J Am Soc Nephrol 10: 2527–2533, 1999 A Mouse Model for Liddle’s Syndrome

SYLVAIN PRADERVAND,* QING WANG,† MICHEL BURNIER,† FRIEDRICH BEERMANN,‡ JEAN DANIEL HORISBERGER,* EDITH HUMMLER,* and BERNARD C. ROSSIER* *Institut de Pharmacologie et de Toxicologie de l’Universite´and †Division d’ et de Me´decine Vasculaire, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; and ‡Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland.

Abstract. Liddle’s syndrome (or pseudoaldosteronism) is an from wild type despite evidence for increased reab- autosomal dominant form of salt-sensitive hypertension, due to sorption in distal colon and low plasma , suggesting abnormal sodium transport by the renal tubule. To study the chronic hypervolemia. Under high salt intake, the Liddle mice pathophysiology of salt sensitivity, a mouse model for Liddle’s develop high BP, , and accom- syndrome has been generated by Cre/loxP-mediated recombi- panied by cardiac and renal hypertrophy. This animal model nation. Under normal salt diet, mice heterozygous (L/ϩ) and reproduces to a large extent a human form of salt-sensitive homozygous (L/L) for Liddle mutation (L) develop normally hypertension and establishes a causal relationship between during the first 3 mo of life. In these mice, BP is not different dietary salt, a gene expressed in and hypertension.

Hypertension is the most common multifactorial cardiovascu- recapitulates most of the clinical features of the human disease lar disorder in human populations (1,2). Both genetic and and represents a useful model for salt-sensitive hypertension. environmental factors are involved. Salt intake is a recognized risk factor, and up to 50% of hypertensive patients are sensitive Materials and Methods to salt (3). Generation of Liddle Mice Direct evidence for the importance of the epithelial sodium The insertion of a stop codon (corresponding to residue R566 in channel in salt homeostasis has come from the molecular human) into the mouse ␤ENaC gene locus, using homologous recom- analysis of genetic diseases disturbing salt reabsorption and bination in HM-1 ES cells (129Ola), was described previously (8). To leading to hypo- or hypertensive phenotypes. Liddle’s syn- generate a mouse model for Liddle’s syndrome, heterozygous mice drome is an autosomal dominant disease characterized by early from the F1 generation (8) for the ␤ENaC mutated allele (m) con- onset of hypertension, associated with hypokalemia, sup- taining the neomycin resistance gene (neo) flanked by two loxP sites pressed plasma activity, and low plasma aldosterone were mated with hemizygous mice ubiquitously expressing the EIIa- levels (pseudoaldosteronism), indicating a constitutive in- Cre transgene (Figure 1A). Mice showing a complete excision of the creased sodium absorption in the kidney (1,4). Investigation of neo gene were then bred with C57BL6/J mice to establish stable Liddle’s syndrome kindreds revealed mutations in the cyto- mouse lines. plasmic C terminus of either ␤ or ␥ENaC leading to a trunca- tion of the proteins (5). Expression studies in Xenopus oocytes Genotyping showed that these mutations result in a constitutive activation Genomic DNA from mouse tails was prepared as described (8). For of the channel in vitro (6). Recently, it has been shown that the genotyping, (to genotype ␤ENaC gene locus), Southern blot analysis target sequence for channel-activating mutations is a proline- was performed on DNA digested with HincII or HincII ϩ KpnI, rich region (PPXY) in the cytoplasmic C terminus of the ␤ and according to standard procedures using probe A (Figure 1A). PCR ␥ subunit (6,7). analyses were performed with primers PLS and PLAS as described (8). To elucidate the causal relationship between dietary salt To genotype the renin gene locus, Southern blot analysis was per- formed on DNA digested with PvuII. A cDNA coding for mouse renin intake, genetically determined salt handling by the kidney, and was used as probe. The Ren-1c gene (from C57Bl6/J strain) was hypertension, a mouse model for Liddle’s syndrome has been detected as a 9-kb band, and the Ren-1d and Ren-2 genes (e.g., 129Ola generated by Cre/loxP-mediated recombination. This model strain) were detected as 10- and 14-kb bands, respectively.

