti-human actin antibody as a loading control. (<b>B</b>) and (<b>D</b>) <i>right-hand panels</i> Immunolabelled ferritin bands from four mice were quantified using a Molecular Imager and ferritin protein levels (means ± S.D.) are plotted in arbitrary units. ** – P<0.01.</p

Decreased copper content and activity/expression of SOD1 in circulating erythrocytes from <i>ms/−</i> mice.

<p>(<b>A</b>) Decreased copper content in erythrocytes of <i>ms/−</i> mice. Values are expressed as the means ± S.D. for erythrocyte samples obtained from 15 control (<i>+/−</i>) and 15 mutant (<i>ms/−</i>) males. (<b>B</b>) <i>left-hand panel</i>, the activity of SOD was measured after resolution by gel electrophoresis using the Nitroblue Tetrazolium (NBT)/riboflavin method as described in the Experimental section. The analyses were performed using erythrocyte total extracts obtained from <i>ms/−</i> and <i>+/−</i> males and representative results are shown. (<b>C</b>) <i>left-hand panel</i>, SOD1 levels in erythrocytes were analyzed by western blotting as described in the Experimental section. The analyses were performed using erythrocyte total extracts obtained from <i>ms/−</i> and control (<i>+/−</i>) males, and representative results are shown. The blot was reprobed with monoclonal anti-actin antibody as a loading control. (<b>B,C</b>) <i>right-hand panels</i>, the intensity of the SOD bands was quantified with a molecular Imager using Quantity One software (Bio-Rad) and is plotted in arbitrary units to present activity (<b>B</b>) and protein level (<b>C</b>). Results are expressed as the mean ± S.D. for 5 mice of both the <i>ms/−</i> and <i>+/−</i> genotypes. Significant differences are indicated (* – P<0.05; ** – P<0.01).</p

Increased expression of haem oxygenase 1 (HO1) in kidneys of <i>ms/−</i> mice.

<p>(<b>A</b>) Real-time quantitative PCR analysis of renal HO1 mRNA expression. The histogram displays HO1 mRNA levels in arbitrary units (means ± S.D., n = 6). (<b>B</b>) <i>left-hand panel</i> Western blot analysis of HO1 protein levels in hepatic membrane fractions prepared from <i>+/−</i> and <i>ms/−</i> males. The blot was reprobed with polyclonal anti-human actin antibody as a loading control. <i>right-hand panel</i> Immunolabelled HO1 bands from six mice were quantified using a Molecular Imager and HO1 protein levels (means ± S.D.) are plotted in arbitrary units. ** – P<0.01. (<b>C</b>) <i>left-hand panel</i> Immunofluorescent staining of HO1 in <i>+/−</i> and <i>ms/−</i> kidneys analyzed by confocal microscopy. RN – renal tubules; RG – renal glomeruli. <i>middle panel</i> Transmitted light image shows the structure of glomeruli and tubules as well as the presence of a large lesion (asterisk) in the kidney of a mutant. <i>right-hand panel</i> To confirm the specificity of HO1 detection, kidney sections of <i>+/−</i> and <i>ms/−</i> males were incubated with only the secondary antibody. No HO1 staining was detected in these negative controls. Nuclei were counterstained with DAPI. Bars correspond to 50 µm.</p

Correlation between decreased ferroportin (Fpn) protein level and increased hepcidin (Hepc) mRNA expression in the liver of <i>ms/−</i> males.

<p>(<b>A</b>) Immunofluorescent staining of Fpn in <i>+/−</i> and <i>ms/−</i> males. <i>top panel</i> Livers analyzed by confocal microscopy. <i>middle panel</i> Tissue morphology observed in transmitted light. <i>bottom panel</i> To confirm the specificity of Fpn detection, liver sections of <i>+/−</i> and <i>ms/−</i> males were incubated with only the secondary antibody. No Fpn staining was detected in these negative controls. Nuclei were counterstained with DAPI. Bars correspond to 50 µm. (<b>B</b>) Colocalization (<i>bottom panel</i>) of Fpn (red channel) and F4/80, a macrophage marker (green channel) in liver from <i>+/−</i> and <i>ms/−</i> males analyzed by confocal microscopy. Nuclei were counterstained with DAPI. Bars correspond to 50 µm. (<b>C</b>) Real-time quantitative PCR analysis of hepatic Hepc mRNA expression in <i>+/−</i> and <i>ms/−</i> males. The histogram displays Hepc mRNA levels in arbitrary units (means ± S.D., n = 6). Significant difference is indicated (** – P<0.01).</p

Haemolysis and acanthocytosis in <i>ms/−</i> mice.

<p>(<b>A</b>) Serum of control (<i>+/−</i>) and mutant (<i>ms/−</i>) males obtained by centrifugation. (<b>B</b>) Peripheral blood smears of <i>ms/−</i> mice showing increased numbers of acanthocytes. High magnification images of a normal and an abnormally-shaped erythrocyte are shown (insets). Six animals from both the control and <i>ms/−</i> male groups were examined and one typical sample of each is shown. Bars correspond to 5 µm. (<b>C</b>) Plasma levels of haptoglobin (Hp) were assessed by western blotting as described in the Experimental section. The blot was reprobed with monoclonal anti-actin antibody as a loading control.</p

Increased expression of haem oxygenase 1 (HO1) in Kupffer cells of <i>ms/−</i> mice.

