(Hypertension. 1997;29:131.)
© 1997 American Heart Association, Inc.
Research Articles (Issue 1, Part 1) |
Role of the 
-, ß-, and 
-Subunits of Epithelial Sodium Channel in a Model of Polygenic Hypertension
the Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, and Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, Mass (R.K., B.S., S.R., N.H., K.L.); Department of Molecular Biology, Université Libre de Bruxelles (Belgium) (J.S., C.S.); Istituto Neurologico Mediterraneo Neuromed, Pozzilli (IS), Italy (S.R.); Max Delbrück Centre for Molecular Medicine, Berlin, Germany (D.G., K.L.); and Division of Biological Sciences, Harvard School of Public Health, Boston, Mass (K.L.)
Correspondence to Klaus Lindpaintner, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Thorn 1103, Boston, MA 02115. E-mail kl@calvin.bwh.harvard.edu.
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Abstract |
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The pathophysiological basis of Liddle's syndrome, a rare autosomal dominant form of arterial hypertension, has been found to rest on missense mutations or truncations of the ß- and
-subunits of the epithelial sodium channel. The hypothesis has been advanced that molecular variants of these genes might also contribute to the common polygenic forms of hypertension. We tested this hypothesis by performing a cosegregation study in a reciprocal cross between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and a Wistar-Kyoto rat (WKY-1HD) reference strain. We carried out genetic mapping and chromosomal assignment of the 
-, ß-, and -subunits of the epithelial sodium channel using both linkage analysis and fluorescent in situ hybridization techniques. We demonstrate that in the rat, the ß- and -subunits, as in humans, are in close linkage; they map to rat chromosome 1 and cosegregate with systolic pressure after dietary NaCl (logarithm of the odds [LOD] score, 3.7), although the peak LOD score of 5.0 for this quantitative trait locus was detected 4.4 cM away from the ß-/-subunit locus. The -subunit was mapped to chromosome 4 and exhibited no linkage to blood pressure phenotype. Comparative analysis of the complete coding sequences of all three subunits in the SHRSPHD and WKY-1HD strains revealed no biologically relevant mutations. Furthermore, Northern blot comparison of mRNA levels for all three subunits in the kidney showed no differences between SHRSPHD and WKY-1HD. Our results fail to support a material contribution of the epithelial sodium channel genes to blood pressure regulation in this model of polygenic hypertension.
Key Words: genetics • sodium channels • rats, inbred strains • rats, inbred WKY • in situ hybridization, fluorescence • cytogenetics
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Introduction |
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Functional studies have long identified the amiloride-sensitive epithelial sodium channel as playing an important role in sodium balance and thus BP regulation.1 2 The channel represents a heterotrimeric or larger unit consisting of
-, ß-, and -subunits that are encoded by three distinct genes.3 Recently, Liddle's syndrome, an autosomal dominant form of hypertension, has been found to be caused by any of a number of molecular variants of either the ß- or -subunit, most of which result in premature termination of the protein.4 5 6 7 8 Whereas Liddle's syndrome is a rare disorder, the hypothesis has been advanced that other perhaps more frequent mutations with less dramatic effects on channel function may contribute to more common and thus epidemiologically more relevant forms of polygenic (essential) hypertension.
This question appeared to be of particular relevance to hypertension in several substrains of the spontaneously hypertensive rat, in which previous cosegregation studies had demonstrated linkage to a marker anchored on the Sa gene,9 10 11 12 which is known to reside in the immediate vicinity of the ß-subunit of the epithelial sodium channel on rat chromosome 1.13 Could it be that the positive linkage data implicating Sa were actually pointing to the ß- or -subunit of the epithelial sodium channel? To study this question, which might shed light, in a more general sense, on the potential contribution of the epithelial sodium channel subunit genes to polygenic hypertension, we characterized the localization of all three subunits, the - (Scnn1a), ß- (Scnn1b), and - (Scnn1g) subunit genes, by genetic mapping and FISH. We then evaluated their possible contribution to hypertension by cosegregation analysis in an experimental rat cross bred from SHRSP (SHRSPHD) and normotensive WKY (WKY-1HD), an appropriate model because the Sa gene locus had been found to be linked to BP regulation in this cross.14 In addition, we performed gene expression studies and complete coding sequence analyses for the three Scnn1 genes in SHRSPHD and WKY-1HD.
