AJP - Renal Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 296: F249-F256, 2009. First published November 26, 2008; doi:10.1152/ajprenal.00612.2007
0363-6127/09 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
296/2/F249    most recent
00612.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mérillat, A.-M.
Right arrow Articles by Hummler, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mérillat, A.-M.
Right arrow Articles by Hummler, E.

CALL FOR PAPERS
Aldosterone and Epithelial Na+ Channel

Conditional gene targeting of the ENaC subunit genes Scnn1b and Scnn1g

Anne-Marie Mérillat,1,2 Roch-Philippe Charles,1 Andrée Porret,2 Marc Maillard,3 Bernard Rossier,1 Friedrich Beermann,4 and Edith Hummler1,2

1Département de Pharmacologie et de Toxicologie, 2Transgenic Animal Facility, and 3Service de Nephrologie du Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne; and 4ISREC (Swiss Institute for Experimental Cancer Research), School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Epalinges, Switzerland

Submitted 24 December 2007 ; accepted in final form 22 November 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial sodium channels (ENaC) are members of the degenerin/ENaC superfamily of non-voltage-gated, highly amiloride-sensitive cation channels that are composed of three subunits ({alpha}-, β-, and {gamma}-ENaC). Since complete gene inactivation of the β- and {gamma}-ENaC subunit genes (Scnn1b and Scnn1g) leads to early postnatal death, we generated conditional alleles and obtained mice harboring floxed and null alleles for both gene loci. Using quantitative RT-PCR analysis, we showed that the introduction of the loxP sites did not interfere with the mRNA transcript expression level of the Scnn1b and Scnn1g gene locus, respectively. Upon a regular and salt-deficient diet, both β- and {gamma}-ENaC floxed mice showed no difference in their mRNA transcript expression levels, plasma electrolytes, and aldosterone concentrations as well as weight changes compared with control animals. These mice can now be utilized to dissect the role of ENaC function in classical and nonclassic target organs/tissues.

transgenic; conditional knockout; epithelial sodium channel; amiloride-sensitive sodium channel; mouse model; β-ENaC; {gamma}-ENaC

THE HIGHLY AMILORIDE-SENSITIVE epithelial sodium channel (ENaC) is composed of three homologous subunits {alpha}, β, and {gamma}, encoded by different genes on human chromosomes 12 (SCNN1A) and 16 (SCNN1B, SCNN1G). In the mouse, the genes have been localized on chromosomes 6 (Scnn1a) and 7 (Scnn1b, Scnn1g) (8, 9, 21, 24). ENaC is found, e.g., in the kidney, colon, lung, and sweat and salivary duct glands where apical membrane localization mediates directional absorption of sodium. In tongue fungiform papillae (33) and the baroreceptor mechanotransducer of the aortic arch (11), ENaC contributes to the electrogenic movement of sodium. Expression of the subunits was also reported in the retina (7), the cochlea (15), and the sensory nerve endings in the rat food pad (2) as well as in the trigeminal mechanosensory neurons (14). Their function in these tissues is not yet well understood. In skin, mice deficient for {alpha}-ENaC dehydrate rapidly after birth and exhibit a severely impaired matrix lipid profile characteristic of a postnatal skin-barrier defect (10).

The constitutive inactivation of β- and {gamma}-ENaC revealed severe early renal dysfunction that results in early postnatal death (4, 20). Failure to thrive and lethargy are associated with urinary Na+ wasting, K+ retention, and increased plasma aldosterone concentration, thus reflecting the renal phenotype found in PHA-1 (for a review, see Refs. 5 and 6). It seems that low residual ENaC activity in these mice is sufficient to circumvent the neonatal lung phenotype, consistent with the assumption that the {alpha}β, and the {alpha}{gamma}-subunit combination can establish some ENaC activity in airway epithelia. Mice with a partial disruption of the β-ENaC gene locus (Scnn1btm1Ipt) show a mild PHA-1 phenotype with reduced ENaC activity and elevated plasma aldosterone levels, but develop an acute PHA1 with continuous weight loss, hyperkalemia, and decreased blood pressure when kept under a low-salt diet (26). Low levels of β-ENaC subunit mRNA might confer sufficient ENaC activity to maintain salt and water homeostasis under normal salt conditions, but be limiting when salt restriction is imposed. The {alpha}-ENaC subunit is important in forming the functional channel, whereas the β- and {gamma}-ENaC subunits are crucial modulators of ENaC channel function. In the lung, β- and {gamma}-ENaC subunits facilitate neonatal lung liquid clearance at birth, whereas in the kidney, where Na+ reabsorption is under the control of aldosterone, β- and {gamma}-ENaC subunits seem to be necessary for proper functioning of ENaC channels. In the Xenopus laevis oocyte expression system, the three ENaC subunits are required for maximal expression and activity, and quantification of ENaC subunits expressed at the cell surface indicates that the subunits assemble preferentially in a heterooligomeric {alpha}-β-{gamma} channel complex. According to such recent findings, ENaC may be composed of totally three single subunits (18).

