From the 1st Department of Internal Medicine, Shiga University of Medical
Science, Otsu, Japan.
Correspondence to Naoharu Iwai, MD, 1st Department of Internal Medicine, Shiga University of Medical Science, Otsu, Japan. E-mail iwai{at}suncuore.shiga-med.ac.jp
We previously isolated a gene, the transcript of which is
expressed in much higher levels in the kidneys of SHR than in those of
Wistar-Kyoto rats (WKY), by differential
screening.3 This gene, designated Sa,
was mapped on rat chromosome 1q near the ß and
Genotype Analysis
The congenic status of the congenic strain established in the
present study was confirmed by genotyping the following markers
that were polymorphic between the SHR and WKY progenitor strains:
D2Mgh6, D2Mgh14, D2Mit3, D2Mit21, D3Mit10, D4Mgh4, D4Mgh8,
D4Mit2, D4IL6, D5Mgh2, D5Mit5, D5Mit10, D6Mit4, D6Mit6, D6IGHE,
D7Mgh10, D7Mgh11, D7Mit4, D7Mit12, D8Mgh8, D8Mit3, D8Mit7, D9Mit9,
D9Mit6, D10Mgh6, D10Mgh8, D11Mgh4, D11Mgh6, D12Mgh2, D12Mgh3, D12Mgh5,
D13Mgh1, D13Mgh2, D13Mgh3, Renin, D14Mgh2, D14Mit2, D15Mit2, ETB,
D16Mit1, D16Mit2, D17Mgh5, D17Mit3, PRLb, D17Mgh8, D18Mgh2, D18Mgh4,
D18Mit7, D19Mit5, D19Mit7, D20Mgh2, UW1, and
DXMgh5.
Cardiovascular Phenotyping
Statistical Analysis
Mean BP was significantly higher in the WKY.SHR-D1Mit3/Rat57 congenic
strain than in the WKY progenitor strain during both the day (7
AM to 7 PM; P=0.0182, t
test; Table
Two weeks of salt loading with a high salt diet increased the mean BP
more in the WKY.SHR-D1Mit3/Rat57 strain than in the WKY progenitor
strain, especially at night. Thus, mean BP was markedly higher in the
WKY.SHR-D1Mit3/Rat57 strain than in the WKY progenitor strain at 18
weeks of age on a high salt diet during both the day
(P=0.0005) and the night (P<0.0001).
Two-way ANOVA showed a significant difference in BP between the 2
strains and a significant difference in the BP response to salt loading
between the 2 strains (Table
To confirm that the Sa gene locus is a QTL for BP and
to perform more fine mapping of a QTL for BP in this region, we
constructed a congenic strain by transferring the SHR chromosome 1
segment around the Sa gene onto the genetic background of
the WKY strain. The present study confirmed that transfer of a
chromosome 1 segment defined by the markers D1Mit3 and
D1Rat57 from SHR to WKY led to a significant increase in BP.
Moreover, salt loading increased BP more in the WKY.SHR-D1Mit3/Rat57
strain than in the WKY progenitor strain.
The present findings are consistent with the results
of previous segregation studies. Polymorphism at the Sa
and/or Acnn1b loci cosegregated with basal BP in
F2 populations derived from SHRxWKY6 9 and SHRxLewis,7 as well as in a
series of recombinant inbred strains derived from SHRxBrown-Norway
(BN) rats.5 In an F2
population derived from SHR-SPxWKY4 and Dahl
salt-sensitivexLewis8 rats, polymorphism at
the Sa locus cosegregated with BP after salt loading.
St. Lezin et al15 recently reported that transfer
of chromosome 1 segment defined by the markers D1Mit3 and
Igf2 from normotensive BN onto the SHR genetic background
was sufficient to induce a significant reduction in
arterial BP. Our present study further confirmed the
existence of a QTL for BP in this chromosome 1 region and more
precisely mapped the QTL for BP between markers D1Mit3 and
D1Rat57. Moreover, we found one of the features of this QTL
for BP, ie, salt sensitivity.
