Original Article

In Liddle Syndrome, Epithelial Sodium Channel Is Hyperactive Mainly in the Early Part of the Aldosterone-Sensitive Distal NephronNovelty and Significance

Viatcheslav Nesterov, Bettina Krueger, Marko Bertog, Anke Dahlmann, Ralf Palmisano, Christoph Korbmacher
https://doi.org/10.1161/HYPERTENSIONAHA.115.07061
Hypertension. 2016;67:1256-1262
Originally published May 11, 2016

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Abstract

The epithelial sodium channel (ENaC) is rate limiting for Na+ absorption in the aldosterone-sensitive distal nephron comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT), and the entire collecting duct. Liddle syndrome (pseudohyperaldosteronism), a severe form of salt-sensitive hypertension, is caused by gain-of-function mutations of ENaC, but the precise tubular site of increased ENaC function is unknown. In the cortical collecting duct (CCD), ENaC is known to be regulated by aldosterone. In contrast, we recently reported aldosterone-independent ENaC regulation in the early part of the aldosterone-sensitive distal nephron. Here, we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice homozygous for Liddle syndrome mutation or from wild-type control mice. Whole-cell patch-clamp recordings were used to measure amiloride-sensitive ENaC currents in nephron fragments from mice maintained on different sodium diets to vary plasma aldosterone levels. Our data indicate that in mice with Liddle syndrome, the primary site of increased Na+ reabsorption is the DCT2/CNT. In addition, increased aldosterone responsiveness of ENaC in CNT/CCD may contribute to salt-sensitive hypertension in Liddle syndrome. Single channel properties of ENaC were similar in Liddle syndrome mutation and wild-type mice, but ENaC expression at the apical membrane was increased in Liddle syndrome mutation when compared with wild-type mice, in particular, in animals maintained on a high salt diet. Our findings highlight the importance of ENaC function and regulation in the early part of the aldosterone-sensitive distal nephron for the maintenance of sodium balance and blood pressure control.

Introduction

Liddle syndrome (pseudohyperaldosteronism) is a monogenic form of salt-sensitive hypertension associated with hypokalemia, metabolic alkalosis, suppressed plasma renin activity, and low plasma aldosterone levels.1 It is caused by gain-of-function mutations in the epithelial sodium channel (ENaC).2 ENaC constitutes the rate-limiting step for sodium absorption in the aldosterone-sensitive distal nephron (ASDN), which comprises the late distal convoluted tubule (DCT2), the connecting tubule (CNT), and the entire collecting duct. The appropriate regulation of ENaC is critically important for the fine tuning of renal sodium excretion and hence for the maintenance of body sodium homeostasis and the long-term control of arterial blood pressure.3 It is generally accepted that Liddle syndrome results primarily from an inappropriately high ENaC activity in the kidney,4 but the precise tubular site of this increased ENaC activity has not been established. In addition, ENaC localized in colon,5 lung,6 and brain7 has been reported to be hyperactive in mouse models of Liddle syndrome. ENaC activity depends on channel open probability and on the number of channels expressed at the cell surface. The latter is determined by the rate of channel insertion and channel retrieval. The majority of mutations causing Liddle syndrome disrupt PY motifs localized in the C termini of the β- or γ-subunit of ENaC.8 These PY motifs serve as binding site for the ubiquitin ligase NEDD4-2, a NEDD4 (neural precursor cell–expressed developmentally down-regulated protein 4) family member.9 Binding at these sites facilitates NEDD4-2–mediated channel ubiquitination with subsequent channel retrieval and proteasomal degradation.10 Mutations in the PY motifs disrupt the physiological NEDD4-2/ENaC interaction. This results in an increase in the number of channels at the cell surface, which is thought to be the main mechanism by which Liddle syndrome mutations cause a gain-of-function effect.2,11–13 In addition, it has been reported that mutations causing Liddle syndrome increase channel open probability (Po),13,14 enhance proteolytic channel cleavage,15 and reduce Na+ feedback inhibition.16

Aldosterone stimulates the expression of the serum and glucocorticoid–regulated kinase 1 (SGK1), which phosphorylates NEDD4-2, and thereby reduces its affinity to ENaC. The resulting inhibition of Nedd4-2–mediated channel retrieval is thought to play a key role in the stimulatory effect of aldosterone on ENaC function.17 This concept predicts that aldosterone should have a reduced stimulatory effect on ENaC with Liddle syndrome mutation because aldosterone and the mutation stimulate ENaC by the same mechanism. However, it has been shown that patients with Liddle syndrome readily reduce their urinary sodium excretion in response to aldosterone1 and that ENaC has an increased rather than decreased responsiveness to aldosterone in a mouse model of Liddle syndrome5,12 and in cultured renal epithelial cells expressing ENaC with Liddle syndrome mutation.18 Moreover, in the cortical collecting duct (CCD)12 of mice homozygous for Liddle syndrome mutation (L/L), enhanced ENaC activity was prominent only when aldosterone levels were high, ie, when animals had a low dietary sodium intake or were treated with aldosterone. In contrast, patients with Liddle syndrome typically have low plasma aldosterone levels, and L/L mice develop significant arterial hypertension on high Na+ diet, when aldosterone levels are suppressed.19 How can this paradox be explained?

The absence of increased ENaC activity in the CCD of L/L mice on standard salt diet suggests that ENaC activity may be enhanced in more proximal parts of the ASDN, ie, in the DCT2 and CNT. These nephron segments have not yet been studied in Liddle mice but are thought to contribute substantially to ENaC-mediated sodium absorption in the kidney.20–22 Previously, we reported that ENaC activity in DCT2/CNT is constitutively high and independent of aldosterone. This is in contrast to the aldosterone dependence of ENaC activity in CNT/CCD.23 We hypothesized that mutations causing Liddle syndrome affect ENaC activity differently in the DCT2/CNT and CNT/CCD and that an increased ENaC activity in the DCT2/CNT may account for sodium retention and hypertension observed in patients with Liddle syndrome. Therefore, the aim of the present study was to investigate ENaC regulation in the DCT2/CNT and CNT/CCD in a mouse model of Liddle syndrome19 using different sodium diets to vary plasma aldosterone levels. The activity and biophysical properties of ENaC were investigated using the whole-cell and outside-out configuration of the patch-clamp technique. In addition, we studied the subcellular distribution of ENaC in isolated fragments of the ASDN by immunohistochemistry.

Methods

Mouse Model

An established mouse model for Liddle syndrome12,19,24 was used, and animals were maintained as previously described.5 All animals used in the present study were from this mouse line and were either homozygous for the R566 Liddle syndrome mutation in βENaC (L/L or Liddle mice) or homozygous for wild-type ENaC (WT mice). Animals routinely were maintained on a standard salt diet (SD; 3.2 g Na+/kg of food). Alternatively, mice were kept on a low-salt diet (LD; 0.13 g Na+/kg) or high salt diet (HD; 16 g Na+/kg and 0.9% saline as drinking water) for 2 weeks prior to the experiments.5 Food was obtained from Altromin (Lage, Germany).

Preparation of Renal Tubules and Electrophysiology

Tubules were prepared from mice aged 9 to 12 weeks, and patch-clamp experiments were performed essentially as previously described.23,25

Immunohistochemical Analysis

To investigate the subcellular localization of ENaC, isolated tubules were stained using subunit specific antibodies against β- and γENaC.26 Specimens were examined by confocal laser scanning microscopy.

Statistics

Values are reported as mean±SEM. Statistical significance was evaluated by Student t test, and factorial ANOVA with post hoc Tukey HSD (honest significant difference) test, as appropriate; P<0.05 was considered significant for the rejection of the null hypothesis. Experimental techniques are described in more detail in the online-only Data Supplement.

Results

In CNT/CCD, the Gain-Of-Function Effect of Liddle Syndrome Mutation on ENaC Is Most Evident in Mice Maintained on an HD

Experiments were systematically performed in 2 distinguishable regions of the ASDN designated DCT2/CNT and CNT/CCD.23 After transfer of the tubular fragments to the perfusion chamber, experiments were routinely started in a bath solution containing 2 μmol/L amiloride to inhibit ENaC and reduce channel rundown.27 The appearance of an inward current component on washout of amiloride, as well as the rapid return of the whole-cell current toward its initial level on readdition of amiloride, indicated that the cell under investigation expressed active ENaC in its apical plasma membrane. Representative whole-cell current recordings obtained from the CNT/CCD of WT and Liddle mice are shown in Figure 1A and 1B, respectively. The middle traces were obtained from mice maintained on SD. As previously reported,23 under these conditions, the amiloride-sensitive ENaC whole-cell current (ΔIami) in CNT/CCD from WT animals is small but clearly detectable (34.0±8.9 pA; n=10; Figure 1A, middle trace). In L/L mice maintained on SD, the current response to amiloride removal and readdition was qualitatively similar to that observed in WT animals (Figure 1B, middle trace). However, the average ΔIami was significantly higher in L/L mice (72.5±11.2 pA; P<0.05; n=12; Figure 1C) when compared with that observed in WT mice. This is consistent with a gain-of-function effect of Liddle syndrome mutation and the previously reported finding that isolated perfused CCD from L/L mice exhibit higher transepithelial potential differences than perfused CCD isolated from WT mice.24 Maintaining the animals on LD substantially increased ΔIami in both, WT and L/L mice (Figure 1A and 1B, right traces), with a nonsignificant trend for a larger average ΔIami in L/L mice (343±60 pA; n=17) compared with that in WT mice (241±69 pA; n=11; Figure 1C). HD largely suppressed the current response to amiloride washout and readdition in the CNT/CCD of WT mice (Figure 1A, left trace) and reduced average ΔIami to 7.8±1.5 pA (n=12; Figure 1C). In contrast, in the CNT/CCD from L/L animals, HD failed to suppress ENaC activity, which was similar to that in L/L animals on SD (Figure 1B, left trace) with an average ΔIami of 71.7±10.8 pA (n=11). Thus, in the CNT/CCD, the gain-of-function effect of Liddle syndrome mutation on ENaC is most evident in animals maintained on HD, whereas the effect is less prominent in L/L and WT animals maintained on SD or LD (Figure 1C).

