Analysis of NaCl transport in thin ascending limb of Henle's
loop in CLC-K1 null mice
Wen
Liu1,
Tetsuji
Morimoto1,
Yoshiaki
Kondo1,
Kazuie
Iinuma1,
Shinichi
Uchida2,
Sei
Sasaki2,
Fumiaki
Marumo2, and
Masashi
Imai3
1 Department of Pediatrics, Tohoku University School of
Medicine, Sendai 980-8574; 2 Second Department of Internal
Medicine, Tokyo Medical and Dental University, Tokyo 113-8519; and
3 Department of Pharmacology, Jichi Medical College, Kawachi
329-0498, Japan
 |
ABSTRACT |
To characterize the nature of NaCl
transport in the thin ascending limb (tAL), we examined the transport
properties of Na+ and Cl
using in vitro
microperfusion of the tAL in CLC-K1 null mice. In the presence of a
transmural NaCl concentration gradient (100 mM higher in the lumen),
the transepithelial diffusion voltage (Vd) was
15.5 ± 1.0 and
7.6 ± 1.4 mV in CLC-K1+/+ and
CLC-K1
/
mice, respectively. Neither Cl
transport inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) nor
acidification of the bathing fluid changed the
Vd values in CLC-K1
/
mice. The addition of 300 µg/ml protamine, a selective blocker of
paracellular conductance, to the bath increased the
Vd values by 5.6 ± 0.7 and 12.6 ± 1.5 mV (P < 0.001) in CLC-K1+/+ and
CLC-K1
/
mice, respectively. Although efflux
coefficients of 36Cl were significantly decreased in
CLC-K1
/
mice (188.3 ± 25.6 in 4 tubules vs.
17.2 ± 7.0 × 10
5 cm/s in 6 tubules), those of
22Na were not different between CLC-K1+/+ and
CLC-K1
/
mice. These results clearly indicate that the
major component of Cl
transport sensitive to NPPB or pH
is mediated by CLC-K1 in the tAL.
gene targeting; renal medulla; urine-concentrating mechanism; chloride channel; paracellular shunt pathway; sodium chloride
 |
INTRODUCTION |
ONE OF THE MOST
SPECIFIC renal functions in mammals is the urine-concentrating
mechanism, a mechanism found only in birds among all other animals
(14). The high level of urine concentration that takes
place in the inner medulla is a unique characteristic of the mammalian
urine-concentrating mechanism. The inner medulla is a novel structure
in which the ascending limb of Henle's loop is shaped like a thin
limb, namely, the thin ascending limb (tAL). The tAL is present only in
the mammalian inner medulla, where the highest osmotic environment in
the kidney is formed. This segment is present, not in the short-looped,
but in the long-looped nephron.
The tAL has long been considered important in the formation of
concentrated urine by means of specific transport properties that
dilute the urine without any movement of water across the epithelium.
The tAL, the first diluting segment of the long-looped nephron in
Henle's loop, dilutes urine concentrated in the descending limbs by
extruding NaCl with simple passive extrusion (6). The
mechanism of NaCl transport in the tAL has been debated since the
1960s. Most of the debate has focused on the presence or absence of
active NaCl reabsorption in the tAL. Gottshalk and Mylle
(2) provided the first direct evidence for very high
permeability of Na+ in the hamster tAL. In 1965, Marsh and
Solomon (12) identified the lumen-negative transepithelial
voltage (Vt) in the tAL under free-flow
conditions, suggesting that Na+ was actively reabsorbed in
this segment. Later in vivo studies using electrodes with increased
reliability found a lumen-positive Vt under
free-flow conditions in the tAL (3, 11, 15), but this did
not solve the issue of whether active Cl
reabsorption was
responsible for lumen-positive potentials.