Whereas a conditional allele of {alpha}-ENaC had been described several years ago (17), no such alleles exist for β- and {gamma}-ENaC. We now describe the generation of "floxed" (and therefore conditional) alleles for the two subunits, β and {gamma}, of the epithelial sodium channel ENaC. This will open the possibility to study the effect of specific molecular defects in vivo and their consequences at the level of aldosterone target cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Generation of the targeting constructs. Design of targeting vectors was performed similarly to the recent generation of a Prss8flox allele (30). To generate a Scnn1b-targeting vector, the vital region (exon 2; 740-bp HindII/HindIII fragment) was inserted into the SmaI site of a vector (K13 pAT FRTneolox antisense) (32), where the Pgk-neomycin (neo) cassette is flanked by two FRT sites, followed 3' by a single loxP site. A 3-kb XhoI/SmaI fragment of a 5' homologous sequence was isolated following different cloning steps and then introduced into the XhoI/ClaI sites of this vector (vector 1). Then, a 3.2-kb 3' homologous sequence (HindIII/SalI fragment) was inserted into the HindIII/PmeI sites 3' of the loxP site of a lox-targeting vector (vector 2) (27). In this vector 2, the herpes simplex virus thymidine kinase gene (Tk) is situated 3' of the 3' homologous sequence. To obtain the final Scnn1b-targeting vector, a XhoI/NotI (blunt) fragment from vector 1 was cloned into SalI/AscI (blunt) sites of vector 2 (Fig. 1A). To generate the Scnn1g-targeting vector, the vital region (exon 2; 550-bp ClaI/KpnI fragment) was inserted into the SmaI site of the plasmid K13 pAT FRTneolox antisense (32) (see above). A 1.85-kb 5' homologous sequence was isolated by different subcloning steps and introduced into the XhoI/ClaI sites of this vector. A 4.4-kb fragment containing a 5' sequence, Pgk-neo, and a floxed vital region was inserted 5' of the loxP site into SalI/AscI sites of a lox-targeting vector neo/Tk (vector 2) (27). The herpes simplex virus thymidine kinase gene (Tk) is situated 3' at the targeting vector. To obtain the final Scnn1g-targeting vector, a 6.4-kb 3' homologous sequence (KpnI/SpeI fragment) was inserted into the PmeI site 3' of the loxP site. The final targeting vector is illustrated in Fig. 3A. Further details on cloning are available on request. The targeting vectors have been electroporated into HM-1 (Scnn1b; 129P2/OlaHsd) and into GS1 (Scnn1g; 129S6/SvEv) mouse embryonic stem (ES) cells, and selection of positive clones and generation of germ line chimeras were done essentially as described (25).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 1. Generation of a conditional Scnn1b allele. Conditional gene targeting strategy for the disruption of the mouse β-ENaC (Scnn1b) gene locus is shown. A: structure of the partial wild-type Scnn1b gene, the targeting vector, and the predicted targeted alleles (not drawn to scale). The precise exon-intron structure has not been determined. Identified exon 2 of the vital region is indicated (black box). Exons 3 and 4 (black boxes) are situated within the targeting vector but outside the loxP site. The 3' external probe (3' probe) was used to illustrate the floxed and the {Delta} allele following HindII digestion of embryonic stem (ES) cell DNA (not shown). Expected fragment sizes of the floxed allele are indicated. B: representative Southern blot analysis of double resistant ES cell clones following electroporation with the targeting vector. ES cell DNA was digested with HindII and hybridized with the 5' external probe (5' probe; A). Fragment sizes of wild-type (wt; 3.5-kb) and mutant (5.5-kb) alleles are indicated. C: DNA-based PCR strategy on positively identified ES cell clones covering the 3'-flanking region (left and middle) and the exon 2 of the targeted allele (right). Note that the different analyses are from different samples. Primers are listed in Table 1.