Figure 1.
Figure 1.

Effect of Liddle syndrome mutation on epithelial sodium channel currents in the connecting tubule/cortical collecting duct (CNT/CCD) from mice maintained on different salt diets. A, Representative whole-cell current (ΔIami) traces obtained from CNT/CCD of wild-type (WT) mice maintained on a high, standard, or low salt diet. Black bars indicate application of amiloride (2 μmol/L). B, Corresponding traces obtained from CNT/CCD of Liddle syndrome mutation (L/L) mice. C, Average ΔIami values (n=10–12 per group) summarized from similar experiments as shown in A. *P<0.05, ***P<0.001. n.s. indicates nonsignificant.

In DCT2/CNT, the Gain-of-Function Effect of Liddle Syndrome Mutation Is Largely Independent of Dietary Salt Intake

As illustrated by the representative current traces shown in Figure 2A, substantial ENaC currents were measured in the DCT2/CNT from WT mice with ΔIami averaging 187±67 pA (n=11), 295±70 pA (n=10), and 305±38 pA (n=13; Figure 2C) in animals maintained on HD, SD, and LD, respectively. Thus, under all dietary conditions, ENaC currents in the DCT2/CNT were of similar magnitude as ENaC currents observed in the CNT/CCD of WT mice maintained on LD. In the DCT2/CNT, LD did not significantly increase ΔIami and HD only partially reduced ΔIami, which is in contrast to the findings in the CNT/CCD. These results are in good agreement with our previous study in which we demonstrated aldosterone-dependent regulation of ENaC in the CNT/CCD and aldosterone-independent regulation in the DCT2/CNT.23 Importantly, in the DCT2/CNT of L/L mice, ENaC currents were much larger than those observed in WT animals under all dietary conditions used (Figure 2B). ΔIami averaged 718±160 pA (n=10), 1136±144 pA (n=12), and 991±116 pA (n=12) in DCT2/CNT of L/L mice maintained on HD, SD, or LD, respectively (Figure 2C). Thus, in DCT2/CNT, Liddle syndrome mutation stimulated ENaC currents ≈3-fold independently of the salt diet.

Figure 2.
Figure 2.

Effect of Liddle syndrome mutation on epithelial sodium channel currents in the distal convoluted tubule/connecting tubule (DCT2/CNT) from mice maintained on different salt diets. A, Representative whole-cell current (ΔIami) traces obtained from the DCT2/CNT of wild-type (WT) mice maintained on a high, standard, or low salt diet. Black bars indicate application of amiloride. B, Corresponding traces obtained from DCT2/CNT of Liddle syndrome mutation (L/L) mice. C, Average ΔIami values (n=10–13 per group) summarized from similar experiments as shown in A.

Single-Channel Properties of ENaC in L/L Mice Are Similar to Those in WT Animals

We compared single-channel current properties of ENaC recorded in L/L mice with those in WT animals. Representative single-channel current traces from a WT and L/L mouse are shown in Figure 3A and 3B, respectively. Measurements were started in the whole-cell configuration (Figure 3A and 3B, left), and then continued in the outside-out mode for the analysis of single-channel properties. In L/L mice, single-channel events could be resolved only in the CNT/CCD but not in the DCT2/CNT because of the high ENaC activity in this latter segment. However, we previously demonstrated that single-channel properties of ENaC in the DCT2/CNT are similar to those in the CNT/CCD.23 Single-channel current amplitudes and NPo values were determined using binned amplitude histograms (Figure 3A and 3B). A binomial fit routine was used to estimate open probability (Po) and number of channels in the patch (N) from NPo and from the observed distribution of the probability of n channels being open simultaneously (Figure 3C and 3D; Methodological details are available in the online-only Data Supplement). Results from similar experiments as those shown in Figure 3 are listed individually in Table S1 in the online-only Data Supplement. On average, the single-channel current amplitude recorded in L/L mice at a holding potential (Vhold) of −60 mV was not significantly different from that in WT (0.31±0.02 pA, n=8 versus 0.32±0.01 pA, n=5). In both genotypes, the estimated Po values varied in a wide range (from <0.1 to 0.84 in WT and from <0.1 to 0.55 in L/L). Importantly, the average Po value in L/L animals (0.31±0.09) was similar to that in WT mice (0.32±0.08), and there was no major effect of diet or segment on Po, albeit in a small sample (Table S1). These findings indicate that the observed differences of ENaC whole-cell currents in WT and in L/L animals mainly reflect differences in the number of channels expressed at the cell surface, whereas the single-channel conductance and Po are similar in both genotypes.

Figure 3.
Figure 3.

