Effects of Mineralocorticoid Receptor Gene Disruption on the Components of the Renin-Angiotensin System in 8-Day-Old Mice
Christine Hubert,
Jean-Marie Gasc,
Stefan Berger,
Günther Schütz and
Pierre Corvol
INSERM U36-Laboratoire de Médecine
Expérimentale (C.H., J.M.G., P.C.) Collège de
France Paris, France 75005
Division Molecular Biology of
the Cell I (S.B., G.S.) German Cancer Research Center
Heidelberg, Germany
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ABSTRACT
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Targeted disruption of mineralocorticoid receptor
(MR) gene results in pseudohypoaldosteronism type I with failure to
thrive, severe dehydration, hyperkalemia, hyponatremia, and high plasma
levels of renin, angiotensin II, and aldosterone. In this study, mRNA
expression of the different components of the renin-angiotensin system
(RAS) were evaluated in liver, lung, heart, kidney and adrenal gland to
assess their response to a state of extreme sodium depletion.
Angiotensinogen, renin, angiotensin-I converting enzyme, and
angiotensin II receptor (AT1 and
AT2) mRNA expressions were determined by
Northern blot and RT-PCR analysis. Furthermore, in situ
hybridization and immunohistochemistry allowed us to identify the cell
types involved in the variation of the RAS component expression. In the
heterozygous mice (MR+/-), compared with wild-type mice (MR+/+), there
was no significant variation of any mRNA of the RAS components. In MR
knockout mice (MR-/-), compared with wild-type mice, there were
significant increases in the expression level of several RAS
components. In the liver, angiotensinogen and
AT1 receptor mRNA expressions were moderately
stimulated. In the kidney, renin mRNA was increased up to 10-fold and
in situ hybridization showed a marked recruitment of
renin-producing cells; however, the levels of angiotensin-I converting
enzyme mRNA and AT1 mRNA were not changed.
Interestingly, in adrenal gland, renin expression was also strongly
up-regulated in a thickened zona glomerulosa, whereas
AT1 mRNA expression remained unchanged.
Altogether, these results demonstrate that in the MR knockout mice
model, RAS component expressions are differentially altered, renin
being the most stimulated component. Angiotensinogen and
AT1 in the liver are also increased, but the
other elements of the RAS are not affected.
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INTRODUCTION
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Aldosterone is a key element of a complex homeostatic mechanism
that preserves the sodium balance and the stability of the
extracellular fluid volume and thereby plays a major role in the
maintenance of blood pressure (1). The actions of aldosterone are
mediated by the occupancy of corticosteroid type I receptors,
i.e. mineralocorticoid receptor (MR) (2), a member of the
nuclear receptor (3). After the binding of aldosterone to its nuclear
receptor, the chaperone proteins dissociate from the receptor, and the
hormone-receptor complex binds to specific DNA sequence as a dimer, to
enhance the expression of aldosterone target (4).
MR is expressed with a much more restricted tissue distribution pattern
than glucocorticoid receptors. Although both aldosterone and
glucocorticoids bind to MR, MR occupancy in aldosterone target cells by
aldosterone is ensured by the 11ß-hydroxysteroid dehydrogenase type
II enzyme, which inactivates glucocorticoids. The presence of this
enzyme is crucial for the specificity of aldosterone effects on target
cells because glucocorticoids and aldosterone have an equivalent high
affinity for the MR and glucocorticoids are much more abundant than
mineralocorticoids (5, 6). Aldosterone acts by stimulating sodium
reabsorption in epithelia of the colon and the kidney, via
amiloride-sensitive epithelial sodium channels (7).
The essential role of MR in the processes of sodium balance has been
recently documented by MR gene disruption in mice (8). The MR knockout
mice MR-/-, obtained by gene targeting, died between day 8 and 13
after birth, with a markedly reduced weight and a severe dehydration
due to failure of sodium reabsorption. The MR knockout mice showed all
signs of pseudohypoaldosteronism, such as hyperkalemia, hyponatremia,
and a strongly activated renin angiotensin system (RAS). Compared with
wild-type mice, plasma renin was 440-fold, angiotensin II 50-fold, and
aldosterone 65-fold increased.
The MR knockout mice also showed conspicious morphological changes at
the glomerular vascular pole. The segment of the distal tubule at the
level of the macula densa was enlarged, and the extraglomerular
mesangium (juxtaglomerular cells) showed prominent hyperplasia.