This chromosome region contains the Sa, Scnn1b, and
Scnn1g genes as candidate genes for hypertension. Although
the expression of the Sa gene was observed in proximal
tubular cells14 and marked differences in the
expression levels were observed between SHR and WKY
strains,3 the functions of the Sa gene
remain to be determined. No sequence variations in Scnn1b
and Scnn1g genes have been reported between SHR and
normotensive BN.5 Thus, we have not yet
identified a gene or genes responsible for salt-sensitive hypertension
in this chromosomal region. Many other genes exist on this chromosomal
region, which is homologous to human chromosomes 16p and 11p and mouse
chromosome 7.16 Recent development in human and
mouse genome projects may reveal a number of other candidate genes
in this chromosomal region in the near future.
The WKY.SHR-D1Mit3/Rat57 strain is a new model for salt-sensitive
hypertension. Analyses of physiological
parameters, including renal hemodynamic and
tubular functions, and hormonal features will help to identify a gene
that contributes to salt-sensitive hypertension. Moreover, the
establishment of congenic sublines will refine the map position of a
QTL for BP.
Received May 1, 1998;
first decision May 20, 1998;
accepted June 8, 1998.
2.
Hilbert P, Lindpaintner K, Beckmann JS, Serikawa T,
Soubrier F, Dubay C, Cartwright P, De Gouyon B, Julier C, Takahashi S,
Vincent M, Ganten D, Georges M, Lathrop GM. Chromosomal mapping of two
genetic loci associated with blood-pressure regulation in hereditary
hypertensive rats. Nature. 1991;353:521–529.[Medline]
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3.
Iwai N, Inagami T. Isolation of preferentially
expressed genes in the kidneys of hypertensive rats.
Hypertension. 1991;17:161–169.
4.
Lindpaintner K, Hilbert P, Ganten D, Nadal-Ginard B,
Inagami T, Iwai N. Molecular genetics of the Sa gene: cosegregation
with hypertension and mapping to rat chromosome 1. J
Hypertens. 1993;44:19–23.
5.
Huang H, Pravenec M, Wang J-M, Kren V, St Lezin E,
Szpirer C, Szpirer J, Kurtz TW. Mapping and sequence analysis
of the gene encoding the beta subunit of the epithelial sodium channel
in experimental hypertension. J Hypertens. 1995;13:1247–1251.[Medline]
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6.
Iwai N, Inagami T. Identification of a candidate gene
responsible for the high blood pressure of spontaneously hypertensive
rats. J Hypertens. 1992;10:1155–1157.[Medline]
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7.
Iwai N, Kurtz TW, Inagami T. Further evidence of the
Sa gene as a candidate gene contributing to the hypertension in
spontaneously hypertensive rat. Biochem Biophys Res Commun. 1992;188:64–69.[Medline]
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8.
Harris EL, Dene H, Rapp JP. Sa gene and blood pressure
cosegregation using Dahl salt-sensitive rats. Am J
Hypertens. 1993;6:330–334.[Medline]
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9.
Samani NJ, Lodwick D, Vincent M, Dubay C, Kaiser
MA, Kelly MP, Lo M, Harris J, Sassard J, Lathrop M, Swales JD. A gene
differentially expressed in the kidney of the spontaneously
hypertensive rat cosegregates with increased blood pressure.
J Clin Invest. 1993;92:1099–1103.
10.
Gu L, Dene H, Deng AY, Hoebee B, Bihoreau M-T, James M,
Rapp JP. Genetic mapping of two blood pressure quantitative trait loci
on rat chromosome 1. J Clin Invest. 1996;97:777–788.[Medline]
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11.
Gu L, Dene H, Rapp JP. Three microsatellites defining
four alleles for the rat Sa gene. Mamm Genome. 1994;5:833.[Medline]
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12.
Kawabe K, Watanabe T, Shiono K, Sokabe H. Influence on
blood pressure of renal isografts between spontaneously hypertensive
and normotensive rats, utilizing the F1 hybrid. Jpn Heart
J. 1979;20:886–894.
13.
Rettig R, Straus H, Folberth C, Ganten D, Waldherr R,
Unger T. Hypertension transmitted by kidneys from stroke-prone
spontaneously hypertensive rats. Am J Physiol. 1989;257:F197–F203.