 
Characterization of positively targeted ES cell clones. Correctly targeted ES cell clones for β-ENaC (Scnn1b) were identified following hybridization of double (G418/gancyclovir)-resistant clones with the external probe β-5' probe (510-bp NotI/StuI fragment) digested with HindII (Fig. 1A). Double-resistant clones for {gamma}-ENaC (Scnn1g) were screened with the external probes {gamma}-5' probe (600-bp EcoRI fragment) and {gamma}-3' probe (1.1-kb BamHI/HindIII fragment) following digestion with HindIII and SalI, respectively (Fig. 1B). As a neo probe, the 0.6-kb EcoRI/BamHI fragment from the pGEMneo vector was used. For the DNA-based PCR analysis of the 3'-flanking region of the β-ENaC (Scnn1b) gene locus, we used primers 1, 2, and 4 (see Table 1). Presence of the exon was tested using primers 1 and 3 (Fig. 1C). {gamma}-ENaC-targeted clones were identified by Southern blot analysis following HindIII (5' probe) and SalI (3' probe) digestion (see Fig. 3B) and by a PCR product of 4.5 kb using primers 8 and 9 followed by digestion with EcoRI and HindIII (see Fig. 3C, left), and by a 1.2-kb PCR product indicating exon 2 (primers 1 and 9) followed by XbaI digestion (550 and 660 bp; see Fig. 3C, middle). The 3'-flanking region was tested by PCR (3.2-kb fragment, primers 1 and 10) followed by digestion with EcoRI (1.7 kb; 640 and 620 bp), XbaI (1.7 kb; 670 and 500 bp) and HindIII (1.7 kb; 760 and 520 bp; see Fig. 3C, right).


View this table:
[in this window]
[in a new window]

  		Table 1. PCR primers (1–19) used for genotyping and characterization of cells and mice, and primers (Scnn1a, Scnn1b, and Scnn1g) used for quantitative real-time PCR

 
Genotyping of β- and {gamma}-ENaC mutant mice. Protocols involving animals were reviewed and approved by the State Authority (Service de la consommation et des affaires vétérinaires, Lausanne, Switzerland). Genotyping was performed by PCR on DNA isolated from tail biopsies. To detect the Scnn1bloxneo, Scnn1blox, and Scnn1b+ alleles, primers 5–7 and 13 (Fig. 2, A and B) were used. To distinguish the Scnn1g+ from the Scnn1gloxneo and Scnn1glox alleles, primers 6, 11, 12, 18, and 19 were used (see Fig. 4B).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 2. Genotyping of β-ENaC (Scnn1b) mutant mice. A: scheme of PCR primers used for genotyping of the Scnn1b alleles before and following Cre/loxP- and Flp-mediated recombination (see also Table 1). B: representative genotyping of mice harboring the wt, loxneo, and lox allele. C: identification of Scnn1blox, Scnn1b+, and Scnn1b{Delta} alleles from breeding of Scnn1blox/lox mice with the Cre-deleter strain (Nestin ::Cre). Expected PCR fragments are indicated. As indicated, primers 5 and 7 are useful to distinguish the lox (Scnn1blox) and wt allele, whereas primers 5 and 13 reveal the presence or absence of the {Delta} allele (Scnn1b{Delta}).

 
Generation of null alleles for the Scnn1b and Scnn1g gene locus. Mice carrying the Scnn1bloxneo and Scnn1glox allele, respectively, were bred to a Cre-deleter strain (12) to constitutively remove the vital regions in vivo. The resulting null alleles were named Scnn1b{Delta}neo, Scnn1b{Delta}, and Scnn1g{Delta}. The Scnn1b{Delta}neo and the Scnn1b{Delta} alleles were detected using primers 5 and 13 (Fig. 2C), The Scnn1g+, Scnn1glox, and Scnn1g{Delta} alleles were distinguished with primers 12, 18, and 19 (see Fig. 4C). The Cre and the Flp transgene were detected by PCR using primers 14 and 15 (0.4 kb) and 16 and 17 (0.5 kb), respectively (not shown). PCR (36 cycles; 1 min, 95°C; 1 min, 56°C; 1 min, 72°C; one final elongation of 10 min. at 72°C) was performed on 1–2 µl tail DNA as previously described (31).

Phenotypic characterization of β-and {gamma}-ENaC mutant mice. Scnn1blox/lox (6 mo old; n = 6), Scnn1glox/lox (3 mo old; n = 7) and age-matched littermates (3 mo old; n = 8) and wild-type C57BL/6N (3 mo old; n = 8) mice were used for the experiments. Animals were either subjected to a regular salt diet (0.23%, 0.95% potassium; Provimi Kliba Nafag, Rotterdam, The Netherlands) or for 5 consecutive days to a sodium-deficient diet (ICN Biomedical, Costa Mesa, CA). Body weight of floxed β- and {gamma}-ENaC and littermate control mice (males and females, 3–6 mo old, 2–6 mice/group) was determined once (time 0) and then on 5 consecutive days (days 1–5) upon a salt-deficient or regular salt diet.

