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Am J Physiol Renal Physiol 287: F932-F939, 2004. First published August 3, 2004; doi:10.1152/ajprenal.00131.2004
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Involvement of cytosolic Cl– in osmoregulation of {alpha}-ENaC gene expression

Naomi Niisato,1 Douglas C. Eaton,2 and Yoshinori Marunaka1,3

Departments of 1Molecular Cell Physiology and 3Respiratory Molecular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; and 2Center for Cell and Molecular Signaling and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322

Submitted 12 April 2004 ; accepted in final form 23 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypotonicity stimulates transepithelial Na+ reabsorption in renal A6 cells, but the mechanism for this stimulation is not fully understood. In the present study, we found that hypotonicity stimulated Na+ reabsorption through increases in mRNA expression of the {alpha}-subunit of the epithelial Na+ channel ({alpha}-ENaC). Hypotonicity decreases cytosolic Cl– concentration; therefore, we hypothesized that hypotonicity-induced decreases in cytosolic Cl– concentration could act as a signal to regulate Na+ reabsorption through changes in {alpha}-ENaC mRNA expression. Treatment with the flavone apigenin, which activates the Na+-K+-2Cl– cotransporter and increases cytosolic Cl– concentration, markedly suppressed the hypotonicity-induced increase in {alpha}-ENaC mRNA expression. On the other hand, blockade of the Na+-K+-2Cl– cotransporter decreases cytosolic Cl– concentration and increased {alpha}-ENaC mRNA expression and Na+ reabsorption. Blocking Cl– channels with 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the hypotonicity-induced decrease in cytosolic Cl– concentration and suppressed the hypotonicity-induced increase in {alpha}-ENaC mRNA expression. Coapplication of NPPB and apigenin synergistically suppressed {alpha}-ENaC mRNA expression. Thus, in every case, changes in cytosolic Cl– concentration were associated with changes in {alpha}-ENaC mRNA expression and changes in Na+ reabsorption: decreases in cytosolic Cl– concentration increased {alpha}-ENaC mRNA and increased Na+ reabsorption, whereas increases in cytosolic Cl– concentration decreased {alpha}-ENaC mRNA and decreased Na+ reabsorption. These findings support the hypothesis that changes in cytosolic Cl– concentration are an important and novel signal in hypotonicity-induced regulation of {alpha}-ENaC expression and Na+ reabsorption.

epithelial sodium channel; sodium transport; epithelial sodium channel regulation; hypotonicity; sodium-potassium-2 chloride cotransporter; chloride channels

PARTS OF THE RENAL EPITHELIUM are among a small number of the tissues routinely exposed to variable extracellular osmolality under physiological conditions that require cellular responses to maintain appropriate cellular volume and osmolality. Hypotonicity causes initial cell swelling followed by regulatory volume decrease (RVD) to return cell volume toward the original volume. The mechanism generating RVD is well understood; i.e., KCl release occurs through volume-sensitive K+ and Cl– channels leading to a substantial decrease in cytosolic Cl– concentration. Generally, the increase in membrane tension associated with hypotonicity-induced increases in cell volume is viewed as the signal that initiates RVD. However, while the release of KCl in the face of a hypotonic challenge efficiently returns cell volume to normal, the changes in the intracellular environment associated with RVD have other consequences. In particular, the large change in cytosolic Cl– concentration has been reported to modify gene expression [e.g., cytosolic Cl– regulates the cyclooxygenase (COX)-2 gene expression in macula densa cells (5, 34), the activity of the Na+-K+-2Cl– cotransporter in dog tracheal cells (13, 14) and in human trabecular meshwork cells (27)].

Many of the renal epithelial cells that are exposed to varying tonicity have an additional problem associated with regulating their volume: they are involved in the transport of large quantities of NaCl and water, often times turning over the total cellular content of sodium in a matter of minutes. Renal Na+ reabsorption plays an essential role in the regulation of total body Na+ balance, extracellular fluid (ECF) volume, and blood pressure (10, 11, 15). Thus it would not be surprising if Na+ transport were altered by the hypotonicity-induced changes in cell volume in a manner consistent with maintaining normal plasma osmolality; i.e., reduced tonicity would promote increased Na+ reabsorption to increase plasma tonicity toward normal. In fact, in A6 cells, a tissue culture model of distal nephron Na+ transport, we and others previously showed that significant reductions in tonicity enhance Na+ transport (6, 26, 33). In our previous work, the acute (~1 h) osmoregulation of Na+ transport is mainly due to the translocation of epithelial sodium channel (ENaC) protein to the apical membrane mediated by a protein tyrosine kinase-dependent pathway (26).

In A6 cells, like many other Na+-transporting epithelia tissues (e.g., kidney, colon, airways, and ducts of several secretory glands), the amiloride-sensitive ENaC is responsible for the majority of transepithelial Na+ absorption. ENaC in the apical membrane of Na+-reabsorbing epithelial cells is, under most circumstances, the rate-limiting step for transepithelial Na+ transport and, therefore, regulation of ENaC is the principle mechanism by which transepithelial Na+ transport is altered. ENaC is composed of three subunits, {alpha}, {beta}, and {gamma}. Each subunit is thought to play a role in determining the transport properties of ENaC with all three subunits forming a heteromultimeric complex (4, 12, 18). However, when each subunit ({alpha}, {beta}, or {gamma} alone) is individually expressed in a Xenopus laevis oocyte system, only the {alpha}-subunit can produce Na+ current (3, 21). Thus the {alpha}-subunit of ENaC ({alpha}-ENaC) plays an essential role in functional expression of ENaC. This fact is supported by a recent study that shows that in Na+-transporting lung alveolar cells {alpha}-ENaC is required for any transport and can even act as a Na+-permeable channel in the absence of other subunits (16). Therefore, in this work, we focused our study on the hypotonicity-induced changes in {alpha}-ENaC mRNA expression. Our aim in the present study was to explore the mechanism by which chronic reductions in extracellular osmolality stimulate Na+ reabsorption. We demonstrate here that hypotonicity-induced changes in cytosolic Cl– concentration are a novel signal regulating {alpha}-ENaC mRNA expression in renal epithelium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. NCTC-109 medium and fetal bovine serum were purchased from Invitrogen (Tokyo, Japan). Permeable tissue culture supports (Nunc Tissue Culture Inserts) were obtained from Nunc (Roskilde, Denmark). Benzamil, bumetanide, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and quercetin were purchased from Sigma (St. Louis, MO). Apigenin was obtained from Calbiochem (San Diego, CA).

Solutions. The 120 NaCl isotonic solution contained 120 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES adjusted to pH 7.4. The 60 NaCl hypotonic solution contained 60 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES adjusted to pH 7.4.

Cell culture. Renal epithelial A6 cells derived from X. laevis were purchased from American Type Culture Collection. A6 cells (passages 76–84) were grown on plastic flasks in NCTC-109 medium modified for amphibian cells and supplemented with 10% fetal bovine serum (osmolality 255 mosmol/kgH2O) (23, 26). The flasks were kept in a humidified incubator at 27°C with 1.5% CO2 in air. Cells were seeded onto Nunc tissue culture supports for Northern blotting or onto Costar supports for electrophysiological measurements at a density of 5 x 104 cells/well and were cultured for 11–15 days. When we studied the effect of hypotonicity in the present report, the cells were incubated in isotonic or hypotonic culture medium for the indicated time period just before starting the short-circuit current (Isc) measurements or Northern blotting. For example, when the Isc was measured, we applied the 120 NaCl isotonic or 60 NaCl hypotonic solution after incubation of the cells in a isotonic or hypotonic culture medium for the indicated time period.

Electrophysiological measurements. Isc and transepithelial conductance (Gt) were measured as previously described (26, 30). The benzamil-sensitive Isc and the benzamil-sensitive Gt were used as a measure of transepithelial Na+ transport and ENaC activity (conductance), respectively (26). The experiments were performed at 24–25°C.

Northern blot analysis. Total RNA was prepared from monolayers of A6 cells grown on Nunc filter by using RNeasy Mini Kit (Qiagen, Tokyo, Japan). Total RNA of 20 µg was separated on 1.2% agarose-formaldehyde gels and blotted onto a nylon membrane (Hybond N+, Amersham Pharmacia Biotech, Tokyo, Japan). The membrane was hybridized with {alpha}-ENaC cDNA probe (28) labeled with a specially developed thermostable alkaline phosphatase enzyme (AlkPhos Direct Labeling and Detection System, Amersham Pharmacia Biotech). Then, the signal in blots was detected by using CDP star chemiluminescent detection reagent (Amersham Pharmacia Biotech). Equal loading of RNA was confirmed by measuring the amount of 18S and 28S rRNA.

Data presentation. Statistical comparisons used Student's t-test or ANOVA as appropriate. A P value <0.05 was considered significant. All data shown in the present study are represented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Our previous study indicates that hypotonicity stimulates Na+ reabsorption mainly by increasing the number of ENaC in the apical membrane via transient tyrosine phosphorylation (26). However, it is unclear whether the hypotonicity-induced increase in apical ENaC protein and Na+ reabsorption is, at least in part, due to an increase in ENaC gene expression. Therefore, we assessed the effect of hypotonicity on mRNA expression of {alpha}-ENaC by exposing monolayers of A6 cells to hypotonic culture medium. We detected a significant hypotonicity-induced increase in {alpha}-ENaC mRNA expression (Fig. 1).



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  		Fig. 1. Hypotonicity induces {alpha}-epithelial sodium channel ({alpha}-ENaC) mRNA expression. A: total RNA was isolated from the A6 cell monolayers, which were exposed to hypotonic culture medium (50% normal osmolality) for the indicated times and subjected to Northern blot analysis with an {alpha}-ENaC cDNA probe. B: relative {alpha}-ENaC mRNA expression induced by a hypotonic challenge. Equal loading of RNA was confirmed by the measurement of 18S and 28S rRNA amount. Data are presented as means ± SE (n = 4).

 
To determine whether a hypotonic-induced increase of {alpha}-ENaC mRNA expression can also functionally stimulate Na+ reabsorption in A6 cell monolayers, we measured the benzamil (a specific blocker of ENaC)-sensitive Isc to estimate the transcellular Na+ reabsorption. As shown in Fig. 2A, the benzamil-sensitive Isc was substantially increased by exposure of A6 cell monolayers to a hypotonic culture medium for 24 h. The benzamil-sensitive Gt also increased (Fig. 2B). These results suggest that hypotonicity can stimulate transcellular Na+ reabsorption through induction of {alpha}-ENaC mRNA expression thereby increasing apical ENaC protein. In contrast, hypotonicity eliminated NPPB-sensitive Isc (Fig. 2C) (NPPB is a blocker of apical Cl– channels) even though hypotonicity still significantly increased the NPPB-sensitive Gt (Fig. 2D). Based on the DISCUSSION below, this indicates that, in the presence of hypotonic solution, the apical Cl– channels contributing to Cl– release are active but because the basolateral membrane potential appears to be near the chloride reversal potential, the Na+-K+-2Cl– cotransporter in the basolateral membrane contributing to Cl– uptake must be relatively inactive. Cl– transport occurs in two steps: 1) Cl– uptake via the basolateral Na+-K+-2Cl– cotransporter from the serosa into the cell and 2) Cl– release via apical Cl– channels from the cell into the apical compartment. The amount of transepithelial Cl– transport can be limited by whichever step has a lower transport activity, channel, or cotransporter. In the present manuscript, we show that hypotonicity increased the conductance but decreased the activity of the cotransporter, suggesting that under hypotonic conditions, the rate-limiting step for Cl– transport would be the cotransporter. Therefore, although the conductance was increased by hypotonicity, the Isc was decreased due to the hypotonicity-induced diminution of the cotransporter activity. On the other hand, for Na+ transport, the rate-limiting step is usually the entry step for Na+ through ENaC. Therefore, the amiloride-sensitive Isc changes in parallel to the conductance. Furthermore, the hypotonic-induced increase in the NPPB-sensitive conductance is due to an increase in the number of the Cl– channels in the apical membrane.



