Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
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ABSTRACT |
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The CLC-K1 chloride channel is a kidney-specific
CLC chloride channel expressed in the thin ascending limb of Henle's
loop (tAL). Recently, we determined that Clcnk1/
mice
show nephrogenic diabetes insipidus (NDI). To investigate the
pathogenesis of impaired urinary concentrating ability, we analyzed
renal functions of Clcnk1
/
mice in more detail. The
osmolar clearance-to-creatinine clearance ratio was not significantly
different between Clcnk1+/
and Clcnk1+/+ mice.
Fractional excretion of sodium, chloride, and urea was also not
significantly affected in Clcnk1
/
mice. These results
indicate that the polyuria observed in Clcnk1
/
mice was
water diuresis and not osmotic diuresis. The papillary osmolarity
in Clcnk1
/
mice was significantly lower than that in Clcnk1+/+ mice under a hydrated condition, and it did not
increase even after water deprivation. Sodium and chloride contents in the inner medulla in Clcnk1
/
mice were at about one-half
the levels observed in Clcnk1+/+ mice. Furthermore, the
accumulation of urea was also impaired in Clcnk1
/
mice,
suggesting that the overall countercurrent system was impaired by a
defect of its single component, chloride transport in the tAL. The
aldose reductase mRNA abundance in Clcnk1
/
mice was
decreased, further evincing that inner medullary tonicity is decreased
in Clcnk1
/
mice. We concluded that NDI in
Clcnk1
/
mice resulted from an impairment in the
generation of inner medullary hypertonicity by a dysfunction of the
countercurrent systems.
chloride channel; knockout mouse; countercurrent system; osmolytes; aquaporin-2 water channel
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INTRODUCTION |
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CLC-K1 AND CLC-K2 are kidney-specific CLC chloride channels (1, 27). Although these channels are highly homologous (90% amino acid identity in humans and 80% in rat), their intrarenal localizations were completely different. We recently demonstrated by in situ hybridization that CLC-K1 is present only in the inner medulla and that CLC-K2 is expressed in the distal tubules, connecting ducts, cortical collecting ducts, and medullary thick ascending limb of Henle's loop (mTAL). Because sodium-dependent chloride transporters [NKCC2 (22) and TSC (9)] are present in the apical surfaces of these nephron segments, it had been speculated that a chloride channel had to be present on the basolateral plasma membrane for a vectorial transepithelial chloride transport to occur in these segments (21, 32). Accordingly, CLC-K2 could be a candidate chloride channel involved in chloride reabsorption in the distal nephron. This hypothesis was recently verified by genetic evidence that mutations of the CLC-Kb gene [a human homolog of rat CLC-K2 (26) cause Bartter's syndrome (23)]. On the basis of these observations and the structural similarity of CLC-K1 to CLC-K2, the former could also be involved in transepithelial chloride transport in the inner medulla. We have already clarified by immunohistochemistry that CLC-K1 is present in both apical and basolateral plasma membranes of the thin ascending limb of Henle's loop (tAL) (28). Because this nephron segment has been reported to have an extraordinarily high chloride permeability among nephron segments (12), we explored whether CLC-K1 was responsible for the high chloride permeability in the tAL by comparing the characteristics of CLC-K1 expressed in Xenopus oocytes with those of a native chloride transport in the tAL (16). Anion selectivity, pH sensitivity, and sensitivities to various inhibitors were quite similar in both transport systems, suggesting that CLC-K1 may be a major chloride channel responsible for the transepithelial chloride transport in the tAL (28). In a passive model proposed by Kokko and Rector (16), the tAL constituted a component of countercurrent systems for urinary concentration, and NaCl transport in the tAL could be an important route for supplying NaCl to the interstitium in the inner medulla. Thus we speculated that CLC-K1 was involved in the countercurrent systems in the inner medulla and important for urinary concentration.
To verify the above hypothesis, we generated CLC-K1-knockout mice
(Clcnk1/
) by targeted disruption of the gene
(17). As a result, the Clcnk1
/
mice showed
overt nephrogenic diabetes insipidus (NDI), clearly demonstrating that
CLC-K1 has an essential role for urinary concentration
(17). In our previous report (17), however,
the exact mechanisms of NDI were not fully studied (17).