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Animals and Genetic Crosses
Rats were obtained from our colonies of SHRSPHD and WKY-1HD at the University of Heidelberg (Germany).14 15 Experimental procedures for animal housing and breeding have previously been reported.15 The WKY-1HD strain has been characterized elsewhere.14 Briefly, WKY-1HD represent a congenic lineage of WKYHD that carry a
6-cM-long SHRSPHD-homologous chromosomal fragment on chromosome 10, which contains a BP relevant locus, BP/SP-1a.14 The reciprocal F2 intercross analyzed in the present work was bred from SHRSPHD and WKY-1HD and has been the subject of previous publications.14 16 This F2 population consisted of 33 males and 42 females with a male SHRSPHD founder and 34 males and 30 females with a male WKY-1HD founder.
BP Measurements
Hemodynamic measurements were obtained at the age of 16 weeks while rats were being fed regular rat chow (Altromin) and tap water ad libitum (baseline) and again after 12 days of exposure to 1% NaCl in the drinking water (NaCl-loaded); two sequential femoral artery cannulations and direct BP recordings were used. All experimental procedures have been described in detail elsewhere.15 Rats were killed at 24 weeks of age.
Sequencing of WKYHD and SHRSPHD cDNAs Encoding Epithelial Sodium Channel Subunit Genes
Total RNA was isolated from WKY-1HD and SHRSPHD kidney by the guanidinium thiocyanate/cesium chloride method.17 First-strand cDNA synthesis was carried out in duplicate on 2 µg total RNA in a 20-µL reaction with Superscript II reverse transcriptase (RT, Life Technologies) following the manufacturer's recommendations. Two microliters of the cDNA reaction mix was subsequently amplified by PCR in a reaction volume of 50 µL. All PCR reactions were carried out in a solution containing 1 µmol/L primers, 200 µmol/L dNTPs, 1.5 mmol/L MgCl, and 1.25 U Taq DNA polymerase (Promega) with the reaction buffer supplied by the manufacturer. PCR reactions were processed on PTC-100 Thermal Controllers (MJ Research) according to the following protocol: initial denaturation at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 15 seconds, annealing for 1 minute, and extension at 72°C for 2 minutes, with a final extension step at 72°C for 7 minutes. Overlapping RT-PCR fragments covering the entire coding sequence as well as the immediate 5' and 3' flanking regions of each of the three genes were generated from primers based on the published sequence information for Scnn1a (GenBank accession No. X70497), Scnn1b (GenBank accession No. X77932), and Scnn1g (GenBank accession No. X77933). Direct PCR product sequencing was carried out with both PCR primers as well as additional nested forward and reverse sequencing primers synthesized to generate overlapping products for sequencing in both directions.
Scnn1b
Three overlapping fragments were generated for the ß-subunit (Scnn1b, X77932): fragment 1, spanning nucleotide positions 37 to 743, forward primer: 5'CTGCACATTGGAGCAGCTTTCTAAAC-3', reverse primer: 5'-ATGTTGGTGGCCTGCAGGATGTAC-3'; fragment 2, spanning positions 654 to 1752, forward primer: 5'-CAGTGCCAATGGGACCGTGTGTAC-3', reverse primer: 5'-CAGGCCTTTACAGGAGGCCACTAG-3'; and fragment 3, spanning positions 1608 to 2307, forward primer: 5'-TATCGTGTGGCTGCTCTCTAAC-3', reverse primer: 5'-ATTCTGGGAGACTGAGGTTGTAAC-3'. In addition, nested sequencing primers were used at nucleotide positions 366, 1021, 1323, 1650, and 1969 (forward) and at nucleotide positions 384, 1050, 1421, and 1986 (reverse).
