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Am J Physiol Renal Physiol 280: F803-F814, 2001;
0363-6127/01 $5.00
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Vol. 280, Issue 5, F803-F814, May 2001

Cellular localization of divalent metal transporter DMT-1 in rat kidney

C. J. Ferguson, M. Wareing, D. T. Ward, R. Green, C. P. Smith, and D. Riccardi

School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that the kidney plays an important role in iron balance and that metabolically significant reabsorption of this ion occurs in the loop of Henle and the collecting ducts [Wareing M, Ferguson CJ, Green R, Riccardi D, and Smith CP. J Physiol (Lond) 524: 581-586, 2000]. To test the possibility that the divalent metal transporter DMT1 (Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Nature 388: 482-488, 1997) could represent the apical route for iron entry in the kidney, we raised and affinity-purified an anti-DMT-1 polyclonal antibody and determined DMT-1 distribution in rat kidney by Western analysis, immunofluorescence, and confocal microscopy. The strongest DMT1-specific (i.e., peptide-protectable) immunoreactivity was found in the collecting ducts, in both principal and intercalated cells. Thick ascending limbs of Henle's loop and, more intensely, distal convoluted tubules exhibited apical immunostaining. Considerable intracellular DMT-1 immunoreactivity was seen throughout the nephron, particularly in S3 segments. The described distribution of DMT-1 protein is in agreement with our previous identification of nephron sites of iron reabsorption, suggesting that DMT-1 provides the molecular mechanism for apical iron entry in the distal nephron but not in the proximal tubule. Basolateral iron exit may be facilitated by a different system.

iron; immunohistochemistry; iron-responsive element; divalent metal transporter 1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IRON IS A VITAL ELEMENT for life. It is an essential metal for a myriad of fundamental biological processes (e.g., electron transfer, oxygen transport). However, because of its ability to generate oxygen species, its reactive nature is also potentially hazardous. Consequently, systems have evolved to transport iron and maintain low concentrations of free ionized iron (Fe2+/Fe3+). This process involves binding of iron to proteins such as transferrin and ferritin. Iron also circulates in plasma and is translocated across plasma membranes via receptor-mediated endocytosis bound to transferrin. Until recently, this means of uptake was considered the main mechanism for iron transport across plasma membranes (2). However, the cloning of divalent metal transporter 1 (DMT-1)/natural resistance-associated macrophage protein 2 from the small intestine of iron-deficient rats (14) demonstrated that free iron can be translocated across membranes by secondary active transport coupled to protons.

DMT-1 is proposed to be the major pathway for uptake of dietary iron by the gastrointestinal tract. Mutations in the DMT-1 gene cause hypochromic, microcytic anemia in rodents (8, 9), which is fatal unless more iron is made available, for example, by increased dietary iron or by direct iron injections (11). The discovery that the anemia associated with this mutation could not be fully corrected by direct iron injections (11) suggested that organs other than the gastrointestinal tract were also affected by this mutation and may thus contribute to total body iron homeostasis. Recently, using in vivo microinjection and renal microperfusion techniques, we have shown that the kidney plays an important role in iron homeostasis. We demonstrated that a metabolically significant amount of iron is filtered at the glomerulus and the majority is reabsorbed (26). Interestingly, in situ hybridization (14) and Western analysis on total kidney membranes (4) showed that DMT-1 mRNA and protein are strongly expressed in the kidney. Therefore, we set out to determine the renal distribution of DMT-1 and whether this protein could account for renal reabsorption of iron. To do this we generated and affinity-purified a polyclonal DMT-1 antiserum and used it for Western blotting, immunofluorescence, and confocal microscopy experiments to assess the overall nephron distribution of DMT-1 in rat kidney.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antibody production and purification. The DMT-1 gene can generate two alternatively spliced transcripts that share 100% homology until the extreme 3'-coding sequence and for the whole of the 3'-untranslated region (UTR). One of the two mRNAs contains an iron-responsive element (IRE) in its 3'-UTR (14). The proteins these transcripts encode are identical at the NH2 terminus but differ at the COOH terminus. We generated a polyclonal antiserum in rabbit to a peptide sequence in the NH2 terminus of DMT-1, common to both isoforms. The antiserum (RA2719) was generated by injecting rabbits with the peptide sequence MVLDPEEKIPDDGASGDHGDS, corresponding to amino acids 1-21 of rat DMT-1 (Sigma Genosys, Cambridge, UK). The antibody was purified in two stages. To isolate the IgG fraction, 3 ml of immunized rabbit serum were mixed with 300 µl of 1 M Tris · HCl, pH 8.0, and added to a column packed with protein A-Sepharose beads (Amersham Pharmacia Biotech, Little Chalfont, UK). The column was washed with 10 vol of 100 mM Tris · HCl, pH 8.0, followed by 10 vol of 10 mM Tris · HCl, pH 8.0. The purified IgGs were eluted from the column with 5 vol of 100 mM glycine, pH 3.0, and immediately neutralized with 1 M Tris · HCl, pH 8.0. The IgG-containing fractions were combined, dialyzed against 1× PBS for 24 h at 4°C, then concentrated by using a Centricon 30 Spin Column (Millipore, Watford, UK) according to the manufacturer's instructions.

