1 Institut National de la Santé et de la Recherche Médicale, Unité 478, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris Cedex 18, France; and 2 Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, Martinistrasse 85, D-20246 Hamburg, Germany
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ABSTRACT |
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ClC-5 is the
Cl channel that is mutated in Dent's disease, an
X-chromosome-linked disease characterized by low molecular weight
proteinuria, hypercalciuria, and kidney stones. It is predominantly expressed in endocytically active renal proximal cells. We investigated whether this Cl
channel could also be expressed in
intestinal tissues that have endocytotic machinery. ClC-5
mRNA was detected in the rat duodenum, jejunum, ileum, and colon.
Western blot analyses revealed the presence of the 83-kDa ClC-5 protein
in these tissues. Indirect immunofluorescence studies showed that ClC-5
was mainly concentrated in the cytoplasm above the nuclei of
enterocytes and colon cells. ClC-5 partially colocalized with the
transcytosed polymeric immunoglobulin receptor but was not detectable
together with the brush-border-anchored sucrase isomaltase. A
subfractionation of vesicles obtained by differential centrifugation
showed that ClC-5 is associated with the vacuolar 70-kDa
H+-ATPase and the small GTPases rab4 and rab5a, two markers
of early endosomes. Thus these results indicate that ClC-5 is present
in the small intestine and colon of rats and suggest that it plays a
role in the endocytotic pathways of intestinal cells.
intestine; endocytosis; H+-ATPase; rab
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INTRODUCTION |
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THE
ClC-5 Cl
channel, encoded by the CLCN5 gene, belongs to the family of
voltage-gated Cl
channels. This gene family is composed
so far of nine members in mammals (see Ref. 19 for
review). The first ClC-0 channel was identified by Jentsch et al.
(20) by expression cloning from the marine ray
Torpedo marmorata. ClC-1 is the major Cl
channel in skeletal muscle, which is mutated in both the dominant and
recessive forms of myotonia (38). ClC-2 is present in many tissues and is thought to play a role in cell volume regulation (14). The rat rClC-K1 and rClC-K2 (hClC-Ka and hClC-Kb in
humans) channels are specific to the kidney (23, 41).
Mutations of the hCLCN-KB gene lead to Bartter's
syndrome type III (35), whereas mice in which the
ClC-K1 gene has been knocked out develop nephrogenic
diabetes insipidus (26). The ClC-3 and ClC-4 channels are
broadly expressed and are very similar (almost 80% identical) to ClC-5
(39). In contrast, the ubiquitous putative ClC-6 and ClC-7
Cl
channels are only ~30% identical to the other CLC
channels (19).
Mutations in the human CLCN5 gene have been found to occur
in Dent's disease, an X-linked hereditary hypercalciuric
nephrolithiasis, causing low-molecular-weight proteinuria,
hypercalciuria, nephrocalcinosis, nephrolithiasis, progressive renal
insufficiency, and in some cases rickets (see Ref. 33 for
review). This discovery has stimulated research to obtain a better
understanding of the function of ClC-5 in the kidney. Cloning and
functional expression studies in Xenopus oocytes have
demonstrated that the rat ClC-5 channel elicits Cl
currents (39). Günther et al. (15)
demonstrated that the 83-kDa rat ClC-5 Cl
channel is
predominantly located in endocytotic vesicles underlying the apical
membrane domain of kidney proximal and intercalated cells of the
collecting duct. Others have found a similar distribution of ClC-5 in
proximal tubule cells and intercalated cells as well as in thick
ascending limb cells in the kidneys of rats, mice, and humans (9,
24, 32).
The colocalization of rClC-5 with vacuolar
H+-ATPases and endocytosed
2-microglobulin (15) strongly
suggests that the ClC-5 Cl
channel is essential for
proximal tubule endocytosis by providing an electrical shunt for the
acidification of endocytotic vesicles. These results also provide a
molecular basis for the proteinuria observed in Dent's disease. More
recently, Luyckx et al. (25) have reported that transgenic
mice with reduced ClC-5 expression due to the introduction
of a ClC-5-inactivating antisense ribozyme transgene were
slightly hypercalciuric when fed a normal Ca2+ diet. These
authors also speculated that ClC-5 is expressed in the
intestine and suggested that the hypercalciuria that occurs in ribozyme
transgenic mice could be due to the hyperabsorption of Ca2+
in the intestine. These results raised the question about the cellular
and subcellular distribution and function of ClC-5 in the intestine.