Scnn1g
Two overlapping RT-PCR products were generated for the analysis of the -subunit gene (Scnn1g, X77933): fragment 1, spanning nucleotide positions 9 to 1800, forward primer: 5'-ACGCGTCCGGCGTCTCCAGACTGTAC-3', reverse primer: 5'-GCCACTGACGGCGGGCGATGATAGAG-3'; and fragment 2, spanning positions 1647 to 2206, forward primer: 5'-ATCATGGAGAGCCCAGCCAACAGTAT-3', reverse primer: 5'-GGGGCAGGTCCCATCAGGTTCTTC-3'. In addition, nested sequencing primers were used at nucleotide positions 312, 625, 988, 1314, and 1905 (forward) and at nucleotide positions 366, 615, 901, 1204, 1487, and 1978 (reverse).
Scnn1a
Four overlapping RT-PCR products were generated for analysis of the -subunit gene (Scnn1a, X70497): fragment 1, spanning nucleotide positions 34 to 753, forward primer: 5'-GGATGCGGGCACGGTCCCGGACAG-3', reverse primer: 5'-GCGCGCCGTGCGGCCGGAGTAG-3'; fragment 2, spanning positions 733 to 955, forward primer: 5'-CTGCGCACTCCACCTCCGCCCTACTC-3', reverse primer: 5'-TCTTCCTCTAGAGCGGGCGAGGTGTC-3'; fragment 3, spanning positions 950 to 2024, forward primer: 5'-TGTCGGACACCTCGCCCGCTCTAG-3', reverse primer: 5'-GGGCAAAGAAGGTGGTGGGGATGTAG-3'; and fragment 4, spanning positions 1964 to 2247, forward primer: 5'-GTGCCAGGGAGGTGGCCTCCACTC-3', reverse primer: 5'-TCGGCGGGGCCGCTGGGGAAGATG-3'. In addition, nested sequencing primers were used at nucleotide positions 373, 1179, 1449, and 1719 (forward) and at nucleotide positions 317, 478, 1295, 1629, and 1935 (reverse).
All PCR fragments were generated in duplicate from independent cDNA samples from WKY-1HD and SHRSPHD and subjected in duplicate to DNA sequence analysis by the fluorescent dideoxy terminator dye method using a DNA automated sequencing apparatus (ABI 373, Applied Biosystems).
Genotype Determination for Scnn1b, Scnn1g, and Scnn1a
Scnn1b
A single base transition in codon 509 of Scnn1b gives rise to a restriction fragment length polymorphism (RFLP) for the restriction enzymes NgoAIV and HaeIII. The genotypes at Scnn1b were determined with primers (forward: 5'-AGTCTGCCTTGCTGCCTCCTAATA-3', and reverse: 5'-AGAGAGCAGCCACACGATCTGTAG-3') based on the genomic sequence generated for WKY-1HD (GenBank accession No. U35177) and SHRSPHD (GenBank accession No. U35176) with 57°C as the annealing temperature. The genotypes in the F2 (WKY-1HDxSHRSPHD) intercross cohort were then analyzed by agarose gel electrophoresis after digestion of the PCR amplification products with the restriction enzyme NgoAIV (Life Technologies), which results in 89- and 133-bp fragments in the case of the WKY-1HD allele; the PCR product from SHRSPHD DNA remains uncleaved (222 bp).