Anti-DMT-1 IgG was purified from the total IgG fraction by using the immunizing peptide. A 2-ml vol of Affi-Gel 15 (pI >6.5, Bio-Rad Laboratories, Hemel Hempstead, UK) was transferred to a scintered glass funnel mounted on a vacuum-filtration apparatus and washed with ice-cold anhydrous isopropanol. The Affi-Gel slurry was then washed with anhydrous DMSO and transferred to a 1-ml column (Bio-Rad). The immunizing peptide was dissolved in 0.5 ml of anhydrous DMSO and added to the Affi-Gel column, which was incubated overnight on a rotating mixer at room temperature. The ligand-binding step was followed by a cycle of washes consisting of 2 column vol of 10 mM Tris · HCl, pH 8.0; 1 vol of 100 mM glycine, pH 2.5; 2 vol of 10 mM Tris · HCl, pH 8.0; 1 vol of 100 mM diethylamine, pH 11.5; and 2 vol of 10 mM Tris · HCl, pH 8.0. The affinity column was then equilibrated in 1× PBS. A 1.5-ml vol of the purified total IgG was applied to the affinity column and allowed to run through into a collection tube. The eluate was reapplied three times to ensure maximum binding of the antibody to the column. The column was washed with 10 vol of 1× PBS, and the purified anti-DMT-1 IgG was eluted with 100 mM glycine, pH 3.0, as described above. Concentration of the antibody was again carried out by using a Centricon 30 Spin Column. Immunoabsorbed serum was prepared by overnight incubation of anti-DMT-1 antiserum with the DMT-1 peptide at 4°C.

Microsomal membrane fraction preparation and Western analysis. Male Wistar rats were killed by cervical dislocation, and the kidneys were removed and dissected into cortex, outer medulla, and inner medulla. Tissue samples were ground in homogenization buffer comprising 12 mM HEPES and 300 mM mannitol, to which 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 1 µg/ml polymethylsulfonyl fluoride were added immediately before use. Homogenization was performed at 4°C, and the homogenate was subsequently spun at 2,500 g for 5 min at 4°C. The postnuclear supernatant was then centrifuged at 100,000 g for 30 min at 4°C to yield a crude membrane pellet (25). The pellet was resuspended in homogenization buffer, and the protein concentration was determined by using the Bradford method (Bio-Rad).

Aliquots of microsomal membrane fraction containing 30 µg of protein were mixed with Laemmli buffer and incubated for 5 min at room temperature before being separated by 8% SDS-PAGE and transferred to BioTrace nitrocellulose membranes (Pall Gelman Sciences, Northampton, UK). The membranes were blocked in Tris-buffered saline-Tween 20 (TBST) solution containing 5% skim milk powder for 30 min at room temperature, washed in several changes of TBST, and incubated for 1 h at room temperature in affinity-purified RA2719 diluted 1:350 in TBST. The membranes were washed again with several changes of TBST, then incubated for 1 h at room temperature in goat anti-rabbit IgG conjugated to horseradish peroxidase (DAKO, Ely, UK) diluted 1:5,000 in TBST. A further washing step was followed by visualization of the signal by ECL Plus (Amersham Pharmacia Biotech) according to the manufacturer's protocol. Experiments were repeated in triplicate. Membranes incubated in the absence of the primary antibody or with antiserum preabsorbed for 1 h at room temperature with an excess of the immunizing peptide served as negative controls.

Tissue preparation for immunofluorescence and confocal microscopy. Six- to eight-week-old male Wistar rats (n = 6) were anesthetized with 5-ethyl-5-(1'-methylpropyl)-2-thiobarbituric acid (Inactin; Sigma-RBI, Poole, UK) at a dose of 100-110 mg/kg (ip). Perfusion was carried out as described elsewhere (28). The left kidney was flushed with PBS and then perfusion-fixed with 4% paraformaldehyde in PBS followed by 750 mosmol/kgH2O PBS-sucrose solution. The kidney was then removed from the animal, and sagittal sections were cryoprotected overnight at 4°C in 30% sucrose in PBS. The cryoprotected kidney was embedded in Tissue-Tek optimum cutting texture compound (Sakura Finetek Europe, Zoeterwoude, The Netherlands) and snap-frozen in N-methylbutane cooled on dry ice. Cryosections (3-4 µm thick for immunofluorescence and 8 µm thick for confocal microscopy) were cut by using a Leica CM3050 cryostat (Leica Instruments, Nussloch, Germany) and thaw-mounted onto Superfrost Plus slides (BDH, Poole, UK).