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  		Fig. 2. Hypotonicity stimulates Na+ reabsorption by increasing the benzamil-sensitive transepithelial conductance (Gt). Benzamil-sensitive short-circuit current (Isc; A), benzamil-sensitive Gt (B), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB)-sensitive Isc (C), and NPPB-sensitive Gt (D) were measured under isotonic and hypotonic conditions. The Isc and Gt were measured in A6 cell monolayers after 24-h exposure to a hypotonic culture medium or an isotonic culture medium. Data are presented as means ± SE (n = 6).

 
Our next question was to clarify how hypotonicity induces {alpha}-ENaC mRNA expression. Hypotonicity causes biphasic cell volume changes; i.e., initial cell swelling followed by RVD (25). Because RVD is associated with a drastic decrease in cytosolic Cl– concentration, we examined the possible role of cytosolic Cl– in regulating Na+ reabsorption and {alpha}-ENaC mRNA expression. Generally, the cytosolic Cl– concentration is determined by the balance between Cl– uptake through the Na+-K+-2Cl– cotransporter at the basolateral membrane and Cl– secretion through Cl– channels at the apical membrane. This implies that cytosolic Cl– concentration will increase when Cl– secretion is inhibited by Cl– channel blockers such as NPPB and will decrease when Cl– uptake is blocked by inhibitors of the Na+-K+-2Cl– cotransporter such as bumetanide. In response to hypotonicity, if KCl release is blocked by the chloride channel blocker NPPB, the decrease in cytosolic Cl– concentration associated with RVD would be impaired and the cytosolic Cl– concentration would remain at a level higher than without NPPB treatment. Therefore, we examined the effect of NPPB on the changes in {alpha}-ENaC mRNA expression induced by hypotonic media. For these experiments, cells were incubated in an isotonic culture medium containing 100 µM NPPB for 30 min. Then, the media was changed to either an isotonic or a hypotonic culture medium containing 100 µM NPPB for 24 h (for a total exposure to 100 µM NPPB of 24 h 30 min). Treatment with NPPB suppressed the {alpha}-ENaC mRNA expression under both isotonic and hypotonic conditions (Fig. 3, A and C). This result suggests that the increased cytosolic Cl– concentration produced by NPPB treatment suppressed basal (isotonic) and hypotonicity-induced {alpha}-ENaC mRNA expression and that cytosolic Cl– plays a key role in the regulation of {alpha}-ENaC mRNA expression. On the other hand, to study effects of the decreased cytosolic Cl– concentration on {alpha}-ENaC mRNA expression, we used a blocker of the Na+-K+-2Cl– cotransporter bumetanide to reduce cytosolic Cl– concentration. Under the isotonic (basal) conditions, treatment with bumetanide increased {alpha}-ENaC mRNA expression (Fig. 3, B and C). However, bumetanide did not affect the hypotonicity-induced {alpha}-ENaC mRNA expression (Fig. 3, B and C). This is consistent with the observation described above (Fig. 2C) that there is no measurable NPPB-sensitive Isc in hypotonic media despite the fact that hypotonicity increased NPPB-sensitive Gt above that in isotonic solution (Fig. 2D). This indicates that the apical Cl– channels are functional under the hypotonic condition even though there is no transcellular Cl– transport. Taken together, these observations suggest that the elimination of transcellular Cl– transport under hypotonic conditions is due to a reduction in activity of the bumetanide-sensitive Na+-K+-2Cl– cotransporter. If the Na+-K+-2Cl– cotransporter is inactive, then bumetanide will have no effect on cytosolic Cl– concentration and will also have no effect on {alpha}-ENaC mRNA expression under the hypotonic conditions. On the other hand, under the isotonic conditions, bumetanide blocks a functional cotransporter and slightly decreases cytosolic Cl– concentration, which induced {alpha}-ENaC mRNA expression.