Scnn1g
Genotype determination at the Scnn1g locus was carried out with an RFLP for the restriction enzyme Taq I in codon 341 (restriction site absent in WKY-1HD and present in SHRSPHD) of the Scnn1g cDNA. PCR amplification was carried out with primers (forward: 5'-CCTCCACTGGAGCCAAGGTGCTTATC-3', and reverse: 5'-GAAGTGGACATTGCTGTCTCGATCTC-3') that were based on the flanking cDNA sequence with 57°C as the annealing temperature. PCR products were subsequently digested with the restriction enzyme Taq I (New England Biolabs) and analyzed by agarose gel electrophoresis. The allele sizes generated from genomic DNA are identical to those generated from cDNA (73 bp in WKY-1HD, 52 and 21 bp in SHRSPHD)
Scnn1a
A single mutation found in codon 75 of the Scnn1a gene created an RFLP for the restriction enzyme EcoRI (restriction site absent in WKY-1HD and present in SHRSPHD). PCR amplification was carried out with primers (forward: 5'-GGGGAAGGGGGACAAACGTGAAGAG-3', and reverse: 5'-GCCCCGTGGATGGTGGTGTTGTTG-3') that were based on the flanking cDNA sequence with 65°C as the annealing temperature. PCR products were subsequently digested with EcoRI (New England Biolabs) and analyzed by agarose gel electrophoresis. The fragments generated thus from genomic DNA are identical to those generated from the cDNA (153 bp in WKY-1HD, 90 and 63 bp in SHRSPHD).
Fluorescent In Situ Hybridization
Cytogenetic localization of the Scnn1 genes was carried out by FISH as previously described.18 19 The probes used were the rat cDNAs encoding each of the three Scnn1 subunits kindly provided by Dr Bernard C. Rossier.3 20
Northern Blot Analysis
Total RNA was prepared from kidney of WKY-1HD and SHRSPHD at the age of 16 weeks (n=6 each). Ten micrograms of total RNA was size-fractionated on a 1.3% formaldehyde-agarose gel and then transferred to Nitro Plus nitrocellulose filters (MSI) by standard techniques.17 From the cDNA fragments generated for sequence analysis, those described as fragment 1 (see above) for each subunit were random labeled with 32P using T7 DNA polymerase (T7 QuickPrime Kit, Pharmacia). Filters were hybridized with the Scnn1a, Scnn1b, and Scnn1g probes, respectively, with the use of QuikHyb Hybridization Solution (Stratagene). Hybridized filters were washed in 30 mmol/L NaCl, 3 mmol/L sodium citrate, and 0.1% sodium dodecyl sulfate at 55°C and were exposed to Kodak XAR films at -80°C for 12 to 18 hours or to phosphor screens for 4 to 12 hours. After washing, the filters were hybridized against a rat GAPDH cDNA probe21 as a reference for differences in RNA loading. The filters were analyzed with a PhosphoImager apparatus and ImageQuant software (Molecular Dynamics).
Statistical Analysis and Linkage Analysis
Statistical evaluation of the effects of a particular locus on phenotype in F2 intercrosses was carried out by ANCOVA to account for sex and reciprocal cross status. Linkage analysis was carried out with the Mapmaker/Exp and Mapmaker/Qtl programs,22 with BP values adjusted to male levels and the first reciprocal cross.23 BP values are expressed as mean±SD. Expression data were compared with a two-tailed Student's t test.
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Sequence Analysis of Epithelial Sodium Channel Subunit Genes
Sequence data for the complete coding sequences of the Scnn1b, Scnn1g, and Scnn1a genes have been deposited under GenBank accession Nos. U35174 (WKY-1HD) and U35175 (SHRSPHD) for Scnn1b, U37540 (WKY-1HD) and U37539 (SHRSPHD) for Scnn1g, and U54699 (WKY-1HD) and U54700 (SHRSPHD) for Scnn1a. A number of differences were found compared with the published sequences3 20 derived from Sprague-Dawley rats. Comparative sequence analysis between WKY-1HD and SHRSPHD cDNAs revealed a number of sequence differences in all three subunit genes. However, the detected variants were conservative mutations without effect on the predicted amino acid sequence. In particular, no differences were found in a recently characterized C-terminal sequence, PPPXYXXL, that is present in all three subunits and shows conservation in human, rat, and Xenopus laevis epithelial sodium channel subunit genes.24
Two single base substitutions, in codons 400 (GGC in WKY-1HD, GGT in SHRSPHD) and 509 (CCG in WKY-1HD, CCA in SHRSPHD), were found for Scnn1b. Three single base differences, in codons 341 (ATT in WKY-1HD, ATC in SHRSPHD), 376 (GAC in WKY-1HD, GAT in SHRSPHD), and 410 (TGC in WKY-1HD, TGT in SHRSPHD), were found for Scnn1g. A single base difference was found in codon 75 (GAG in WKY-1HD, GAA in SHRSPHD) of the Scnn1a cDNA.