The differentiated epithelial cells lining the crypt-villus axis of the
small intestine and colon have many morphological and functional
features that are similar to the renal proximal tubule cells. They
possess a well-developed apical brush border rich in membrane
transporters and membrane-anchored hydrolases (22). A
large number of in vitro studies have also demonstrated that epithelial
polarized intestinal and kidney tubule cells have similar endocytotic
systems for the uptake of peptides and the turnover of proteins
residing in the apical membrane (4, 16, 17).
The present study was designed to analyze the expression of the ClC-5
gene and the cellular and subcellular distribution of this
Cl channel in rat intestinal and colon epithelial cells.
RT-PCR and immunohistochemical studies showed ClC-5 mRNA and
protein in the small intestine and colon cells. This Cl
channel was located in a vesicle-rich region beneath the apical brush
border of enterocytes. The coexpression of ClC-5 with the 70-kDa
subunit of the vacuolar-type H+-ATPase
(29) and small GTPase rabs and with internalized polymeric immunoglobulin receptors (pIgRs) responsible for the transepithelial transport of dimeric immunoglobulin A (dIgA; see Refs. 2
and 36) also suggests that this Cl
channel plays a role
in the endocytotic pathway(s) of epithelial intestinal cells.
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MATERIALS AND METHODS |
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Rat tissues.
Experiments were performed on gastrointestinal and kidney tissues from
adult male rats fed a standard diet with free access to tap water. All
tissue samples were rapidly frozen in liquid nitrogen and kept at
80°C until used.
RNA extraction and RT-PCR.
Total RNAs were extracted from rat duodenum, jejunum, ileum, proximal
and distal colon, kidney, and skeletal muscle using the RNA-PLUS
extraction kit (Quantum Biotechnologies, Illkirch, France). RNA (2 µg) was reverse-transcribed with Moloney murine leukemia virus RT at
42°C for 45 min. cDNA (100 ng) and non-reverse-transcribed RNA were
amplified for 28 cycles in 100 µl total volume of PCR buffer (50 mM
KCl and 20 mM Tris · HCl, pH 8.4) containing 40 µM dNTP, 2 mM
MgCl2, 1 µCi [-32P]dCTP, 1 unit
Taq polymerase, 36 pmol of rat ClC-5 primers, and 11.5 pmol of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH; internal standard) primers. The rat ClC-5 primers used were those described by Steinmeyer et al. (39). The hGAPDH primers
were those described by Hummler et al. (18). The thermal
cycling program was 94°C for 30 s, 55°C for 30 s, and
72°C for 60 s. Amplified products were run on a 4%
polyacrylamide gel and autoradiographed.
Protein extraction and immunoblot analysis. ClC-5 was detected by Western blotting using a rabbit polyclonal anti-ClC-5 antiserum, PEP5A2, previously characterized by Günther et al. (15). Briefly, PEP5A2 was raised in rabbits against a synthetic peptide (KSRDRDRHREITNKS) representing a part of the amino terminus of ClC-5. This peptide was coupled to BSA by 3-maleimidobenzoic acid N-hydroxysuccinimide ester via a cysteine added to the amino terminus of the peptide and injected several times (in intervals of 3 wk) in rabbits. The serum obtained from the final bleed was purified by affinity chromatography against the peptide. The specificity was checked in Western blots using membranes from Xenopus oocytes previously injected with ClC-3, ClC-4, or ClC-5 and additionally in indirect immunofluorescence experiments using COS-7 cells transiently transfected with either one of these related channel cDNAs (15). The abdominal cavity of the killed rats was opened. Kidneys and lungs were removed and pulverized in liquid nitrogen. The intestine and colon were cannulated and rinsed with ice-cold PBS. Five- to seven-cm-long pieces of duodenum, jejunum, ileum, and colon were removed and placed in ice-cold PBS. The lumen was opened with a scalpel, and cells were gently scraped off, collected, centrifuged (150 g for 5 min at 4°C), and kept in liquid nitrogen until used. Tissue samples and pelleted cells were homogenized in a hypertonic sucrose solution (0.25 M sucrose, 3 mM imidazole, and 1 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 100 µg/ml protease inhibitor cocktail (Boehringer Mannheim) in a glass Dounce homogenizer (10 strokes at 4°C). All samples were kept at 4°C for 30-45 min and centrifuged at 150 g for 5 min at 4°C to remove nuclei and any remaining intact cells. The supernatant was then centrifuged (105,000 g for 1 h at 4°C). Pelleted enriched membrane preparations were then suspended in 250 µl sucrose buffer and were used for Western blotting.