Renin-producing granular cells extended upstream along the afferent
arteriole, and renin granules were seen in almost all extraglomerular
mesangium cells in MR knockout mice.
In heterozygous mice MR+/-, the RAS was moderately activated. Compared
with wild-type mice, plasma levels of renin, angiotensin II, and
aldosterone were 2- to 3-fold increased, showing the adapted feedback
response of the circulating RAS elements in an attempt to counteract
the lack of aldosterone effects by partial MR deficiency (8).
This condition of extreme sodium depletion is unique in many aspects.
Gene disruption of other components of the RAS do not produce such a
severe and lethal phenotype. Angiotensinogen (AGT) knockout and
angiotensin I-converting enzyme (ACE) knockout mice have a low
perfusion pressure, renal insufficiency, and urinary concentration
defect but can survive although Unemara et al. (9) reported
that about 60% of the AGT knockout mice in their study did not survive
until weaning. The stimulation of the RAS observed in the MR knockout
mice is also much stronger than that observed in rodent models in which
the RAS is stimulated, such as salt depletion induced by low sodium
diet, furosemide, and (or) renovascular hypertension, reflecting the
crucial role of salt reabsorption in the early days of life when
the sodium intake from milk is limited (10).
It was therefore of interest 1) to further describe morphological
changes induced by MR gene disruption in the kidney and the adrenal
gland, two organs involved in sodium reabsorption and steroid
biosynthesis, respectively; 2) to evaluate the expression of the
different components of the RAS in these organs, but also in tissues
where other components of the RAS are synthesized, such as the liver,
lung, and heart; and 3) to determine whether the animals harboring a
single MR allele exhibited any of the changes observed in MR knockout
mice.
In MR knockout mice at age 8 days, at a time when the RAS is already
functional, a marked increase in renin expression was found in the
kidney and the adrenal gland, using mRNA semiquantification, in
situ hybridization, and immunohistochemistry. An up-regulation of
angiotensinogen in the liver was also observed, whereas ACE mRNA levels
in the lung as well as in the heart were not altered. Except in the
liver, angiotensin II receptor AT1 mRNA remained stable in
heart, kidney, and adrenal gland.
The heterozygous mice exhibited a limited impairment of renal sodium
reabsorption and a moderate 3-fold stimulation in plasma of renin,
angiotensin II, and aldosterone levels compared with their
concentrations in plasma of wild-type mice (8). However, compared with
wild-type mice, the heterozygous mice showed no significant difference
in any of the tested mRNA levels of the RAS components.
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RESULTS
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The expression of the RAS components was determined in liver,
lung, heart, kidney, and adrenal gland of 8-day-old wild-type MR+/+,
heterozygous MR+/-, and MR knockout MR-/- mice. The main results are
outlined below.
Liver
AGT is a unique renin substrate and is mainly synthesized in the
liver. Expression of its mRNA was semiquantified by Northern blot and
normalized to the glyceraldehyde-3 phosphate dehydrogenase (G3PDH) mRNA
signal. In 8-day-old mice, AGT mRNA was increased by 12% in MR+/-,
and by 49% in MR-/-, compared with MR+/+ mice. Only the difference
between MR+/+ and MR-/- mice was statistically significant
(P < 0.01) (Fig. 1A
).
AT1 receptor is the only angiotensin II receptor expressed
in liver. Its mRNA level was evaluated by RT-PCR in the three
genotypes. AT1 expression increased by 145% in MR-/-
vs. MR+/+ mice (P < 0.01), whereas no
variation was observed in MR+/- mice (Fig. 1B
).

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Figure 1. Normalized Levels of Angiotensinogen (A) and
AT1 (B) mRNA in Liver of 8-Day-Old Mice of MR+/+, MR+/-,
and MR-/- Genotypes
A, For angiotensinogen determination, Northern blots were
initiated and hybridized with a rat angiotensinogen probe. The
intensity of the autoradiographic signal was normalized to the value of
G3PDH mRNA signal, as indicated in Materials and
Methods. B, Incorporation of [3H]dCTP in amplicon
for AT1 detection was conducted by PCR and was also
normalized to G3PDH amplicon from the same RT. The amplified products
were subjected to electrophoresis and the bands were excised for
radioactivity measurement. Columns represent mean
± SEM from number of organs indicated in the
circle. The P values of Mann-Whitney test are
represented between groups.