Localization of DMT-1 along the nephron was performed by using markers for specific nephron segments and for intracellular compartments. Proximal tubular brush-border and intercalated cells of the collecting ducts were identified by using an anti-rabbit polyclonal antibody raised against the 31-kDa subunit of the H+-ATPase (1:100 dilution) (21). Principal cells of the collecting duct were identified by using an affinity-purified rabbit polyclonal antibody raised against aquaproin-2 (AQP2; 1:50 dilution) (18). Distal convoluted tubules were stained with an affinity-purified rabbit polyclonal antibody raised against the rat thiazide-sensitive Na+-Cl- cotransporter (rNCCT, 1:1,000 dilution) (19). For intracellular localization of DMT-1 in lysosomal compartments, a mouse anti-lysosome-associated membrane protein 1 (LAMP-1) monoclonal antibody was utilized (1:50 dilution, Bioquote, York, UK) (15).

Experimental conditions were optimized for each antibody, and double-staining experiments were performed on the same kidney cryosections. All sections were brought to room temperature, rehydrated in 1× PBS for 5 min, and then blocked for 30 min with 5% normal goat serum in PBS with 1% BSA. The blocking solution was removed, replaced with the appropriate antibody or combination of antibodies (i.e., DMT-1 alone and DMT-1+LAMP-1), diluted in 1% BSA in PBS with 5% normal goat serum added, and the slides were then incubated overnight at 4°C in a humidified box. Whenever double staining was performed using a rabbit and a mouse antibody, both primary antibodies were added at the same time. The primary antibody was removed, and the sections were washed in high-salt PBS (2.8% wt/vol NaCl), followed by two washes with PBS. The slides were then incubated for 1 h at room temperature with the appropriate secondary antibodies, which were goat anti-rabbit IgG conjugated to Cy3 or Texas red (DMT-1 immunostaining) and goat anti-mouse IgG conjugated to FITC (LAMP-1 immunodetection). All secondary antibodies were diluted in 5% normal goat serum in 1% BSA in PBS and applied simultaneously.

Costaining of DMT-1 with NCCT1, AQP2, or H+-ATPase antibodies was performed by using the protocol described in detail elsewhere for double labeling using two rabbit polyclonal antibodies (20). Secondary antibodies were rhodamine red-conjugated Fab fragment goat anti-rabbit IgG for DMT-1 and FITC goat-anti rabbit IgG for NCCT1, AQP2, and H+-ATPase.

All secondary antibodies were affinity purified and purchased from Jackson Immunoresearch Laboratories (Luton, UK). After incubation with the secondary antibodies, all sections were washed twice for 5 min in high-salt PBS, followed by two washes in PBS, and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Negative controls were carried out by preincubating the DMT-1 antiserum with an excess of antigenic peptide and by omitting the primary antibodies.

Immunofluorescence was visualized by using a Zeiss Axioplan 2 microscope with ×20-100 objectives. Images were acquired by using a Hamamatsu digital camera and processed by using a KS300 version 3.0 software package (Carl Zeiss, Welwyn Garden City, UK). Confocal images were taken by using an Ultraview confocal optical scanner with a Kr/Ar laser (PerkinElmer Life Sciences, Cambridge, UK) mounted on an Olympus Ix70 microscope (Olympus, London, UK). Images were acquired with an Ultrapix charge-coupled device digital camera and processed by using an Ultraview software package. Spatial colocalization of DMT-1 immunoreactivity (red fluorescence) with other antibodies (green fluorescence), resulting in yellow, was obtained by overlaying separately recorded images on a color image by using Adobe Photoshop.

RNA extraction, generation of isoform-specific probes, and Northern analysis. Male Wistar rats were killed by cervical dislocation, and the kidneys were removed and dissected into cortex, outer medulla, and inner medulla. Total RNA was extracted by the acid guanidinium isothiocyanate-phenol method (5). DMT-1 transcript-specific probes were generated by PCR, and both strands were sequenced by using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Warrington, UK). Probe 1 corresponded to nucleotides 85-1600 of rat DMT-1. These nucleotides are shared by the +IRE and -IRE transcripts (GenBank accession no. AF029757). Probe 2 corresponded to nucleotides 2238-2819 for the +IRE transcript of rat DMT-1 (GenBank accession no. AF008439), and probe 3 corresponded to nucleotides 1630-2038 of the -IRE transcript of rat DMT-1(GenBank accession no. AF029757). Messenger RNA was enriched from total RNA by using oligo d(T) cellulose type 7 (Amersham Pharmacia Biotech), and 3 µg of polyadenylated RNA were separated on 1% agarose gels containing 2.2 M formaldehyde and blotted onto Duralon membranes (Stratagene Europe, Amsterdam, The Netherlands). Membranes were hybridized at 42°C overnight with 5× standard sodium citrate (SSC)-50% formamide-3× Denhardt's solution-0.4% SDS-200 µg/ml of salmon sperm DNA and 10% wt/vol dextran sulfate. Membranes were washed at 65°C with 0.1% SSC-0.1% SDS and exposed to film (Kodak Biomax, Hemel, Hempstead, UK) for 12-48 h.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Production and characterization of DMT-1 antiserum. Alternative splicing of the DMT-1 gene can generate two transcripts, one of which contains an IRE in its 3'-UTR, and two corresponding proteins that differ only in their COOH terminus (16). We have raised a rabbit antibody against a 21-amino acid region in the NH2 terminus of rat DMT-1 sequence, which, it was hypothesized, should recognize both DMT-1 isoforms. The anti-DMT-1 polyclonal antibody (RA2719) was affinity purified, and Western analysis was performed to determine its specificity and the general distribution of DMT-1 in the rat kidney. An immunoblot representative of three independent experiments is shown in Fig. 1. The antibody detected a predominant immunoreactive species of ~70-90 kDa in microsomal membrane fractions from kidney cortex (lanes 3 and 6), outer medulla (lanes 4 and 7) and, to a lesser extent, inner medulla (lanes 5 and 8). In addition, in the outer medulla a faint lower molecular weight signal was detected at ~30 kDa (lane 4). The specificity of the anti-DMT-1 antiserum was confirmed by probing total kidney membranes in the absence of the primary antibody (lane 1) or with DMT-1 antiserum preabsorbed with an excess of the immunizing peptide (lane 2).


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  		Fig. 1.   SDS-PAGE on an 8% acrylamide gel showing divalent metal transporter 1 (DMT-1) immunoreactivity in microsomal membrane fractions from rat kidney. Crude membrane extracts from whole kidney (lanes 1 and 2), cortex (lanes 3 and 6), outer medulla (lanes 4 and 7), and inner medulla (lanes 5 and 8) are shown. The DMT-1 antiserum recognized a broad immunoreactive protein band of between 70 and 90 kDa (using long film exposure times; lanes 6-8). At low-exposure times, we observed that most of the DMT-1 immunoreactivity in the cortex and outer medulla was concentrated at ~70 kDa (lanes 3 and 4). A 30-kDa protein was also detected in the outer medulla (lanes 4 and 7). Omitting the DMT-1 antiserum abolished the signal (lane 1). The specificity of the DMT-1 antiserum was confirmed by incubation with peptide-preabsorbed antiserum (lane 2). Sizes (in kDa) of molecular mass markers are shown on the right.

Renal distribution of DMT-1. Northern analysis and in situ hybridization have previously shown the presence of DMT-1-related transcripts in rat kidney (14). However, the cellular distribution of DMT-1 protein in the kidney remains unknown. To determine the intrarenal localization of DMT-1, the affinity-purified antiserum RA2719 was used in immunofluorescence experiments on paraformaldehyde-fixed rat kidney cryosections. Figure 2B shows that the anti-DMT-1 immune serum recognized distinct structures in rat kidney and that no significant fluorescence was observed in sections incubated with immunizing peptide-absorbed antiserum (Fig. 2D) or when the primary antibody was omitted (Fig. 2F). Furthermore, the specific pattern revealed by the anti-DMT-1 antiserum was unchanged when secondary antibodies conjugated to different fluorescent labels were utilized (i.e., Cy3, Texas red or rhodamine, data not shown). No DMT-1-specific immunofluorescence was detected in the glomeruli, in the parietal Bowman's epithelium, thin descending and ascending limbs of the loop of Henle, or in the blood vessels. From these results and our Western analysis, we conclude that the affinity-purified anti-DMT-1 antibody produces specific immunostaining (i.e., peptide-protectable) in rat kidney.


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  		Fig. 2.   Low-magnification (×85) images of 4-µm cryosections from paraformaldehyde-perfused rat kidney. A and B: phase-contrast and DMT-1-specific fluorescence in kidney cortex, respectively. C and D: phase-contrast and fluorescence with peptide-preabsorbed immune serum, respectively. E and F: phase-contrast and fluorescence after incubation in the absence of anti-DMT-1 antibody, respectively. All immunofluorescence micrographs were taken using the same exposure time.

The overall distribution of DMT-1 immunoreactivity in rat kidney cortex and outer medulla is shown in Figs. 3 and 4. Low-magnification images show that the strongest immunofluorescence was observed in the kidney cortex (Fig. 3, A and B) and outer stripe of the outer medulla (Fig. 3C). The signal decreased abruptly at the boundary between the outer and inner stripe of the outer medulla (Fig. 3C). Consistent with the Western blot studies, the inner medulla showed only a weak signal, although this was above the background (Fig. 3D). In the kidney cortex, DMT-1 immunoreactivity was present intracellularly in the proximal tubules, with immunostaining increasing along the length of the tubule. The signal was weak and diffuse throughout the cytosol in S1 segments (Fig. 4A) and progressively increased in the S2 and S3 segments (Fig. 4D). To further characterize the cellular localization of DMT-1 in the proximal tubule, we carried out double immunostaining with DMT-1 (Fig. 5A, red fluorescence) and H+-ATPase (Fig. 5B, green fluorescence), and colocalization is shown in yellow (Fig. 5C). As previously described (3), H+-ATPase immunoreactivity in the proximal tubule was present at the base of the brush border. DMT-1 did not colocalize to the brush border, although cytosolic colocalization, mainly surrounding the nuclei, was present in some proximal tubules (Fig. 5C).