In the 1980s, more extensive studies revealed that Na+ and
Cl
may be separately transported across the tAL
epithelium (7, 9, 20). These reports suggested that
luminal Na+ is passively diffused via tight junctions
(16), whereas luminal Cl
is transported
across the cell membranes (5, 18, 20). Indeed, extensive
studies in the 1990s further clarified this issue. In 1993, Uchida et
al. (17) revealed the presence of the specific
Cl
channel protein CLC-K1 in this segment. Later studies
clarified that CLC-K1 is expressed only in the tAL and that the major
properties of this channel resemble those of the segment itself
(18). Taken collectively, these studies make it plain that
CLC-K1 is a kidney-specific chloride channel that mediates
transepithelial chloride transport in the tAL. Our recent research
collaboration demonstrated that the deletion of CLC-K1 by gene
targeting results in a severe nephrogenic diabetes insipidus in mice
(13). This observation strongly suggests that CLC-K1
in the tAL plays an important role in urine concentration and that the
countercurrent system in the inner medulla is involved in the
generation and maintenance of hypertonic medullary interstitium (13).
To further clarify the mode of NaCl reabsorption in the tAL in
the present study, we conducted a series of experiments in CLC-K1 null
mice. Our results strongly suggest that the indirect electrical
coupling of Na+ and Cl
is a basic mechanism
of NaCl reabsorption in the tAL.
 |
METHODS |
Isolation and microperfusion of renal tubules.
Knockout mice deficient in CLC-K1 were generated by targeted gene
disruption (13). Genotype analysis of tail DNA was
performed by PCR. Homozygous wild-type (CLC-K1+/+) and null
(CLC-K1
/
) mice resulting from breeding of heterozygotes
were maintained on a regular diet and tap water until the day of the
experiment. The ages of the mutant animals were matched with their
wild-type controls. The mice were anesthetized by intraperitoneal
injection of 50 mg/kg of pentobarbital sodium, and their left
kidneys were removed. The renal tubule segment was microdissected with
fine forceps under a stereoscopic microscope. A fragment of the tubule was transferred to a chamber on the stage of an inverted microscope and
microperfused in vitro using Burg's method (1) with some modifications (6) at 37°C. Both sides of the tubule were
initially microperfused with HEPES-buffered Ringer solution containing
(in mM) 135 NaCl, 3.0 KCl, 2.0 KH2PO4, 1.5 CaCl2, 1.0 MgCl2, 10 HEPES, 100 urea, 1.0 sodium acetate, 5.5 glucose, and 5.0 L-alanine
(solution 1, Table 1). The
solution was titrated to pH 7.4 with NaOH at 37°C.
Measurement of transepithelial potentials.
The Vt was measured by connecting the bath and
perfusion pipette with a saturated KCl flowing boundary and an agar
bridge containing NaCl-Ringer solution, respectively. A high-input
impedance electrometer was used to monitor the
Vt (Duo773; WP Instruments, New Haven, CT). To
measure diffusion potential evoked by the transepithelial electrolyte
gradient, the bath solution was exchanged, and the deflection of the
Vt was observed. The KCl flowing boundary
minimized the changes in the liquid junction potential caused by the
solution exchange.
Measurement of relative permeability for Cl
.
Because the tAL is more permeable to Cl
than to
Na+, the lumen-positive diffusion voltages
(Vd values) are generated when a NaCl
concentration gradient is imposed across the tAL. The orientation of
the voltage was compatible with the preferential conductance for
electrolytes. Thus the NaCl Vd provides a good
approximation for the relative permeability of Cl
over
Na+
(PCl/PNa). The NaCl
Vd was measured by reducing the NaCl
concentration of the bathing fluid by 100 mM while the lumen was
perfused with solution 1 (solution 2, Table 1).
The PCl/PNa was
calculated from Goldman's equation
where Vd is diffusion voltage,
R is the gas constant, T is absolute temperature,
F is the Faraday constant; ab and
al are the ion activities in the bath and
luminal fluid, respectively; and PCl and
PNa are the permeabilities of the epithelium to
Cl
and Na+, respectively.
Direct measurements of efflux coefficients for 22Na
and 36Cl.