Western blotting was also performed on subcellular fractions from rat jejunum prepared by differential centrifugation following the procedures described by Devuyst et al. (9) for the isolation of subcellular fractions from kidney. The jejunum was perfused with ice-cold PBS, and scraped-off cells were homogenized in a glass Dounce homogenizer (40 strokes at 4°C) in ice-cold homogenization buffer (300 mM sucrose and 25 mM HEPES, adjusted to pH 7.0 with 1 M Tris) containing 1 mM PMSF and 100 µg/ml protease inhibitor cocktail. All subsequent steps were performed at 4°C. The cell homogenate was centrifuged at 500 g for 20 min to remove debris. The resulting supernatant (S1) was centrifuged at 80,000 g for 30 min. The pellet, corresponding to the enriched membrane fraction, was suspended in the ice-cold homogenization buffer and centrifuged at 19,000 g for 20 min. Two steps of centrifugation were then performed on the pellet (P1) and supernatant (S2). The P1 pellet was suspended in homogenization buffer, layered on top of a 1.12 M sucrose solution, and centrifuged again at 100,000 g for 60 min. The interface layer was centrifuged at 40,000 g for 20 min, and the resulting pelleted fraction (fraction I) containing plasma membranes was kept. The S2 supernatant was centrifuged at 42,000 g for 20 min. The resulting low-speed pellet (fraction II) was kept atImmunofluorescence studies.
Rats were anesthetized by an intraperitoneal injection of nembutal.
Fragments (1 cm long) of duodenum, jejunum, ileum, and a midportion of
the colon were rinsed in PBS, snap-frozen in liquid nitrogen, and
stored at 80°C until used. Kidneys were removed rapidly, cut along
the longitudinal axis of the medullary rays, and processed as described
above. Frozen tissues were sectioned (5-7 µm thick) with a
cryostat (Bright). Tissue sections were mounted on superfrost glass
slides and fixed in ice-cold methanol for 8 min. Samples were incubated
with the anti-PEP5A2 ClC-5 antibody for 2 h at room temperature,
rinsed three times with PBS, and incubated with goat anti-rabbit IgG
coupled to Cy3 reactive dye (Cy3; Jackson Immunoresearch) for 1 h
at room temperature. The sections were rinsed in PBS and mounted. The
specificity of the labeling was checked by indirect immunofluorescence
on jejunum sections by diluting the primary PEP5A2 antibody with an
excess (1.15-11.5 µg/ml) of the synthetic peptide used to
produce PEP5A2. Negative controls were also performed by omitting the
primary antibody. Tissue sections were also double labeled using PEP5A2 and a polyclonal antibody against sucrase isomaltase (30)
or a polyclonal antibody raised against the ectodomain of pIgR
(3). Samples were incubated with PEP5A2 as above, rinsed
with PBS, and incubated for 30 min or 1 h at room temperature with
the anti-sucrase isomaltase antibody or the anti-pIgR antibody. Binding
was detected with anti-species FITC- and Cy3-conjugated IgG antibodies.
All specimens were examined under a Zeiss photomicroscope equipped with
epifluorescence optics (Zeiss, Oberkochen, Germany) and photographed. Double-labeled specimens were also examined by confocal laser scanning
microscopy (CLSM; Leica, Wetzlar, Germany). Tissue sections were viewed
in the x-y plane, and the images were photographed.
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RESULTS |
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ClC-5 mRNA and protein in tissues.
RT-PCR experiments using a set of primers specific for the rat
rClC-5 (39) were performed to study the
expression of the ClC-5 gene in the intestine and colon.
Similar amounts of ClC-5 transcripts were detected in the
rat duodenum, jejunum, ileum, proximal and distal colon, and kidney
compared with the amount of hGAPDH transcripts, used as
internal standard (Fig. 1). No ClC-5 mRNA was detected in skeletal muscle or in
non-reverse-transcribed colon and kidney RNAs or by omitting cDNA (Fig.
1).