On the basis of results from a 1-deamino-8-D-arginine vasopressin (dDAVP) infusion test, we speculated that NDI in
Clcnk1
/
mice resulted from the impairment of generating
hypertonicity in the inner medulla (17). Accordingly, we
directly measured tissue osmolarity and solute contents in the inner
medulla of Clcnk1
/
mice in this study. We also performed
detailed studies on renal function of Clcnk1
/
mice
because our previous data on urine were based on urine collected over
24 h, a limited period, which might not have allowed accuracy
because of evaporation and contamination with stool, especially when
urine volumes were small. Accordingly, we performed an exact
clearance study of Clcnk1
/
mice under normal and
dehydrated conditions.
We found that the polyuria in the Clcnk1/
mice is water
diuresis and that the Clcnk1
/
mice cannot accumulate
osmotic solutes in the inner medulla even in dehydration.
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METHODS |
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Animals.
Wild-type (Clcnk1+/+), heterozygous
(Clcnk1+/), and homozygous mutant (Clcnk1
/
)
littermates were generated from F6 heterozygous mice
(Clcnk1+/
) (18). Only male mice (5-6 wk
old) were used in this study.
Clearance study. A single experiment consisted of 6 mice (2 wild, 2 heterozygous, and 2 homozygous mutant) divided into a normal group (free access to water) and a dehydrated group (24-h water deprivation). The mice were kept awake during the clearance study because anesthetics significantly affected blood pressure and kidney function in initial attempts to perform the experiment. Urination was induced at the beginning of the clearance period by abdominal tapping. Over a 4-h clearance period, each mouse was kept into a clean plastic cage and its urine was collected at every urination. After the clearance period, the mice were anesthetized deeply by nebulization of diethyl ether, blood was collected from their hearts, and their kidneys were isolated for Northern hybridization. Urine in the bladder was also recovered for clearance analysis.
The serum sodium, chloride, and urea concentration were measured by i-STAT (i-STAT, East Windsor, NJ). Urinary concentration of sodium and chloride was measured by a 9180 electrolyte analyzer (AVL Scientific, Roswell, GA). Serum and urinary creatinine and urea concentration were measured by the Creatinine Test Wako and Urea Test Wako, respectively, (Wako, Tokyo, Japan). Osmolarity of serum and urine was measured by a One-Ten Osmometer (Fiske, Norwood, MA).Tissue analysis. The animals were anesthetized with diethyl ether, and their kidneys were removed. To obtain enough tissue for the analyses for sodium, chloride, and urea contents and osmolarity, eight papillae from four mice were pooled, weighed, and homogenized in 200 µl of water. The supernatants were analyzed after centrifugation at 15,000 rpm for 10 min. Sodium and chloride concentrations were measured by a 9180 electrolyte analyzer, and osmolarity was measured by using a One-Ten Osmometer. The urea content was measured by a Urea Test Wako kit.
Measurements of urinary vasopressin and plasma aldosterone. Urinary vasopressin was measured with a radioimmunoassay kit (AVP-kit Mitsubishi; Mitsubishi Kagaku, Tokyo, Japan) and corrected by creatinine concentration. Plasma aldosterone concentrations were measured with a radioimmunoassay kit (SPAC-S aldosterone kit; Daiichi Radio-isotope, Tokyo, Japan).
Probes.
For Northern hybridization, the mouse aldose reductase (mAR) probe was
prepared by PCR using kidney cDNA library in gt10 (Clontech, Palo
Alto, CA) as a template. The primer sequences were as follows: sense
strand, 5'-GTGAACCAGATCGAGTGCCAC-3' (nucleotides 560-580,
in mAR cDNA), and antisense strand,
5'-AGTCGTTACGCGTGACCACAG-3' (nucleotides 831-811). The PCR
product was subcloned into pGEM-T easy vector (Promega, Madison, WI)
and then sequenced. The cDNAs for probe of mouse betaine/GABA
transporter (mBGT) and rat sodium/myo-inositol cotransporter
(rSMIT) were kindly provided by M. Takenaka (Osaka Univ.). The probe of
mouse aquaporin-2 (mAQP2) was obtained as described previously
(7).
