1. Hepcidin regulates intrarenal iron handling at the distal nephron
Boualem Moulouel, Dounia Houamel, Constance Delaby, Dimitri Tchernitchko, Sophie Vaulont, Philippe Letteron, Olivier Thibaudeau, Hervé Puy, Laurent Gouya, Carole Beaumont, Zoubida Karim 
Kidney International 
Volume 84, Issue 4, Pages (October 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Parameters of iron status in Hepc -/- mice. (a) Physiological variations in iron status were evaluated by the measurement of non-heme iron, ferritin, and transferrin levels in serum using an AU400 automate (Olympus, Tokyo, Japan). Transferrin saturation (TS) was calculated using a standard formula: TS (%)=[plasma iron concentration/(25 × concentration of transferrin)] × 100. (b) Urinary iron excretion was evaluated by the measurement of non-heme iron in the collected urine, as described in Material and Methods. The values were normalized per mole of creatinine. Bars are the means±s.e.m. ***P<0.0001; **P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Iron accumulation in Hepc -/- kidneys. Tissue iron content was determined by Perl’s staining of kidney sections. The upper panel (wild type, WT) and lower panel (Hepc -/-) are representative images showing iron deposition mostly in the medulla in Hepc -/- mice. At higher magnification ( × 40 and × 60), iron was observed in selective renal epithelial cells and in the lumen of the collecting duct (CD). G, glomerulus.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Ferroportin (FPN) expression in Hepc -/- kidneys. The mRNA and protein levels of FPN were determined by reverse transcription-quantitative PCR (a) and western blot (b) experiments. Total RNA and proteins were extracted from cortex and medulla preparations. In a, data are normalized by the quantities of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and in b by actin protein level. The results are the mean±s.e.m. of at least five individual mice of each group. ***P<0.0001; *P<0.05. NS, not significant. FPN localization was performed by immunofluorescence (c) using anti-FPN antibody and green fluorescent Alexa Fluor 488 (goat anti-rabbit secondary antibody). The images are focal planes at magnification × 5 of samples analyzed by confocal microscopy. In Hepc -/-, A and B are high-magnification images ( × 20) showing FPN abundance in the cortex (A) and in the medulla (B).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Ferroportin (FPN) distribution in Hepc -/- kidneys. Staining of FPN and of specific tubular markers lectin Lotus tetragonolobus (LTL), aquaporin 2 (aq2), and TH was performed in consecutive sections. FPN is shown in green and the markers in red. LTL-positive tubules are proximal tubules (PTs). Tubules expressing TH are TAL of Henle’s loop and the ones expressing aq2 are the collecting duct (CD). All tubular markers exhibit apical staining. FPN-negative tubules are shown with asterisks. The upper panel represents the cortex region and the middle and lower panels represent the medulla. FPN immunofluorescence showed no tubular overlap with LTL or aq2 staining. Images are taken by confocal microscopy ( × 40). G, glomerulus; TAL, thick ascending limb; TH, Tamm Horsefall.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

6. Figure 5
Transferrin receptor1 (TFR1) and divalent metal transporter1 (DMT1) expressions in Hepc -/- kidneys. The mRNA and protein levels of TFR1 (a) and DMT1 (b) were determined by reverse transcription-quantitative PCR and western blot experiments. The data are normalized by the quantities of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA or by actin protein level, respectively. In b2, kidney slices from Hepc -/- and wild-type (WT) mice were incubated during 4h in Hank’s solution (pH 7.4 at 37°C, 5% CO2/95% O2) containing 200mmol/l hepcidin (Hepc.; black columns) or vehicle (control (Con.), white columns), and total proteins were prepared for SDS-polyacrylamide gel electrophoresis to detect DMT1 protein level. The results are the mean±s.e.m. of at least five individual mice of each group. ***P<0.0001; **P<0.001; *P<0.05. NS, not significant.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Iron transport in opossum kidney (OK) cells and regulation by hepcidin. OK cells grown on Transwell inserts were incubated for 4h with 200nmol/l hepcidin or with water (control) added at the basolateral side. (a) The 55Fe transepithelial transport was measured by challenging cells at the apical side with 55FeNTA and quantifying the 55Fe at the basolateral side. (b) Hepcidin-treated cells were challenged for 30min with 55FeNTA, and then their 55Fe contents were quantified and normalized per mg of proteins. The amount of counts per minute (cpm) in control cells was set as 100% (white column). (c) OK cells were incubated with different hepcidin concentrations, and 55Fe transepithelial transport at t=30min was measured. Data are mean±s.e.m. from triplicate samples of four independent experiments. **P<0.001; *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

8. Figure 7
Divalent metal transporter1 (DMT1) expression in opossum kidney (OK) cells and regulation by hepcidin. (a) Confocal analysis of DMT1 protein transiently expressed as green fluorescence protein (GFP) fusion protein. Actin was stained using the phalloidine-Texas Red marker. (A) Focal plane at the apical (1) and subapical (2) levels. (B) Cross-section from the apical (top) to basolateral (bottom) side. (b) Immunofluorescence imaging of live OK cells overexpressing DMT1-GFP and treated with 200nmol/l hepcidin or with vehicle (H2O). Images were obtained at time 0, 1, 2, and 4h after treatment.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

9. Figure 8
Handling of iron in the kidney in different hemochromatosis models. (a) Perl’s staining of kidney sections from Hepc -/-, Hjv -/-, and phenylhydrazine (PHZ) mice. (b) Representative images of protein abundances of ferritin (Ft) and ferroportin (FPN) detected by western blot analysis using protein preparation from cortex and medulla. At least five individual mice of each group were used. C: control mice. (c) Hepcidin mRNA quantification in the kidney in different hemochromatosis models. Total RNAs were extracted from cortex and medulla preparations of each group. The mRNA levels of hepcidin were determined by reverse transcription-quantitative PCR. Data were normalized by the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level. The results are the mean±s.e.m. of at least five individual mice. ***P< NS, nonsignificant; WT, wild type.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

10. Figure 9
Speculative representation of iron handling in kidney. (a) In normal conditions, plasma iron concentration is low and transferrin (TF) is only one-third saturated. A small portion of TF-iron may be filtered by the glomerulus (G) and dissociates at acidic pH in the tubular fluid. TF is reabsorbed via megalin (M)/cubulin (C) complex or via transferrin receptor 1 (TFR1) and degraded by the proximal tubule. Released iron is rapidly excreted in urine because of an inhibition of iron reabsorption by hepcidin in the distal nephron. (b) Lack of hepcidin induces iron overload conditions; TF is fully saturated and free iron is concentrated in the tubular fluid. The thick ascending limb (TAL) has a major role in absorbing and sequestrating iron via DMT1 and ferritin, respectively. Under conditions of hemolysis, the massive wasting of hemoglobins through the kidney is offset by their reabsorption in the proximal tubule and recycling of iron to the circulation. Plasma iron is consumed by active erythropoiesis. DMT1, divalent metal transporter1; FPN, ferroportin; PT, proximal tubule.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions