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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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.
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RESULTS |
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.
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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.
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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.
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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.
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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.
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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 |
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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