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Lung
The lung is the major source of ACE because of its very large
surface covered with endothelial cells. In this tissue, most of plasma
angiotensin I is converted to angiotensin II. Lung ACE mRNA levels,
measured by RT-PCR and normalized to G3PDH mRNA levels, were not
statistically different between the three genotypes (Table 1
). Similarly, in situ
hybridization revealed no difference in expression of the ACE mRNA
among the three genotypes (data not shown).
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Table 1. Effect of MR Genotype in 8-Day-Old Mice, on
mRNA Levels of Renin, ACE, and AT1 Receptor in Lung, Heart, and
Kidney
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Heart
A fetal and neonatal expression of the components of the RAS has
been reported in different species (Ref. 11 and references therein).
Since the RAS could have been stimulated in MR knockout mice, the
levels of renin, ACE, and AT1 mRNA were evaluated. In
situ hybridization, however, did not reveal any variation in the
expression of renin mRNA or AT1 mRNA. RT-PCR analysis did
not detect any changes in ACE or AT1 mRNA expression (Table 1
).
Kidney
In addition to the morphological changes at the glomerular
vascular pole in MR-/- mice described by Berger et al.
(8), we made the following observations. The wall of interlobar
branches of renal arteries was conspicuously thickened in MR-/- (Fig. 3
, A and B). The degree of maturation of the kidney appeared
similar in mice of the three genotypes and, in particular, the number
and maturation stages of glomeruli did not grossly differ. In MR+/+
kidney, in addition to the juxtaglomerular apparatuses, immunoreactive
renin was detected in the innermost segments of proximal convoluted
tubules (PCT), whereas in MR-/-, renin was detected in PCTs in the
whole cortex, including the outermost region (not shown).
Interestingly, in MR-/- kidneys, renin-producing cells labeled by
immunohistochemistry (Fig. 3
, panel D vs. panel C) as well
as by in situ hybridization (Fig. 3
, panels I and J
vs. panels F and G), showed considerably enlarged
juxtaglomerular apparatuses, exhibiting an intensity of immunostained
renin not obviously above that observed in MR+/+ (Fig. 3
, panel D
vs. panel C). No signal of renin mRNA could be detected in
the PCTs, whatever the genotype (Fig. 3
, F and I). By RT-PCR analysis,
renin mRNA expression was 2.5-fold increased in MR-/- compared with
MR+/+ mice (P < 0.05), but not in MR+/-
(P = 0.7) (Table 1
). Renin mRNA level, evaluated by
Northern blot and normalized to G3PDH mRNA level, showed a 10-fold
increase in MR-/- (P < 0.01) and again no change in
MR+/- mice (Fig. 2
). No other
significant variations of ACE and AT1 mRNA between
genotypes were found by RT-PCR (Table 1
). Similarly, using in
situ hybridization, there was no difference in the expression
level of AT1 receptor mRNA according to genotype (not
shown).

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Figure 3. Histopathological Phenotype of the Kidney of MR+/+
(A, C, E, F, and G) and MR-/- (B, D, H, I, and J) Mice
The wall of intrarenal arteries appear thickened in homozygous mutant
(B) when compared with the homozygous wild-type mouse (A).
Juxtaglomerular apparatuses, immunostained with a renin antibody (C and
D) or labeled by in situ hybridization with a riboprobe
to renin mRNA (F, G, I, and J), appear enlarged in MR-/- (D and J)
compared with MR+/+ (C and G) mice. At lower magnification, a kidney
section shows the difference between MR+/+ (EG) and MR-/- (HJ) in
size and number of juxtaglomerular apparatuses labeled with the renin
mRNA riboprobe. All photomicrographs are by bright field illumination,
except F and I which are darkfield. Scale bar, 100 µm
in panels E, F, H, and I; 20 µm in panels A, B, C, D, G, and J.
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Figure 2. Renin mRNA in Kidney of Mice from the Three
Genotypes, MR+/+, MR+/-, and MR-/-
Total RNA were subjected to Northern blot analysis, hybridized with
-32P-labeled mouse renin cDNA. The blots were
rehybridized with a labeled G3PDH cDNA probe to normalize RNA integrity
and RNA load. A, Autoradiographic signals were quantified by scanning
densitometry. B, Columns represent the ratio mean ±
SEM of number of animals indicated in the
circle. The P values of Mann-Whitney test
are indicated between groups.