Single-channel activity of epithelial sodium channel in the aldosterone-sensitive distal nephron of wild-type (WT) and Liddle syndrome mutation (L/L) mice. A, Representative current traces from an isolated distal convoluted tubule/connecting tubule (DCT2/CNT) region of a WT mouse maintained on high salt diet. Whole-cell current trace is on the left, subsequent outside-out recording from the same cell is on the right. Vhold=−60 mV. B, Similar recordings from a connecting tubule/cortical collecting duct (CNT/CCD) of a L/L mouse maintained on low salt diet. Black bars above the traces indicate presence of amiloride. Different channel open levels are indicated by dotted horizontal lines and are numbered. The current level at which all channels are closed is marked with a c. Lines below the outside-out recordings represent portions used for further analysis. Binned current amplitude histograms of these portions are shown on the right of the traces. C, Observed density of level distribution (open bars) and corresponding binomial distribution predicted by maximum likelihood estimate (black bars) for the recording shown in A. D, Observed and predicted level distributions from the recording, shown in B. To the right of the bar diagrams in C and D, the estimated values for number of channels in the patch (N) and open probability (Po) are shown.

The Liddle Syndrome Mutation Enhances the Apical Localization of ENaC in the ASDN in Particular in the DCT2/CNT

Using an antibody directed against γ-ENaC, typical ENaC staining28 was detected along the entire ASDN of WT and L/L animals under all dietary conditions used (Figures 4; Figure S1). Only a minority of ASDN cells, likely to be intercalated cells, did not show ENaC staining. Control experiments were performed with an antibody directed against β-ENaC to demonstrate the absence of ENaC staining in the ASDN of L/L mice that lack the C terminus of β-ENaC and therefore lack the epitope against which the antibody was raised (Figure S2).

Figure 4.
Figure 4.

Subcellular distribution of the γ subunit of epithelial sodium channel (γENaC) in microdissected aldosterone-sensitive distal nephron (ASDN) of wild-type (WT) and Liddle (L/L) mice maintained on low salt (A–F) or high salt diet (G–L). Red, γENaC staining; blue, DAPI (4',6-diamidino-2-phenylindole) staining. Open or filled arrow heads indicate cells with prominent apical ENaC staining or cells with predominant perinuclear ENaC localization, respectively. In A, B, G, and H, the dotted rectangles delineate the portions of the distal convoluted tubule/connecting tubule (DCT2/CNT) (C, E, I, and K) and connecting tubule/cortical collecting duct (CNT/CCD) (D, F, J, and L) shown at higher magnification in the smaller panels labeled accordingly.

In WT and L/L mice maintained on SD, prominent apical ENaC staining was limited to the DCT2/CNT in most tubular fragments investigated (Figures S1 and S3). Apical ENaC staining extended to the CNT/CCD region in WT and L/L mice maintained on LD (Figure 4; Figure S3), which is in good agreement with the observed increase in ENaC currents in the CNT/CCD under these conditions. In contrast, in mice maintained on HD, ENaC staining in the CNT/CCD of WT and L/L mice was mainly perinuclear (Figure 4; Figure S3) and was similar to the staining pattern observed in animals on SD. Importantly, in animals maintained on HD, prominent apical ENaC staining was clearly detectable in >70% of ENaC-positive cells in the DCT2/CNT of L/L mice but only in ≈10% of ENaC-positive cells in the DCT2/CNT of WT mice (Figure 4; Figure S3). Overall, these findings are consistent with our ENaC current measurements and indicate that Liddle syndrome mutation enhances the apical localization of ENaC in particular in the DCT2/CNT.

Discussion

Hyperactivity of ENaC in the DCT2/CNT Is Likely to Be the Primary Pathophysiological Mechanism of Liddle Syndrome

ENaC is expressed along the entire ASDN, but previous studies of ENaC regulation have mainly focused on the CCD. The key hormonal regulator of ENaC activity in the CCD is aldosterone. Indeed, ENaC activity is barely detectable in the CCD unless animals are kept on LD or treated with mineralocorticoids.12,29,30 Thus, ENaC-mediated sodium absorption in the CCD is probably essential in states of sodium depletion but plays a minor role when dietary sodium intake is normal or high. In contrast, ENaC activity in the DCT2/CNT is largely aldosterone independent23 and selective inactivation of ENaC in the CNT22 but not in the collecting duct20 severely compromises sodium balance in mice. In the present study, we demonstrate that in the DCT2/CNT, the Liddle syndrome mutation has a profound gain-of-function effect on ENaC in mice maintained on LD, SD, or HD. Thus, our findings solve the conundrum that in the CCD, high ENaC activity was detectable only in L/L mice maintained on LD or treated with aldosterone,12 whereas aldosterone levels typically are suppressed in patients with Liddle syndrome.1,3,4 Our results establish the DCT2/CNT as the main nephron site where ENaC is hyperactive in Liddle syndrome. This supports the emerging concept that appropriate regulation of ENaC activity in the DCT2/CNT is critically important for renal sodium homeostasis and the long-term control of blood pressure,31 namely, under low plasma aldosterone conditions. The specific hormonal, local, and molecular factors involved in ENaC regulation in the DCT2/CNT remain to be determined. The finding that ENaC activity in the DCT2/CNT is preserved in aldosterone synthase–deficient animals23 suggests that glucocorticoids and angiotensin II may play a role because these hormones are upregulated in this mouse model. In addition, differences in subunit availability, channel trafficking, and channel activation by proteases may contribute to the differential regulation of ENaC in the DCT2/CNT versus the CNT/CCD. A detailed analysis of these mechanisms will be necessary to understand why Liddle syndrome mutation has a predominant stimulatory effect in the DCT2/CNT.

