J Am Soc Nephrol 11: S129–S134, 2000 Scnn1 Sodium Channel Gene Family in Genetically Engineered Mice

EDITH HUMMLER* and FRIEDRICH BEERMANN† *Institut de Pharmacologie et de Toxicologie, Universite´de Lausanne, Lausanne, and †Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland.

Abstract. The amiloride-sensitive epithelial sodium channel is have been obtained by introducing specific mutations or using the limiting step in salt absorption. In mice, this channel is transgenic rescue experiments. In this report, these approaches composed of three subunits (␣, ␤, and ␥), which are encoded are summarized and a current gene-targeting strategy to obtain by different genes (Scnn1a, Scnn1b, and Scnn1c, respectively). conditional inactivation of the channel is illustrated. This latter The functions of these genes were recently investigated in approach will be indispensable for the investigation of channel transgenic (knockout) experiments, and the absence of any function in a wide variety of organ systems. subunit led to perinatal lethality. More defined phenotypes

The highly amiloride-sensitive epithelial sodium channel epithelial potential differences in ␣ENaCϪ/Ϫ (Scnn1atm1/ (ENaC) is a membrane constituent of many salt-reabsorbing Scnn1atm1) mice revealed that ENaC activity was completely epithelia that facilitates Naϩ movement across the tight epi- abolished (Table 1). This suggests that channels composed of ␤␥ thelia that line the distal renal tubule, the distal colon, the ducts subunits alone do not confer sufficient activity to compensate for of salivary and sweat glands, and the lung (1). ENaC is a loss of the ␣ subunit in the lung. In human subjects, mutations in heterotetramer composed of three homologous subunits, i.e., ␣, all three Scnn1 genes do result in ENaC hypofunction and are ␤, and ␥; each subunit contains intracellular amino and car- expected to induce a salt-wasting syndrome similar to pseudohy- boxyl termini, two membrane-spanning domains, and a large poaldosteronism type 1 (PHA-1) (9). In contrast to the ␣ENaC extracellular loop (2–4). cDNA encoding the three homolo- deficiency in mice, PHA-1 variants in human subjects might gous subunits of this channel have been characterized in sev- retain sufficient residual ENaC activity to rescue or attenuate the eral species, including humans and mice (5–7). Here we sum- lung phenotype (10,11). In an in vitro expression system, none of marize transgenic experiments addressing channel function in the mutations causing PHA-1 has been shown to be a null muta- mice and present the three different approaches that we have tion and those tested still confer ENaC-mediated sodium transport used, i.e., classic knockout experiments, transgenic rescue ex- activity (albeit diminished) (11). However, pulmonary ENaC dys- periments, and the introduction of a specific mutation in the function and excess airway liquid have been demonstrated in ␤-subunit. Finally, we discuss an additional tool for analyzing patients with PHA-1 (12). Species-specific differences, such as the channel in vivo, namely conditional gene inactivation. anatomic immaturity of newborn mouse lungs (13) or the pres- ence of additional ENaC subunits in human subjects (e.g., Role of the Channel Subunit Genes ␦ENaC) (14), might also explain the phenotypic differences. Gene targeting is defined as the introduction of site-specific Scnn1b-deficient (15) and Scnn1c-deficient (16) mice lack a modifications into the genome. It allows the in vivo analysis of pulmonary phenotype but exhibit defects in renal function that diverse aspects of gene function. The classic gene-targeting cause perinatal death. In both knockout types, hyponatremia and (knockout) approach leads to inactivation of a gene in all tissues hyperkalemia indicated a defect in renal collecting duct Naϩ of the body, from the onset of development through the entire channel activity and Kϩ secretion. In addition, Scnn1b- and lifespan. Phenotypes of targeted mouse mutants cannot always be Scnn1c-deficient mice exhibited small but significant increases in predicted from the presumed function of the given gene product wet/dry lung weight ratios, although this was not the primary and/or the pattern of expression of the gene. For example, con- cause of their death. We suggest that, in contrast to the ␤␥ channel stitutive inactivation of the ␣ subunit of ENaC revealed an im- subunits in Scnn1a-deficient mice, ␣␤ channels (Scnn1c-defi- portant role for the channel in lung liquid clearance after birth (8). cient) or ␣␥ channels (Scnn1b-deficient) provide low but suffi- ␣ENaC knockout mice died within 40 h after birth, as a result of cient levels of Naϩ absorptive capacity in lung to account for the respiratory failure. Measurements of the amiloride-sensitive trans- transient slower liquid clearance a few hours after birth. We suggest that, for neonatal survival, low levels of ENaC activity are sufficient in lung but not in kidney. Correspondence to Dr. Edith Hummler, Institut de Pharmacologie et de Toxi- cologie, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Phone: 41-21- 692-5357; Fax: 41-21-692-5355; E-mail: [email protected] Introduction of Specific Mutations into Scnn1 1046-6673/1111-0129 Genes Journal of the American Society of Nephrology Direct evidence that ENaC dysfunction is involved in patho- Copyright © 2000 by the American Society of Nephrology logic processes has come from the molecular analysis of two S130 Journal of the American Society of Nephrology J Am Soc Nephrol 11: S129–S134, 2000