Northern Blot Received September 24, 1999. Accepted October 13, 1999. Drs. Hummler and Rossier contributed equally to this work. Total RNA from colon and kidney was isolated according to Correspondence to Dr. Bernard C. Rossier, Institut de Pharmacologie et de standard procedures. Northern blots were performed with 15 ␮g 32 Toxicologie, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Phone: ϩ41 loaded in each lane and hybridized with ␤ENaC-specific P-labeled 21/692-5351; Fax: ϩ41 21/692-5355; E-mail: [email protected] probes as described previously (8). For quantification of RNA tran- 1046-6673/1012-2527 scripts, loading was normalized by hybridization with a PCR-ampli- Journal of the American Society of Nephrology fied cDNA probe from mouse GAPDH gene. RNA from four inde- Copyright © 1999 by the American Society of Nephrology pendent animals were analyzed for each group. 2528 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2527–2533, 1999

Bioelectric Measurements The rectal potential difference (PD) was measured in vivo on adult mice (3 to 5 mo old) before and after addition of amiloride as described previously (8). The amiloride-induced change in rectal PD ⌬ ( PDamil) was determined between 10 a.m. and noon. The light cycle of the animal house was from 7 a.m. to 7 p.m.

Analytical Procedures and BP Measurements Adult mice (3 to 5 mo old) were fed with a normal Naϩ diet (3 g/kg Naϩ) and plain drinking water or a high-sodium diet consisting of 16gNaϩ/kg chow in the food (A03, UAR; France) plus 0.9% saline as drinking water ad libitum for 8 to 10 wk. Plasma aldosterone levels were determined in adult mice by 125I RIA (Coat-A-Count Aldoste- rone kit, Diagnostic Products Corp., Los Angeles, CA) between 10 a.m. and noon or between 4 p.m. and 6 p.m. All assays were run in duplicate. BP was measured with carotid catheterization using a single-blind protocol (9). Four hours after surgery, mean BP from resting mice was recorded over 12 min with a computerized data acquisition system (8,9). Samples were then collected from arterial blood taken from the carotid artery to determine electrolyte values.