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Adrenal Gland
A conspicuous histological difference was observed between MR+/+
and MR-/- mice at 8 days. In MR-/- mice (Fig. 4C
), based on histological criteria, the
zona glomerulosa appeared thicker and extended more deeply toward the
medulla than in MR+/+ mice (Fig. 4A
), to the detriment of the zona
fasciculata, which was reduced in thickness up to the point of being
histologically undetectable. This difference was not observed in
heterozygous animals. The expression of renin was investigated by
immunodetection and in situ hybridization. In MR-/-, a
clear hybridization signal for renin was located in the enlarged zona
glomerulosa (Fig. 4D
). The labeling was not uniform and appeared in
cell clusters distributed within the thickened zona glomerulosa. In
heterozygous and wild-type (Fig. 4B
) mice, renin mRNA remained below
detectable level. Renin was not immunodetected in the same adrenal
glands in which the renin mRNA level was high, although renin was
readily noticeable in glomeruli of the adjacent kidney (Fig. 3C
). Because the adrenals were small,
particularly in 8-day-old mice, only two or three pools of organs could
be used for the RT-PCR and, therefore, no statistical analysis were
performed. A 10-fold increase of the normalized renin mRNA levels to
G3PDH was detected in MR-/- mutants compared with MR+/+ or MR+/-
mice (2.02, 0.22, and 0.17, respectively). No variation in
AT1 mRNA expression in 8-day-old mice was evaluated by
RT-PCR in MR+/+, +/-, and -/-, with relative values of 1.28, 1.28,
and 1.32, respectively, nor was there a variation in AT1
receptor mRNA levels by in situ hybridization. The other
isoform of angiotensin II receptors, AT2, appeared highly
expressed only in the medullary zone of the adrenal gland in MR+/+
(Fig. 4E
) and to a lower level in MR-/-
animals (Fig. 4F
). A 57% decrease in AT2 expression level
was measured by RT-PCR between MR+/+ and MR-/- with relative values
of 7.57 and 4.32, respectively. Incidentally, no expression of
AT2 mRNA was detected in the zona glomerulosa of mice of
the three genotypes.

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Figure 4. Renin and AT2 Receptor mRNAs Expression
in Adrenal Glands
Renin mRNA is not detectable in MR+/+ mice (A and B) and appears in
clusters of cells of the enlarged zona glomerulosa in MR-/- mutants
(C and D). The AT2 receptor mRNA is detected by in
situ hybridization exclusively in the medullary zone of MR+/+
wild type (E) and MR-/- mutant (F) mice, with decreased level of
expression in the mutant. A and C, Bright field illumination; B, D, E,
and F: dark field illumination. Scale bar, 100 µm.
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DISCUSSION
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Mineralocorticoid receptor-deficient mice exhibit a major
dehydration marked by an elevated hematocrit, hyperkalemia, and urinary
sodium loss. Such a severe phenotype has not been observed in animals
where angiotensin production or action is defective when AGT, ACE, or
AT1A and AT1B receptors genes are inactivated
(12, 13, 14, 15, 16, 17, 18). This is likely due to the persistence of some aldosterone
secretion regulated by factors other than angiotensin II, such as
plasma potassium and corticotropin. For example, the mean plasma
aldosterone level of AGT-/-mice was only suppressed to 30% of that
of wild-type mice (19). Similarly, marmosets whose renin activity has
been totally suppressed by active renin immunization still have a
noticeable circulating aldosterone (20). To our knowledge, this
phenomenon of neonatal extreme dehydration is more drastic than any
other experimental procedures reported in laboratory animals, such as
sodium-restricted diet associated or not with diuretic treatment. The
sodium loss observed in these animals markedly decreases body sodium
content and extracellular fluid volume. The observed changes reported
in this study are, therefore, the result of both sodium depletion and
hypovolemia, but this experimental setting precluded the
differentiation of these two parameters.
In addition to conspicuous changes at the glomerular vascular pole and
the enlargement of the segment of the distal tubule at the macula densa
level, we found, in MR-/- mice, a hyperplasia of the renal
interlobar arteries (8). In animals with disruption of AGT (12, 21), ACE (14, 15), or both AT1A and AT1B genes
(22), such renal artery hyperplasia was also noticed. These mice and
MR-/- mice are likely to suffer from major renal hypoperfusion. The
increase in vascular wall thickness is unlikely due to an increase in
angiotensin II since AGT-/- and ACE-/- mice exhibit the same
pattern without any detectable angiotensin II. This phenomenon may
result rather from the stimulation of vascular growth factors provoked
by the profound renal hemodynamic anomalies.