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  		Fig. 3. NPPB (a Cl– channel blocker) inhibits the hypotonicity-induced increase in {alpha}-ENaC mRNA expression. {alpha}-ENaC mRNA expression was determined by Northern blot analysis. A: effects of NPPB (100 µM) on the basal (isotonic) and hypotonicity-induced {alpha}-ENaC mRNA expression. {alpha}-ENaC mRNA was measured in lysates from A6 cell monolayers after 24-h exposure to a hypotonic culture medium (50% diluted) or an isotonic culture medium (normal). B: effects of bumetanide (BMT; 100 µM) on the basal (isotonic) and hypotonicity-induced {alpha}-ENaC mRNA expression. C: relative {alpha}-ENaC mRNA expression under the conditions described above. HYPO (–), isotonic condition; HYPO (+), hypotonic condition; NS, not significant. Data are presented as means ± SE (n = 3). *P < 0.05 compared with HYPO (–). #P < 0.05 compared with HYPO (+).

 
As mentioned above, treatment with NPPB for 24 h significantly diminished the expression of {alpha}-ENaC mRNA. Therefore, we examined whether NPPB also reduces the hypotonicity-stimulated Na+ reabsorption. NPPB of 100 µM after which treatment of A6 cell monolayers for 24 h markedly decreased the hypotonicity-induced, benzamil-sensitive Isc (Fig. 4A) and benzamil-sensitive Gt (Fig. 4B); however, treatment with bumetanide had no effect on the hypotonic-induced benzamil-sensitive Isc (Fig. 4A) or the benzamil-sensitive Gt (Fig. 4B). On the other hand, under the isotonic conditions, treatment of A6 cells with 100 µM bumetanide for 24 h increased the benzamil-sensitive Isc from 0.19 ± 0.02 to 0.42 ± 0.03 µA/cm2 (n = 4, P < 0.05). These observations were consistent with the bumetanide action on {alpha}-ENaC mRNA expression as described above.



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  		Fig. 4. Effects of NPPB and BMT on the hypotonicity-stimulated Na+ reabsorption. The benzamil-sensitive Isc (A) and benzamil-sensitive Gt (B) were measured in A6 cell monolayers after 24-h exposure to a hypotonic culture medium (50% diluted). HYPO, hypotonic treatment without NPPB or BMT; HYPO + NPPB, hypotonic treatment with 100 µM NPPB; HYPO + BMT, hypotonic treatment with 100 µM BMT. NPPB significantly reduced the benzamil-sensitive Isc associated with a decrease in the benzamil-sensitive Gt. Data are presented as means ± SE (n = 6). *P < 0.05 compared with HYPO.

 
Apigenin activates the Na+-K+-2Cl– cotransporter (22) and thereby promotes accumulation of cytosolic Cl–. Therefore, we applied apigenin to increase cytosolic Cl– concentration under both hypotonic and isotonic conditions. Treatment with 100 µM apigenin in the apical and basolateral culture media inhibited the hypotonicity-induced increase in {alpha}-ENaC mRNA expression (Fig. 5). Furthermore, pretreatment with both apigenin and NPPB (to prevent secretion) strongly inhibited hypotonicity-induced increases in {alpha}-ENaC mRNA expression to a level less than 30% of control (Fig. 5). We also examined the effects of apigenin (100 µM) on the {alpha}-ENaC mRNA expression under basal (isotonic) conditions with or without 100 µM NPPB. Under basal (isotonic) conditions, apigenin treatment without NPPB showed a tendency to suppress the {alpha}-ENaC mRNA expression (Fig. 5). Furthermore, the simultaneous treatment with apigenin and NPPB was more effective in the suppression than the sole treatment with apigenin or NPPB alone (Fig. 5), because cytosolic Cl– concentration should become much higher after the simultaneous application of apigenin and NPPB rather than the sole application of either apigenin or NPPB.



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  		Fig. 5. Apigenin and NPPB inhibit the hypotonicity-induced increase in {alpha}-ENaC mRNA expression. A: {alpha}-ENaC mRNA was measured in lysates from A6 cell monolayers after 24-h exposure to a hypotonic culture medium (50% diluted) or an isotonic medium (normal) with or without 100 µM NPPB and 100 µM apigenin. B: relative {alpha}-ENaC mRNA expression induced by hypotonicity with or without 100 µM apigenin and 100 µM NPPB. HYPO (–), isotonic condition; HYPO (+), hypotonic condition. Data are presented as means ± SE (n = 3). *P < 0.05 compared with HYPO (–). #P < 0.05 compared with HYPO (+).