Results Generation of the Liddle Mutation in the Mouse Liddle’s syndrome arises from deletions or missense muta- tions (reviewed in references (2) and (10) of the amiloride- sensitive epithelial sodium channel (ENaC) (11). In vitro, these mutations result in a constitutive activation of sodium transport (6,7). To reproduce the Liddle’s syndrome in mice, we used a gene replacement strategy. In the first step, we introduced a stop codon in the mouse ␤ENaC gene locus, a mutation cor- ␤ responding to residue R566 in human ENaC (2,10) as found in the original pedigree described by Liddle (1). This mutation was followed by neomycin selection marker (neo), flanked with two loxP sites (8). In the second step, the neo gene was excised in vivo by Cre/loxP-mediated recombination. Mice carrying a ␤ENaC-mutated allele containing neo from the F1 generation (8) were mated with hemizygous mice ubiquitously expressing the EIIa-Cre transgene. (Figure 1A) (12). Southern blot (Figure 1B) and PCR (data not shown) analyses on tail DNA of the progeny revealed that four (3%) of the 125 mice Figure 1. Establishment of a mutant mouse line carrying the Liddle showed a complete excision of the neo gene. These four mice ␤ ␤ mutation ENaC R566Stop. (A) Structure of the wild-type ENaC were then bred with C57BL6/J mice to establish stable mouse gene, the targeted locus (allele m) described previously (8), and the ␤ lines. Mice heterozygous for the Liddle mutation 566Stop predicted recombined locus mediated by Cre-recombinase enzyme were interbred. The expected Mendelian ratio (1:2:1) was (allele L). The neomycin resistance gene (neo) was flanked by two loxP sites (grey triangles). Allele L possesses the R566Stop mutation followed by 100 exogenous bp containing the residual loxP site. Expected fragment sizes of wild-type, m, and L ␤ENaC alleles are wild-type, m, and L alleles are indicated in Panel A. Genomic DNA indicated after digestion with HincII or HincII plus KpnI and hybrid- from mouse tails was prepared as described (8). Southern blot analysis ization with probe A. H2 ϭ HincII, K1 ϭ KpnI. The precise exon- was performed on DNA digested with HincII or HincII ϩ KpnI intron structure has not been determined. Identified exons are indi- according to standard procedures using probe A (Panel A). (C) cated (grey box). (B) Southern blot analysis of offspring following Amount of ␤ENaC RNA transcripts from kidney and colon of ␤ENaC breeding of ␤ENaC m/ϩ mice with EIIa-Cre transgenic mice. Tail L/ϩ (light gray bar) and L/L (dark gray bar) compared with wild type DNA was digested with HincII or HincII ϩ KpnI and hybridized with (white bar). Total RNA from colon and kidney was isolated according Ј ␮ a5 flanking probe (probe A). Lane 1, mouse carrying the R566Stop standard procedures. Northern blots were performed with 15 g mutation (indicated by the marker KpnI site, which was introduced 6 loaded in each lane and hybridized with ␤ENaC-specific 32P-labeled bp after the mutation), without the neo gene; lane 2, mouse carrying probes as described previously (8). For quantification of RNA tran- the R566Stop mutation and the neo gene; lanes 3 and 4, mouse scripts, loading was normalized by hybridization with a PCR-ampli- carrying the R566Stop mutation with a partial deletion of the neo gene; fied cDNA probe for mouse GAPDH gene. RNA from four indepen- lane 5, mouse with two wild-type alleles. Expected fragment sizes of dent animals were analyzed for each group. J Am Soc Nephrol 10: 2527–2533, 1999 A Mouse Model for Liddle’s Syndrome 2529 observed, suggesting that embryonic and fetal development was not impaired by the mutation. In ␤ENaC L/ϩ and L/L mice, Northern blot analysis revealed that ␤ENaC expression was significantly reduced in colon (L/ϩ:45Ϯ 10%, L/L: 28 Ϯ 10% compared to wild type) and kidney (L/ϩ:61Ϯ 6%, L/L: 13 Ϯ 2% compared to wild type) (Figure 1C). By contrast, no significant difference was found in these organs for ␣ENaC and ␥ENaC mRNA expression (data not shown). We propose two explanations for our observations. (1) The Liddle muta- tion, as a missense stop codon mutation, destabilized ␤ENaC mRNA. It has been suggested that missense mutations in the coding region of some genes can lead to mRNA instability, but the molecular mechanism is not well documented. (2) The loxP sites that have been left in the mouse genome in the 3Ј untranslated region of the transcript could lead to mRNA instability. It is known that the 3Ј untranslated region of mRNA contains sequences that are involved in mRNA stability. It is therefore quite possible that the loxP sites left over have just been inserted into such important sequences. At present, and not knowing the consequence of the Liddle mutation on mRNA stability in human patients, we favor the second explanation.