Increased Aldosterone Responsiveness of ENaC in CNT/CCD May Contribute to Salt-Sensitive Hypertension in Liddle Syndrome

Liddle mice become hypertensive on HD,19 and preliminary data from telemetric blood pressure measurements indicate that blood pressure values are significantly elevated in Liddle mice even under SD (Q. Wang and E. Hummler, personal communication, University of Lausanne). Moreover, plasma aldosterone is suppressed in Liddle mice on SD,5,24 suggesting chronic hypervolemia resulting from increased ENaC-mediated renal sodium absorption. As discussed above, our findings suggest that this is mainly because of an increased ENaC activity in the DCT2/CNT. In Liddle syndrome, ENaC has been reported to have an increased responsiveness to aldosterone.5,12,18 Our present data confirm increased aldosterone responsiveness of ENaC localized in the CNT/CCD of Liddle mice. This becomes apparent when the data are interpreted in the context of previously reported plasma aldosterone values (Paldo) from mice of the same strain and maintained under essentially identical conditions.5 In animals maintained on SD, ENaC currents in the CNT/CCD of L/L mice are slightly higher than those in CNT/CCD of WT mice (Figure 1), despite a lower Paldo of ≈0.15 nmol/L in L/L mice than that of ≈0.55 nmol/L in WT mice. Interestingly, in L/L mice maintained on LD, Paldo was ≈0.54 nmol/L, which is similar to Paldo of WT mice maintained on SD. However, despite the similar Paldo, ENaC currents in L/L mice are much higher than those in WT mice (Figure 1). Taken together, these findings support the concept that ENaC localized in the CNT/CCD has an increased responsiveness to aldosterone in Liddle syndrome. Moreover, our findings indicate that in L/L mice, ENaC activity in the CNT/CCD cannot be reduced sufficiently to compensate for the increased ENaC activity in the DCT2/CNT, which is present under all dietary conditions. This inability to compensate is most prominent when L/L mice are maintained on HD because their aldosterone levels are already suppressed to near-minimal values on SD.5 Because of the increased aldosterone sensitivity of ENaC in Liddle syndrome, some residual ENaC activity is maintained in the CNT/CCD even when aldosterone levels are maximally suppressed by HD. Thus, the failure of L/L mice to downregulate ENaC activity in the CNT/CCD when challenged with HD is likely to contribute to the salt-sensitive hypertension observed in Liddle syndrome. It is tempting to speculate that pathophysiologically increased ENaC activity in the DCT2/CNT and increased aldosterone sensitivity of ENaC in the CNT/CCD may contribute to other forms of salt-sensitive hypertension.

Increased ENaC Activity in Liddle Syndrome Is Mainly Caused by an Increased Expression of the Channel at the Apical Cell Surface in Particular in the DCT2/CNT

Immunocytochemical data reported in this study support the established view that the main cause of the gain-of-function effect of Liddle syndrome is an increased expression of the mutant channel at the cell surface. This does not exclude the possibility that an increased Po of the mutant channel may contribute to the gain-of-function effect.13,14 However, in our outside-out patch-clamp recordings, we did not find any evidence for an increased Po of ENaC in L/L mice. This is in agreement with the conclusion reached in a previous study using the same mouse model of Liddle syndrome and a noise analysis approach to estimate ENaC Po in whole-cell recordings from isolated CCD.12 Our immunohistochemical data indicate that in L/L mice, ENaC expression is increased at the apical membrane, in particular, in the DCT2/CNT consistent with our ENaC current measurements.

In the kidney, long-term regulation of ENaC activity is mainly achieved by changes in the cell surface abundance of the channel.32–34 The number of ENaC channels in the luminal membrane is the net result of exocytotic delivery and the endocytotic retrieval of channels from the membrane. Liddle syndrome mutations have been shown to reduce the retrieval of ENaC from the membrane35–37 probably by interfering with Nedd4-2–dependent ubiquitylation and subsequent endocytosis.37 Under physiological conditions, Nedd4-2–mediated ENaC retrieval is thought to be inhibited by aldosterone via Nedd4-2 phosphorylation by SGK1. The resulting increase in ENaC expression at the cell surface is considered to be a major mechanism of ENaC stimulation by aldosterone. This concept predicts a reduced stimulatory effect of aldosterone on ENaC with Liddle syndrome mutation. In contrast, we demonstrate an increased aldosterone responsiveness of ENaC in L/L mice consistent with previous studies.1,5,12,18 Moreover, we demonstrate that the stimulatory effect of Liddle syndrome mutation is most prominent in the DCT2/CNT, where ENaC regulation is not dependent on aldosterone.23 Therefore, reduced Nedd4-2–mediated ENaC retrieval is probably not the only mechanism by which Liddle syndrome mutations increase the number of ENaC channels at the cell surface.

In conclusion, this study identifies the DCT2/CNT as the primary nephron site where the disease-causing gain-of-function effect of Liddle syndrome mutation occurs. This highlights the importance of ENaC regulation in the DCT2/CNT for long-term blood pressure control. The present study also confirms that in Liddle syndrome, the aldosterone responsiveness of ENaC is increased, rather than decreased.1,5,12,18 This may contribute to the development of salt-sensitive hypertension by impeding compensatory downregulation of ENaC activity in the CNT/CCD. Finally, our data confirm the concept that the gain-of-function effect of Liddle syndrome mutation is mainly the result of an increased channel expression at the cell surface, in particular, in the DCT2/CNT.

Perspectives

Increased ENaC activity may be an important contributing factor to the pathophysiology of salt-sensitive arterial hypertension not only in Liddle syndrome.3,8 Thus, the identification of the DCT2/CNT as the major nephron site for increased ENaC activity in Liddle syndrome may have implications for our understanding of the pathophysiology of other forms of salt-sensitive and low aldosterone hypertension. Moreover, our findings suggest that inhibiting ENaC activity in the DCT2/CNT may be a therapeutic strategy to treat patients with salt-sensitive hypertension. Finally, a better understanding of systemic and local mechanisms that contribute to ENaC regulation in the DCT2/CNT may reveal novel insights into the pathophysiology of salt-sensitive hypertension.

Acknowledgments

We thank Prof. Winfried Neuhuber (Lehrstuhl für Anatomie I, FAU) for his help with the analysis and interpretation of the immunocytochemical data and Jessica Rinke (Institut für Zelluläre und Molekulare Physiologie, FAU) for her expert technical assistance.

Sources of Funding

This study was supported by grant from the Deutsche Forschungsgemeinschaft (DFG, SFB423, TP-A12; to C. Korbmacher) and the Interdisciplinary Center for Clinical Research (IZKF) at the Universitätsklinikum Erlangen (to A. Dahlmann and C. Korbmacher)

Disclosures

None.

Footnotes

  • Received December 23, 2015.
  • Revision received January 4, 2016.
  • Accepted March 16, 2016.

References

  1. 1.↵
    1. Liddle GW,
    2. Bledsoe T,
    3. Coppage WS
    . A familial renal disorder simulating primary aldosteronism but with negligible aldosterone secretion. Trans Assoc Am Physicians. 1963;76:199–213.
    OpenUrl
  2. 2.↵
    1. Schild L,
    2. Canessa CM,
    3. Shimkets RA,
    4. Gautschi I,
    5. Lifton RP,
    6. Rossier BC
    . A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci U S A. 1995;92:5699–5703.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Rossier BC
    . Epithelial sodium channel (ENaC) and the control of blood pressure. Curr Opin Pharmacol. 2014;15:33–46. doi: 10.1016/j.coph.2013.11.010.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Warnock DG
    . Liddle syndrome: an autosomal dominant form of human hypertension. Kidney Int. 1998;53:18–24. doi: 10.1046/j.1523-1755.1998.00728.x.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Bertog M,
    2. Cuffe JE,
    3. Pradervand S,
    4. Hummler E,
    5. Hartner A,
    6. Porst M,
    7. Hilgers KF,
    8. Rossier BC,
    9. Korbmacher C
    . Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for Liddle’s syndrome. J Physiol. 2008;586:459–475. doi: 10.1113/jphysiol.2007.140459.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Randrianarison N,
    2. Escoubet B,
    3. Ferreira C,
    4. Fontayne A,
    5. Fowler-Jaeger N,
    6. Clerici C,
    7. Hummler E,
    8. Rossier BC,
    9. Planès C
    . β-Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice. J Physiol. 2007;582(Pt 2):777–788. doi: 10.1113/jphysiol.2007.131078.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Van Huysse JW,
    2. Amin MS,
    3. Yang B,
    4. Leenen FH
    . Salt-induced hypertension in a mouse model of Liddle syndrome is mediated by epithelial sodium channels in the brain. Hypertension. 2012;60:691–696. doi: 10.1161/HYPERTENSIONAHA.112.193045.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Rossier BC,
    2. Schild L
    . Epithelial sodium channel: mendelian versus essential hypertension. Hypertension. 2008;52:595–600. doi: 10.1161/HYPERTENSIONAHA.107.097147.
    OpenUrlFREE Full Text
  9. 9.↵
    1. Kamynina E,
    2. Debonneville C,
    3. Bens M,
    4. Vandewalle A,
    5. Staub O
    . A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. FASEB J. 2001;15:204–214. doi: 10.1096/fj.00-0191com.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Malik B,
    2. Price SR,
    3. Mitch WE,
    4. Yue Q,
    5. Eaton DC
    . Regulation of epithelial sodium channels by the ubiquitin-proteasome proteolytic pathway. Am J Physiol Renal Physiol. 2006;290:F1285–F1294. doi: 10.1152/ajprenal.00432.2005.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Snyder PM,
    2. Price MP,
    3. McDonald FJ,
    4. Adams CM,
    5. Volk KA,
    6. Zeiher BG,
    7. Stokes JB,
    8. Welsh MJ
    . Mechanism by which Liddle’s syndrome mutations increase activity of a human epithelial Na+ channel. Cell. 1995;83:969–978. doi: 10.1016/0092-8674(95)90212-0.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Dahlmann A,
    2. Pradervand S,
    3. Hummler E,
    4. Rossier BC,
    5. Frindt G,
    6. Palmer LG
    . Mineralocorticoid regulation of epithelial Na+ channels is maintained in a mouse model of Liddle’s syndrome. Am J Physiol Renal Physiol. 2003;285:F310–F318. doi: 10.1152/ajprenal.00016.2003.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Firsov D,
    2. Schild L,
    3. Gautschi I,
    4. Mérillat AM,
    5. Schneeberger E,
    6. Rossier BC
    . Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci U S A. 1996;93:15370–15375.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Anantharam A,
    2. Tian Y,
    3. Palmer LG
    . Open probability of the epithelial sodium channel is regulated by intracellular sodium. J Physiol. 2006;574(pt 2):333–347. doi: 10.1113/jphysiol.2006.109173.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Knight KK,
    2. Olson DR,
    3. Zhou R,
    4. Snyder PM
    . Liddle’s syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage. Proc Natl Acad Sci U S A. 2006;103:2805–2808. doi: 10.1073/pnas.0511184103.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Kellenberger S,
    2. Gautschi I,
    3. Rossier BC,
    4. Schild L
    . Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system. J Clin Invest. 1998;101:2741–2750. doi: 10.1172/JCI2837.
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Kamynina E,
    2. Staub O
    . Concerted action of ENaC, Nedd4-2, and Sgk1 in transepithelial Na+ transport. Am J Physiol Renal Physiol. 2002;283:F377–F387. doi: 10.1152/ajprenal.00143.2002.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Auberson M,
    2. Hoffmann-Pochon N,
    3. Vandewalle A,
    4. Kellenberger S,
    5. Schild L
    . Epithelial Na+ channel mutants causing Liddle’s syndrome retain ability to respond to aldosterone and vasopressin. Am J Physiol Renal Physiol. 2003;285:F459–F471. doi: 10.1152/ajprenal.00071.2003.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Pradervand S,
    2. Wang Q,
    3. Burnier M,
    4. Beermann F,
    5. Horisberger JD,
    6. Hummler E,
    7. Rossier BC
    . A mouse model for Liddle’s syndrome. J Am Soc Nephrol. 1999;10:2527–2533.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Rubera I,
    2. Loffing J,
    3. Palmer LG,
    4. Frindt G,
    5. Fowler-Jaeger N,
    6. Sauter D,
    7. Carroll T,
    8. McMahon A,
    9. Hummler E,
    10. Rossier BC
    . Collecting duct-specific gene inactivation of αENaC in the mouse kidney does not impair sodium and potassium balance. J Clin Invest. 2003;112:554–565. doi: 10.1172/JCI16956.
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Ronzaud C,
    2. Loffing J,
    3. Bleich M,
    4. Gretz N,
    5. Gröne HJ,
    6. Schütz G,
    7. Berger S
    . Impairment of sodium balance in mice deficient in renal principal cell mineralocorticoid receptor. J Am Soc Nephrol. 2007;18:1679–1687. doi: 10.1681/ASN.2006090975.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Christensen BM,
    2. Perrier R,
    3. Wang Q,
    4. Zuber AM,
    5. Maillard M,
    6. Mordasini D,
    7. Malsure S,
    8. Ronzaud C,
    9. Stehle JC,
    10. Rossier BC,
    11. Hummler E
    . Sodium and potassium balance depends on αENaC expression in connecting tubule. J Am Soc Nephrol. 2010;21:1942–1951. doi: 10.1681/ASN.2009101077.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Nesterov V,
    2. Dahlmann A,
    3. Krueger B,
    4. Bertog M,
    5. Loffing J,
    6. Korbmacher C
    . Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron. Am J Physiol Renal Physiol. 2012;303:F1289–F1299. doi: 10.1152/ajprenal.00247.2012.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Pradervand S,
    2. Vandewalle A,
    3. Bens M,
    4. Gautschi I,
    5. Loffing J,
    6. Hummler E,
    7. Schild L,
    8. Rossier BC
    . Dysfunction of the epithelial sodium channel expressed in the kidney of a mouse model for Liddle syndrome. J Am Soc Nephrol. 2003;14:2219–2228. doi: 10.1097/01.ASN.0000080204.65527.E6.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Nesterov V,
    2. Dahlmann A,
    3. Bertog M,
    4. Korbmacher C
    . Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron. Am J Physiol Renal Physiol. 2008;295:F1052–F1062. doi: 10.1152/ajprenal.00031.2008.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Krueger B,
    2. Haerteis S,
    3. Yang L,
    4. Hartner A,
    5. Rauh R,
    6. Korbmacher C,
    7. Diakov A
    . Cholesterol depletion of the plasma membrane prevents activation of the epithelial sodium channel (ENaC) by SGK1. Cell Physiol Biochem. 2009;24:605–618. doi: 10.1159/000257516.
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Volk T,
    2. Konstas AA,
    3. Bassalaý P,
    4. Ehmke H,
    5. Korbmacher C
    . Extracellular Na+ removal attenuates rundown of the epithelial Na+-channel (ENaC) by reducing the rate of channel retrieval. Pflugers Arch. 2004;447:884–894. doi: 10.1007/s00424-003-1193-x.
    OpenUrlCrossRefPubMed
  28. 28.↵
    1. Loffing J,
    2. Schild L
    . Functional domains of the epithelial sodium channel. J Am Soc Nephrol. 2005;16:3175–3181. doi: 10.1681/ASN.2005050456.
    OpenUrlFREE Full Text
  29. 29.↵
    1. Frindt G,
    2. Sackin H,
    3. Palmer LG
    . Whole-cell currents in rat cortical collecting tubule: low-Na diet increases amiloride-sensitive conductance. Am J Physiol. 1990;258(3 pt 2):F562–F567.
    OpenUrl
  30. 30.↵
    1. Loffing J,
    2. Pietri L,
    3. Aregger F,
    4. Bloch-Faure M,
    5. Ziegler U,
    6. Meneton P,
    7. Rossier BC,
    8. Kaissling B
    . Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets. Am J Physiol Renal Physiol. 2000;279:F252–F258.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Loffing J,
    2. Korbmacher C
    . Regulated sodium transport in the renal connecting tubule (CNT) via the epithelial sodium channel (ENaC). Pflugers Arch. 2009;458:111–135. doi: 10.1007/s00424-009-0656-0.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Butterworth MB,
    2. Edinger RS,
    3. Johnson JP,
    4. Frizzell RA
    . Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool. J Gen Physiol. 2005;125:81–101. doi: 10.1085/jgp.200409124.
    OpenUrlPubMed
  33. 33.↵
    1. Rossier BC
    . Hormonal regulation of the epithelial sodium channel ENaC: N or P(o)? J Gen Physiol. 2002;120:67–70.
    OpenUrlFREE Full Text
  34. 34.↵
    1. Snyder PM
    . Minireview: regulation of epithelial Na+ channel trafficking. Endocrinology. 2005;146:5079–5085. doi: 10.1210/en.2005-0894.
    OpenUrlCrossRefPubMed
  35. 35.↵
    1. Abriel H,
    2. Loffing J,
    3. Rebhun JF,
    4. Pratt JH,
    5. Schild L,
    6. Horisberger JD,
    7. Rotin D,
    8. Staub O
    . Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle’s syndrome. J Clin Invest. 1999;103:667–673. doi: 10.1172/JCI5713.
    OpenUrlCrossRefPubMed
  36. 36.↵
    1. Goulet CC,
    2. Volk KA,
    3. Adams CM,
    4. Prince LS,
    5. Stokes JB,
    6. Snyder PM
    . Inhibition of the epithelial Na+ channel by interaction of Nedd4 with a PY motif deleted in Liddle’s syndrome. J Biol Chem. 1998;273:30012–30017. doi: 10.1074/jbc.273.45.30012.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Lu C,
    2. Pribanic S,
    3. Debonneville A,
    4. Jiang C,
    5. Rotin D
    . The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool. Traffic. 2007;8:1246–1264. doi: 10.1111/j.1600-0854.2007.00602.x.
    OpenUrlCrossRefPubMed