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Cellular distribution of ClC-5 in the intestine and colon.
Indirect immunofluorescence was used to determine the cellular
distribution of ClC-5 in frozen tissue sections of the rat intestine
and colon. All sections from the duodenum, jejunum, ileum, and colon
were stained with PEP5A2 (Fig. 3,
A-D). The staining appeared to be mainly in the cytoplasm
of epithelial intestinal cells, above nuclei, and to a lesser extent in
the cytoplasm near the basal side of the cells (Fig. 3,
A-C). The cytoplasm was stained more diffusely in colon
cells (Fig. 3D). There appeared to be no clear differences
in staining intensity along the crypt-villus axis of the intestine and
colon walls (Fig. 3, A and D). The pattern of
PEP5A2 immunostaining in frozen kidney sections of the same rats was
identical to that previously reported by Günther et al.
(15) using the same PEP5A2 antiserum. The region of the cytoplasm underlying the apical brush border from proximal tubule sections and some cells from the collecting duct, shown to correspond to intercalated cells (15), were positively stained (Fig.
3E). Like the control, no staining was observed when the
primary antibody was omitted from kidney sections (data not shown) or
colon sections (Fig. 3F). To further assess the specificity
of the labeling provided by PEP5A2, indirect immunofluorescence was
performed on jejunum sections using PEP5A2 without or with increasing
amounts of the synthetic peptide against which it was raised (Fig.
4). The PEP5A2 labeling was specific, as
it could be blocked by preincubation with increasing amounts of
synthetic peptide (Fig. 4, A-D for comparison).
Taken together, the immunostaining provided by PEP5A2 was specific and
revealed the presence of ClC-5 in digestive tissues.
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Subcellular distribution of ClC-5 in small intestine and colon
cells.
CLSM analyses of frozen sections of jejunum and colon stained with
PEP5A2 showed a striking distribution of ClC-5 within the small
intestine and colon cells (Fig. 5). The
staining of enterocyte cytoplasm gradually increased from the region
underlying the apical membrane to reach a maximum intensity in the
cytoplasm just above the nucleus (Fig. 5A). The staining of
the cytoplasm of colon cells was more diffuse (Fig. 5C).
CLSM analyses of immunostained sections from jejunum and colon with
PEP5A2 revealed a fine punctate staining in the cytoplasm,
predominantly concentrated above the nuclei (Fig. 5, B and
D), and a weak punctate cytoplasmic staining in the basal
compartment of the cytoplasm of enterocytes and colon cells (Fig. 5,
B and D).
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Subcellular distributions of ClC-5 and vacuolar
H+-ATPase, rab4, and rab5a.
Previous studies have shown that ClC-5 is colocalized with
vacuolar H+-ATPase and rab5a in renal proximal tubule cells
(9, 15, 24, 32). We therefore examined the subcellular
distributions of ClC-5 and several organelle-specific markers in a
jejunum cell preparation using differential centrifugation
(9). This was done because indirect immunofluorescence
studies using the available antibodies directed against the vacuolar
proton ATPase and small GTPase rabs did not provide satisfactory
results. Western blotting was performed on the plasma membrane-enriched
fraction (fraction I), low-speed (fraction II)
and high-speed (fraction III) pelleted fractions using an
anti-E-cadherin antibody, PEP5A2, an anti-vacuolar H+-ATPase antibody, and three different anti-rab
antibodies (Fig. 7). E-cadherin, an
adhesion molecule expressed in basolateral membranes of epithelial
cells, was mainly found in fraction I and to a much lesser
extent in fraction II and was almost not detectable in
fraction III (Fig. 7). ClC-5 was mainly found in pelleted fractions II and III and was almost
undetectable in the E-cadherin-enriched plasma membrane fraction
I. The 70-kDa subunit of the vacuolar H+-ATPase was
also almost undetectable in fraction I but had almost the same pattern of subcellular distribution as ClC-5 in
pelleted fractions II and III (Fig. 7). Rab4,
which is located in the early endosome and plasma membrane recycling
pathway (8, 42), was almost equally distributed in
fractions I, II, and III. Rab5a, which
is found in the plasma membrane, clathrin-coated vesicles, and early
endosomes (6, 7), was also detected in all three fractions, mainly in fraction II (Fig. 7). Rab6, a
ubiquitous small GTPase associated with the membranes of the Golgi
complex (1, 11), was mainly detected in plasma
membrane-enriched fraction I and to a lesser extent in
fraction II, with almost none in fraction III
(Fig. 7). Although the separation of membrane fractions was not
optimal, especially for the plasma membrane fraction I
contaminated with Golgi membranes, these results clearly indicate that
the ClC-5 Cl channel is mainly in the subcellular
fractions that are highly enriched in rab4, rab5a, and
H+-ATPase. Both the punctate cytoplasmic immunostaining
obtained with the PEP5A2 antibody (see Fig. 6C) and the
detection of the ClC-5 Cl
channel in subcellular
fractions highly enriched in rab4 and rab5a involved in regulating the
membrane traffic of the endocytotic pathway strongly suggest that ClC-5
is concentrated in vesicular organelles corresponding to early and/or
recycling endosomes.