No Liddle Phenotype under Normal Salt Diet but Evidence for a Hyperactive Channel in Vivo Since Liddle mutations generate hyperactive channels in vitro (6), we tested in vivo ENaC-mediated sodium transport in distal colon by measuring transepithelial amiloride-sensitive potential difference (PD) in the rectum (8). In mice fed a Figure 2. Measurements of sodium transport and plasma aldosterone ␤ ϩ normal salt diet, ENaC L/ and L/L mice exhibited a signif- content. (A) In vivo measurements of rectal potential difference (PD) icantly larger baseline PD than ␤ENaC ϩ/ϩ mice (Figure 2A). before (white bar) and after (black bar) inhibition by amiloride. ⌬ ϭ ϩ ϩ ϭ ϩ ϭ These differences were mainly due to (1) a large increase of the PDamil amiloride-sensitive PD. / : n 16; L/ : n 18, L/L: ⌬ ϩ ϩ Ϫ Ϯ ϭ Ͻ Ͻ amiloride-sensitive PD ( PDamil:[ / ] 16.1 2.6 mV; n 15. *P 0.05; **P 0.01. The rectal PD was measured in vivo [L/ϩ] Ϫ22.8 Ϯ 2.5 mV, P ϭ 0.07; [L/L] Ϫ27.9 Ϯ 2.2 mV, in adult mice (3 to 5 mo old) before and after addition of amiloride as ⌬ P Ͻ 0.01) and (2) an increase of the amiloride-insensitive PD. described previously (8). The PDamil was determined between In Liddle’s patients, constitutive increased channel activity 10 a.m. and noon. The light cycle of the animal house was from 7 a.m. leads to high blood volume (hypervolemia) and suppressed to 7 p.m. (B) Morning and afternoon plasma aldosterone concentra- ␤ tions (nmol/L) in ␤ENaC ϩ/ϩ (white bar), L/ϩ (light gray bar), and aldosterone secretion (4). In ENaC L/L mice, plasma aldo- ϭ ϩ ϩ ϭ ϩ Ͻ L/L (dark gray bar). a.m.: each group, n 7; p.m.: / : n 15; L/ : sterone was threefold lower in the morning (P 0.05) and ϭ ϭ Ͻ Ͻ Ͻ n 8, L/L: n 7. *P 0.05; **P 0.01. Plasma aldosterone levels 7.5-fold lower in the afternoon compared to wild type (P were determined in adult mice by 125I RIA (Coat-A-Count Aldoste- ␤ ϩ 0.01), whereas ENaC L/ mice showed intermediate levels rone kit, Diagnostic Products Corp., Los Angeles, CA) between (Figure 2B). Liddle’s patients exhibit hypokalemia and meta- 10 a.m. and noon or between 4 p.m. and 6 p.m. All assays were run bolic alkalosis as a consequence of a constitutive increased in duplicate. sodium reabsorption in the cortical collecting duct of the kid- ney (13). Under a normal salt diet, there were no significant ϩ ϩ Ϫ Ϫ differences in plasma pH, Na ,K ,Cl ,orHCO3 concen- lemia and showed a compensated metabolic alkalosis when trations between mutant and wild-type mice (data not shown). compared with L/L or ϩ/L mice. BP was not significantly different from ϩ/ϩ mice (data not As shown in Table 2, diastolic and systolic BP, heart rate, shown). Fed a normal salt diet, L/ϩ or L/L animals develop and cardiac and kidney indexes were measured. Systolic BP low plasma aldosterone levels, suggesting salt retention and was significantly higher in L/ϩ animals (ϩ15 mmHg,). Dia- hypervolemia but no other changes, which is characteristic of stolic BP increased by ϩ8 mmHg, a difference that did not the disease (i.e., hypokalemia, metabolic alkalosis). reach statistical significance. Likewise, heart rate tended to decrease (Ϫ14 beats/min) but this was not statistically signif- Salt-Induced Liddle Phenotype icant. Hypertension leads to congestive heart failure and car- To test the hypothesis of a salt-induced Liddle phenotype, a diac hypertrophy that can be assessed by the cardiac index second group of mice was maintained on a high salt diet for 8 (heart weight:body weight ratio). The cardiac index of ␤ENaC to 10 wk. Under these conditions, ␤ENaC L/ϩ mice were L/L showed a significant increase of their cardiac index com- hypokalemic (Table 1). ␤ENaC L/L mice developed hypoka- pared with ␤ENaC ϩ/ϩ mice (ϩ10%). In heterozygotes, the 2530 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2527–2533, 1999

Table 1. Plasma electrolytes and acid-base status under high salt dieta

Electrolyte ϩ/ϩ L/ϩ L/L

[Naϩ] (mmol/L) 147.7 Ϯ 0.9 144.6 Ϯ 3.2 146.6 Ϯ 2.1 (n ϭ 11) (n ϭ 7, P ϭ 0.28) (n ϭ 11, P ϭ 0.64) [Kϩ] (mmol/L) 4.3 Ϯ 0.1 3.7 Ϯ 0.1 3.2 Ϯ 0.1 (n ϭ 11) (n ϭ 7, P ϭ 0.004) (n ϭ 11, P Ͻ 0.001) [CIϪ] (mmol/L) 123.0 Ϯ 1.7 121.4 Ϯ 3.3 116.5 Ϯ 2.1 (n ϭ 11) (n ϭ 7, P ϭ 0.65) (n ϭ 11, P ϭ 0.025) Ϫ Ϯ Ϯ Ϯ [HCO3 ] (mmol/L) 19.5 0.3 20.6 0.8 25.4 1.3 (n ϭ 11) (n ϭ 6, P ϭ 0.15) (n ϭ 11, P Ͻ 0.001) pH 7.31 Ϯ 0.02 7.32 Ϯ 0.01 7.38 Ϯ 0.02 (n ϭ 11) (n ϭ 6, P ϭ 0.62) (n ϭ 11, P ϭ 0.027)