Novelty and Significance

What Is New?

  • The distal convoluted tubule 2/connecting tubule (DCT2/CNT) is the primary nephron site where epithelial sodium channel (ENaC) is hyperactive in a mouse model of Liddle syndrome independent of dietary salt intake.

  • Liddle syndrome mutation increases aldosterone responsiveness of ENaC in the connecting tubule/cortical collecting duct (CNT/CCD) that may contribute to salt-sensitive hypertension in Liddle syndrome.

What Is Relevant

  • ENaC regulation in DCT2/CNT, where ENaC regulation is largely independent of aldosterone, is critically important for long-term blood pressure control.

  • Hyperactive ENaC in DCT2/CNT is the key mechanism of Liddle syndrome and may contribute to other forms of salt-sensitive hypertension.

Summary

Hyperactive ENaC in the early part of the aldosterone-sensitive distal nephron is the underlying mechanism of Liddle syndrome.

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  • In Liddle Syndrome, Epithelial Sodium Channel Is Hyperactive Mainly in the Early Part of the Aldosterone-Sensitive Distal NephronNovelty and Significance
    Viatcheslav Nesterov, Bettina Krueger, Marko Bertog, Anke Dahlmann, Ralf Palmisano and Christoph Korbmacher
    Hypertension. 2016;67:1256-1262, originally published May 11, 2016
    https://doi.org/10.1161/HYPERTENSIONAHA.115.07061
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