Localization of rat CLC-K2 chloride channel mRNA in the kidney
Momono
Yoshikawa1,
Shinichi
Uchida1,
Atsushi
Yamauchi2,
Akiko
Miyai2,
Yujiro
Tanaka1,
Sei
Sasaki1, and
Fumiaki
Marumo1
1 Second Department of Internal
Medicine, Tokyo Medical and Dental University, Tokyo 113-8519; and
2 First Department of Medicine,
Osaka University, School of Medicine, Osaka 565-0871, Japan
 |
ABSTRACT |
To gain insight into the physiological role of a
kidney-specific chloride channel, CLC-K2, the exact
intrarenal localization was determined by in situ hybridization. In
contrast to the inner medullary localization of CLC-K1, the signal of
CLC-K2 in our in situ hybridization study was highly evident in the
superficial cortex, moderate in the outer medulla, and absent in the
inner medulla. To identify the nephron segments where CLC-K2 mRNA was expressed, we performed in situ hybridization of CLC-K2 and
immunohistochemistry of marker proteins
(Na+/Ca2+
exchanger,
Na+-Cl
cotransporter, aquaporin-2 water channel, and Tamm-Horsfall
glycoprotein) in sequential sections of a rat kidney. Among the tubules
of the superficial cortex, CLC-K2 mRNA was highly expressed in the
distal convoluted tubules, connecting tubules, and cortical collecting ducts. The expression of CLC-K2 in the outer and inner medullary collecting ducts was almost absent. In contrast, a moderate signal of
CLC-K2 mRNA was observed in the medullary thick ascending limb of
Henle's loop, but the signal in the cortical thick ascending limb of
Henle's loop was low. These results clearly demonstrated that CLC-K2
was not colocalized with CLC-K1 and that its localization along the
nephron segments was relatively broad compared with that of CLC-K1.
in situ hybridization; sodium/calcium exchanger; aquaporin-2 water
channel; sodium-chloride cotransporter; Tamm-Horsfall glycoprotein
 |
INTRODUCTION |
CLC-K2, A KIDNEY-SPECIFIC member of the CLC
chloride channel family (11), possesses ~80% amino acid identity
with rat CLC-K1 (1, 12). RT-PCR using dissected nephron segments
suggested that the localization of CLC-K2 was completely different from that of CLC-K1 (1), but the exact intrarenal localization of CLC-K2 has
yet to be established. Previously, we determined the intrarenal and
cellular localization of CLC-K1 in rat kidney by immunohistochemistry
(10). CLC-K1 is exclusively localized to both the apical and
basolateral plasma membranes of the ascending thin limb of Henle's
loop (ATL). Moreover, since the functional characteristics
of CLC-K1 expressed in Xenopus oocyte
match those of chloride transport in ATL observed in in vitro perfusion
experiments (10), CLC-K1 can be considered a major chloride channel
mediating the transepithelial chloride transport in ATL. Considering
its structural similarity to CLC-K1, it has been speculated that CLC-K2 shares a similar role as a route for transepithelial
chloride transport in the thick ascending limb of Henle's loop (TAL)
and collecting ducts (CD) (1). The presence of chloride channels in the
basolateral surface of these nephron segments has been postulated by
several physiological and biochemical studies (5-7, 14-16,
18, 19). Recently, Zimniak et al. (20) demonstrated that treatment of
TAL cells with the antisense oligonucleotide for rabbit homolog of
CLC-K2 decreased the reconstituted chloride channels from the cells,
thus suggesting that CLC-K2 might provide the basolateral chloride
conductance in TAL. Simon et al. (8) also recently reported that
mutations in CLCNKB, the gene encoding the human homolog of rat CLC-K2
(9), led to Bartter's syndrome. This evidence strongly suggested that
CLC-K2 had an important role in the transepithelial reabsorption of
chloride ions. To understand how CLC-K2 is involved in the pathogenesis
of Bartter's syndrome, the exact intrarenal localization of CLC-K2
must be determined. However, since CLC-K1 and CLC-K2 are highly
homologous proteins, the generation of a specific antibody for each
channel has been very difficult. In a report on the
immunohistochemistry of rat CLC-K channels by Vandewalle et al. (13),
the antiserum used recognized both CLC-K1 and -K2 and was not specific
for CLC-K2. They showed the relatively wide distribution of
immunoreactivity in the basolateral surfaces of the ATL and other
distally located nephron segments. Recently, Winters et al. (17)
reported the immunolocalization of rbClC-Ka, a gene that
may be the rabbit homolog of rat CLC-K2. Their antiserum recognized the
basolateral surface of the TAL and cytoplasm of intercalated cells in
the cortical CD (CCD) (17). The antiserum in their study was raised against a 156-amino acid COOH-terminal fragment of rbClC-Ka protein, but they did not mention the specificity of the antiserum to rbClC-Ka against a rabbit homolog of rat CLC-K1 protein. Thus there
were still wide discrepancies concerning the exact intrarenal
localization of CLC-K2 between them. To overcome this situation, a
highly specific probe for each channel must be prepared. Since the
3'-untranslated regions of these two channels are virtually not
homologous and we were able to prepare a specific cRNA probe for each
channel, we adopted in situ hybridization to precisely determine the
sites of CLC-K2 expression in this study.