Quantitative RT-PCR of ENaC subunits. Kidney and lung samples were isolated and snap frozen, and total RNA was extracted with Qiazol (Qiagen, Basel, Switzerland). The reverse transcription was performed with 1 µg RNA mixed in a final volume of 20 µl with oligo dT(20) primer (5 µM), dNTP (500 µM), buffer (50 mM Tris·HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2; 5 mM DTT), and 40 U of Superscript II (Invitrogen, Basel, Switzerland). The reaction was incubated at 42°C for 1 h and finally stopped by denaturing the enzyme for 10 min at 70°C. The cDNA was diluted five times before measurements. Quantitative real-time PCRs were done by TaqMan PCR using Applied Biosystems 7500. The 20x primer/probe mix (4352341E for β-actin) was purchased with the Universal TaqMan mix (2x) and used according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Sequences of primers and probes for Scnn1a, Scnn1b, and Scnn1g are shown in Table 1. The quantification was done by measuring the {Delta}{Delta}Ct normalized to β-actin. All measurements were done in duplicate.

Plasma electrolyte and aldosterone measurements. At the time of dissection, blood samples were collected from the aorta under anesthesia. Plasma sodium and potassium concentrations were measured in the Laboratory of Clinical Chemistry, Centre Hospitalier Universitaire Vaudoise (CHUV), University Hospitals, Lausanne, Switzerland. Plasma aldosterone levels (pg/ml) of retroorbitally collected blood were determined according to standard procedures.

Calculation and statistics. All data are expressed as means ± SE. Values (n) refer to the number of mice in each group. Individual groups were compared by using an unpaired t-test. A level of P < 0.05 was accepted as statistically significant for all comparisons.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Analysis of the in vivo function of genes involved in aldosterone-dependent sodium transport allows dissection of complex processes and to draw conclusions on the functional redundancy or absolute requirement of various genes involved in salt absorption along the nephron. Since complete gene inactivation of all three Scnn1 genes (encoding for {alpha}-, β-, and {gamma}-ENaC) leads to early postnatal death, consequences of ENaC deficiency in tissues such as kidney, lung, or skin of adult mice cannot be addressed properly (4, 16, 20). Therefore, in this study, we decided to generate conditional alleles at the gene loci encoding for β- and {gamma}-ENaC (Scnn1b and Scnn1g). The vital regions of the Scnn1b (exon 2; 740-bp fragment) and the Scnn1g gene (exon 2; 550-bp fragment) were flanked by loxP sites (floxed; Figs. 1A and 3A). We chose this strategy since these exons have been targeted by the Pgk-neo cassette in the previously described constitutive knockouts of Scnn1b and Scnn1g (4, 20). Following electroporation of mouse ES cells and injection of targeted Scnn1b and Scnn1g ES cell clones into C57BL/6N donor blastocysts (25), male chimeras were obtained which transmitted the floxed Scnn1b and Scnn1g alleles retaining the Pgk-neo cassette (Scnn1bloxneo, Scnn1gloxneo) through the germ line. Breeding of mice carrying the Scnn1bloxneo and Scnn1gloxneo alleles resulted in expected frequencies of genotypes (Table 2). We further crossed mice carrying the Scnn1bloxneo allele with Nestin::Cre-deleter mice to remove the vital region. Since the Pgk-neo cassette is outside of the floxed region (Fig. 1), it is still retained in this breeding, and the resulting Scnn1b{Delta}neo allele was identified by Southern blot analysis and PCR to corroborate removal of exon 2 (not shown). As expected, interbreeding of Scnn1b{Delta}neo/+ mutant mice resulted in the absence of living Scnn1b{Delta}neo/{Delta}neo homozygous mutant mice (Table 2). We therefore conclude that 1) exon 2 can be efficiently removed by Cre recombinase in vivo, and 2) the complete absence of exon 2 leads to a lethal phenotype as described for the β-ENaC deficiency (20). With respect to Scnn1g, we obtained several Scnn1glox/{Delta} mice following breeding of Scnn1glox/+ mice (see below) with the Nestin::Cre deleter strain (Fig. 4C), and we so far identified one dead newborn that exhibited two null alleles (Scnn1g{Delta}/{Delta}; not shown).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 3. Conditional gene targeting of the {gamma}-epithelial Na channel (ENaC; Scnn1g) allele. A: structure of the partial wt Scnn1g allele, the targeting vector (PGK-neo; neomycin resistance cassette; Tk, thymidine kinase), the predicted targeted allele (loxneo; Scnn1gloxneo), and the floxed (lox; Scnn1glox) allele (not drawn to scale). B: Southern blot analysis of positive ES cell clones. Expected fragments of the wt and Scnn1gloxneo allele following digestion with HindIII ({gamma}-5' probe) and SalI ({gamma}-3' probe) are indicated. C: DNA-based PCR strategy on identified targeted ES cell clones covering the 5'-flanking region (left), exon 2 (middle), and the 3'-flanking region (right). Amplified PCR fragments were further characterized by digestion of PCR products with EcoRI and HindIII (PCR5'; left), XbaI (PCRexon; middle), and EcoRI, XbaI, and HindIII (PCR3'; right). See Table 1 and MATERIALS AND METHODS for details of PCR reaction.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Genotype analysis of 3-wk-old mice from heterozygous matings