 
To assess the effects of apigenin on the Na+ reabsorption, the benzamil-sensitive Isc and Gt were measured in A6 cells treated with or without apigenin. Incubation of A6 cell monolayers with 100 µM apigenin diminished the benzamil-sensitive Isc and Gt in the hypotonic culture medium (HYPO in Fig. 6), but not in the isotonic medium (ISO in Fig. 6). These results suggest that the increased cytosolic Cl– concentration diminished the hypotonicity-induced transcellular Na+ reabsorption by suppressing the ENaC activity and that cytosolic Cl– controls not only the {alpha}-ENaC mRNA expression level but also the functional level of the ENaC.



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  		Fig. 6. Reduction of the hypotonicity-induced, benzamil-sensitive Isc by apigenin (APIG). The benzamil-sensitive Isc (A) and benzamil-sensitive Gt (B) were measured under isotonic (ISO) and hypotonic conditions. The Isc and Gt were measured in A6 cell monolayers after 24-h exposure to a hypotonic culture medium (50% diluted) or an isotonic medium (normal) with or without 100 µM apigenin. Data are presented as means ± SE (n = 6). *P < 0.05 compared with ISO. **P < 0.05 compared with HYPO.

 
Furthermore, to confirm whether a flavone, in general, diminishes the {alpha}-ENaC mRNA expression and Na+ reabsorption by activating the Na+-K+-2Cl– cotransporter, we examined the effect of another flavone, quercetin, on the {alpha}-ENaC mRNA expression and the Na+ reabsorption, because quercetin, like apigenin, activates the Na+-K+-2Cl– cotransporter (24), resulting in an increase in cytosolic Cl– concentration. Treatment with 100 µM quercetin under basal (isotonic) and hypotonic conditions abolished the {alpha}-ENaC mRNA expression (Fig. 7) and Na+ reabsorption (Fig. 8). This result strongly suggests that apigenin and quercetin suppressed the {alpha}-ENaC mRNA expression and the Na+ reabsorption by increasing cytosolic Cl– concentration via activation of the Na+-K+-2Cl– cotransporter.



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  		Fig. 7. Quercetin and NPPB inhibit the hypotonicity-induced increase in {alpha}-ENaC mRNA expression. A: {alpha}-ENaC mRNA expression was measured in lysates from A6 cell monolayers after 24-h exposure a hypotonic culture medium (50% diluted) or an isotonic medium (normal) with or without 100 µM NPPB and 100 µM quercetin. B: relative {alpha}-ENaC mRNA expression induced by hypotonicity with or without 100 µM quercetin and 100 µM NPPB. HYPO (–), isotonic condition; HYPO (+), hypotonic condition. Data are presented as means ± SE (n = 3). *P < 0.05 compared with HYPO (–). #P < 0.05 compared with HYPO (+).

 


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  		Fig. 8. Suppression of the hypotonicity-induced benzamil-sensitive Isc by quercetin (QUER). The benzamil-sensitive Isc (A) and benzamil-sensitive Gt (B) were measured under isotonic and hypotonic conditions. The Isc and Gt were measured in A6 cell monolayers after 24-h exposure to a hypotonic culture medium (50% diluted) or an isotonic medium (normal) with or without 100 µM quercetin for 24 h. Data are presented as means ± SE (n = 4). *P < 0.05 compared with ISO. **P < 0.05 compared with HYPO.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrate a possible mechanism by which a long-term reduction in tonicity can regulate renal Na+ reabsorption. Our experiments suggest that hypotonicity reduces cytosolic Cl– concentration, which in turn increases {alpha}-ENaC mRNA expression and functional ENaC activity. We and others described acute osmoregulation of Na+ reabsorption in renal epithelium (6, 26, 33), and we reported that hypotonicity stimulates Na+ reabsorption mainly by increasing the number of functional ENaC channels via a PTK-dependent pathway (26). However, the question remained open of how cells sense the extracellular osmolality and regulate Na+ transport. In the present study, we focused our study on changes that are associated with hypotonic RVD. During RVD, several events occur. First, KCl release occurs through volume-sensitive K+ and Cl– channels, so that the absolute amount of Cl– loss is identical to that of K+ loss; but because the initial cytosolic Cl– concentration is much lower than cytosolic K+, the relative magnitude of Cl– loss is much larger than that of K+ and water loss [refer to our previous article (19, 20)]. For these reasons, cytosolic Cl– concentration is known to decrease during RVD. In the present study, we show that a hypotonic-induced change in cytosolic Cl– concentration might act as a signal to regulate Na+ reabsorption by altering ENaC gene expression. In particular, we demonstrate that 1) elevation of cytosolic Cl– concentration (produced by either blocking apical Cl– efflux channels or activating basolateral influx via the Na+-K+-2Cl– cotransporter) suppresses the hypotonicity-induced {alpha}-ENaC expression, 2) reduction of cytosolic Cl– concentration (produced by blocking the Na+-K+-2Cl– cotransporter) enhances {alpha}-ENaC expression, and 3) an increase in Na+ reabsorption is associated with the hypotonicity-induced increases in {alpha}-ENaC expression.