14.
Yang T, Hassan SA, Singh I, Smart A, Brosius FC,
Holzman LB, Schnermann JB, Briggs JP. Sa gene expression in the
proximal tubule of normotensive and hypertensive rats.
Hypertension. 1996;27:541–545.
15.
St Lezin E, Liu W, Wang J-M, Wang N, Kren V, Krenova D,
Musilova A, Zdobinska M, Zidek V, Lau D, Pravenec M. Genetic isolation
of a chromosome 1 region affecting blood pressure in the spontaneously
hypertensive rat. Hypertension. 1997;30:854–859.
16.
Szpirer C, Szpirer J, Tissir F, Stephanova E, Vanvooren
P, Kurtz TW, Iwai N, Inagami T, Pravenec M, Kren V, Klinga-Levan K,
Levan G. Mamm Genome. 1997;8:657–660.[Medline]
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© 1998 American Heart Association, Inc.
Scientific Contributions
Isolation of a Chromosome 1 Region That Contributes to High Blood Pressure and Salt Sensitivity
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—Linkage
analyses in the spontaneously hypertensive rat (SHR) suggest
that a gene involved in blood pressure regulation may be located on rat
chromosome 1, in the Sa region. To confirm this
possibility, we replaced a region of chromosome 1 in the Wistar-Kyoto
rat (WKY) defined by the markers D1Mit3 and
MTPA with the corresponding chromosome segment from SHR.
Genotyping using 65 polymorphic microsatellite markers throughout
the entire genome confirmed the congenic status of this new strain
designated WKY.SHR-D1Mit3/Rat57. In male WKY.SHR-D1Mit3/Rat57, mean
blood pressures in the daytime and in the nighttime assessed by
radiotelemetry were significantly higher than those in male progenitor
WKY. Moreover, salt loading significantly increased the mean blood
pressure in male WKY.SHR-D1Mit3/Rat57 but not in male progenitor WKY.
The present study confirmed the existence of a gene that
contributes to high blood pressure and salt sensitivity in this
chromosomal segment. This congenic strain represents a new
animal model for fine mapping and characterization of the gene in this
region involved in salt-sensitive hypertension.
Key Words: blood pressure • rats, inbred SHR • genetics • congenic strain • chromosome 1
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The spontaneously hypertensive rat (SHR) is the most
widely used animal model of human essential hypertension. Although
recent developments in molecular genetics have revealed several
candidate loci that influence blood pressure (BP) in SHR, the distinct
genes that contribute to SHR hypertension have not been
identified.1 2 
subunits of the
epithelial sodium channel (Scnn1b and
Scnn1g),4 5 and several linkage studies in
second filial generation (F2) populations
have suggested that a blood pressure quantitative trait locus (BP QTL)
may exist in this chromosomal region in the vicinity of
Sa.6 7 8 9 10 However, a linkage study in
segregating populations cannot be used to precisely identify a gene or
a BP QTL. To confirm the presence of a putative BP QTL on rat
chromosome 1 and to perform fine genetic mapping of a BP QTL in this
region, we replaced a WKY chromosome 1 region defined by the markers
D1Mit3 and MTPA with the corresponding chromosome
region from SHR. Using this congenic strain, we confirmed the existence
of a gene in this chromosomal segment that contributes to high BP and
salt sensitivity in SHR.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Strains
The WKY congenic strain was derived by a selective breeding
protocol in which a segment of chromosome 1 from SHR was transferred
onto the genetic background of the progenitor WKY. The progenitor SHR
and WKY were obtained from Charles River Laboratories (Atsugi,
Japan), and the inbred status of these strains was confirmed with 65
microsatellite markers throughout the genome. After 10 generations of
selective back-crossing to the WKY progenitor strain, during which the
presence of the SHR allele of the Sa gene in the
heterozygous state and absence of the SHR allele in the
D1Mit18 and D1Mit17 loci were confirmed, we
selected a litter that had the WW genotype in D1Mit17,
D1Rat89, MTPA, and RCA0120 markers and SW
genotype in D1Mit3, Sa, and D1Rat57. By
brotherxsister mating and selective breeding of the offspring, the
chromosomal segment around Sa was fixed and maintained.