 

Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 4. Generation of {gamma}-ENaC (Scnn1g) floxed mice. A: strategy to identify mice harboring the loxneo (Scnn1gloxneo) and the lox (Scnn1glox) allele. The primers used are as described in MATERIALS AND METHODS and indicated (see Table 1). B and C: representative genotype analysis of mice harboring the Scnn1gloxneo and the Scnn1glox allele (B) and following breeding with Nestin::Cre and hACTB::Flpe mice (C). Exon 2 is excised, and mice harboring the {Delta} allele (Scnn1glox/{Delta}, Scnn1g{Delta}/+) are identified. The genotype is indicated above each line.

 
To avoid any effect of the Pgk-neo cassette on wild-type Scnn1b and Scnn1g expression, we removed the Pgk-neo cassette after breeding to a FlpE-deleter strain, resulting in mice carrying the floxed Scnn1b and Scnn1g allele (Scnn1blox, Scnn1glox; Figs. 2 and 4). The resulting offspring were genotyped by Southern blot and/or PCR analysis, and viable heterozygous mutant Scnn1blox/+ and Scnn1glox/+, and homozygous mutant Scnn1blox/lox and Scnn1glox/lox mice, were obtained by interbreeding, suggesting that the position of the remaining loxP sites does not interfere with survival of the animals (Figs. 2 and 4; Table 2). Homozygous mutant mice for the Scnn1bloxneo, Scnn1blox, and Scnn1gloxneo and Scnn1glox alleles are viable and are detected in concordance with the expected Mendelian frequency (Table 2).

To demonstrate that these floxed mice have normal physiological characteristics upon a regular-salt diet, we analyzed the mRNA transcript expression level by quantitative RT-PCR analysis of kidneys from Scnn1blox/lox and Scnn1glox/lox mice. Data obtained from pooled RNA samples showed that the relative expression of Scnn1a, Scnn1b, and Scnn1g is not affected by the presence of loxP sites (data not shown). We then analyzed mRNA transcript levels of the targeted alleles. Here, neither Scnn1b nor Scnn1g mRNA levels in kidneys from Scnn1blox/lox and Scnn1glox/lox mutant mice, respectively, were different from littermates and C57BL/6N controls (Fig. 5, A and B). The slight variation in the absolute expression levels might be due to strain differences (129Sv vs. C57BL/6N). Following 5 days of salt-deficient treatment, Scnn1blox/lox and Scnn1glox/lox mice showed comparable levels of Scnn1b and Scnn1g expression, respectively. Interestingly, we found an overall twofold increase in expression levels in the kidney upon a salt-deficient diet compared with a regular-salt diet (Fig. 5, A and B). Previous studies in rats reported rather an increase in the abundance of mRNA coding for {alpha}-ENaC in the kidney without affecting the mRNAs encoding the β- and {gamma}-ENaC subunits (1, 13, 23), although data as obtained by Northern blot analysis cannot be directly compared with quantitative RT-PCR measurements.


Figure 5
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 5. Measurements of the relative mRNA transcription level of in kidneys of control, littermates, and Scnn1blox/lox (A) and Scnn1glox/lox (B) mice upon a regular-salt (black bars) vs. a salt-deficient diet (grey bars). For every genotype, kidneys from 3–4 individual C57BL/6N controls, littermates, and Scnn1blox/lox or Scnn1glox/lox mice were analyzed. *P < 0.05, ***P < 0.001 (zero-salt vs. regular-salt condition).