Our preliminary observations on the cytosolic Cl– and K+ concentrations using Cl–- and K+-sensitive fluorescent dyes indicate that the cytosolic Cl– concentration was changed by application of bumetanide or flavones but that the cytosolic K+ concentration was not significantly altered. This observation is not surprising because, in epithelial cells, the initial cytosolic K+ concentration is much higher than the initial cytosolic Cl– concentration. Our measurements show that the cytosolic K+ concentration is ~120 mM (meq/l) and the cytosolic Cl– concentration is 40–60 mM (meq/l). When the same amounts of Cl– and K+ move into or out of the cytosol, the cytosolic Cl– concentration can change substantially without a proportionally large change in cytosolic K+ concentration (19). Therefore, although, in theory, the effect of bumetanide or flavones on {alpha}-ENaC mRNA expression and/or Na+ transport could be secondary to a change in intracellular potassium concentration, because of the magnitude of the changes in ion concentrations, we suggest that the effects of the two agents are most likely mediated through changes in cytosolic Cl–. Nevertheless, an examination of the effects of cytosolic K+ on {alpha}-ENaC mRNA expression and/or Na+ transport may be appropriate.

In this paper, we only examined the effect of hypotonicity and cytosolic Cl– on {alpha}-ENaC expression. However, the {alpha}-ENaC subunit alone can form an amiloride-sensitive cation channel (2, 17). Therefore, an increase in {alpha}-ENaC by itself is sufficient to increase Na+ transport. On the other hand, {alpha}-ENaC often forms together with {beta}- and {gamma}-ENaC subunits an amiloride-sensitive cation channel, increasing the Na+ transport. Nonetheless, the {alpha}-subunit is the ionophoric component so that an increase in expression of {alpha}-ENaC will increase transport regardless of the nature of the channel formed from the new {alpha}-ENaC.

A literature report (29) indicates that hypotonicity increases Na+ transport in A6 cells by augmenting SGK1 expression. This stimulation is observed within 15 min after exposure to hypotonic solutions. We also previously reported that hypotonicity increased Na+ transport in A6 by translocating ENaC channel preexisting in a subapical intracellular pool to the apical membrane via a calmodulin-dependent pathway (25, 30) and that this stimulation of Na+ transport was observed within 15 min after application of hypotonicity. Taken together, these observations suggest that hypotonicity increases Na+ transport by augmenting SGK1 expression at early times after exposure to hypotonicity and by increasing ENaC expression if the hypotonicity is sustained for a longer period of time.

As mentioned above, we focused our present study on the hypotonic action on ENaC mRNA expression and Na+ transport, which relatively slowly appears compared with the hypotonic action on the ENaC protein preexisting in the cytosolic space (e.g., trafficking of the ENaC protein). Some previous studies including ours (26, 29, 30) indicate that hypotonicity increases the Na+ transport within 15 min after application of hypotonicity. Specifically, Rozansky and colleagues (29) showed the effect on ENaC within 15 min after application of hypotonicity does not depend on transcription of mRNA but if hypotonicity persists for longer than 30 min, mRNA transcription is increased including SGK1 mRNA. In the present study, we indicate that hypotonicity began to elevate {alpha}-ENaC mRNA 1 h after hypotonic shock. Therefore, although the SGK1 action on {alpha}-ENaC mRNA is still unclear, it is possible that hypotonicity, besides increasing {alpha}-ENaC mRNA, could also increase sodium transport by activating SGK1.