a Adult mice (3 to 5 mo old) were fed with a high sodium diet consisting of 16 g Naϩ/kg chow in food (A03, UAR; France) plus 0.9% saline as drinking water ad libitum for 8 to 10 wk. Samples were collected from arterial blood taken from carotid artery to determine electrolyte values. Results shown are mean Ϯ SEM. P values for ␤ENaC L/ϩ and ␤ENaC L/L animals compared with wild type (ϩ/ϩ)(t test, unpaired).

Table 2. Blood pressure and cardiac and renal indexes under high salt dieta

Parameter ϩ/ϩ L/ϩ L/L

Systolic BP (mmHg) 155.0 Ϯ 4.4 170.5 Ϯ 6.1 168.8 Ϯ 6.3 (n ϭ 17) (n ϭ 13, P ϭ 0.042) (n ϭ 17, P ϭ 0.082) Diastolic BP (mmHg) 107.8 Ϯ 4.0 115.0 Ϯ 5.1 114.0 Ϯ 5.4 (n ϭ 17) (n ϭ 13, P ϭ 0.27) (n ϭ 17, P ϭ 0.36) Heart rate (bpm) 636 Ϯ 18 621 Ϯ 15 619 Ϯ 12 (n ϭ 17) (n ϭ 13, P ϭ 0.56) (n ϭ 17, P ϭ 0.44) Cardiac index (mg/g) 4.49 Ϯ 0.09 4.73 Ϯ 0.11 4.92 Ϯ 0.10 (n ϭ 17) (n ϭ 13, P ϭ 0.096) (n ϭ 19, P ϭ 0.004) Renal index (mg/g) 12.81 Ϯ 0.29 13.81 Ϯ 0.49 15.67 Ϯ 0.40 (n ϭ 17) (n ϭ 13, P ϭ 0.073) (n ϭ 19, P Ͻ 0.001)

a Adult mice (3 to 5 mo old) were fed with a high sodium diet as described in Table 1 for 8 to 10 wk. Blood pressure (BP) was measured blinded with carotid catheterization. Four hours after surgery, systolic and diastolic BP were recorded over 12 min from resting mice with a computerized data acquisition system (9). Cardiac and renal indexes were measured as organ weight: body weight ratios. Results shown are mean Ϯ SEM. P values for ␤ENaC L/ϩ and ␤ENaC L/L animals compared with wild type (ϩ/ϩ)(t test, unpaired).