 
Moreover, the same group of Scnn1blox/lox and Scnn1glox/lox mice, which followed a regular-salt or salt-deficient diet, exhibited plasma sodium and potassium (Fig. 6, A and B), and plasma aldosterone levels (Fig. 6, C and D) not different from their controls. Upon regular-salt treatment, the plasma potassium levels are slightly, but not significantly elevated, which is most likely due to increased hemolysis in these samples. The plasma potassium level from the salt-deficient group is in the normal range, with the exception of a sample from C57BL/6N mice that might be due to the genetically different background. As expected, in all groups, plasma aldosterone levels were generally lower upon a regular-salt diet and, although not significantly, increased after a salt-deficient diet (Fig. 6, C and D). The variation in the basal aldosterone levels is most probably due to the mixed genetic background with differing contributions of 129Sv and C57BL/6N, and hence by the presence of either one renin (Ren-1c; C57BL/6N) or two renin genes (Ren-1d, Ren-2; 129Sv), which influences the basal plasma aldosterone levels (36). In conclusion, physiological data and weight measurements (Fig. 7, A and B) in a comparison of regular-salt and salt-deficient diets clearly illustrate that mice carrying the floxed alleles for both β- and {gamma}-ENaC are not different from control littermates.


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 6. Measurements of plasma sodium (A), potassium (B), and aldosterone concentrations (C and D) in mice following a regular-salt (black bars) or a salt-deficient diet (grey bars). Plasma sodium and potassium concentrations (in mM) were determined in C57BL/6N, wt littermate controls, and Scnn1blox/lox and Scnn1glox/lox mice. Note that the plasma sodium concentration is the same among all groups. Even though the plasma potassium concentration is about the same in all groups upon the same salt-diet, the difference between the groups from different salt diets is most likely explained by higher blood hemolysis in the regular-salt-diet group (black bars). C and D: plasma aldosterone levels (in pg/ml) were determined in wt littermates and in mice heterozygously floxed and homozygously floxed for β-ENaC (Scnn1b; C) or {gamma}-ENaC (Scnn1g; D) upon a regular-salt (black bars) and a salt-deficient diet (grey bars). Note that basal plasma aldosterone levels might vary due to the genetic background of the mouse colony.

 

Figure 7
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 7. Body weight loss (%) in Scnn1b+/+ (regular-salt diet; normal salt, n = 3; salt-deficient diet, no salt, n = 3), Scnn1blox/+ (normal salt, n = 2; no salt, n = 3), and Scnn1blox/lox (normal salt, n = 6; no salt, n = 4) mice (A) and in Scnn1g+/+ (normal salt, n = 3; no salt, n = 4), Scnn1glox/+ (normal salt, n = 6; no salt, n = 6), and Scnn1glox/lox (normal salt, n = 5; no salt, n = 5) mice (B) upon a regular-salt (solid lines) and salt-deficient (dashed lines) diet. Note that the weight loss measurements do not differ among groups.

 
Genetic dissection of β- and {gamma}-ENaC subunits in vivo will help our understanding of how and why mineralocorticoid specificity can be achieved in aldosterone target cells. The tissue distribution of ENaC subunits is compatible with its involvement in Na+ transport in the aldosterone-sensitive distal nephron (ASDN), with ENaC being the main effector for aldosterone action through mineralocorticoid receptor action and 11β-hydroxysteroid dehydrogenase type II (11-βHSD2) activity (3). So far, by using the conditional Scnn1aloxlox mice following breeding to the HoxB7::Cre mice, we tested the importance of the early ASDN vs. late ASDN for the maintenance of sodium balance. Surprisingly, mice with targeted inactivation of {alpha}-ENaC in the whole collecting duct do not develop a PHA-1 phenotype (29).

The Scnn1blox/lox and Scnn1glox/lox mice presented here can now be used to assess the role of these missing ENaC subunit genes in sodium absorption in a temporally and/or tissue-specific manner. This tool to genetically dissect the role of the three ENaC subunit genes ({alpha}-β-{gamma}) will also become important in studying the interaction with negative and positive regulators of ENaC, e.g., channel-activating serine protease (CAPs) (34, 35). One report suggests that this ENaC-activating mechanism is influenced by aldosterone induction of urinary serine protease Prss8/CAP1 (prostasin) (22). With the availability of conditional knockout mice for β- and {gamma}-ENaC, we can now proceed with the experimental analysis of specific gene defects in vivo in the whole organism and also at the level of target cells.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This work has been supported by grants from the Swiss National Science Foundation to E. Hummler and B. Rossier.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Hummler, Département de Pharmacologie et de Toxicologie, Univ. of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland (e-mail: Edith.Hummler{at}unil.ch)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 