Table 1. Transgenic models targeting ENaC functiona

ENaC Activity Allele and Genotype (%)b Organ Phenotype Reference

Null alleles Scnn1atm1/Scnn1atm1 0 Lung (kidney) Perinatal lethal 8 ␤ENaCϪ/Ϫ ND Kidney Perinatal lethal 15 Scnn1ctm1/Scnn1ctm1 Ͻ15 Kidney Perinatal lethal 16 Mutant alleles Scnn1bneo/Scnn1bneo ϳ20 Kidney PHA-1 22 Scnn1bLid/Scnn1bLid Ͼ100 Kidney Liddle’s syndrome 23 Transgenic rescue Scnn1atm1/Scnn1atm1 Tgr␣ENaC ϳ15 Kidney PHA-1 29

a According to the International Committee on Standardized Genetic Nomenclature, the actual gene symbol for the epithelial sodium channel (ENaC) is Scnn1 (sodium channel, non-voltage-gated 1). Genes encoding the three subunits are Scnn1a,-b, and -c (41). The ␤ENaC null allele was not indicated in the naming of the different alleles, because it has been produced elsewhere. PHA-1, pseudohypoaldosteronism type 1; ND, not determined. b The values for resting ENaC activity were estimated from in vitro experiments. human genetic diseases, i.e., Liddle’s syndrome (aldosteron- ism) and PHA-1 (9,17). In patients with Liddle’s syndrome, mutations in the Scnn1b and Scnn1c genes are responsible for a monogenic form of salt-sensitive hypertension (for review, see reference (10). These mutations are clustered in the last exons of the Scnn1b and Scnn1c genes, and, in the resulting protein, PPxY motifs are affected or deleted. It has been shown that these PPxY motifs act as binding sites for a protein called Nedd4 (18). Normally, ENaC is ubiquitinated and degraded, and the number of channels at the cell surface is tightly controlled. In patients with Liddle’s syndrome, lack of this protein-protein interaction has been proposed as a cause for the regulatory defect. Interference of the mutant ENaC proteins Figure 1. Strategy for generating two new alleles at the Scnn1b with the clathrin-mediated endocytosis pathway has also been epithelial sodium channel (␤ENaC) gene locus. A schematic repre- discussed (19). As a consequence of the mutations, increased sentation of the Scnn1b targeting vector, the 3Ј portion of the wild- sodium channel activity is obtained through increased channel type allele (including exons 12 and 13), and the two newly generated numbers and greater open probability. This renders the channel alleles is presented. Arrow, premature termination codon introduced constitutively active (20). In patients with PHA-1 (a salt- by the targeting vector. Black triangles, loxP sites. Black lines, ho- wasting syndrome), mutations have been identified in all three mologous sequences included in the targeting vector. Gray boxes, exons. HSV-Tk, herpes simplex virus thymidine kinase; neo, neomy- Scnn1 genes. In the variant proteins, mutations are well spread cin selection marker. throughout the functional domains, including the gating do- main, which renders the channel hypoactive (for review, see references (10 and 21). The introduction of a Liddle mutation into mice should in appearance, growth rate, and fertility (22). However, the permit elucidation of the causal relationship between dysfunc- introduction of this mutation resulted in large decreases in tion of the Scnn1 genes, dietary salt intake, and hypertension. Scnn1b mRNA and protein levels in all organs tested and We recently introduced the Liddle mutation R566STOP into reduced in vivo ENaC activity in the colon. This was unex- the mouse Scnn1b gene locus and thus established two mouse pected, because this mutation should not affect RNA levels and models, i.e., a model for salt-dependent PHA and a model for instead should lead to increased ENaC density and activity salt-induced