cardiac index change (ϩ5%) was intermediate and did not same mutation can induce an opposite phenotype. The level of reach the level of significance. ␤ mRNA in the kidney of L/L animals (present study) is Under a high salt diet, we also observed the presence of approximately 13-fold higher than that measured in the kidney renal hypertrophy (Table 2) in heterozygous ␤ENaC L/ϩ mice of ␤ m/m mice (13% versus 1% of ϩ/ϩ animals), as estimated with a significantly elevated kidney index (kidney weight:body by Northern blot analysis in the previous study (8). In the colon weight ratios in mg/g) (ϩ8%). The kidney index was even of L/L mice, the level of ␤ mRNA is sevenfold higher than in higher in homozygotes (ϩ23%). Because chronic hypokalemia the ␤ m/m animals (28% versus 4% of ϩ/ϩ animals). If we is known to lead to renal hypertrophy (14), this observation assume that the mRNA abundance reflects protein levels, we may be explained by the lower plasma level in the propose the following explanation for the generation of two ␤ENaC L/L animals exposed to a high salt diet. Thus, under models with opposite phenotypes but carrying the same muta- ␤ high salt diet, the Liddle mutation ENaC R566Stop severely tion. Extremely low (1% in kidney and colon, and 4% in lung) affects renal and cardiovascular functions in mice. but physiologically significant levels (partial knock out) of ␤ ENaC protein (presumably “hyperactive” since carrying the Discussion Liddle mutation) are sufficient to ensure survival at birth and to From a PHA-1 to a Liddle Phenotype allow a normal life under normal salt diet. Under salt restric- The present Liddle mouse model (␤ L/L) was derived from tion, however, a severe and lethal PHA-1 phenotype is induced a previously described inducible PHA-1 animal model (␤ m/m) (8). We suggest that normal principal cells express a consid- (8) using Cre/loxP technology. It raises the question of how the erable reserve pool (up to 100-fold) of ␤ENaC mRNA (and J Am Soc Nephrol 10: 2527–2533, 1999 A Mouse Model for Liddle’s Syndrome 2531 protein). We assume that the regulation between a small “ac- Table 3. Effects of ␤ENaC and renin locus on blood tive” pool (at the apical membrane) and a large “inactive” pressure under high salt dieta reserve pool (mainly intracellular) is extremely tight since a Category Systolic BP Diastolic BP 13-fold increase in ␤ L/L ENaC mRNA(or protein) is sufficient to switch from a PHA-1 to a Liddle phenotype. We estimate Ren-1 ϩ/ϩ 146 Ϯ 297Ϯ 3 that a normal native principal cell of CCD may express 1000 (n ϭ 10) (n ϭ 10) copies of ENaC protein per cell. If this estimate is correct, we L/ϩ 152 Ϯ 498Ϯ 3 come up with the striking speculation that maybe less than 10 (n ϭ 6) (n ϭ 6) copies of mutant but hyperactive channels per principal cell is L/L 154 Ϯ 599Ϯ 3 sufficient for survival, but 100 copies of the same mutated (n ϭ 10) (n ϭ 10) protein can lead to a salt-sensitive hypertensive phenotype. Ren-1/Ren-2 ϩ/ϩ 168 Ϯ 7 123 Ϯ 4 (n ϭ 7) (n ϭ 7) Effect of the Liddle versus Renin Locus L/ϩ 187 Ϯ 5 130 Ϯ 2 An animal model mimicking a human monogenic disease (n ϭ 7) (n ϭ 7) provides a good scientific basis for understanding in molecular L/L 190 Ϯ 8 136 Ϯ 6 and pathophysiologic terms the development of salt-sensitive (n ϭ 7) (n ϭ 7) hypertension. We describe here a novel animal model, which Two-way ANOVA expresses a gain-of-function mutation in the ␤ subunit of the P, ␤ENaC genotypes 0.024 0.163 epithelial sodium channel (Liddle mutation). Interestingly, this P, renin genotypes Ͻ0.001 Ͻ0.001 animal model remains normotensive under normal salt diet, P, interaction 0.405 0.397 despite evidence of hypervolemia and increased sodium reab- a sorption in the large intestine. Under high salt diet, the Liddle Systolic and diastolic blood pressure (BP) were measured as Ϯ phenotype is induced. This animal model should help in elu- described in Table 2. Results shown are mean (mmHg) SEM. Renin genes Ren-1 and Ren-2 are indicated. Statistical differences cidating in the future the pathophysiology of hypervolemic were assessed using two-way ANOVA. P values are indicated. salt-sensitive hypertension. The relevance of the animal model to human physiology or pathophysiology of BP control is supported by two recent studies. Wong et al. found significant Liddle mutation onto various inbred mouse background should linkage between systolic BP and chromosome 16p12 (Liddle help in identifying modifier genes that can potentiate or antag- locus) in a cohort comprising 286 white families from the onize the Liddle mutation and thus modulate salt sensitivity. general population in Victoria, Australia. There was no corre- lation with diastolic BP or body mass index (15). Nagy et al. A Mouse Model for Salt-Sensitive Hypertension: studied 66 pairs of normotensive healthy dizygotic twin sub- Interaction between Genetic and Environmental jects and their parents in a sibpair analysis to look for linkage Factors of selected candidate genes to the quantitative trait (BP). The Genetically engineered mouse models for hypertension have authors reported