  1. Asher C, Wald H, Rossier BC, Garty H. Aldosterone-induced increase in the abundance of Na+ channel subunits. Am J Physiol Cell Physiol 271: C605–C611, 1996.[Abstract/Free Full Text]
  2. Askwith CC, Benson CJ, Welsh MJ. DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature. Proc Natl Acad Sci USA 98: 6459–6463, 2001.[Abstract/Free Full Text]
  3. Bachmann S, Bostanjoglo M, Schmitt R, Ellison D. Sodium transport-related proteins in the mammalian distal nephron—distribution, ontogeny and functional aspects. Anat Embryol 200: 447–468, 1999.[CrossRef][Medline]
  4. Barker PM, Ngugen MS, Gatzy JT, Grubb B, Norman H, Hummler E, Rossier B, Boucher RC, Koller B. Role of {gamma}ENaC subunit in lung liquid clearance and electrolyte balance in newborn mice: insights into perinatal adaptation and pseudohypoaldosteronism. J Clin Invest 102: 1634–1640, 1998.[Web of Science][Medline]
  5. Bonny O, Hummler E. Dysfunction of epithelial sodium transport: from human to mouse. Kidney Int 57: 1313–1318., 2000.[CrossRef][Web of Science][Medline]
  6. Bonny O, Rossier BC. Disturbances of Na/K balance: pseudohypoaldosteronism revisited. J Am Soc Nephrol 13: 2399–2414, 2002.[Free Full Text]
  7. Brockway LM, Zhou ZH, Bubien JK, Jovov B, Benos DJ, Keyser KT. Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits. Am J Physiol Cell Physiol 283: C126–C134, 2002.[Abstract/Free Full Text]
  8. Canessa CM, Horisberger JD, Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467–470, 1993.[CrossRef][Medline]
  9. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463–467, 1994.[CrossRef][Medline]
  10. Charles R, Guitard M, Leyvraz C, Breiden B, Haftek M, Haftek-Terreau Z, Stehle J, Sandhoff K, Hummler E. Postnatal requirement of the epithelial sodium channel ENaC for the maintenance of the epidermal barrier function. J Biol Chem 283: 2622–2630, 2008.[Abstract/Free Full Text]
  11. Drummond HA, Price MP, Welsh MJ, Abboud FM. A molecular component of the arterial baroreceptor mechanotransducer. Neuron 21: 1435–1441, 1998.
  12. Dubois N, Hofmann D, Kaloulis K, Bishop J, Trumpp A. Nestin-Cre transgenic mouse line Nes-Cre1 mediates highly efficient Cre/loxP mediated recombination in the nervous system, kidney, and somite-derived tissues. Genesis 44: 355–360, 2006.[CrossRef][Web of Science][Medline]
  13. Escoubet B, Coureau C, Bonvalet JP, Farman N. Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone. Am J Physiol Cell Physiol 272: C1482–C1491, 1997.[Abstract/Free Full Text]
  14. Fricke B, Lints R, Stewart G, Drummond H, Dodt G, Driscoll M, Düring M. Epithelial Na+ channels and stomatin are expressed in rat trigeminal mechanosensory neurons. Cell Tissue Res 299: 327–334, 2000.[Web of Science][Medline]
  15. Gründer S, Müller A, Ruppersberg JP. Developmental and cellular expression pattern of epithelial sodium channel alpha, beta and gamma subunits in the inner ear of the rat. Eur J Neurosci 13: 641–648, 2001.[CrossRef][Web of Science][Medline]
  16. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, Rossier BC. Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nat Genet 12: 325–328, 1996.[CrossRef][Web of Science][Medline]
  17. Hummler E, Merillat AM, Rubera I, Rossier BC, Beermann F. Conditional gene targeting of the Scnn1a (alphaENaC) gene locus. Genesis 32: 169–172, 2002.[CrossRef][Medline]
  18. Jasti J, Furukawa H, Gonzales E, Gouaux E. Structure of acid-sensing ion channel 1 at 19A resolution and low pH. Nature 449: 316–322, 2007.[CrossRef][Medline]
  19. Magin TM, McWhir J, Melton D. A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Res 20: 3795–3796, 1992.[Free Full Text]
  20. McDonald FJ, Yang B, Hrstka RF, Drummond HA, Tarr DE, McCray PB Jr, Stokes JB, Welsh MJ, Williamson RA. Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype. Proc Natl Acad Sci USA 96: 1727–1731, 1999.[Abstract/Free Full Text]