To elucidate the permeabilities of Na+ and Cl
in the tAL directly, the efflux coefficient (Ke)
of 22Na or 36Cl was measured in the in vitro
microperfused tAL in CLC-K1+/+ and CLC-K1
/
mice. 22Na (370 mBq/ml) or 36Cl (185 mBq/ml)
was added to the perfusate (final concentration). The lumen-to-bath
efflux coefficient for isotope x
(Ke x) was calculated as
where Vo is the collection
velocity rate, L is the length of the tubule, and
C*i and
C*o are radioactivity (concentrations)
of isotopes in the perfusate and collected fluid, respectively.
Chemicals.
The compositions of the solutions used in this study are listed in
Table 1. HEPES was obtained from Sigma (St. Louis, MO). All other
chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Statistical analyses.
The data for the Vd represent the values
obtained when the voltage became stable for at least 30 s. On rare
occasions when the voltages were not stable, the maximal voltage
deflections were chosen as representative values. Data are expressed as
means ± SE. Comparisons within a group with one treatment used
the paired Student's t-test. Comparisons between two groups
employed the unpaired Student's t-test. A value of
P < 0.05 was accepted as statistically significant.
 |
RESULTS |
Effects of NPPB on NaCl Vd.
In the absence of a transmural NaCl concentration gradient, the
Vt values were not different from zero,
as Imai and Kokko (4) reported previously. To characterize
the passive ionic permeabilities of Na+ and
Cl
, the transepithelial diffusional potential
(Vd) was measured when the basolateral NaCl
concentration was decreased by 100 mM. When the NaCl in the basolateral
solution was reduced from 135 to 35 mM by isotonic replacement with
urea, the Vd sharply turned toward the
lumen-positive direction in CLC-K1+/+ and
CLC-K1+/
mice, indicating the preferential permeation of
Cl
, whereas in CLC-K1
/
mice it continued
moving in the lumen-negative direction. The steady-state
Vd values were 15.5 ± 1.0 (n = 10), 12.8 ± 0.7 (n = 11),
and
7.6 ± 1.4 mV (n = 16) in
CLC-K1+/+, CLC-K1+/
, and
CLC-K1
/
mice, respectively (Fig.
1). These values in wild-type and
heterozygous mice were very different from those in knockout mice
(P < 0.0001, Student's unpaired t-test).
The values were also slightly different between wild-type and
heterozygous mice (P = 0.048).

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Fig. 1.
Diffusion voltage (Vd) caused by
transepithelial NaCl concentration gradient. Reduction of NaCl in the
basolateral solution from 135 to 35 mM by isotonic replacement with
urea caused marked positive deflections of the
Vd values in CLC-K1+/+ and
CLC-K1+/ mice and negative deflection in
CLC-K1 / mice. The steady-state
Vd values were 15.5 ± 1.0 (n = 10), 12.8 ± 0.7 (n = 11),
and 7.6 ± 1.4 mV (n = 16) in
CLC-K1+/+, CLC-K1+/ , and
CLC-K1 / mice, respectively. The
Cl -to-Na+ permeability ratio
(PCl/PNa) values are also
depicted at the bottom of each bar.
|
|
To characterize the electrical properties of the
Vd values, we tested the sensitivities of the
Vd values to various transport inhibitors. As
shown in Fig. 2, we examined the effects
of 0.1 mM 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) on the
Vd values evoked by making the basolateral NaCl
concentration 100 mM lower than that of the perfusate in the tAL. NPPB
is the most potent inhibitor of Cl
transport in the
hamster tAL (5). The Vd values were
suppressed by
17.3 ± 1.8 (n = 9),
13.8 ± 0.5 (n = 11), and
0.2 ± 0.1 mV (n = 11) in CLC-K1+/+,
CLC-K1+/
, and CLC-K1
/
mice, respectively.