In loading gels, we attempted to carefully load equal amounts of RNA. We apparently succeeded because, even if we normalized our mRNA amounts based the density of the 18S and 28S rRNA bands, the results were not significantly different than uncorrected values.

The Isc value shown in the present report is identical to that previously reported from our laboratory (e.g., 26). This low value compared with that reported from other laboratories (29) is due to the culture medium and filter substrate.

Interestingly, cytosolic Cl– regulates {alpha}-ENaC mRNA expression under both the isotonic and hypotonic conditions. This implies that cytosolic Cl– always has a crucial function in controlling {alpha}-ENaC mRNA expression and is not just a special signal involved only in hypotonicity-mediated responses. Recent studies demonstrate the importance of cytosolic Cl– for several fundamental cellular responses, regulation of ion transporter function, and control of gene expression; e.g., in macula densa cells, lowering Cl– stimulates PGE2 release and COX-2 expression through activation of MAP kinases (5, 34), and a decrease in cytosolic Cl– concentration and/or cell volume plays a key role in regulation of the Na+-K+-2Cl– cotransporter in dog tracheal cells (13, 14) and human trabecular meshwork cells (27).

Usually inhibitors of ion transporters and ion channels are developed and widely used for studies of ion transport. However, to our knowledge, activators for ion transporters and ion channels are more rarely available and used. However, recent reports describe the action of flavones, multifunctional compounds extracted from soy beans and other plants, on cell cycle, apoptosis, ion transport, and gene expression (22, 32, 35). In our previous report, we showed that flavones have stimulatory effects on Cl– secretion by activating the cystic fibrosis transmembrane conductance regulator Cl– channel and/or the Na+-K+-2Cl– cotransporter depending on the flavone (22). Recent reports indicated that flavones, especially quercetin, play a role in decreasing blood pressure in the hypertensive rats, although the mechanism is not well understood (8, 9). On the other hand, Na+ reabsorption in the distal nephron contributes to the fine control of total body Na+ content and ECF volume, which are important factors in regulating blood pressure. Indeed, quercetin treatment significantly suppressed the {alpha}-ENaC mRNA expression under both the isotonic and hypotonic conditions. Therefore, we hypothesize that quercetin controls blood pressure by activating the Na+-K+-2Cl– cotransporter, increasing cytosolic Cl– concentration, thereby suppressing ENaC gene expression with a subsequent reduction in blood pressure. Therefore, quercetin, in particular, and flavones, in general, may be candidates for pharmacological regulators of blood pressure through regulation of ENaC expression. Indeed, our recent study reveals that quercetin intake diminished {alpha}-ENaC mRNA expression in the kidney associated with a reduction of blood pressure elevated by high-salt diet in Dahl salt-sensitive rats (1).

In the present report, we indicate the possible role of cytosolic Cl– in the regulation of {alpha}-ENaC mRNA expression. However, the specific mechanism by which cytosolic Cl– can regulate {alpha}-ENaC mRNA expression is unclear. One report (29) indicates that SGK1, the expression of which is regulated by hypotonic stress, controls ENaC; therefore, the action of cytosolic Cl– on {alpha}-ENaC mRNA expression might be mediated by SGK1, but the answer to this question awaits additional experiments.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grants-in-Aid from Japan Society of the Promotion of Science (15590189), the Ministry of Education, Culture, Sports, Science and Technology (15659052), The Salt Science Research Foundation (0241), the Ministry of Health, Labor and Welfare for Nervous and Mental Disorders (15A-4) and for Child Health and Development (14C-6), the grant of a leading project for Biosimulation from the Ministry of Education, Culture, Sports, Science and Technology to Y. Marunaka and N. Niisato, and United States Public Health Service Grants DK-037963 and DK-064399 to D. C. Eaton.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Marunaka, Dept. of Molecular Cell Physiology, Kyoto Prefectural Univ. of Medicine, Kyoto 602-8566, Japan (E-mail: marunaka{at}koto.kpu-m.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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