Liddle’s syndrome (22,23). First, to reproduce (24). The only difference between our mutated mouse Scnn1b Liddle’s syndrome in mice, we introduced the stop codon in locus and that in human subjects is the presence of the selection the last exon of the mouse Scnn1b gene. This mutation corre- marker neo controlled by the phosphoglycerate kinase (PGK) sponds to the R566STOP mutation in human subjects (17) promoter, which are needed to select for stable transfection in (Figure 1). In the targeting vector, the neomycin selection embryonic stem cell culture (Figure 1). It is possible that the marker (neo), flanked by two loxP sites, followed this muta- insertion of an additional selection marker (neo) into 3Ј un- tion. Phenotypically, mice homozygous mutant for this new translated sequences resulted in destabilization and/or degra- allele (Scnn1bneo) were indistinguishable from wild-type mice dation of the Scnn1b mRNA transcripts, thus interfering with J Am Soc Nephrol 11: S129–S134, 2000 ENaC Genes S131 the expression of the corresponding gene locus (25,26). Alter- Transgenic Rescue of ␣ENaC Deficiencies natively, the PGK-neo cassette might have directly affected the By introducing a transgenic Scnn1a gene into the Scnn1a transcriptional activity of the nearby Scnn1b promoter region genetic knockout background, we were able to rescue the (27). With a low-salt diet, these mice (Scnn1bneo) developed perinatal lethal pulmonary phenotype at birth and partially clinical symptoms of acute PHA-1, including weight loss, restored sodium transport in renal, colonic, and pulmonary hyperkalemia, and decreased BP (22). epithelia (29). This provided further insights into the role of the Nevertheless, we could derive a Liddle mouse model from Scnn1a subunit in defective Naϩ absorption in the kidney. this Scnn1bneo allele. Because the PGK-neo cassette was Young mice (5 to9dofage) exhibited clinical features of flanked by loxP sites, it could be deleted by mating Scnn1bneo severe PHA-1, with metabolic acidosis, urinary salt-wasting, mice with transgenic mice ubiquitously expressing Cre recom- growth retardation, and 50% mortality rates. Adult transgenic binase (EIIa-Cre) (used as a so-called deleter strain) (28). This rescue mice exhibited compensated PHA-1, with normal acid/ breeding converted the Scnn1bneo allele into the Scnn1bLid base and electrolyte values but sixfold elevations of plasma allele (Figure 1) and the salt-wasting phenotype observed in aldosterone levels, compared with wild-type littermate control Scnn1bneo mice into a salt-retaining phenotype (23) (Figure 2). animals. We propose that, as in patients with PHA-1, ENaC Interestingly, the mice remained normotensive with a normal- activity derived from transgene expression provides similar ϩ salt diet, despite evidence of hypervolemia and increased so- levels of Na -absorbing function in the critical organs (lung, dium reabsorption in the large intestine. However, a Liddle kidney, and colon). The clinical course observed for these phenotype, characterized by higher BP, metabolic alkalosis, rescued animals was similar to that observed for children with and hypokalemia accompanied by cardiac and renal hypertro- PHA-1. Such affected children have no apparent problem with phy, was induced with a high-salt diet. Mice carrying the clearance of fetal lung liquid in the perinatal period and gen- Scnn1bLid allele thus largely reproduce the clinical symptoms erally present with clinical symptoms related to metabolic observed for human patients with Liddle’s syndrome (23). dysfunction only after the first 48 h of life (30).