The changes in the Vd values of
CLC-K1
/
mice brought about by applying NPPB were not
significantly different from zero. NPPB (10
4 M) changed
PCl/PNa from 4.02 ± 0.36 to 0.99 ± 0.09 and from 0.63 ± 0.06 to 0.67 ± 0.08 in CLC-K1+/+ and CLC-K1
/
mice,
respectively (Table 2).

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Fig. 2.
Effect of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)
on the NaCl Vd values. The
Vd values were measured in the presence of a
transmural NaCl concentration gradient (100 mM higher in the lumen).
When 0.1 mM NPPB was added to the bath solution, the
Vd values were suppressed by 17.3 ± 1.8 (n = 9), 13.8 ± 0.5 (n = 11),
and 0.2 ± 0.1 mV (n = 11) in
CLC-K1+/+, CLC-K1+/ , and
CLC-K1 / mice, respectively. The changes in the
Vd values of CLC-K1 / mice
brought about by applying NPPB were not significantly different from 0. n.s., Not significant.
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Effects of ambient pH on Vd.
It has been reported that Cl
conductance in the tAL is
regulated by H+. The Cl
conductance is
suppressed by acidic pH (8, 9). Briefly, we observed the
effects of the pH of the bathing fluid on the Vd
across the tAL generated when a transmural NaCl gradient was imposed.
As shown in Fig. 3, the decrease in the
pH of the bathing fluid from 7.4 to 6.0 sharply reduced the
Vd values by
6.9 ± 2.0 (n = 10) and
4.2 ± 0.5 mV (n = 11) in CLC-K1+/+ and CLC-K1+/
mice,
respectively. In CLC-K1
/
mice, however, the same
maneuver changed the Vd values by only
0.1 ± 0.1 mV (n = 9), and this was not
significant.

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Fig. 3.
Effect of acidification of bathing fluid on the NaCl
Vd values. The Vd values
was measured in the presence of a transmural NaCl concentration
gradient (100 mM higher in the lumen). The decrease in pH of the
bathing fluid from 7.4 to 6.0 changed the Vd
values by 6.9 ± 2.0 (n = 10) and 4.2 ± 0.5 mV (n = 11) in CLC-K1+/+ and
CLC-K1+/ mice, respectively. The same maneuver,
however, showed no effect in CLC-K1 / mice ( 0.1 ± 0.1 mV, n = 9).
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Effects of protamine on Vd.
We also examined the effect of the Na+ channel blocker
protamine on the NaCl Vd across the tAL. When
the composition of the perfusate and bathing fluid was identical,
Vt was zero. Replacement of the bathing fluid
with solution 2 caused a marked positive deflection of the
Vd values in CLC-K1+/+ and
CLC-K1+/
mice and a negative deflection in
CLC-K1
/
mice. Under this condition, the addition of 300 µg/ml protamine into the bath caused a rapid lumen-positive
deflection of the Vd in all three types of mice
that reached a plateau in a few minutes. The Vd
did not recover in any of the experiments when protamine was eliminated
from the bath, but it recovered rapidly when 30 U/ml heparin was added
under this condition. Figure 4 shows the
results of experiments conducted to observe the effect of protamine on
the NaCl Vd. Representative data showing the
effects of protamine and heparin in the tAL of CLC-K1+/+
and CLC-K1+/
mice are depicted in Figs.
5 and 6,
respectively.

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Fig. 4.
Effect of protamine on the NaCl Vd
values. The Vd values were measured in the
presence of a transmural NaCl concentration gradient (100 mM higher in
the lumen). The addition of 300 µg/ml protamine into the bath caused
a rapid lumen-positive deflection of the Vd by
5.6 ± 0.7 (n = 9), 5.7 ± 1.1 (n = 9), and 12.6 ± 1.5 mV (n = 9) in CLC-K1+/+, CLC-K1+/ , and
CLC-K1 / mice, respectively.
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Fig. 5.