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ClC-5 partially colocalizes with transcytosed pIgRs.
The striking colocalization of ClC-5 with H+-ATPase and
endocytosed 2-microglobulin in renal proximal tubule
cells strongly suggested that this Cl
channel plays an
important role in the dissipation of the electrical gradient generated
by the vacuolar H+-ATPase in endosomes (15,
32). The similar distribution of ClC-5 in intestinal cells
prompted us to test whether it was associated with endocytosed or
transcytosed proteins via endosomal vesicles. Previous studies have
demonstrated that pIgR, which can bind its ligand dIgA, represents a
useful model system for studying transcytosis (2, 36).
With or without dIgA, the pIgR is internalized from the basolateral
plasma membrane and delivered to early endosomes (2). The
pIgR then moves through several endosome compartments before reaching
the apical plasma membrane. At this site, the extracellular binding
domain of the pIgR is cleaved (2). This cleaved fragment,
also called the secretory component (SC), is normally released with
dIgA. We have previously shown the presence of pIgRs mainly in the
cytoplasm from mouse intestinal crypt cells by using anti-pIgR
antibodies (3). Cryosections of rat jejunum processed for
indirect immunofluorescence using an antibody raised against the
ectodomain of pIgR showed a similar distribution of pIgRs (Fig.
8A). Double indirect
immunofluorescence with PEP5A2 and the anti-pIgR antibody analyzed by
CLSM clearly showed that the endocytosed pIgRs detected in the region
above the nuclei were colocalized with ClC-5 (Fig. 8,
B-D). In contrast, the immunostaining provided
by the anti-pIgR antibody, which was detected in the apical region
beneath the intestinal brush border, presumably corresponding to
cleaved SC released from vesicles, was not associated with the ClC-5
Cl
channel (Fig. 8E). Thus these results
provided more direct evidence that ClC-5 is present in endosomes from
the endocytotic and transcytotic pathways of intestinal cells.
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DISCUSSION |
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The ClC-5 Cl channel is predominantly expressed in
kidney epithelial cells and is probably involved in the endocytosis of low-molecular-weight proteins (9, 15). Previous mRNA and Western blot analyses also suggest that this Cl
channel
is present in other tissues, including the brain, testis, and liver
(15, 39). We have now shown that the ClC-5
Cl
channel is present in the epithelial cells lining the
villi of the rat small intestine and colon. ClC-5 mRNA was
detected by RT-PCR in all of the segments of the anteroposterior axis
of the small intestine and the proximal and distal parts of the colon examined, and Western blot analyses revealed ClC-5 in these tissues. Few Cl
channels have been identified in intestinal and
colon cells. The cystic fibrosis transmembrane conductance regulator
(CFTR) gene, belonging to the superfamily of
ATP-binding cassette transporters, is expressed in crypt cells
(40) and plays an important role in the cAMP-dependent
regulated secretion of Cl
(34). The ClC-2
Cl
channel is also present in the mouse duodenal
epithelium (21). The exact function of ClC-2 is still not
clear. Its mRNA expression declines during late gestation
(27) and is unaltered in mice with cystic fibrosis
(21). These results indirectly suggest that ClC-2 has some
housekeeping function. ClC-3 is present in colonic myocytes
(10), but it has not been shown to occur in colon and/or
small intestinal epithelial cells. Recently, a potential human
Ca2+-sensitive Cl
channel, and its murine
counterpart, has been shown to be expressed in basal crypt epithelia
and goblet cells (12, 13). Thus, to our knowledge, ClC-5
is the fourth potential Cl
channel to be identified in
intestinal epithelial cells. As in renal proximal tubule and
intercalated collecting duct cells, the ClC-5 Cl
channel
in intestinal and colon epithelial cells is very predominantly, if not
exclusively, intracellular.