<p>(<b>A</b>) Real-time quantitative PCR analysis of hepatic HO1 mRNA expression. The histogram displays HO1 mRNA levels in arbitrary units (means ± S.D., n = 6). (<b>B</b>) <i>left-hand panel</i> Western blot analysis of HO1 protein levels in hepatic membrane fractions prepared from <i>+/−</i> and <i>ms/−</i> males. The blot was reprobed with polyclonal anti-human actin antibody as a loading control. <i>right-hand panel</i> Immunolabelled HO1 bands from six mice were quantified using a Molecular Imager and HO1 protein levels (means ± S.D.) are plotted in arbitrary units. * – P<0.05. (<b>C</b>) <i>top panel</i> Immunofluorescent staining of HO1 in <i>+/−</i> and <i>ms/−</i> livers analyzed by confocal microscopy. <i>middle panel</i> Tissue morphology observed in transmitted light. <i>bottom panel</i> To confirm the specificity of Fpn detection, liver sections of <i>+/−</i> and <i>ms/−</i> males were incubated with only the secondary antibody. No HO1 staining was detected in these negative controls. Nuclei were counterstained with DAPI. Bars correspond to 50 µm. (<b>D</b>) Colocalization (<i>bottom panel</i>) of HO1 (red channel) and F4/80, a macrophage marker (green channel) in the livers of <i>+/</i>− and <i>ms/−</i> males analyzed by confocal microscopy. Nuclei were counterstained with DAPI. Bars correspond to 50 µm.</p

Correlations between urine hepcidin and hepatic/plasma hepcidin expression, iron transferrin saturation and hepatic non-heme iron content.

<p>Urine hepcidin-25 levels were measured in control and iron supplemented piglets, and plotted against: (A) hepcidin mRNA levels, Spearman correlation r = 0.8061, p = 0.0049; (B) hepatic non-heme iron content, Spearman correlation r = 0.8061, p = 0.0049; (C) hepatic non-heme iron content, Spearman correlation r = 0.6242, p = 0.0537. In each correlation 12 couples of examined parameters on day 28 were used for the statistical calculations.</p

Hemoglobin levels, transferrin saturation, plasma iron level and hepatic non-heme iron content/distribution in control and iron supplemented piglets.

<p>(A) Hemoglobin level. Values are expressed as the mean ± S.D. for liver samples obtained from 7 piglets from each group. (B) Plasma iron levels and (C) iron transferrin saturation were measured and calculated as described in Material and Methods. Values are expressed as the mean ± S.D. for liver samples obtained from 7 piglets from each group. (D) Hepatic non-heme iron content and distribution were measured and examined as described in Materials and Methods. A high magnifications present deposits of iron or its absence in iron dextran injected and control piglets, respectively. Values are expressed as the mean ± S.D. for liver samples obtained from 7 piglets from each group. Statistically significant differences are indicated (*P≤0.05; **P≤0.01).</p

WCX-TOF MS analysis of urine porcine hepcidin.

<p>(A) Urine and plasma pig Hepcidin-25 quantification by mass spectrometry. Hepcidin-25 measurements in piglet plasma were performed by peptide enrichment through weak cation exchange chromatography coupled to time-of-flight mass spectrometry (WCX-TOF MS) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136695#pone.0136695.ref015" target="_blank">15</a>]. The spectra (1–4) illustrate the appearance of hepcidin-25 in plasma on day 3, 14, 21 and 28 upon iron dextran injection to piglets on day 3 after birth. Spectra 5 and 6 show hepcidin-25 in the urine of piglets on day 28. Spectrum 5 corresponds to piglet injected with iron dextran on day 3 and spectrum 6 corresponds to non-supplemented, control piglet. IS—internal standard. (B) Immune capture of pig hepcidin-25 and isoform hepcidin-20 in piglet urine. Pig urine was incubated with anti-hepcidin overnight at 4°C, and subsequently hepcidin was measured by WCX-TOF MS. Hepcidin was almost not detectable in the urine sample incubated with anti-hepcidin antibody (upper spectrum); in the spectrum below (pig urine without anti-hepcidin antibody) both a high hepcidin-25 peak and a hepcidin-20 peak (mass 2152 Da) are present. (C) Correlation of pig hepcidin-25 in plasma with pig hepcidin-25 in urine measured with WCX-TOF-MS in piglets injected with iron dextran. Data from 4 measurements of urine and 4 measurements of plasma samples from day 28 were used for the statistical calculations. Spearman correlation r 0.9063, p = 0.0067.</p

Induction of HO1 expression and increased iron status in duodenal enterocytes from hemoglobin-supplemented piglets.

<p><b>(A)</b> RT-qPCR analysis of HO1 mRNA expression. The histogram displays HO1 mRNA levels in arbitrary units (means ± S.D., n = 7). <b>(B and C)</b> Western blot analysis of HO1 protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(B)</b>. Immunolabelled HO1 bands from separate blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager, and HO1 protein levels (means ± S.D.) are plotted in arbitrary units <b>(C)</b>. <b>(D)</b> Immunofluorescent staining of HO1 in the duodenum. To confirm the specificity of the HO1 detection, duodenum sections of piglets were incubated with only the secondary antibody. No HO1 staining was detected in these negative controls. Counterstaining of nuclei was performed with DAPI. <b>(E and F)</b> Western blot analysis of L-ferritin protein levels in membrane fractions prepared from duodenal scrapings. A representative immunoblot is shown <b>(E)</b>. Immunolabelled L-ferritin bands from separate blots performed on scrapings isolated from 6 piglets were quantified using a Molecular Imager, and L-ferritin protein levels (means ± S.D.) are plotted in arbitrary units <b>(F)</b>. <b>(G)</b> Histological examination of iron loading in duodenum sections. Non-heme iron deposits (indicated by arrows) were detected by staining with Perls’ Prussian blue and counterstained with nuclear red. Duodenum morphology is shown in transmitted light.</p