Chromosomal Localization and Positional Analysis
We used the FISH technique to determine the regional localization of the Scnn1 genes. Double fluorescent spots (two labeled sister chromatids) were found only on chromosome 1 for the Scnn1b and Scnn1g genes and on chromosome 4 for Scnn1a. Several metaphases showed labeling of the two homologous chromosomes. The positions of the spots along the chromosomes were measured and found to be between the following values: 66% to 78% for Scnn1b and Scnn1g and 80% to 90% for Scnn1a, starting from the end of the p arm in the case of chromosome 1 or from the centromere in the case of chromosome 4. As illustrated in Fig 1, these measurements allow us to place the genes at the following positions: Scnn1b and Scnn1g at 1q36-q42 and Scnn1a at 4q42. Comparison of the positions of the fluorescent spots with the positions of the bands obtained after staining with 4'-6-diamidino-2-phenylindole (DAPI) supports these conclusions.
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Multilocus linkage analysis revealed the expected colocalization of the Scnn1b and Scnn1g locus5 in close vicinity to Sa13 on chromosome 1 (Fig 2a). Linkage analysis with chromosome 4 markers confirmed the results obtained by FISH, indicating that Scnn1a is tightly linked to the enolase 2 (Eno2) locus on rat chromosome 4 (Fig 2b).
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BP Linkage Analysis
Quantitative trait linkage analysis carried out by both ANCOVA and multipoint likelihood methods revealed a significant cosegregation of the Scnn1b/Scnn1g locus with systolic and diastolic BPs at baseline and after NaCl loading (Table). Further analysis with additional markers revealed a 12-cM-wide 1:100 confidence interval for placement of this QTL (Fig 2a). Maximum placement was found at a homologous marker from mouse chromosome 7, D1M7Mit236 (logarithm of the odds [LOD] score 5.0), 4.4 cM away from the Scnn1b/Scnn1g locus (LOD score 3.7). Thus, the two Scnn1 genes mapped closely to the confidence interval for placement of this BP QTL (Fig 2a). No linkage to BP phenotypes was seen for the Scnn1a locus (Table).
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Kidney mRNA Analysis
Northern blot analysis of kidney mRNA revealed no difference in gene expression among WKY-1HD and SHRSPHD for Scnn1a (P=.52), Scnn1b (P=.27), and Scnn1g (P=.12), as shown in Fig 3.
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Discussion |
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The mutations initially identified in patients with Liddle's syndrome cause truncation of the C-terminus of either the ß- or
-subunit of the epithelial sodium channel, resulting in the formation of constitutively activated channels and thus increased sodium reabsorption.4 5 The spectrum of Liddle's syndrome has since been broadened by in vitro experiments showing that nontruncating mutations in a conserved proline-rich motif in the C-terminus in any of the three subunits of the human epithelial sodium channel reproduce the defect seen in Liddle's syndrome.24 More recently, the relevance of these in vitro observations has been highlighted by the identification of families in whom Liddle's syndrome results from such missense mutations within the C-terminal motif in the ß-subunit.7 8 Thus, both the ß- and -subunits, as well as the -subunit gene, have to be considered as candidate genes for Liddle's syndrome and, by extension, for other forms of genetic hypertension.