The adrenal gland of MR-/- mice was abnormal, with a marked increase
in the zona glomerulosa, which may result from the trophic effect of
high levels of angiotensin II, whereas the zona fasciculata was hardly
detectable.
In this condition of unique neonatal sodium and volume depletion, it
was of interest to evaluate the variations of expression of the
components of the RAS in their main organs of production and in their
target tissues. mRNA levels were assayed by Northern blot and/or RT-PCR
and by in situ hybridization. The semiquantitative
estimation of the different mRNAs was in good agreement with the
qualitative in situ hybridization observations, although its
main purpose was to identify the cells types affected.
The 0.5-fold increase in AGT mRNA expression in the liver of MR-/-
mice is comparable to that observed in rat by a low sodium diet (23).
This increase in AGT mRNA likely results from angiotensin II positive
feedback, which has been shown to stimulate both AGT transcription and
mRNA stability (24, 25).
The significant increase in renal renin mRNA expression observed in
MR-/- animals varied between 2.5 and 10-fold according to the
technique used. In AGT-/- mice, Tanimoto et al. (13)
reported, by Northern blot analysis, a 6- to 8-fold increase in renin
mRNA when compared with wild type animals. Renin mRNA is abundant in
the hypertrophic juxtaglomerular apparatuses in MR-/- mice.
Immunoreactive renin was detected in PCTs of the whole cortex,
suggesting an intense renin reabsorption (26), as no renin mRNA was
found in these structures. In this situation, renin mRNA is stimulated
despite a marked increase in angiotensin II that is unable to
fully down-regulate renin production (23, 27). In the MR-/- mice, as
in salt-depleted rats, plasma renin levels are remarkably much higher
than intrarenal renin content or mRNA level (28, 29). This discrepancy
between intrarenal and circulating renin could be explained by a rapid
release of newly synthesized renin related to a long half-life of renin
mRNA.
The renin gene was intensely expressed in the adrenal gland in
8-day-old MR-/- mice and particularly in the zona glomerulosa. These
observations are comparable to the effects of salt restriction in
nephrectomized rats, where adrenal renin mRNA is increased (30), thus
implicating the adrenal RAS in the regulation of mineralocorticoid
biosynthesis (31, 32). Again, there was a discrepancy between the high
level of renin mRNA and the absence of immunodetectable renin, which is
likely due to the rapid secretion of renin by the adrenal gland. In the
MR-/- mice, two factors may contribute to the marked expression of
the renin gene, the increase in angiotensin II and (or) plasma
potassium, but the relative contribution of these two mechanisms is
still to be elucidated. Furthermore, how much adrenal renin could
contribute to plasma renin is still unknown.
Opposite variations in rat AT1 mRNA expression have been
reported in the literature during dietary sodium restriction, either in
the liver, kidney, or adrenal gland (23, 33, 34, 35, 36). These discrepancies
were still noticeable when the AT1A and AT1B
subtypes were differentiated during the experiments. In 8-day-old
MR-/- mice, the expression of AT1 receptor was slightly
increased in the liver and unchanged in the kidney and the adrenal
gland. These results are not in favor of a major effect of salt
depletion on AT1 mRNA level. In this study the two
AT1 receptors were not analyzed individually, and one
cannot exclude a shift of one receptor isoform to another. However,
such a phenomenon may have little physiological consequence since gene
disruption of each isoform of AT1 receptor reveals that the
two isoforms are interchangeable, at least to some extent (18, 37).
The changes in mRNA levels of the components of the RAS in 8-day-old
MR-/- mice were not apparent in the heterozygous MR+/- mice.
However, these animals have an increased urinary sodium loss, a 3-fold
increase in sodium fractional excretion, and a modest compensatory
stimulation of the circulating RAS, revealed by a 3-fold increase in
renin, angiotensin II, and aldosterone levels compared with those in
MR+/+. Altogether, this suggests a modest neonatal sodium loss,
compatible with survival in the MR+/- mice. This mild sodium loss
exhibited by these heterozygous mice is somewhat similar to the
phenotype observed in patients with autosomal dominant
pseudohypoaldosteronism type I (38). In these patients, with
heterozygous defect in the MR gene, a modest form of neonatal renal
salt wasting, with hyperkalemia and acidosis, was observed. The disease
remits with age indicating the crucial importance of
aldosterone-dependent sodium reabsorption in the postnatal period and
its decreasing role with age (39).