A representative tracing of the Vd
in response to protamine and heparin in the thin ascending limb (tAL)
of CLC-K1+/+ mice. The Vd was
measured in the presence of a transmural NaCl concentration gradient
(100 mM higher in the lumen) in CLC-K1+/+ mice. The
addition of protamine immediately increased the
Vd by ~7 mV. Subsequent removal of protamine
from the bath did not alter the Vd. The addition
of 30 U/ml heparin then caused rapid recovery of the
Vd.
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Fig. 6.
Representative tracing of the Vd
in response to protamine and heparin in the tAL of
CLC-K1 / mice. Although the same protocol described in
Fig. 5 was performed in the tAL of CLC-K1 / mice, the
difference of the basal Vd, protamine, and
heparin had similar effects on the Vd in
CLC-K1 / mice.
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Effect of protamine in the presence of NPPB.
Isozaki et al. (5) reported that NPPB suppresses
Cl
conductance in this segment. If NPPB selectively
inhibits Cl
transport, Na+ conductance can be
expected to become dominant in the presence of NPPB. We examined the
effect of protamine under this condition. At first, we observed the
Vd by the same methods used in the first protocol. When we added 0.1 mM NPPB to the bathing fluid,
CLC-K1+/+ and CLC-K1+/
mice showed a rapid
change and negative deflection of the diffusion potential, whereas
CLC-K1
/
mice showed no such change. Under this
condition, 300 µg/ml protamine significantly increased this
Vd to the same extent in all three types of
mice. This was also reversed by 30 U/ml heparin. Table 2 summarizes the
results of various Cl
and Na+ transport
inhibitors on the NaCl Vd. The calculated
Cl
/Na+ permeability ratios are also given.
The NPPB and ambient pH clearly decreased the positively oriented the
NaCl Vd in CLC-K1+/+ and
CLC-K1+/
mice while imparting no such effect in
CLC-K1
/
mice. The addition of protamine to the bath
increased the NaCl Vd in all three types of
mice, and the presence of NPPB did not affect this protamine-induced increase.
Measurements of efflux coefficients for 22Na and
36Cl.
To elucidate the characteristics of NaCl transport in the tAL of
CLC-K1+/+ and CLC-K1
/
mice directly,
permeabilities of Cl
and Na+ were measured
using 36Cl and 22Na. As depicted in Fig.
7, the efflux coefficient for
36Cl (Ke Cl) was strongly impaired
in CLC-K1
/
mice, whereas the efflux coefficient for
22Na (Ke Na) in
CLC-K1
/
mice was not different from that in wild-type
mice. These results strongly indicate that targeted disruption of the
CLC-K1 gene in mice leads to selective impairment of Cl
transport in the tAL of the renal medulla.

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Fig. 7.
Efflux coefficients of 36Cl and
22Na in the tALs of CLC-K1+/+ and
CLC-K1 / mice. Efflux coefficients of 36Cl
(Ke Cl) and 22Na
(Ke Na) were measured in the tALs of
CLC-K1+/+ and CLC-K1 / mice. Although
Ke Cl was significantly decreased in
CLC-K1 / mice (188.3 ± 25.6 in 4 tubules vs.
17.2 ± 7.0 × 10 5 cm/s in 6 tubules),
Ke Na was not significantly different between
CLC-K1+/+ and CLC-K1 / mice (56.2 ± 15.7 in 4 tubules vs. 31.3 ± 2.3 × 10 5 cm/s
in 6 tubules). The results also imply that the preparation of the tAL
of CLC-K1+/+ and CLC-K1 / mice in the
present studies was reliable for obtaining good viability.
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Effect of NPPB on Ke Cl and Ke Na.
To characterize the Ke Cl and
Ke Na of the tALs in CLC-K1+/+ and
CLC-K1
/
mice, effects of 10
4 M NPPB in
the basolateral solution were examined. As clearly shown in Fig.
8, NPPB only inhibited the
Ke Cl in the CLC-K1+/+ mice. This
result indicates that NPPB-sensitive Cl
permeability in
the tAL is predominantly mediated by CLC-K1, whereas Na+
permeability is entirely unaffected by NPPB.

