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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
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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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
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Table 1.   Composition of artificial solutions

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
V<SUB>d</SUB><IT>=R∗T/F∗</IT>ln {[<IT>a</IT><SUP><IT>b</IT></SUP><SUB>Na</SUB><IT>+</IT>(<IT>P</IT><SUB>Cl</SUB><IT>/P</IT><SUB>Na</SUB>)<IT>a</IT><SUP><IT>l</IT></SUP><SUB>Cl</SUB>]<IT>/</IT>[<IT>a</IT><SUP><IT>l</IT></SUP><SUB>Na</SUB><IT>+</IT>(<IT>P</IT><SUB>Cl</SUB><IT>/P</IT><SUB>Na</SUB>)<IT>a</IT><SUP><IT>b</IT></SUP><SUB>Cl</SUB>]}
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
K<SUB>e <IT>x</IT></SUB>(<IT>×</IT>10<SUP><IT>−</IT>7</SUP> cm<SUP>2</SUP><IT>/</IT>s)<IT>=</IT><FR><NU><IT>V</IT><SUB>O</SUB></NU><DE><IT>L</IT></DE></FR> ln <FR><NU>C<SUP>*</SUP><SUB>i</SUB></NU><DE>C<SUP>*</SUP><SUB>o</SUB></DE></FR>
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.


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REFERENCES

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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Table 2.   Effect of various Cl- and Na+ transport inhibitors on NaCl diffusion voltage in tAL

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).

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.

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.

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.


    DISCUSSION
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INTRODUCTION
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DISCUSSION
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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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Burg, M, Grantham J, Abramow M, and Orloff J. Preparation and study of fragments of single rabbit nephrons. Am J Physiol 210: 1293-1298, 1966[ISI][Medline].

2.   Gottshalk, CW, and Mylle M. Micropuncture study of the mammalian urinary concentrating mechanism. Am J Physiol 196: 927-936, 1959[ISI].

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