Our in situ hybridization study clearly demonstrated that CLC-K1 and
CLC-K2 are localized in different areas of the kidney, and that the
latter is most abundantly expressed in distal convoluted tubules (DCT),
connecting tubules (CNT), and CCD. Moderate expression was also
observed in the medullary TAL (MTAL).
 |
EXPERIMENTAL PROCEDURES |
Animals. Male Wistar rats weighing
~150 g received water and standard rat chow for several days before
the experiments. The rats were deeply anesthetized by an
intraperitoneal injection of pentobarbital (50 µg/g) and
transcardially perfused with a solution of 4% paraformaldehyde for in
situ hybridization and immunohistochemistry.
In situ hybridization. After the
perfusion with paraformaldehyde, the kidneys were placed in a solution
of 4% paraformaldehyde for 2 h and 15% sucrose overnight. Cryostat
sections (5 µm) were mounted on siliconized slides. Initially, the
slides were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for
20 min, washed with 0.1 M phosphate buffer, and treated with a solution
of 10 µg/ml proteinase K in 50 mM Tris · HCl, pH
7.5, and 5 mM EDTA, pH 8.0, for 1 min at room temperature. Next, they
were postfixed in 4% paraformaldehyde, treated with 0.25% acetic
anhydride for 10 min, rinsed in phosphate buffer (28 mM
NaH2PO4,
72 mM
Na2HPO4), and dehydrated in increasing concentrations of ethanol.
In in situ hybridization using radiolabeled probes, tissue sections
were hybridized for 24 h at 55°C in a buffer [50% deionized formamide, 10% dextran sulfate, 0.3 M NaCl, 1× Denhardt's
solution, 20 mM Tris · HCl (pH 8.0), 5 mM EDTA (pH
8.0), 0.2% sarcosyl, 200 µg/ml salmon sperm DNA, and 500 µg/ml
yeast tRNA] containing one of the
35S-labeled RNA probes. The probe
concentration was 1 × 106
cpm/200 µl per slide. After hybridization, the sections were immersed
in 5× SSC at 55°C and rinsed in 50% deionized formamide with
2× SSC at 65°C for 30 min. The sections were then rinsed with
RNase buffer [0.5 M NaCl, 10 mM Tris · HCl (pH
8.0), 5 mM EDTA (pH 8.0)] for 10 min three times each at
37°C, incubated with 1 µg/ml RNase A in RNase buffer for 10 min
at 37°C, washed in 50% formamide with 2× SSC at 50°C for
30 min, rinsed with 2× SSC and 0.1× SSC for 10 min each at
room temperature, dehydrated in alcohol, and air dried. After the
slides were initially exposed to X-ray film for 3 days to provide an
indication of the intensity of the hybridization signal, they were
coated with Kodak NTB-2 emulsion diluted 1:1 with water. The sections
were exposed at 4°C for 2-3 wk in tightly sealed dark boxes,
developed in Kodak D-19, fixed with photographic fixer, washed with
water, and then counterstained with hematoxylin and eosin to allow
morphological identification.