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MATERIALS AND METHODS
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Animals and Dissection of Organs
Inactivation of the mouse MR gene was achieved by gene targeting
as described by Berger et al. (8). The major part of exon 3
(last 110 of 140 bp) encoding the first zinc finger of the DNA-binding
domain was deleted, and a ß-galactosidase/neomycin cassette was
inserted in the MR reading frame by homologous recombination in
embryonic stem (ES) cells. Homologous recombinant ES cell clones were
injected into C57Bl/6 blastocysts to generate chimeric mice. Germline
transmission of the mutant allele was identified by Southern blot
analysis. Heterozygotes were bred to obtain the three genotypes,
wild-type MR+/+, heterozygous MR+/-, and MR knockout MR-/- mice. All
animal experiments were done in accordance with the institutional
guidelines. The MR knockout mice die around day 10 after birth. The
genotyped 8-day-old mice were killed by decapitation, and organs devoid
of fat were rapidly removed, frozen in liquid nitrogen, and stored at
-80 C until RNA isolation. For histological techniques, organs were
fixed in 4% buffered paraformaldehyde and dehydrated and processed
following a routine preparation protocol.
RNA Preparation
Total RNA was isolated from various organs by the acid
guanidinium thiocyanate-phenol chloroform method (40). The
concentration of RNA was determined by measuring the absorbance at 260
nm, and its integrity was assessed by agarose gel electrophoresis.
Northern Blot
Total RNA was electrophoresed on 1.2% agarose denaturing gels
containing 2.2 M formaldehyde (41). The RNA was transferred
to charged Biohylon membrane (Bioprobe Systems, Montreuil, France) by
capillary blotting in 20 x SSC (1 x SSC: 0.15 M
sodium chloride, 0.015 M sodium citrate, pH 7).
Hybridization was carried out at 42 C for 18 h in 50% formamide,
40 mM Tris-HCl (pH 7.5), 350 mM NaCl, 1% SDS,
8 x Denhardts solution, 10% dextran, and 200 µg/ml salmon
sperm DNA, with full-length rat angiotensinogen cDNA (42) or mouse
renin cDNA (43). The probes were labeled with
[
-32P]dCTP (Amersham, Les Ulis, France) by the
multiprimer technique (44). The filters were washed at room temperature
for 30 min in 2 x SSC, 0.1% SDS, and finally in 1 x SSC,
0.1% SDS at 65 C. The filters were exposed to x-ray film at -80 C
with intensifying screen.
To normalize angiotensinogen and renin mRNA levels, the membranes were
rehybridized with a
-32P-labeled G3PDH cDNA probe
(nucleotide position from 71 to 1053) (45). Autoradiographic signals
were quantified by scanning densitometry (Scanjet IICX; Hewlett
Packard, Palo Alto, CA). The results are expressed as the mean ±
SEM of triplicate determinations of the ratio of
angiotensinogen or renin mRNA to G3PDH mRNA levels.
Reverse Transcriptase Reaction and PCR Amplification
Total RNA (400 ng) of each organ or renal RNA (40 ng) for renin
expression were reverse transcribed in the presence of 50
mM Tris-HCl (pH 8.3), 30 mM KCl, 6
mM MgCl2, 10 mM dithiothreitol,
1.25 mM deoxynucleoside triphosphate, 20 U
ribonuclease (RNase) inhibitor (Promega, Charbonnieres, France), 100
pmol of random hexamer (Pharmacia, Orsay, France), and 20 U MuLV
reverse transcriptase (Boehringer Mannheim, Meylan, France) in 20 µl
at 37 C for 90 min. To minimize the variability between samples, for
each organ, all reverse transcriptions were conducted
simultaneously, including the three different genotypes. Primers used
for PCR were 2024 nucleotides long, homologous to mouse species, and
did not exhibit 3'-complementary ends between primer pairs. The
sequences listed in Table 2
include
the respective positions in the cDNA sequences and the expected sizes
of the amplicons (43, 46, 47, 48, 49). The amplicons were further characterized
by rapid Southern blotting using internal oligonucleotides specific for
each mRNA (data not shown). For each set of primers, duplicate samples
were performed in a total volume of 25 µl. Three microliters of RT
reaction product were incubated in the presence of 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 35.5 mM
MgCl2, taking into account the DNA to be amplified,
200 nM of each 5'- and 3'-primer pairs, 0.5 mM
deoxynucleoside triphosphate, 3 µCi [
-3H]dCTP
(Amersham), and 2.5 U of Taq polymerase (Boehringer
Mannheim). The appropriate cycle number, primer-annealing step, and
polymerization step were optimized for each selectioned cDNA. All
experiments were performed within the exponential phase of the reaction
taking into account the number of cycles and within the linear
curve response concerning the initial RNA amount.