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  		Fig. 3.   Indirect immunofluorescence micrographs of DMT-1 distribution in 4-µm cryosections from rat kidney (magnification ×85). A: superficial cortex showing extensive DMT-1-specific fluorescence in proximal tubule within the cortical labyrinth (cl) and extending along the collecting ducts (within area outlined by white dotted line). B: micrograph of midcortical region showing more intense, punctate DMT-1-specific labeling in the proximal tubule of deeper nephrons. C: outer medulla showing marked decrease in fluorescence, indicating a reduction in DMT-1-specific immunostaining at boundary between the outer stripe (os) and inner stripe (is), which is indicated by white dotted arc. D: inner medulla showing faint DMT-1-specific signal in collecting ducts. Thin limbs are negative for DMT-1.



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  		Fig. 4.   High-magnification (×340) images of DMT-1-specific fluorescence in 4-µm cryosections from rat kidney. A: superficial cortex showing diffuse cytosolic staining in S1 proximal tubule and no staining in the glomerulus (g). B: superficial cortex showing collecting ducts (c) that express apical, intracellular, and bipolar DMT-1 immunoreactivity. C: midcortical region showing extensive intracellular labeling in proximal tubules (p), apical staining in thick ascending limb (TAL; t), and distal convoluted tubule (DCT; d). Collecting ducts (c) exhibit apical, basolateral, and intracellular staining. g, Glomerulus. D: junction between outer stripe and inner stripe of outer medulla showing extensive intracellular DMT-1 fluorescence in S3 segments, which ends abruptly at the transition to thin descending limbs (arrow).



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  		Fig. 5.   Indirect immunofluorescence confocal micrographs of DMT-1 localization in proximal tubules (magnification ×520). Eight-micrometer cryosections were dual labeled with DMT-1 (rhodamine fluorescence; A) and H+-ATPase (FITC fluorescence; B). C: superimposition of images in A and B showing perinuclear colocalization (yellow punctate, arrows) of DMT-1 and H+-ATPase. Note the absence of DMT-1-specific staining in the brush border of proximal tubules, positively stained for H+-ATPase.

To exclude the possibility that the intracellular signal detected in proximal tubules was due to autofluorescence, we performed double staining by using DMT-1 in combination with an antiserum against a lysosomal and late endosomal marker, LAMP-1 (Fig. 6). Although most of the LAMP-1 immunostaining does not colocalize with DMT-1, there is a partial degree of overlap (Fig. 6C, bottom center). The latter is probably due to the late endosomal localization of LAMP-1 and is reminiscent of the recent findings for DMT-1 distribution reported by Tabuchi et al. (23).


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  		Fig. 6.   Confocal micrographs of DMT-1 intracellular localization in proximal tubules (8-µm cryosections). A and B: colocalization of DMT-1 (Texas red fluorescence) with lysosome-associated membrane protein 1 (LAMP-1; a lysosomal marker; FITC fluorescence), respectively. C: superimposition of images in A and B showing minimal colocalization of DMT-1 and LAMP-1 (arrow). Magnification ×520.

Thick ascending limbs (TAL) exhibited punctate, DMT-1-specific immunoreactivity at the apical membrane and, more intensely, in the cytoplasm (Figs. 4C and 7, A and B). The intensity of staining increased progressively toward the distal convoluted tubules (DCT), where a stronger and more defined apical localization could be detected (Fig. 7A). Previously, it has been shown that immunoreactivity for the thiazide-sensitive Na+-Cl- cotransporter NCCT is present from the initial DCT until early connecting segments (19). The results of the double-staining experiments are shown in Fig. 7, where NCCT immunofluorescence is shown in green (D), DMT-1 immunofluorescence in red (C), and spatial colocalization in yellow (E). Our results show that, in the DCT, NCCT and DMT-1 colocalized to the apical membrane. In addition, DMT-1 immunoreactivity was also observed intracellularly. As previously reported (19), the NCCT signal was found to decrease at the transition from DCT to the connecting segment. In these regions, DMT-1 immunoreactivity was also found to decrease (Fig. 7C), indicating little or no DMT-1 protein expression in late DCT-early connecting segments.


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  		Fig. 7.   Indirect immunofluorescence micrographs of DMT-1 localization in TAL and DCT. A: apical and subapical punctate-like DMT-1 immunoreactivity in TAL (t) and DCT (d). DMT-1 immunoreactivity in DCT appears more intense. The arrow indicates strong immunoreactivity in an intercalated cell (magnification ×340). B: high-magnification (×850) image of TAL. Punctate staining at the plasma membrane and more intense cytoplasmic signal are shown. C-E: confocal images of DCT costained with anti-DMT-1 [red (C)] and anti-thiazide-sensitive Na+-Cl- cotransporter [NCCT; green (D)], a marker for DCT. E: spatial colocalization of green and red fluorescence is shown (yellow). There is extensive colocalization of DMT-1 and NCCT in the apical membrane of DCTs. NCCT and DMT-1 fluorescence are absent in late DCT-to-early connecting segments (arrowhead). p, Proximal tubule. Magnification ×520.