In in situ hybridization using digoxigenin-labeled riboprobes, slides
were hybridized in a buffer [50% deionized formamide, 0.24%
SDS, 0.3 M NaCl, 1 mM EDTA (pH 8.0), 10 mM Tris · HCl
(pH 8.0), 0.7 mg/ml salmon sperm DNA, and 1 mg/ml yeast tRNA, 1×
Denhardt's solution, and 10% dextran sulfate] at 42°C in a
moist chamber for 16 h. Concentration of a digoxigenin-labeled sense or
antisense probe was 8 ng/µl. After hybridization, the sections were
immersed in 5× SSC at 42°C, rinsed in 50% deionized
formamide with 2× SSC at 42°C for 30 min, and incubated with
1 µg/ml RNase A in RNase buffer [0.5 M NaCl, 10 mM
Tris · HCl (pH 8.0)] for 30 min at 37°C. Washing procedures included a first washing step in RNase buffer for 10 min at 37°C, followed by three washes in 2× SSC, once at 42°C for 15 min and twice at room temperature for 10 min each. The
slides were then equilibrated for 5 min in buffer
1 (100 mM maleate, 150 mM NaCl, pH 7.5) for
immunohistochemical detection of digoxigenin-labeled probes, immersed
in buffer 2 (buffer
1 containing 1% blocking reagent; Boehringer,
Mannheim, Germany), and incubated for 30 min at room temperature.
Blocking solution was drained from the slides and a polyclonal alkaline
phosphate-coupled sheep anti-digoxigenin antibody (diluted 1:500 in
buffer 1) was applied to the
sections. The sections were then incubated in a moist chamber for 2 h
at room temperature, washed twice for 2 min each in
buffer 1, and equilibrated for 2 min
in buffer 3 (100 mM
Tris · HCl, 100 mM NaCl, and 50 mM
MgCl2, pH 9.5). For signal development, the slides were immersed in a substrate solution (buffer 3 containing 0.404 mM
nitroblue tetrazolium chloride, 0.384 mM 5-bromo-4-chloro-3-indolyl
phosphate, 4-toluidine salt, and 1 mM levamisol) at 37°C. Color
reaction was observed under microscope and terminated by immersing the
slides in TE buffer [10 mM Tris · HCl (pH 8.0),
1 mM EDTA]. After staining of nuclei with methyl green, sections
were mounted in PBS-buffered glycerol.
To prepare the probes specific for rat CLC-K1 and -K2, the
3'-untranslated regions of rat CLC-K1 and -K2 were amplified by PCR using K1- and K2-specific primer sets. The primer sequences were as
follows
The underscored sequences are EcoR I and
BamH I sites introduced for
subcloning. Nucleotide identity between K1 and K2 probes was less than
30%. PCR fragments of expected sizes (K1 for 220 bp and K2 for 159 bp)
were subcloned into pSPORT1 (Life Technologies), and their sequences
were verified. To make 35S- or
digoxigenin-labeled sense or antisense cRNA probes, in vitro transcription was performed using T7 RNA polymerase and SP6 RNA polymerase after linearizing by cutting with
BamH I and
EcoR I, respectively.
Immunohistochemistry.
Immunohistochemistry of aquaporin-2 (AQP-2) water channel (2),
Tamm-Horsfall glycoprotein (Biomedical Technologies, Stoughton, MA),
thiazide-sensitive
Na+-Cl
cotransporter (TSC; the antiserum was a generous gift from Dr. S. C. Hebert in Vanderbilt University), and
Na+/Ca2+
exchanger (the antiserum kindly provided by Dr. R. F. Reilly at Yale
University) was performed in cryostat sections of rat kidney using a
TSA-Indirect kit (NEN Life Science Products, Boston, MA).
 |
RESULTS |
In situ hybridization of rat CLC-K2.
In film autoradiograms generated from sections hybridized with rat
CLC-K1 and CLC-K2 antisense probes, the most intense CLC-K1 signals
were localized in the inner medulla and the most intense CLC-K2 signals
were localized in the superficial cortex (Fig.