quantitative trait loci (QTL) near the Liddle been recently obtained by inactivating “hypotensive” genes syndrome locus (systolic BP) and the renin locus (systolic and (19–22) such as eNOS, proANP, ANP receptor, bradykinin diastolic BP) (16). receptor B2, EP2 receptor, and more recently 11␤-hydroxys- We were intrigued by the relatively high diastolic BP of the teroid dehydrogenase type 2 (23) or, alternatively, by increas- ϩ/ϩ mice (Table 2) and wondered whether the genetic back- ing the copy number of “hypertensive” genes such as angio- ground could play a role in this observation. Due to the tensinogen (reviewed in reference (22). These models are breeding with EIIa-Cre transgenic outbred mice, offspring useful in defining the physiologic role of these genes but are of have a heterogeneous genetic background for renin locus, a less relevance for understanding human hypertension, because factor that could influence BP. In particular, they could possess they do not reproduce well-characterized human mutations, either one (Ren-1, i.e., C57Bl6/J strain) or two renin genes with the exception of the recently reported targeted disruption (Ren-1 and Ren-2, 129 strain) (17,18) with possible distinct of the 11␤HSD2 gene (23). effects on BP. Renin is a key factor modulating the BP re- Hypertension is the most frequent disease in human popu- sponse to changes in sodium balance in normal and hyperten- lations. Genetic and nongenetic factors are involved and high sive animals (19). To distinguish the effects of Liddle’s muta- salt intake has been proposed as a major risk factor. Large tion from that of the renin locus and their potential interaction epidemiologic and clinical studies, however, have led to con- on BP, animals were genotyped for the Ren-1 and Ren-2 locus troversial conclusions (24,25). We propose that these contra- genes. The data were assessed by using two-way ANOVA dictory results may be explained by two kinds of confounding (Table 3). Liddle mutation affected systolic BP (P Ͻ 0.05), factors. First, the study of low salt or high salt intake as a single whereas renin locus affected both diastolic and systolic pres- experimental variable in human populations is obviously dif- sures (P Ͻ 0.01). No significant interaction between the two ficult if not impossible. Second, epidemiologic studies have not variables could be detected. This analysis suggests that this taken into account the possibility that susceptibility genes mouse model exhibits both sodium channel and renin locus could confer salt sensitivity in a large proportion of the popu- dependency for BP control as in humans (14,15). Breeding the lation. Regarding the first factor, Denton et al. (26) were 2532 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2527–2533, 1999 recently able to demonstrate in chimpanzees (the species ge- syndrome: Heritable human hypertension caused by mutations in netically the closest to us) that the addition of salt within the the ␤ subunit of the epithelial sodium channel. Cell 79: 407–414, human dietetic range causes a highly significant rise in their 1994 BP. Interestingly, the effect of salt differed between individu- 6. Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, als, and only 60% of the cohort developed high BP, defining Rossier BC: A mutation in the epithelial sodium channel causing two subpopulations of salt-sensitive or salt-resistant animals. Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci USA 92: 5699– Regarding the second factor, the identification of mutations in 5703, 1995 the epithelial sodium channel ␤ subunit as a cause of a mono- 7. Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, genic form of salt-sensitive hypertension in humans (Liddle’s Zeiher BG, Stokes JB, Welsh MJ: Mechanism by which Liddle’s syndrome) has recently highlighted the importance of a single syndrome mutations increase activity of a human epithelial so- mutated gene as sufficient to induce large changes in BP. Our dium channel. Cell 83: 969–978, 1995 study demonstrates that a form of salt-induced hypertension in 8. Pradervand S, Barker PM, Wang Q, Ernst SA, Beermann F, mice recapitulates many, but not all, aspects of the human Grubb BR, Burnier M, Schmidt A, Bindels RJ, Gatzy JT, Rossier disease. The relative salt resistance of the mouse model (only BC, Hummler E: Salt restriction induces pseudohypoaldosteron- the L/L animals have the full-blown Liddle phenotype) may be ism type 1 in mice expressing low levels of the ␤-subunit of the due to either low expression of the Liddle allele at RNA amiloride-sensitive epithelial sodium channel. Proc Natl Acad (Figure 1C) and protein level and/or some species-specific Sci USA 96: 1732–1737, 1999 factors. It is also important to stress that the Liddle mutation 9. Wiesel P, Mazzolai L, Nussberger J, Pedrazzini T: Two-kidney, does not have full penetrance in humans, since some members one clip and one-kidney, one clip hypertension in mice. Hyper- of the original pedigree that carry the mutated allele are nor- tension 29: 1025–1030, 1997 10. 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