Strong DMT-1-specific immunofluorescence was present in the cortical and outer medullary collecting ducts (Fig. 8, A and B). The signal gradually decreased in intensity from the cortex (Fig. 8A) to the outer stripe (Fig. 8B) and inner stripe of the medulla (Fig. 8C). Interestingly, DMT-1 cellular distribution varied considerably. In some of the cells, DMT-1 was present at the apical membrane, in others in the cytosol, often adjacent to the apical and/or basolateral membranes or surrounding the nuclei. To determine the type of cells in which DMT-1 was expressed, we performed double labeling of DMT-1 with AQP2 and of DMT-1 with H+-ATPase. AQP2 is localized in the apical membranes of principal cells of both cortical and medullary collecting ducts (18). In the cortical collecting ducts, the 31-kDa subunit of H+-ATPase is expressed at the apical membrane of A-intercalated cells, and at the basolateral membrane or with diffuse bipolar localization in B-intercalated cells (3). Representative photomicrographs of superficial cortex are shown in Fig. 9, A-F, whereas a section of the outer stripe of the outer medulla is shown in Fig. 9, G-H. Colocalization with AQP2 shows that DMT-1-specific immunoreactivity was present apically and intracellularly in principal cells (Fig. 9, A-C). Similar results were obtained in outer medullary regions (not shown). In superficial cortex, DMT-1 colocalized with H+-ATPase at the apical membrane in A-intercalated cells and with a bipolar distribution in some, but not all, B-intercalated cells (Fig. 9, D-F). In the outer medullary region, DMT-1 was less intense at the apical membrane and more diffuse throughout the cytosol in intercalated cells (Fig. 9, G-H). Furthermore, we also observed colocalization of DMT-1 with H+-ATPase at the apical surface of DCT (Fig. 9C).


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  		Fig. 8.   DMT-1-specific immunoreactivity in collecting ducts of superficial cortex (A), outer medulla (B), and at the junction between outer and inner stripe of the outer medulla (C, white dotted line). Note the decrease in DMT-1 immunoreactivity although the cellular distribution remains unchanged. cd, Collecting duct. Magnification × 340. D: high-magnification micrograph (×850) shows DMT-1-specific signal predominantly apically (most likely to be A cells) and intracellularly.



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  		Fig. 9.   Confocal micrographs of indirect DMT-1 immunofluorescence in cortical (A-F) and outer stripe of outer medullary collecting ducts (G-I). A-C: dual staining of DMT-1 with aquaporin-2 (AQP2). Eight-micrometer cryosections were dual stained with anti-DMT-1 [red fluorescence (A)] and anti-AQP2 [green fluorescence (B)], a marker for collecting duct principal cells. C: spatial colocalization of red and green fluorescence is shown (yellow). DMT-1 is localized to principal cells. Predominantly intracellular DMT-1 localization (arrow) and strong apical colocalization of DMT-1 and AQP2 (arrowhead) in principal cells. p, Proximal tubule. D-F: dual staining with DMT-1 and H+-ATPase. Eight-micrometer cryosections were dual stained with anti-DMT-1 [red fluorescence (D)] and anti-H+-ATPase [green fluorescence (E)], a marker for collecting duct intercalated cells. F: spatial colocalization of red and green fluorescence is shown (yellow). In the cortex, DMT-1 is expressed apically in A cells (arrowhead) and intracellularly/basolaterally in some (filled arrow), but not all, B cells (open arrow). H+-ATPase and DMT-1 immunostaining colocalize in a DCT (d). G-I: dual staining with DMT-1 and H+-ATPase. Eight-micrometer cryosections were dual stained with anti-DMT-1 [red fluorescence (G)] and anti-H+-ATPase [green fluorescence (H)]. I: spatial colocalization of red and green fluorescence is shown (yellow). Outer medullary region shows diffuse, cytosolic DMT-1 immunoreactivity that only partly colocalizes with H+-ATPase. Filled arrow, intracellular DMT-1 localization; open arrow, absence of DMT-1 and H+-ATPase colocalization; arrowhead, colocalization of DMT-1 and H+-ATPase in an A-intercalated cell. p, Proximal tubule. Magnification ×520.

DMT-1 immunoreactivity decreased progressively along the length of the collecting ducts, and, consistent with our Western blot experiments (Fig. 1), inner medullary collecting ducts showed only faint DMT-1-specific staining (Fig. 3D). Others have reported DMT-1 mRNA to be present in inner medulla at levels similar to those in other regions of the kidney (14). Therefore, to investigate whether levels of mRNA in our animals were the same, we performed Northern analysis on mRNA extracted from the kidney cortex and inner medulla.

Northern analysis using probe 1 (common for both +IRE and -IRE transcripts) detected two transcripts in the cortex (2.4 and 4.4 kb) and three transcripts in the inner medulla (2.4, 4.0, and 4.4 kb, Fig. 10, lanes 1 and 2). Levels of the 4.4-kb transcript were comparable in the cortex and inner medulla. This was confirmed by Northern analysis using probe 2 (+IRE) that only recognized the 4.0- and 4.4-kb transcripts (Fig. 10, lanes 3 and 4). There was more of the 2.4-kb transcript in the cortex than in inner medulla. This was confirmed by Northern analysis with probe 3 (-IRE) that only recognized the 2.4-kb transcript (Fig. 10, lanes 5 and 6).