1). There were also significant CLC-K2
signals in the outer medulla (Fig. 1), but there was virtually no
signal of CLC-K2 expression in the inner medulla. No hybridization
signal was detected using sense probes. As shown in Fig.
2, A and
B, based on the hematoxylin-eosin
staining, microscopic examination of emulsion-coated kidney sections
identified a high grain density in the distal nephron segments in the
cortex, but not in the proximal tubules. In the outer medulla, a
moderate signal was also detected in the TAL (Fig. 2,
C and
D). In contrast to the inner
medullary localization of CLC-K1, CLC-K2 was not present in the inner
medulla. To identify the nephron segments where CLC-K2 is expressed,
the signal of CLC-K2 mRNA in the in situ hybridization study was
compared with the immunohistochemistry of several marker proteins. TSC
was used to identify DCT,
Na+/Ca2+
exchanger was used for CNT, AQP-2 water channel was used for CD, and
Tamm-Horsfall glycoprotein was used as a marker of the TAL. In the
superficial cortex, the tubules positive for TSC, Na+/Ca2+
exchanger, and AQP-2 water channel (indicated by white arrows, Fig. 3)
were all positive for CLC-K2 expression (Fig.
3,
A-F). Like 35S-labeled sense probe,
there was no signal when sense digoxigenin probe was used for
hybridization (Fig. 3I), confirming
that the signals detected by digoxigenin probe were also specific to
CLC-K2. These results demonstrated that the most intense signals of
CLC-K2 in the kidney were present in DCT, CNT, and CCD. The signal of CLC-K2 expression was faintly observed in the AQP-2-positive tubules in
the inner cortex and the outer stripe of the outer medulla (Fig. 3,
G and
H), but almost absent in the inner
stripe of the outer medulla and the inner medulla (data not shown).
This indicated that the CLC-K2 expression in the CD significantly
decreased as it entered into the medulla.

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Fig. 1.
Film autoradiograms illustrating CLC-K1 and -K2 mRNA distribution in a
rat kidney. The hybridization signal of rat CLC-K1 was detected in
inner medulla. Strongest signals of rat CLC-K2 hybridization were
detected in outer cortex and inner stripe of outer
medulla.
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Fig. 2.
In situ hybridization of rat CLC-K2 in cortex
(A and
B) and outer medulla
(C and
D) of rat kidney. Bright-field
photomicrographs demonstrating the pattern of in situ hybridization of
35S-labeled rat CLC-K2 antisense
probe. Note that signals in distal nephron segments in outer cortex are
stronger than those in outer medulla. Magnifications:
A and
C, ×20;
B, ×80; and
D, ×100.
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Fig. 3.
Sequential sections of paraformaldehyde-fixed superficial cortex
hybridized with digoxigenin-labeled rat CLC-K2 antisense probe
(A,
C, and
E) or immunostained with an anti-TSC
antibody (a marker of distal convoluted tubules;
B), an
anti-Na+/Ca2+
exchanger antibody (a marker of connecting tubules;
D), or an anti-AQP-2 water channel
antibody (a marker of collecting ducts and connecting tubules;
F). Sequential sections of inner
cortex hybridized with digoxigenin-labeled rat CLC-K2 antisense probe
(G) and immunostained with an
anti-AQP-2 water channel antibody
(H).
I: hybridization with
digoxigenin-labeled CLC-K2 sense probe. In each pair of sequential
sections (A and
B, C
and D,
E and
F, G
and H), the specific nephron
segments identified in the immunostaining
(B,
D, F,
and H) are marked with white arrows
on in situ hybridization (A,
C, E,
and G). All the first antibodies and
the second antibodies [horseradish peroxidase (HRP)-conjugated
anti-rabbit IgG for the detection of TSC and AQP-2, and HRP-conjugated
anti-guinea pig IgG for
Na+/Ca2+
exchanger] were diluted to 1:300. AQP-2, aquaporin-2; TSC,
thiazide-sensitive Na+-Cl cotransporter. Magnification,
×100.