Northern blots.
Total RNA (20 µg/lane) separated by electrophoresis in an agarose gel
containing formamide was transferred to nylon membranes (MagnaGraph,
MSI, Westborough, MA). The filters were hybridized for 24 h at
42°C in 50% formamide, 6× sodium chloride-sodium phosphate-EDTA, and 5× Denhardt's solution containing 1% SDS and 100 µg/ml salmon sperm DNA, with cDNAs of mAR, mBGT, Rsmit, and mAQP2 labeled with [-32P]dCTP. The filters were washed once in 0.1% SDS
and 1× standard sodium citrate (SSC) at room temperature for 20 min
and then twice in 0.1% SDS and 0.2× SSC at 50°C for 20 min. They
were exposed to film (RX-U, Fujifilm, Tokyo, Japan) at
80°C.
Immunohistochemistry.
Clcnk1+/+ and Clcnk1/
mice were perfused and
fixed in a PLP solution (6% paraformaldehyde, 25 mM
L-lysine monohydrochloride, and 0.1 M sodium periodate in
PBS). Frozen sections were incubated with 1:100 dilution of anti-rat
AQP2 antibody obtained as described previously (8) and
visualized with cyanine 3-conjugated anti-rabbit IgG antibody (Sigma,
St. Louis, MO) under a LMS-5 laser scanning microscope (Carl Zeiss,
Thornwood, NY).
Statistics. ANOVA was used to analyze the differences in data among groups. P values <0.05 were considered to be significant.
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RESULTS |
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Clearance study and hormonal data.
Figure 1 shows creatinine clearance
(Ccr) of Clcnk1+/+, Clcnk1+/ and
Clcnk1
/
mice in normal and dehydrated conditions, and
the ratio of osmolar clearance to Ccr (FEosm)
in Clcnk1+/+, Clcnk1+/
, and
Clcnk1
/
mice under normal and dehydrated conditions. Ccr of Clcnk1+/+, Clcnk1+/
, and
Clcnk1
/
mice was 5.8 ± 2.1, 5.8 ± 1.2, and
7.7 ± 1.6 (SE) ml · min
1 · g body
wt
1 (n = 3) under normal conditions, with
no significant difference among groups. These Ccr values
are compatible with those previously reported (15),
suggesting that a 4-h clearance period was long enough to evaluate
overall renal function. Clcnk1
/
mice could preserve
normal glomerular filtration rate (GFR) under an euhydrated condition.
Ccr after 24-h water deprivation significantly decreased in
Clcnk1
/
(1.0 ± 0.2) compared with
Clcnk1+/+ (5.4 ± 0.8) and Clcnk1+/
mice
(5.8 ± 0.6), indicating that GFR in Clcnk1
/
mice was impaired only by 24-h dehydration. As shown in Fig. 1B,
FEosm values were not significantly different among the
three groups in normal conditions, clearly demonstrating that polyuria
in Clcnk1
/
mice was not osmotic diuresis but water
diuresis. Figure 2 shows fractional
excretion of sodium (FENa), chloride (FECl),
and urea (FEurea) of mice in six groups. These values were
not significantly different among the three groups under a normal
euhydrated condition, clearly demonstrating that a loss of chloride
transport in the tAL did not result in chloride diuresis.
FENa significantly decreased after 24-h dehydration in
Clcnk1
/
mice (Fig. 2A), a finding that also
showed, along with the decreased GFR (Fig. 1A), that 24-h
dehydration was sufficient to cause a severe reduction in circulating
blood volume in Clcnk1
/
mice.
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Tissue solute contents.