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  		Fig. 10.   High-stringency Northern analysis of poly A+ RNA (3 µg/lane) isolated from kidney cortex (lanes 1, 3, and 5) and inner medulla (lanes 2, 4, and 6). Poly A+ RNA from cortex (lane 1) and inner medulla (lane 2) was probed with probe 1, corresponding to both +iron-responsive element (IRE) and -IRE transcripts of DMT-1. Poly A+ RNA from cortex (lane 3) and inner medulla (lane 4) was probed with probe 2, corresponding to the +IRE transcript. Poly A+ RNA from cortex (lane 5) and inner medulla (lane 6) was probed with probe 3, corresponding to the -IRE transcript. Molecular mass markers (in kb pairs) are shown on the right.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we have shown that the kidney plays an important role in iron homeostasis and that >98% of the iron filtered at the glomerulus is reabsorbed (26). In the present study, we set out to determine the nephron location of the divalent metal transporter DMT-1 and to correlate expression of DMT-1 with our previous microinjection and microperfusion studies (26) to determine whether DMT-1 is implicated in iron reabsorption by the kidney.

Two isoforms of the DMT-1 protein, encoded by the +IRE and -IRE transcripts, have been identified (16). The antibody we generated was designed to recognize both of these proteins. The predicted size of the two isoforms, calculated from their primary sequences, are 61 (+IRE) and 63 kDa (-IRE). We found the anti-DMT-1 antibody recognized proteins of 70-90 kDa in the kidney cortex, outer medulla, and inner medulla. Appropriate controls showed that this immunoreactivity was specific for the immunizing peptide, and we therefore conclude that the proteins we detected were DMT-1 isoforms. The discrepancy between the predicted size and the size we observed is probably due to posttranslational modification because DMT-1 glycosylation has been previously reported in mouse (13).

Our antiserum detected an additional 30-kDa protein in kidney outer medulla. Because the signal could be abolished when the antiserum was preincubated with the immunizing peptide, we suggest that the 30-kDa protein contains the epitope used to immunize our animals and may represent a COOH-terminal truncated form of DMT-1. Another possibility is that it is an unrelated protein containing a DMT-1 epitope. However, after searching GenBank using the BLASTP algorithm, we were unable to find a protein with homology to the immunizing peptide other than DMT-1. In any case, this 30-kDa species represents only a minor portion of total DMT-1 immunoreactivity in the outer medulla.

At the level of the nephron, DMT-1 was strongly expressed in the cytosol of proximal tubules, with increasing intensity from the S1 to the S3 segment. Colocalization with H+-ATPase showed that DMT-1 staining was not present on the apical brush-border membrane. Consistent with our previous functional studies (26), this suggests that DMT-1 is not involved in translocation of iron across the apical membrane of the proximal tubule.

We explored the possibility that the apparent high levels of DMT-1 expression in the cytosol of the proximal tubule were not representative of DMT-1 expression but were due to autofluorescence of lysosomal compartments. In our experiments, we found minimal colocalization of DMT-1 with the lysosomal protein LAMP-1. Therefore, the DMT-1 expression in the proximal tubule was not an artifact due to proximal tubular autofluorescence. In addition, this indicates that DMT-1 in the kidney is not present in lysosomes, which is in agreement with a previous study carried out in several different mouse cell lines (13). Although we do not presently know the exact subcellular location of DMT-1, the high level of expression in the proximal tubule suggests that DMT-1 plays an important role in these cells. DMT-1 is involved in the transferrin receptor-mediated endocytic pathway for cellular iron uptake (7, 8, 12). The proximal tubule is metabolically very active and would therefore be expected to require iron for energy-generating pathways such as electron transfer. Other groups have shown that DMT-1 is expressed in transferrin-positive recycling endosomes in nonintestinal cells (4, 13, 22), and therefore the subcellular expression we observed may represent a similar distribution. Double-labeling studies at the ultrastructural level are required to further define DMT-1 localization in H+-ATPase and/or in the transferrin-expressing vesicles. In addition, intracellular DMT-1 could be involved in a non-transferrin-bound iron uptake similar to the one identified in rat reticulocytes (10).

In the segments that make up the loop of Henle, DMT-1 expression was only expressed in the TAL. In this segment, DMT-1 was present at the apical membrane and in the subapical cytosolic region. Expression of DMT-1 in the apical membrane was higher toward the DCT. In early and mid-DCT, DMT-1 was strongly expressed in the apical membrane, and expression decreased at the transition into the connecting segment. This suggests that DMT-1 may play a role in apical membrane transport of iron into TAL and DCT cells.

This distribution agrees with our previous findings, where we used renal microperfusion studies, in which we found that iron was transported out of the nephron segments between the late proximal tubule (late S2) and early to mid-DCT (26). These segments, from a microperfusion standpoint, equate with the loop of Henle. We showed that iron transport in this group of segments had characteristics of DMT-1; i.e., transport of iron was inhibited by copper and, to a lesser extent, manganese. Because we do not think that DMT-1 is responsible for movement of iron into the S2 and S3 segments and no DMT-1 expression could be detected in the thin limbs of the loop of Henle, we suggest that DMT-1 in the TAL and DCT is responsible for the transport of iron out of these nephron segments.