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In the inner stripe of the outer medulla, CLC-K2 expression matched the
staining pattern of Tamm-Horsfall glycoprotein (Fig. 4,
A-D),
thereby confirming that the moderate expression of CLC-K2 in the outer
medulla was present in the MTAL. In the cortex, the tubules positive
for Tamm-Horsfall glycoprotein were faintly positive for CLC-K2
expression (Fig. 4, E and
F), suggesting that the level of
expression of CLC-K2 in the cortical TAL (CTAL) was significantly lower
than that in the MTAL. This pattern of expression of CLC-K2 in the TAL
is opposite to that in the CD.

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Fig. 4.
In situ hybridization of rat CLC-K2
(A,
C,
E) and immunohistochemistry of
Tamm-Horsfall glycoprotein (B,
D,
F) in sequential sections of rat
kidney. A and
B: inner stripe of outer medulla
(×50). C and
D: inner stripe of outer medulla
(×100). E and
F: cortex (×100). Tubules
strongly positive for Tamm-Horsfall glycoprotein in inner stripe of
outer medulla (B and
D) were also moderately positive for
rat CLC-K2 expression (A and
C). In
C, moderate expression of CLC-K2 was
observed in the tubules positive for Tamm-Horsfall glycoprotein
(indicated by white arrows). In contrast, rat CLC-K2 expression was
faint in the tubules in the cortex (indicated by white arrows)
(E) that were positive for
Tamm-Horsfall glycoprotein
(F).
|
|
 |
DISCUSSION |
We previously showed by RT-PCR using dissected nephron segments that
the intrarenal localization of CLC-K2 is completely different from that
of CLC-K1 (1). However, the technical difficulty of dissecting
objective nephron segments without contamination by other nephron
segments and the high sensitivity of the PCR method often resulted in
false positive data or data that were not physiologically relevant to
the localization of the message along the nephron segments. For
example, CLC-K1 mRNA was detected in the CD in our initial report (12),
but immunohistochemical findings clearly demonstrated that CLC-K1
protein was exclusively localized to the ATL (10). In a study using the
RT-PCR technique, Kieferle et al. (3) also reported that rat CLC-K1 and
CLC-K2 were present relatively broadly along the nephron segments and possibly colocalized in some nephron segments. In a recent RT-PCR analysis of rat CLC-K1 and -K2 by Vandewalle et al. (13), both clones
were found to be widely expressed along the nephron segments. To
precisely determine where CLC-K1 and CLC-K2 are expressed in the
kidney, we prepared CLC-K1- and CLC-K2-specific riboprobes for in situ
hybridization. The nucleotide sequences of coding regions of CLC-K1 and
-K2 cDNAs are very homologous, so we chose the 3'-untranslated
region of each cDNA to generate clone-specific riboprobes. Since the
nucleotide identity between K1- and K2-specific probes was less than
30%, there was no cross-hybridization under the condition used in this
study. Using these probes, our in situ hybridization study clearly
demonstrated that although CLC-K1 and CLC-K2 are highly homologous
proteins, they are not colocalized in the kidney. As in our previous
RT-PCR study (1), the main sites of CLC-K2 expression were identified
to be TAL and the distal nephron segments, including the DCT, CNT, and
CCD. On the basis of the in situ hybridization results using
35S-labeled cRNA probe, the
intensity of the signal in the distal nephron segments was about an
order of magnitude higher than that in the TAL (Fig. 2). Accordingly,
the main site of CLC-K2 expression in the kidney is the distal nephron
segments in the superficial cortex. This contradicts the result of our
previous Northern blot (1), in which CLC-K2 expression was highest in
the inner medulla, moderate in the outer medulla, and faint in the
cortex. We reevaluated the previous record and came to the conclusion
that the developed film had been mistakenly aligned with the blot. As a
result, the lane of the inner medulla was labeled as the cortex, and
that of the cortex was labeled as the inner medulla. We performed
Northern blot again and confirmed that the signal of CLC-K2 in the
kidney was highest in the cortex, moderate in the outer medulla, and faint in the inner medulla (data not shown), which is consistent with
the present in situ hybridization data.
The expression of CLC-K2 mRNA was clearly detected in glomeruli using
digoxigenin probe in the Bowman's capsule and epithelial cells (Fig.