The potential colocalization of the ß- and -subunit genes with a BP QTL identified by the Sa locus on rat chromosome 1,5 13 29 along with the speculation that less dramatic mutations in the Scnn1 genes may contribute to polygenic forms of hypertension,5 spurred our interest in examining these interesting candidate genes further. As expected from the human data, the rat ß- and -subunits map in close vicinity to Sa and to a BP QTL that contributes to the genetic variance of systolic BP in the WKY-1HD/SHRSPHD intercross as well as other previously described crosses.9 10 11 12 13
The lack of clear evidence that abnormalities in sodium handling play a prominent pathogenic role in SHRSPHD hypertension does not detract from the possibility that perhaps more subtle functional mutants of the epithelial sodium channel subunit genes than those operative in Liddle's syndrome may contribute to BP regulation in this strain, analogous to arguments advanced regarding human essential hypertension.5 Our findings, however, supported by complete cDNA sequence analysis of Scnn1b and Scnn1g genes in WKY-1HD and SHRSPHD, exclude the presence of biologically relevant mutations in the coding sequences of these genes as a possible substrate for the BP QTL on chromosome 1. Recently, near full-length cDNA analysis of Scnn1b in spontaneously hypertensive and Dahl salt-sensitive and salt-resistant rats also failed to reveal any relevant coding sequence mutations.13 In addition, in the current investigation, no differences in Scnn1b and Scnn1g gene expression in the kidney between the parental strains were found by Northern blot analysis of RNA in WKY-1HD and SHRSPHD. Likewise, our study excluded a role of Scnn1a.
The mapping of the three Scnn1 genes in the rat is interesting with respect to anchoring the rat linkage and cytogenetic maps and comparative mapping in humans and mice, because all three genes are now mapped in these three species.30 31 An additional gene, Spn (=Lsn), known to reside within the same linkage group as Scnn1b and Scnn1g, is found on rat chromosome 1, human chromosome 16p, and mouse chromosome 7.32 The localization of the two Scnn1 genes to rat chromosome 1 thus defines a new synteny group conserved in three species. Furthermore, the gene order, Scnn1b/g–Spn–Igf2, is conserved in rat and mouse.13 30 The Sa gene is part of the same linkage group on rat chromosome 1 and human chromosome 16p29 33 34 ; it has not been localized in the mouse. Likewise, Scnn1a is localized, along with Eno2, in a conserved region on rat chromosome 4 and human chromosome 12p13.31 In the mouse, it has been mapped to chromosome 6.30 31
The speculation is intriguing that genes to which rare single-gene syndromes have been mapped might represent, on the basis of mutations with less severe functional consequences, candidate genes that contribute to common, polygenic manifestations of the same morbid phenotype. Despite the increased odds for such a possibility in the WKY-1HDxSHRSPHD intercross because of prior linkage of BP to the Scnn1b/Scnn1g locus, our data fail to support a role relevant to BP for either the Scnn1b or Scnn1g gene. As is true for all studies examining the genetics of complex traits, our data are a priori only applicable to the specific strains, the specific developmental phase, and the specific gene environment constellations studied. Additional experiments, including the creation of congenic strains, are currently in progress to further characterize the chromosome 1 QTL, identify the operative gene, and more definitively rule out a contribution of the Scnn1b and Scnn1g genes to hypertension in SHRSPHD.
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Acknowledgments |
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This work was supported in part by National Institutes of Health SCOR grant P50HL5580, the Deutsche Forschungsgemeinschaft (to R.K., Kr1152/1-2, and to D.G., Ga175/8-1), by the EURHYPGEN concerted action of the European Economic Community, by the FRSM (Belgium), and the Communauté Française de Belgique. R.K. is the recipient of a Howard Hughes Medical Institute Research Fellowship for Physicians, C.S. is a research director of the FNRS (Belgium), and K.L. is the recipient of National Institutes of Health Research Career Development Award K04HL-03039. We are grateful to Pascale Vanvooren for technical assistance and to Lily Eng and David Mycue for support with automated DNA sequencing.
Received May 3, 1996;
first decision May 29, 1996; first decision July 25, 1996;
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