A caveat of this conclusion is that the prevailing pH of the TAL and DCT is not compatible with optimal DMT-1 function. Iron transport by DMT-1 is coupled to the movement of protons and is optimal at acid pH in the region of 5.5 (14). In the lumen of the TAL, the pH is 7.4 and in the late DCT, 6.6 (27). At these pH values DMT-1 would be expected to transport iron suboptimally, and therefore, from a teleological viewpoint, the observed DMT-1 distribution does not seem appropriate. However, measurement of the luminal pH does not necessarily reflect the pH of the microenvironment at the surface of the apical membrane. This can be modified by the presence of transporters such as H+-ATPase. In this study we have colocalized DMT-1 and H+-ATPase in DCT cells. In addition, Brown et al. (3) have also reported that DCT expresses H+-ATPase and that this segment has high carbonic anhydrase activity and can secrete protons. This suggests that in this segment the H+-ATPase might provide the proton-rich milieu necessary for optimal DMT-1 function.

In the collecting ducts, expression of DMT-1 was highest in cortical and outer medullary segments, where it colocalized with the H+-ATPase in A-intercalated and some, but not all, of the B-intercalated cells. Because DMT-1 transports iron with the symport of a single proton in a pH-dependent manner, the colocalization with H+-ATPase in intercalated cells correlates well with the functional requirements of a proton-coupled iron transporter. In addition, cytosolic DMT-1 immunoreactivity could be seen in principal and intercalated cells. The presence of DMT-1 in these compartments might be related to cellular metabolism and is similar to DMT-1 expression in the proximal tubule.

The overall distribution of DMT-1 protein expression we have observed in rat kidney matches the distribution of DMT-1 mRNA reported by Gunshin et al. (14) using in situ hybridization. They reported DMT-1 mRNA levels to be highest in the cortex and outer medulla, and this correlates well with our findings of DMT-1 protein expression. However, we were surprised to find only low amounts of DMT-1 protein in the inner medullary collecting duct because previous studies had reported that comparable levels of mRNA were present in the inner medulla and cortex (14), an observation that is clearly not reflected at the protein level. Therefore, we compared the amount of DMT-1 mRNA between the kidney cortex and inner medulla in our animals and showed that overall levels were equal between the regions, although there were differences in the distribution of DMT-1 transcripts. The inner medulla contained less of the -IRE transcript than did the cortex and an additional DMT-1 homolog that was recognized by a probe to the +IRE transcript but not to the -IRE transcript. The discrepancy between mRNA and protein levels could be due to translational regulation of DMT-1, and therefore mRNA levels would not necessarily be expected to reflect levels of protein expression. Alternatively, the novel transcript we observed in the inner medulla may represent a DMT-1 isoform that differs, at the NH2 terminus, from the isoforms to which we generated our antiserum. Therefore, although this transcript would be detected by in situ hybridization employing DMT-1 nucleotide probes, our antiserum would not detect it. The molecular characteristics of this transcript need to be determined.

We did not detect DMT-1 expression in the basolateral membrane of any nephron segment. This suggests that DMT-1 does not mediate basolateral membrane translocation of iron into or out of cells lining the nephron. In the gut, two proteins, ferroportin 1 (IREG-1/MTP-1) (1, 6, 17) and hephaestin (24), are required for iron movement out of duodenum and into the circulation via the basolateral membrane. Ferroportin 1 is an iron transporter proposed to account for basolateral iron exit from mammalian duodenal enterocytes. Hephaestin is a multicopper ferroxidase that has been predicted to catalyze the conversion of extruded Fe2+ to Fe3+ for binding to plasma transferrin (2). Both ferroportin 1- and hephaestin-related transcripts are expressed in the kidney and may provide a means of basolateral iron exit.

Overall, our results indicate that the localization of DMT-1 is consistent with translocation of iron across the apical membrane in TAL, DCT, and some cells in the cortical and outer medullary collecting ducts. In our experimental conditions (i.e., normal levels of dietary iron), considerable DMT-1 immunostaining is expressed in the cytosol of proximal and distal nephron segments, where it may play a role in transferrin receptor-mediated endocytosis. In addition, intracellular DMT-1 could also facilitate transport of iron into organelles functioning as reservoirs for excess iron and/or its distribution to other intracellular compartments through the secretory pathway. Finally, DMT-1 is not expressed in the basolateral membranes of the nephron, indicating that this protein is not involved in basolateral iron movement.


    ACKNOWLEDGEMENTS

We thank Drs. S. C. Hebert, S. Nielsen, and E. Roussa for the kind gifts of NCCT, AQP2, and H+-ATPase antibodies, respectively.


    FOOTNOTES

This work was supported by the Wellcome Trust and Royal Society (C. P. Smith is a Royal Society University Fellow).

Address for reprint requests and other correspondence: D. Riccardi, G38 Stopford Bldg., Oxford Rd., Manchester M13 9PT, UK (E-mail: riccardi{at}fs1.scg.man.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 August 2000; accepted in final form 17 January 2001.


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DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 280(5):F803-F814
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