  21. Meisler MH, Barrow LL, Canessa CM, Rossier RC. SCNN1, an epithelial cell sodium channel gene in the conserved linkage group on mouse chromosome 6 and human chromosome 12. Genomics 24: 185–186, 1994.[CrossRef][Web of Science][Medline]
  22. Narikiyo T, Kitamura K, Adachi M, Miyoshi T, Iwashita K, Shiraishi N, Nonoguchi H, Chen LM, Chai KX, Chao J, Tomita K. Regulation of prostasin by aldosterone in the kidney. J Clin Invest 109: 401–408, 2002.[CrossRef][Web of Science][Medline]
  23. Ono S, Kusano E, Muto S, Ando Y, Asano Y. A low-Na+ diet enhances expression of mRNA for epithelial Na+ channel in rat renal inner medulla. Pflügers Arch 434: 756–763, 1997.[CrossRef][Web of Science][Medline]
  24. Pathak BG, Shaughnessy JD, Meneton P, Greeb J, Shull GE, Jenkins NA, Copeland NG. Mouse chromosomal location of three epithelial sodium channel subunit genes and an apical sodium chloride cotransporter gene. Genomics 33: 124–127, 1996.[CrossRef][Medline]
  25. Porret A, Mérillat AM, Guichard S, Beermann F, Hummler E. Tissue-specific transgenic and knockout mice. Meth Mol Biol 337: 185–205, 2006.[Medline]
  26. Pradervand S, Barker P, Wang Q, Ernst SA, Beermann F, Grubb B, Burnier M, Schmidt A, Bindels RJM, Gatzy J, Rossier BC, Hummler E. Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel. Proc Natl Acad Sci USA 96: 1732–1737, 1999.[Abstract/Free Full Text]
  27. Radtke F, Wilson A, Stark B, Bauer M, vanMeerwijk J, MacDonald HR, Aguet M. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10: 547–558, 1999.[CrossRef][Web of Science][Medline]
  28. Reis LF, Ruffner H, Stark G, Aguet M, Weissmann C. Mice devoid of interferon regulatory factor (IRF-1) show normal expression of type I interferon genes. EMBO J 13: 4798–4806, 1994.[Web of Science][Medline]
  29. Rubera I, Loffing J, Palmer LG, Frindt G, Fowler-Jaeger N, Sauter D, Carroll T, McMahon A, Hummler E, Rossier BC. Collecting duct-specific gene inactivation of {alpha}ENaC in the mouse kidney does not impair sodium and potassium balance. J Clin Invest 112: 554–565, 2003.[CrossRef][Web of Science][Medline]
  30. Rubera I, Meier E, Vuagniaux G, Merillat AM, Beermann F, Rossier BC, Hummler E. A conditional allele at the mouse channel activating protease 1 (Prss8) gene locus. Genesis 32: 173–176, 2002.[CrossRef][Medline]
  31. Schmidt A, Tief K, Foletti A, Hunziker A, Penna D, Hummler E, Beermann F. LacZ transgenic mice to monitor gene expression in embryo and adult. Brain Res Prot 3: 54–60, 1998.[CrossRef][Medline]
  32. Trumpp A, Refaeli Y, Oskarsson T, Gasser S, Murphy M, Martin GR, Bishop JM. c-myc regulates mammalian body size by controlling cell number but not cell size. Nature 414: 768–773, 2001.[CrossRef][Medline]
  33. Vandenbeuch A, Clapp TR, Kinnamon SC. Amiloride-sensitive channels in type I fungiform taste cells in mouse. BMC Neurosci 9: 1–13, 2008.[Medline]
  34. Vuagniaux G, Vallet V, Jaeger NF, Hummler E, Rossier BC. Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus oocytes. J Gen Physiol 120: 191–201, 2002.[Abstract/Free Full Text]
  35. Vuagniaux G, Vallet V, Jaeger NF, Pfister C, Bens M, Farman N, Courtois-Coutry N, Vandewalle A, Rossier BC, Hummler E. Activation of the amiloride-sensitive epithelial sodium channel by the serine protease mCAP1 expressed in a mouse cortical collecting duct cell line. J Am Soc Nephrol 11: 828–834, 2000.[Abstract/Free Full Text]
  36. Wang Q, Hummler E, Nussberger J, Clément S, Gabbiani G, Brunner HR, Burnier M. Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of renin genes. J Am Soc Nephrol 13: 1509–1516, 2002.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
296/2/F249    most recent
00612.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mérillat, A.-M.
Right arrow Articles by Hummler, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mérillat, A.-M.
Right arrow Articles by Hummler, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2009 by the American Physiological Society.