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Fig. 8.
Effects of NPPB on efflux coefficients of
36Cl and 22Na in the tALs of wild-type and
knockout (KO) mice. The effects of 0.1 mM NPPB in bath on
Ke Cl and Ke Na
were examined in wild-type and KO mice. Ke Cl
was decreased by 79.7 ± 19.3 (n = 4) and 2.7 ± 3.6 × 10 5 cm/s (n = 6) in
CLC-K1+/+ and CLC-K1 / mice, respectively.
Ke Na was increased by 6.7 ± 8.1 (n = 4) and 8.2 ± 3.9 × 10 5
cm/s (n = 6) in CLC-K1+/+ and
CLC-K1 / mice, respectively.
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 |
DISCUSSION |
Urine concentration is achieved by water reabsorption in the
collecting duct according to the osmolar gradient across the tubule. In
this process, the tAL plays an important role in the inner medulla by
diluting urine and by maintaining the hypertonicity of the interstitium
through reabsorption of NaCl. Whether NaCl is reabsorbed actively in
the tAL has long been an important question. Kondo et al. (7,
9) and Yoshitomi et al. (20) provided indirect and
direct evidence for the presence of stilbene-sensitive Cl
channels in both luminal and basolateral membranes of the tAL. Furthermore, molecular biological studies demonstrated the presence of
the CLC-K1, a kidney-specific chloride channel that mediates transepithelial Cl
transport, in the tAL (17,
18). In the present attempt to identify the mechanism of
Na+ transport across the tAL, the fragile properties of the
tAL in CLC-K1 null mice unfortunately thwarted our attempts to obtain the direct data on the lumen-to-bath 22Na and
36Cl flux. However, in conjunction with previous
characterizations of the tAL, the present data on the electrical
properties of the tAL in CLC-K1 null mice probably already suffice to
elucidate the nature of Na+ and Cl
transport.
The results reported here support the view that the paracellular shunt
pathway of this segment has, in fact, selective Na+ permeability.
Characterization of Cl
transport in the tAL.
As shown in Fig. 1, CLC-K1+/+ and CLC-K1+/
mice showed lumen-positive deflection on reduction of basolateral NaCl
concentration, whereas CLC-K1
/
mice generated an
opposite deflection in the same condition. This phenomenon supports the
view that the tAL is more permeable to Cl
than to
Na+ in normal mice. To further characterize the mechanism
of this Cl
conductance across the tAL, we examined
effects of various Cl
transport inhibitors. NPPB is a
specific transport inhibitor of the Cl
channel CLC-K1 in
the tAL (5), and the ambient pH regulates the
Cl
conductance (8, 9). As shown in Figs. 2
and 3, the reduction of the pH or application of NPPB suppressed the
Vd strongly and to approximately the same extent
in CLC-K1+/+ and CLC-K1+/
mice, whereas the
same maneuvers for CLC-K1
/
mice scarcely changed the
Vd at all. As shown in Table 2, acidification of
the bathing solution or application of NPPB induced a suppression of
the NaCl Vd that corresponded to the decreases
in PCl/PNa in CLC-K1+/+ and CLC-K1+/
mice but showed no
effect in CLC-K1
/
mice. Theoretically, the decrease in
the PCl/PNa ratio
reflects the following changes: 1) decrease in
PCl, 2) increase in
PNa, 3) a combination of 1 and 2, or 4) decrease in both parameters with a
preferential decrease in PCl. In the previous
studies, it was proved that the reduction of the pH or application of
NPPB selectively decreased Cl
permeability without
affecting Na+ permeability (8, 9). Therefore,
we conclude that acidic pH or NPPB decreases Cl
transport, not in CLC-K1
/
, but in CLC-K1+/+
and CLC-K1+/
mice. As the sensitivities to acidic pH or
NPPB are characteristics of a major Cl
transport pathway
in the tAL, our results indicate that Cl
transport in the
tAL is indeed abolished in CLC-K1
/
mice.