Figure 4 shows the contents of
osmolarity, sodium, chloride, and urea, in the tissue of inner medulla
of Clcnk1+/+ and Clcnk1/
mice. Total
osmolarity in the inner medulla in Clcnk1
/
mice was
about one-half of that of Clcnk1+/+ mice, and it did not
increase after the dehydration period (Fig. 4A). These
results clearly showed that Clcnk1
/
mice could not
generate hypertonicity in the inner medulla even under the dehydrated
conditions. Because the tAL was potentially a main site for the supply
of NaCl to the interstitium, the decrease in NaCl content in the
Clcnk1
/
mice had been anticipated. In fact, the inner
medullary contents of sodium and chloride in Clcnk1
/
mice in both normal and dehydrated conditions were significantly
smaller than those in Clcnk1+/+ mice (Fig. 4, B
and C). Sodium osmolarity in the inner medulla in
Clcnk1+/+ and Clcnk1
/
mice under normal
conditions was 0.50 ± 0.09 and 0.25 ± 0.05 (SE) mmol/g wet
wt, n = 3, respectively. Those in a dehydrated
condition were 0.46 ± 0.04 and 0.28 ± 0.02 mmol/g wet wt,
respectively. Chloride osmolarity in the inner medulla in
Clcnk1+/+ and Clcnk1
/
under normal conditions
were 0.23 ± 0.03 and 0.11 ± 0.05 mmol/g wet wt,
n = 3, respectively. Those in a dehydrated condition
were 0.25 ± 0.02 and 0.07 ± 0.01 mmol/g wet wt,
respectively. These values of sodium and chloride contents in wild-type
mice under normal conditions are consistent with those in a previous
report (3). Furthermore, urea accumulation in the inner
medulla in Clcnk1
/
mice was also impaired under normal
conditions (Fig. 4D). In contrast to the marked increase in
urea content under dehydrated conditions in Clcnk1+/+ mice, urea content in Clcnk1
/
mice was not increased by
dehydration (Fig. 4D).
|
Expression of osmolyte-related genes.
Figure 5 shows Northern blots of kidney
total mRNA (20 µg/lane) from normal and dehydrated
Clcnk1+/+, Clcnk1+/, and Clcnk1
/
mice probed with AR, BGT, and SMIT. Although all three genes were known
to be regulated by tonicity (6), their sites of expression in kidney were different. In previous in situ hybridization studies (19), AR expression was found to be localized in the inner
medulla, whereas BGT and SMIT were mainly expressed in the outer
medulla. As shown in Fig. 5, AR expression in Clcnk1
/
mice was significantly lower [66.0 ± 9.1 (SE) % of
Clcnk1+/+ in normal conditions, n = 3, P < 0.05] than that in Clcnk1+/+ mice in
normal conditions, and it was not increased by dehydration (75.7 ± 3.1% of Clcnk1+/+ in normal conditions,
n = 3, not significantly different from Clcnk1
/
mice in normal conditions), whereas that in
Clcnk1+/+ mice apparently was 140.4 ± 10.8% of
Clcnk1+/+ in normal conditions, n = 3, P < 0.05. This result also supports the relative
hyposmolarity in the inner medulla of Clcnk1
/
mice. BGT
and SMIT mRNA in Clcnk1
/
mice was significantly
increased [1.3 ± 0.02 (SE)-fold increase in BGT and 1.2 ± 0.03-fold increase in SMIT, n = 3, P < 0.05] by dehydration, suggesting that the abnormality in the tissue osmolarity in Clcnk1
/
mice only occurred in the inner
medulla.
|
AQP2 expression and cellular localization.
To confirm that NDI in Clcnk1/
mice is not due to the
dysregulation of the AQP2 water channel, we checked the expression of
AQP2 in Clcnk1+/+ and Clcnk1
/
mice by
Northern analysis and the cellular localization by
immunohistochemistry. As shown in Fig. 6,
AQP2 expression was slightly increased in Clcnk1
/
mice [1.6 ± 0.1 (SE)-fold increase, n = 3, P < 0.05] than in Clcnk1+/+ mice.
Fluorescence immunohistochemical study also revealed that AQP2 was
localized in apical membranes of inner medullary collecting ducts in
Clcnk1
/
mice as well as in Clcnk1+/+ mice
(Fig. 7, A and B).
These results suggest that NDI in Clcnk1
/
mice may not
be due to the apparent abnormal regulation of AQP2.
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DISCUSSION |
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We previously demonstrated that the Clcnk1/
mice
showed an exogenous vasopressin-insensitive urinary concentrating
defect (18). We speculated that this NDI could result from
the dysfunction of the countercurrent system because the tAL is thought
to be an important component of this system (16).