Classic Approach for Investigating ENaC Function in Other Tissues Mice deficient for ENaC (␣ENaCϪ/Ϫ or Scnn1atm1/ Scnn1atm1) can now be used as tools to study the role of ENaC in tissues such as eye, skin, or inner ear. The role of ENaC expression is less clear in those tissues; for example, a role for ENaC in differentiation or mechanoperception has been pos- tulated. In skin, ENaC expression has been found in amphib- ians (31) and mammals (32,33), specifically in hair follicles, interfollicular epidermis, and sweat glands. For the latter, a role in mediating Naϩ absorption has been proposed (34). The function of ENaC in the mammalian epidermis, which is a nonabsorbing epithelium, is not clear. Preliminary studies on skin sections from newborn Scnn1atm1 homozygous mutant and wild-type mice demonstrated abnormal epidermal differ- entiation, with hyperplasia and vacuolization of the epidermis, accompanied by premature lipid secretion. These results indi- cate that ENaC is essential for normal epidermal differentiation and barrier formation, presumably through adjustment of ion transport required for normal epidermal development (E.H., M.G., T.M., unpublished observations). Furthermore, a role for ENaC in the cochlea has been suggested (35–37). It has been proposed that ENaC might be Figure 2. Development of a mouse model for Liddle’s syndrome. (A) implicated in mechanically gated transducer channels, which Schematic diagram of the wild-type exon 13 and the mutant exon 13, are involved in hearing. To test this hypothesis, we examined carrying the R566STOP mutation within the Scnn1b gene locus. (B) mice deficient in Scnn1a (Scnn1atm1/Scnn1atm1) and therefore ␣ ␤ Wild-type and mutant ENaC, composed of two subunits, one in ENaC function. First, neonatal Scnn1atm1/Scnn1atm1 mice subunit, and one ␥ subunit. Note the lack of the carboxyl-terminal exhibited vestibular reflexes not different from those of wild- segment in the mutant ␤ subunit. (C) ENaC activity. Hormones such as aldosterone normally tightly control ENaC activity. In patients with type littermates, indicating normal vestibular function (38). In Liddle’s syndrome, ENaC activity (the number of channels at the cell organotypic cultures of cochlear outer hair cells, we could surface as well as the open probability of the channel) is increased show that hearing function, as monitored by measurement of despite low plasma renin and aldosterone levels, leading to a salt- transducer currents in whole-cell voltage-clamp experiments, retention phenotype. was not impaired in these mice (38). Therefore, the mechani- S132 Journal of the American Society of Nephrology J Am Soc Nephrol 11: S129–S134, 2000 cally gated transducer channel is different from ENaC, but ENaC might play a role in regulating the Naϩ concentration within the endolymph, thus being directly or indirectly in- volved in auditory function. Because Scnn1a knockout mice (with the Scnn1atm1 allele) die soon after birth, conditional knockout of the Scnn1a gene is one of the possible methods to address this topic in the future.