ClC-5 has been shown to colocalize with the proton pump in renal
proximal tubule and -intercalated cells (9, 15, 32). The results from fractionation studies on jejunum cell preparations also indicate that CIC-5 and vacuolar H+-ATPase are
detected in both low- and high-speed pelleted subcellular fractions
containing rab4 and rab5a, two ras-like GTP-binding proteins associated
with early endosomes (7, 42). Consistent with the fact
that the vacuolar-type H+-ATPase is absent from the
brush-border membrane vesicles of enterocytes (31),
neither the 70-kDa H+-ATPase subunit nor ClC-5 was clearly
detectable in the plasma membrane fraction of jejunum cells. Thus the
distribution of ClC-5 in intestinal cells closely resembles that of
ClC-5 in renal epithelial tubule cells. The results from subcellular
fractionated studies and immunohistochemical studies also strongly
suggest that ClC-5 is predominantly, if not exclusively, located in
densely packed endocytic vesicles. The intravesicular acidification of
these vesicles mediated by vacuolar-type H+-ATPases is
essential for various sorting processes (28). The parallel
distributions of ClC-5, H+-ATPase, and rab GTPase proteins
suggest that ClC-5 provides the electrical shunt necessary for the
acidification of vesicles from the intestinal endocytotic pathway
(15).
The endocytotic and transcytotic capacities of epithelial cells have been demonstrated in various cell systems (2, 16, 17, 36, 37). Interestingly, we found that the internalized pIgR, known to be delivered from basolateral endosomes to the apical endosomes in epithelial polarized cells (2, 36), is colocalized with ClC-5 in the intestinal secreting cells bordering the base of the villi. Thus these results provided further evidence that ClC-5 is expressed in some, but may be not all, of the endosomal compartments involved in the endocytosis and transcytosis of proteins.
It has also been suggested that ClC-5 plays a role in Ca2+ absorption. Patients suffering from Dent's disease caused by inactivating mutations in ClC-5 have low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, and nephrolithiasis (33). The primary mechanism by which hypercalciuria occurs remains a matter of debate. Sufferers from Dent's disease often have low plasma parathyroid hormone concentrations and elevated plasma 1,25-dihydroxyvitamin D, a situation that contrasts with renal insufficiency of other origins (33). The hypercalciuria that occurs in Dent's disease could result from abnormal regulation of vitamin D in the kidney or from the hyperabsorption of Ca2+ by the gut. Luyckx et al. (25) have recently shown that transgenic mice with reduced ClC-5 expression are hypercalciuric when fed a normal Ca2+ diet but do not exhibit other altered renal functions. These authors also mention that ClC-5 is expressed throughout the mouse intestinal epithelium and hypothesize that ClC-5 regulates the absorption of Ca2+ across the gut mucosa. However, we did not detect ClC-5 at the apical brush-border membrane of intestinal cells. Thus it seems unlikely that ClC-5 is directly involved in the apical intestinal absorption of Ca2+. Furthermore, the primary cause of abnormal Ca2+ transport and regulation in Dent's disease remains to be determined.
In summary, we have demonstrated the presence of the ClC-5
Cl channel in the small intestine and colon cells of the
rat. Our data also suggest that this CLC channel, which is exclusively intracellular, is involved in some of the endocytotic processes occurring in intestinal cells.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. K. Stone, R. Riby, B. Courtesy, and M. Kedinger for generously providing us with valuable antibodies. We thank E. Pringault for stimulating discussions. We also thank S. Roger and P. Disdier for photographic work and Dr. O. Parkes for editing assistance.
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FOOTNOTES |
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This study was supported by Institut National de la Santé et de la Recherche Médicale (INSERM). Dr. K.-C. Peng holds an INSERM postdoctoral (Poste vert) fellowship supported by the Conseil Régional de l'Ile de France.
Address for reprint requests and other correspondence: A Vandewalle, INSERM U478, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris Cedex 18, France (E-mail: vandewal{at}bichat.inserm.fr)
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 9 May 2000; accepted in final form 6 September 2000.
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