Generation and maintenance of a hypertonic environment in the inner
medulla is thought to be a major mechanism of urinary concentration,
together with a switching mechanism of water permeability in the
collecting ducts. The significance of the latter has been verified by
the identification of mutations of vasopressin V2 receptor
and AQP2 genes in patients with NDI. However, there has been no direct
evidence that a countercurrent system(s) in the inner medulla is indeed
functioning to generate hypertonicity in vivo. To prove that the
deletion of the Clcnk1 resulted in the dysfunction of a
countercurrent system and impairment of generating hypertonicity in the
inner medulla, in this study we directly measured solute contents in
the inner medulla in the Clcnk1
/
mice. In addition, we
performed a clearance study to confirm that the polyuria observed in
the Clcnk1
/
mice is water diuresis.
In our previous report (17), the urine osmolarity in the
Clcnk1/
mice was not proportionally lower than the value
expected from the increased urinary volume. If the data was truly
accurate, the increased urinary volume in the Clcnk1
/
mice could be partly due to osmotic diuresis, and this could be
expected because the chloride reabsorption in the tAL was thought be to
blocked by the Clcnk1 deletion. However, our previous data
were based on 24-h collected urine, so the urine volume might have been
affected by evaporation, especially in the case of the
Clcnk1+/+ and Clcnk1+/
mice due to their scant
urine output (~1-2 ml/day). Although we initially tried the
clearance study according to the method described by Okubo et al.
(20), we could not maintain the blood pressure during
operation and clearance period when anesthesia was used. Thus we
performed the clearance study while the mice were awake. First, we
confirmed that creatinine clearance of the Clcnk1
/
mice
was not significantly different from those of the Clcnk1+/+ and Clcnk1+/
mice (Fig. 1A), indicating that
the deletion of Clcnk1 gene did not affect the glomerular
filtration rate under normal conditions. Next, we measured the osmolar
clearances (Fig. 1B) of the Clcnk1+/+,
Clcnk1+/
, and Clcnk1
/
mice and found that
they were not significantly different. This clearly indicated that
osmotic diuresis was not observed in the Clcnk1
/
mice. FECl, as well as FENa and FEurea,
did not significantly differ under normal conditions among the three
groups. This confirmed the data on osmolar clearance, and it also
indicated that, unlike the mutations of CLC-Kb in humans
(23), the transepithelial chloride transport in the tAL
did not affect chloride clearance. The decrease in chloride
reabsorption in the tAL may be compensated for in the thick ascending
limb, where there is a powerful, active chloride reabsorption system
(10). We previously reported that the
Clcnk1
/
mice appeared remarkably lethargic even after
24-h water deprivation. Our clearance study also clearly demonstrated the consequence of water deprivation in the kidney of the
Clcnk1
/
mice. FENa decreased significantly
in the Clcnk1
/
mice after dehydration (Fig. 2). This
showed that the Clcnk1
/
mice were in a state of volume
depletion, a finding consistent with the severe hemoconcentration and
reduction in body weight reported previously (17).
Because our clearance study confirmed that the polyuria in the
Clcnk1/
mice is water diuresis, we know that the
mechanism of NDI could be either a loss of water permeability in the
collecting ducts or an impairment of the generation of hypertonicity in
the inner medulla. There is no reason to suspect that the molecules involved in regulating water permeability in the collecting ducts were
impaired in the knockout of CLC-K1 in the tAL, and accordingly there
was no need in the gene-knockout experiments to check whether all
molecules other than the targeted molecule are intact in the body, nor
is there in fact a way to perform such an investigation. However, we
did check the expression and cellular localization of AQP2, a major
molecule regulating water permeability in the collecting ducts, in the
Clcnk1
/
mice (Figs. 6 and 7). The expression of AQP2 in
the Clcnk1
/
mice confirmed by Northern analysis was even
higher than that in the Clcnk1+/+ mice. We and others
reported that AQP2 expression was mainly regulated by vasopressin
through the V2 receptor and a cAMP-responsive element in
the AQP2 gene promoter (11, 18, 31). Because we observed
that U-AVP was higher in the Clcnk1
/
mice (Fig.