Properties of Na+ transport in tAL.
It has been reported that protamine increases the resistance of the
paracellular shunt pathway in various epithelia. Because Koyama et al.
(10) reported that protamine selectively inhibits Na+ conductance of the paracellular pathway, we decided to
examine the effects of protamine on the Vd
values during in vitro microperfusion.
In our study, the addition of protamine to the bath caused a marked
increase in the Vd values in all three types of
mice, indicating that the protamine-sensitive Na+
conductance in the tAL is independent of CLC-K1. The effect of protamine persisted even after the bathing fluid was exchanged for a
solution that did not contain protamine (Fig. 4, Table 2). The addition
of heparin, an agent believed to be sufficient to neutralize charges of
protamine, rapidly reversed the voltage toward the control level. In
the presence of a transmural NaCl concentration gradient (100 mM higher
in the lumen), CLC-K1 channel knockout mice showed the lumen-negative
deflection (Fig. 1) described above. Because Cl
transport
in the tAL is practically abolished in the CLC-K1
/
mice, this lumen-negative potential is evidently induced by a major
outflow of Na+.
Given the large contribution of transcellular Cl
conductance, simple NaCl Vd does not reflect
selectivity of paracellular permeability. NPPB was first reported by
Wangemann et al. (19), who characterized it as the most
potent inhibitor of the Cl
conductance of the basolateral
membrane of the thick ascending limb of Henle's loop. Isozaki et al.
(5) reported that, in hamster tAL, NPPB added to the bath
caused reversible suppression of Cl
permeability, as
determined by the Vd, and Cl
flux.
Furthermore, because the CLC-K1 null mouse is manifested with
deprivation of a chloride channel by targeted gene disruption, we
studied the effect of protamine on the NaCl Vd
in all three groups of mice in the presence of NPPB. The reversal of
the orientation of the Vd by NPPB in
CLC-K1+/+ and CLC-K1+/
mice indicates that
the Na+ conductance became dominant by this maneuver,
whereas the lack of any comparable change with the
CLC-K1
/
mice indicates that NPPB imparted no such
effect on this latter group of animals. Under this condition, the
comparable strength of the inhibitory effect of protamine on the
diffusion potential in all three types of mice indicates that the
Na+ conductance in CLC-K1
/
mice is more or
less the same as that in CLC-K1+/+ and
CLC-K1+/
mice (Table 2).
There is another possibility, that the abolishment of Cl
transport in the tAL for the CLC-K1 null mice may be caused by damage to the tubular cells through the deletion of CLC-K1 by gene targeting. In fact, we observed that the tAL of CLC-K1 null mice showed relative fragility compared with that in normal mice. However, the studies on
Na+ transport in the tAL have proved that normal
Na+ conductance can be observed even in CLC-K1 null mice if
the experimental period can be kept sufficiently brief. However, it can
be conjectured that such fragility of the tubule cells might lead to
chronic damage to the tubules and, ultimately, to renal dysfunction.
Further studies will be needed to demonstrate this type of renal damage caused by the disruption of CLC-K1.
In conclusion, we confirmed that CLC-K1 null mice have the same
Na+ conductance compared with wild-type mice. We propose
that this Na+ shunt is conferred by the paracellular shunt
pathway and is only electrically coupled with the transport of
Cl
in an indirect manner.
 |
ACKNOWLEDGEMENTS |
We thank Naoko Sato for excellent technical assistance.
 |
FOOTNOTES |
This work was supported in part by a grant-in-aid from the Ministry of
Education, Science, and Culture of Japan and by a research grant from
the Salt Science Research Foundation (Japan).
Address for reprint requests and other correspondence: Y. Kondo, Dept. of Pediatrics, Tohoku Univ. School of Medicine,
Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan (E-mail:
ykondo{at}ped.med.tohoku.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.
10.1152/ajprenal.0192.2001
Received 2 July 2001; accepted in final form 27 October 2001.
 |
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