Twenty microliters of the PCR reactions were electrophoresed on a low
melting agarose gel (1.5% or 2%, according to the length of the
amplified DNA). The bands, vizualized with ethidium bromide, were
excised, solubilized in 10 mM Tris-HCl (pH 8.0), 1
mM EDTA, at 65 C, and counted by liquid scintillation
spectrometry.
Within the same RT reaction, the ubiquitously expressed G3PDH mRNA was
used to correct tube-to-tube variations in RT efficiencies. The ratio
of the radioactivity of a considered cDNA to the radioactivity obtained
for the G3PDH signal was calculated to express semiquantitatively the
different mRNA levels.
In Situ Hybridization
The riboprobes used in this study were obtained by in
vitro transcription of cDNAs cloned or recloned in the laboratory.
We used a human renin cDNA (50), a rat angiotensinogen cDNA (42), a rat
ACE cDNA (51), and rat AT1 and AT2 cDNA (52, 53). The rat AT1 probe did not allow us to discriminate
between AT1A and AT1B mRNA since the coding
sequence of the available transcript subtypes is 90% identical in
nucleotide sequence. Consequently, the hybridization signal detected
both AT1 receptor subtype mRNAs. The transcription was made
from the T3, T7, or SP6 RNA polymerase promoter site of the plasmid
vector, after linearization with the proper enzyme, and in the presence
of [35S]UTP (Amersham). The technique of in
situ hybridization has been described in detail (54). Briefly,
paraffin sections were rehydrated and submitted to microwave heating at
100 C and to proteinase K digestion. Each section received 3050 µl
of hybridization mixture containing approximately
104cpm/µl of 35S-labeled antisense or sense
riboprobe. Hybridization was at 50 C for 16 h. The
posthybridization washes included solutions of varying degrees of
stringency (from 5 xSSC with 50% formamide at 55 C to 0.1 xSSC at
room temperature) and a digestion with RNase A (20 µg/ml). The slides
were exposed on a Biomax MR film (Kodak, Marne La Vallee, France) to
obtain macroscopic autoradiographic pictures (13 days) and then were
dipped into NTB2 liquid emulsion (Kodak). At the end of the exposure
time (14 weeks), the slides were photographically processed and
stained with toluidine blue.
Immunohistochemistry
Renin and ACE were detected in kidney, lung, heart, and adrenal,
using primary antibodies produced in the laboratory (55, 56) and a
biotinylated secondary antibody revealed with the ABC-peroxidase
complex (Vector Laboratories, Burlingame, CA).
Statistical Analysis
Results are expressed as mean ± SEM.
Differences between means were analyzed by the nonparametric
Mann-Whitney U test for the comparison of wild-type MR+/+
animals and heterozygous MR+/- mutant mice and for comparison of MR+/+
and homozygous MR-/- mutant mice. Differences were considered
statistically significant at the P < 0.05 level.
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ACKNOWLEDGMENTS
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We thank E. Schmid, A. Klewe-Nebenius, A. Angelosanto, M.
T. Morin, and F. Mongiat for technical assistance.
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FOOTNOTES
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Address requests for reprints to: C. Hubert, INSERM U36-Collège de France, 3 rue dUlm, 75005 Paris, France. E-mail: chubert{at}infobiogen.fr
This work was supported in France by the Institut National de la
Santé et de la Recherche Médicale and the Association
Claude Bernard and in Germany by the Deutsche
Forschungsgemeinschaft through Sonderforschungsbereich 229 and
Boehringer Ingelheim 422/11, by the Fonds der Chemischen Industrie,
by the European Community through Grant PL 960179, by the
Bundesministerium für Bildung und Forschung through the Human
Genome Project Grant 01 KW 9606/7, and by the Volkswagen-Stiftung.
Received for publication September 2, 1998.
Revision received November 4, 1998.
Accepted for publication November 9, 1998.
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