3B), it is conceivable that AQP2 expression was increased by
the increased secretion of vasopressin. As for cellular localization,
AQP2 was clearly stained on the apical surface of collecting ducts, and
the staining appeared to be even stronger than that in the
Clcnk1+/+ mice. These results at least show that NDI in the
Clcnk1
/
mice is not due to the secondary effect of the
CLC-K1 knockout on AQP2.
Measurement of total osmolarity in the inner medulla clearly showed
that levels of osmotic substances in the inner medulla of the
Clcnk1/
mice were lower than those in the
Clcnk1+/+ mice (Fig. 4A), suggesting the
inability of generation of hypertonicity in the inner medulla in the
Clcnk1
/
mice. These results were more impressive when
the osmolarity at the tip of papilla (Ctip) was calculated
from the measured osmolarity in papilla homogenates (Cpap)
on the basis of the equation by Kettyle and Valtin (14). To make this calculation we need to determine the distance from the
papillary tip to the corticomedullary junction (hm), that distance from
the papillary tip to the papilla base (hp), and the osmolarity at the
corticomedullary junction (Cm)
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(14) |
|
The next problem to solve was to identify the accumulation of which
solute was impaired in the Clcnk1/
mice. Decreased
sodium and chloride accumulations were expected to occur because the tAL is a nephron segment that has high NaCl permeability
(13). As shown in Fig. 4, B and C,
sodium and chloride accumulations were impaired in the
Clcnk1
/
mice even under dehydrated conditions. In
addition, urea accumulation was also impaired in the
Clcnk1
/
mice (Fig. 4D) under both normal and
dehydrated conditions. Because a marked increase in urea accumulation
was observed in the Clcnk1+/+ mice under dehydrated
conditions, urea must be an important solute that constitutes
hypertonicity in the inner medulla in mice. The absence of any increase
of urea accumulation by dehydration in the Clcnk1
/
mice
was surprising, and the exact mechanisms of this phenomenon remain to
be determined. Impairment of urea accumulation as well as NaCl in the
Clcnk1
/
mice implies that the mechanisms for generation
of hypertonicity in the inner medulla is not a sum of transport systems
independently functioning, but rather a system consisting of various
transport systems interacting with each other, i.e., countercurrent
systems. From this viewpoint, this study for the first time verified
that a countercurrent system(s) is really functioning in vivo.
Finally, we confirmed the relative hypoosmolarity in the inner medulla
of the Clcnk1/
mice by examining the expression of osmolyte-related genes. The genes involved in the accumulation of
osmolytes are known to be regulated by tonicity in vitro and in vivo
(24, 29). Accordingly, the expressions of these genes are
good markers of tissue osmolarity. The major organic osmolyte in the
inner medulla was sorbitol generated by AR from glucose (2,
5). Previous reports identified that the AR gene is present only
in the inner medulla in the kidney (4), whereas myo-inositol and betaine are major osmolytes located mainly
in the outer medulla (19, 30). Figure 5 clearly showed
that AR expression was lower in the Clcnk1
/
mice than in
Clcnk1+/+ mice and that the upregulation by dehydration
observed in the Clcnk1+/+ mice was minimal in the
Clcnk1
/
mice. This coincides with the measured tissue
osmolarity (Fig. 4A) and further supports the relative
hypoosmolarity in the inner medulla of the Clcnk1
/
mice.
In contrast to AR, expression of SMIT and BGT was increased under dehydrated conditions in the Clcnk1
/
mice.
Considering that the main site of expression of SMIT and BGT is in the
outer medulla (19), these data suggest that the reduction
of tissue osmolarity in the Clcnk1
/
mice only occurs in
the inner medulla. The increase in SMIT and BGT in the
Clcnk1
/
under normal and dehydrated conditions may be
caused by the dehydration and/or increased vasopressin secretion.
In conclusion, we demonstrated that the gene knockout of the CLC-K1 chloride channel impaired the solute accumulation in the inner medulla without affecting water permeability in the collecting duct and that the proposed countercurrent system(s) is actually functioning in vivo.
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ACKNOWLEDGEMENTS |
---|
This work was supported by the Salt Science Research Foundation and Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan.
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: S. Uchida, Second Dept. of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail: suchida.med2{at}med.tmd.ac.jp).
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 7 February 2000; accepted in final form 29 August 2000.
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