Animals of the N10F3 generation were used in the present
study.
To determine the length of the differential chromosome 1
segment, we genotyped the congenic strain using the following
genetic markers that were polymorphic between the SHR and WKY
progenitor strains: D1Mgh14, D1Mgh13, D1Mit7, D1Mit18, RCA0120,
MTPA, D1Rat139, D1Rat57, Sa,11 D1Mit3,
D1Rat89, and D1Mit17. The map positions of these
markers were determined with Mapmanager by genotyping 68 male
F2 rats derived from the SHR and WKY progenitor
strains.
Pulsatile arterial pressures and heart rates
were measured in unanesthetized, unrestrained male rats at 16
and 18 weeks of age. Indwelling radiotelemetry transducers, connected
to catheters implanted in the lower abdominal aorta, were implanted in
rats at 12 weeks of age. Pulsatile pressures and heart rates were
recorded in 5-second bursts every 5 minutes for 24 hours in
16-week-old rats on a standard diet (NaCl; 0.35%) and in 18-week-old
rats after 2 weeks of salt loading with a high salt diet (NaCl; 8.0%).
Rats were kept at a controlled room temperature with light from 7
AM to 7 PM (daytime) and were given tap water
ad libitum. The BP of 8 male progenitor WKY and 8 male congenic rats
was measured in the present study. This study was conducted in
accordance with the current guidelines for the care and use of
experimental animals of Shiga University of Medical Science.
Two-way ANOVA was used to delineate the effects of strain and
salt loading, as well as the interaction between these
parameters, on BP.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Genotype analysis of markers on chromosome 1
confirmed the successful transfer of a defined segment of chromosome
from the SHR strain onto the WKY genetic background. The maximum size
of the transferred segment was defined by the markers
D1Rat89 and MTPA, and the minimum size was
defined by the markers D1Mit3 and D1Rat57
(Figure
). Genotype
analysis using 65 genetic markers throughout the genome
confirmed the congenic status of the new strain, designated
WKY.SHR-D1Mit3/Rat57.
View larger version (12K):
[in a new window]
Figure 1. Linkage map showing the transferred segment of chromosome 1
in WKY.SHR-D1Mit3/Rat57. The solid bar indicates the chromosome region
transferred from the SHR strain, and open regions indicate the flanking
segments of WKY chromosomes. The shaded portions indicate boundaries of
the transferred chromosomes. ) and the night (7
PM to 7 AM; P=0.0105) at 16 weeks of
age under a standard diet. The difference in BP levels was greater
during the night.
View this table:
[in a new window]
Table 1. Mean Blood Pressure Levels Determined by
Radiotelemetry 
).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The kidney plays a crucial role in the pathogenesis and
maintenance of hypertension in the SHR. Renal transplantation
experiments have suggested that the kidneys of SHR may have primary
genetic defects.12 13 On the basis of the
hypothesis that a gene that contributes to hypertension in SHR might be
differentially expressed in the kidneys of SHR and its control strain
WKY, we previously identified a gene, designated Sa, by
differential screening. The expression levels of the Sa gene
transcript were about 10 times higher in the kidneys of SHR than in
those of WKY from the 4th week of age.3 In situ
hybridization analysis has indicated that the transcript of the
Sa gene is present in proximal
tubules,14 which suggests that a product of
the Sa gene may be involved in body fluid homeostasis.
However, differential expression and/or tubular expression do not
necessarily mean that the gene is involved in the pathogenesis of
hypertension. Subsequently, cosegregation analyses of the
genotype of the Sa gene with BP were carried out in
several F2 populations and confirmed that the
Sa gene was in the region of a QTL for BP. However, it
remains to be determined whether the Sa gene itself is the
gene responsible for SHR hypertension.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Jacob HJ, Lindpaintner K, Lincoln SE, Kusumi K,
Bunker RK, Mao Y-P, Ganten D, Dzau VJ, Lander ES. Genetic mapping of a
gene causing hypertension in the stroke-prone spontaneously
hypertensive rat. Cell. 1991;67:213–224.[Medline]
[Order article via Infotrieve]
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