Investigating ENaC Function with Conditional Gene Targeting More recently, methods aimed at controlling gene targeting in a time- and tissue-dependent manner have been developed. This approach is appropriate in cases where complete gene inactivation leads to a lethal or complex phenotype or where it may be difficult to distinguish cell-autonomous lesions from more complex lesions, as in the case of the Scnn1 genes. Conditional gene targeting involves the use of the site-specific recombinase Cre (“causes recombination”) from phage P1, which recognizes and binds to a 34-bp, partly palindromic, target sequence called loxP (“locus of crossover in P1”). Cre recombinase has the ability to efficiently excise any sequence placed between two loxP sites (“floxed”) of the same relative orientation by intramolecular recombination. As a result, one loxP site remains within the genome (39). Gene inactivation can be restricted to a particular cell type in vivo by crossing a mouse strain harboring the floxed allele with a transgenic strain expressing Cre recombinase under the control of a cell type- Figure 3. Conditional targeting of ENaC. (A) Generation of different ␣ tm1 specific promoter, e.g., the aquaporin 2 promoter (40). More- alleles at the Scnn1a ( ENaC) gene locus. The Scnn1a allele represents the original ␣ENaC knockout mouse model (8). The new over, tissue-specific gene inactivation may define physiologic allele (Scnn1aneo) is generated by first using a modified targeting roles of Scnn1 gene products in a given tissue, without com- vector, with exon 1 and phosphoglycerate kinase (PGK)-neo flanked promising other functions in the organism. by loxP sites (floxed). Scnn1aflox and Scnn1atm2 are obtained from Therefore, we have planned a targeting vector that contains embryonic stem cell clones expressing the Scnn1aneo allele after homologous sequences of the endogenous Scnn1a locus, the transient transfection with Cre recombinase. Scnn1atm2 can also be vital coding exon (here exon 1) flanked by loxP sites, and a obtained from mice carrying Scnn1aneo after breeding with a germline neomycin selection marker (neo), followed by a third loxP site deleter strain (e.g., EIIa-Cre). (B) Schematic diagram of specific (Scnn1aneo allele) (Figure 3). In embryonic stem cells, the inactivation of Scnn1a in the kidney (e.g., the distal cortical collecting flox neomycin selection marker is removed by transient transfection duct). Mice homozygous for the mutant Scnn1a allele are crossed using Cre recombinase, leaving the floxed exon 1 (Scnn1aflox with transgenic mice expressing the Cre recombinase under the con- allele) untouched. In mice, mating of mice carrying the trol of a kidney-specific promoter (e.g., aquaporin 2) (40). This results in inactivation exclusively in cortical collecting duct cells. All other Scnn1aneo or Scnn1aflox allele with a germline deleter strain cell types remain unaffected and retain 100% ENaC activity. (e.g., EIIa-Cre) (28) induces excision of the flanked sequences, thereby creating the Scnn1atm2 mutated allele. This should inactivate the Scnn1a gene locus and abolish ENaC activity (Figure 3). Conditional gene targeting will be possible using experiments. This work was supported by grants from the Swiss Cre-expressing mouse strains and mice homozygous for either National Science Foundation (Grant 31-52943.97 to E. Hummler and the Scnn1aflox or Scnn1aneo allele. Control of gene targeting Grant 31-43384.95 to B. C. Rossier). will allow differentiation of the effects of chronic versus acute References depletion of proteins and analysis of functions at different time ϩ points in development. Gene inactivation at a specific time 1. Verrey F, Hummler E, Schild L, Rossier BC: Control of Na point, leaving gene function intact throughout development, transport by aldosterone. In: The Kidney: Physiology and Patho- physiology, edited by Seldin DW, Giebisch G, 2000, in press should prevent adaptive responses; therefore, phenotypes 2. Canessa CM, Horisberger JD, Rossier BC: Epithelial sodium might be different in conditional, compared with conventional, channel related to proteins involved in neurodegeneration. Na- knockout mice. ture (Lond) 361: 467–470, 1993 3. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horis- Acknowledgments berger JD, Rossier BC: Amiloride-sensitive epithelial Naϩ chan- We thank Bernard C. Rossier for continuous support of the project nel is made of three homologous subunits. Nature (Lond) 367: and Sylvain Pradervand and Anne-Marie Merillat for help with the 463–467, 1994 J Am Soc Nephrol 11: S129–S134, 2000 ENaC Genes S133

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