3). The absence of signal in glomeruli using
35S-labeled probe may be explained
by the difference in sensitivity between
35S-labeled and
digoxigenin-labeled probes. The existence of CLC-K2 mRNA in glomeruli
was consistent with our previous study, in which human CLC-K2 message
was detected in glomeruli by the RT-PCR method (9). However, the
physiological significance of CLC-K2 in the glomeruli remains to be
determined. In contrast, previous physiological data suggested the
existence of chloride conductance in the basolateral plasma membranes
of the TAL and CD (4). Therefore, we speculate that CLC-K2 may be
present in the basolateral plasma membranes of the DCT, CNT, CCD, and
MTAL. This is in agreement with the evidence that the
loss-of-function mutations of ClC-Kb (the human homolog of rat CLC-K2)
lead to a loss of chloride ions from the body, i.e.,
Bartter's syndrome.
Although previous immunohistochemistry studies (13, 17) coincided with
this in situ hybridization study in general, there are some
discrepancies in the details. Winters et al. (17) reported that the
rabbit homologue of CLC-K2 was present in the basolateral plasma
membrane of the MTAL. In the cortex, intercalated cells of the CCD had
cytosolic immunoreactivity. Our in situ hybridization detected strong
signals of the CLC-K2 message in the principal cells of the CNT and CCD
that far exceeded the signal detected in the MTAL. Regardless of
whether the staining is basolateral or cytoplasmic, much
stronger staining is expected in the cortex. This discrepancy could be
partly due to the species difference between rat and rabbit. In a
recent report on the immunohistochemistry of rat CLC-K channels by
Vandewalle et al. (13), their antibody could recognize both CLC-K1 and
-K2, since the antigen peptide, the carboxy-terminal end of CLC-K2,
differed from that of CLC-K1 by only one amino acid. Accordingly, their
immunostaining could not show where each channel was present in the
kidney. Their RT-PCR analysis showed almost identical expression
profiles of K1 and K2 along the nephron segments, suggesting that both
clones could be colocalized. We clearly showed in this study that
CLC-K1 is almost exclusively expressed in the inner medulla, further
confirming our previous immunohistochemistry of CLC-K1 (10). In
contrast to the inner medullary localization of CLC-K1, CLC-K2 is
expressed in the cortex and outer medulla, but not in the inner
medulla. This clearly showed that CLC-K1 and -K2 are not colocalized
and may have different roles in different nephron segments in the kidney. Given that rat CLC-K1 is expressed only in the ATL (10), the
staining in tubules other than the ATL in the study of Vandewalle et
al. (13) should be the staining of CLC-K2. Although the staining of the
DCT, CD, MTAL, and CTAL in their study mostly agrees with our present study, they clearly showed the staining of the inner medullary CD, where we could find no signal of CLC-K2
expression. Since the antibody in the study of Vandewalle et al. (13)
could not be used for Western blot of kidney membrane, it is still
possible that their antibody cross-reacted with proteins other than
CLC-K channels in the kidney.
There has been no clear determination of localization of CLC-K1 and -K2
expression in the kidney. By clearly showing in this study that CLC-K1
is expressed only in the inner medulla, we could confirm our previous
RT-PCR analysis and immunohistochemistry. We also were able to clearly
determine the sites and the relative abundance of CLC-K2 expression
along the nephron segments. CLC-K2 is moderately expressed in the MTAL
and highly expressed in the DCT, CNT, and CCD. Based on this study and
studies by Vandevalle et al. (13) and Simon et al. (8),
it is highly conceivable that CLC-K2 is a chloride channel that serves
as a route for transcellular chloride transport (an exit for chloride
ions in the basolateral plasma membrane of the TAL, DCT, CNT, and CCD).
 |
ACKNOWLEDGEMENTS |
This work was supported by grants-in-aid from the Ministry of
Education, Science, Sports and Culture of Japan.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Uchida,
Second Dept. of Internal Medicine, Tokyo Medical and Dental Univ.,
Yushima Bunkyo-ku Tokyo 113-8519, Japan (E-mail:
suchida.med2{at}med.tmd.ac.jp).
Received 24 September 1998; accepted in final form 31 December
1998.
 |
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