 |
INTRODUCTION |
An essential feature of complex organisms is the ability to
maintain near constancy of their internal environments. Homeostasis is
maintained by the operation of sophisticated systems that permit desirable physiological changes in biological variables, but that also
act homeostatically if external factors cause undesirable changes in
the variables. Genetic heterogeneity, such as is inherent to all
outbred species including humans, also tends to cause variation in the
internal environment. Yet the extent and types of homeostatic changes
induced by naturally occurring genetic differences have not received
much attention. We have recently been carrying out experiments aimed at
identifying genes whose quantitative expression affects an important
biological variable, blood pressure. To this end, we have used gene
targeting in mice to alter the number of functional copies of several
candidate genes, and so to produce systematic changes in their
expression of the same order of magnitude as those occurring naturally
in humans. Since the resulting mice are in other respects wild-type,
their homeostatic systems are intact, and the mice can be used to
investigate what compensations have been induced by the genetically
determined differences in expression of the "titrated" genes.
Because the causative genetic changes are life long, any induced
compensations are categorically comparable with the lifelong
adjustments that individual humans make in adjusting to the genetic
heterogeneity inherent to our species.
The renin-angiotensin system
(RAS)1 is critical for
controlling blood pressure and salt balance in mammals. Angiotensinogen (AGT) is the sole source of angiotensin II (AngII), the major active
peptide of the system. AGT is synthesized primarily in the liver and is
secreted constitutively into the blood stream. It is the substrate for
renin, a highly specific protease whose only known substrate is AGT.
The majority of renin synthesis and secretion into the blood stream in
normal mature animals is by modified smooth muscle cells in the
juxtaglomerular apparatus (JGA) of the kidney. The action of renin on
AGT produces the decapeptide angiotensin I (AngI), which has no
significant cardiovascular activity. Angiotensin-converting enzyme,
ACE, a dipeptidase present in the blood stream as a circulating protein
and in tissues as a membrane bound protein, converts AngI to the
vasoactive octapeptide AngII. Genetic heterogeneity has been
demonstrated at the angiotensinogen locus in humans (1, 2), and two
common alleles are associated with quantitative differences in the
plasma concentration of AGT and with differences in blood pressure. In
previous experiments (3, 4) quantitative differences in expression in
mice of the angiotensinogen gene (Agt) have been shown to
directly cause modest changes in blood pressure. Here we explore the
long term homeostatic adjustments that occur in mice attempting to
restore their blood pressures to normal in the face of inheriting below normal expression of the angiotensinogen gene (Agt).
 |
EXPERIMENTAL PROCEDURES |
Animals--
Except as indicated, all mice used were F1 hybrids
between the inbred strains 129 and B6 with or without a disruptive
mutation in the 129-derived copy of the Agt gene. The
mutation in the Agt gene was generated in embryonic stem
cells from the substrain 129/OlaHsd (5). Prior to the matings to
produce the F1 hybrids, the Agt gene mutation had been
maintained for several generations on the closely related substrain
129/J. The mice were fed regular chow and handled following the
National Institutes of Health guidelines for the care and use of
experimental animals.
Protein Studies--
Blood samples were rapidly withdrawn from
the descending aorta of mice after exposure to an atmosphere of
CO2 (less than 1 min from loss of consciousness to the end
of collection). The blood was collected into ice-cold microcentrifuge
tubes containing EDTA and was immediately centrifuged to isolate
plasma. Plasma AGT and (active) renin concentrations were determined by
radioimmunoassay as described previously (4). Plasma prorenin
concentrations were determined after conversion to active renin by the
trypsin-Sepharose 4B method (6). ACE activity was measured by the
cleavage of a chromogenic tripeptide (7), using serum isolated from
blood collected retro-orbitally without anticoagulant.
Plasma Peptide Measurement--
Blood handling and
radioimmunoassays followed published methods (8, 9) with slight
modifications. Peptides were extracted with ethanol as described in the
assay procedure from the Nichols Institute (San Juan Capistrano, CA)
using 600 µl of plasma pooled from three individuals matched by
genotype and gender. The extracted peptide samples were divided into
three equal portions and dried in a vacuum centrifuge. Single portions
were used for measurement of AngI, AngII, or bradykinin. Recoveries of
each peptide at completion of the extraction procedure were determined
by 1125-labeled tracers to be approximately 80%. By using
highly specific monoclonal antibodies for the measurements, the
radioimmunoassays could be carried out without further separations. The
radioimmunoassays were performed with commercially available kits for
AngI (DuPont), AngII (Nichols), and bradykinin (Peninsula, Belmont, CA).
RNA Isolation--
Tissues were rapidly dissected after
withdrawing blood. Total RNA was isolated conventionally (10) using the
TRI REAGENTTM procedure (Molecular Research Inc.,
Cincinnati, OH).
Riboprobes and Sense RNA Preparation--
DNA fragments from the
Agt, renin, and
-actin genes were prepared by PCR using
strain 129/OlaHsd mouse genomic DNA as the template and the following
probes designed from published sequences: a 390-bp exon 2 fragment from
the Agt gene (11); a 290-bp exon 9 fragment from the mouse
renin gene Ren-1d (12); and a 250-bp fragment
from the mouse
-actin gene (13). A 418-bp fragment corresponding to
nucleotides 2523-2931 of mouse ACE cDNA (14) was cloned after
RT-PCR using total RNA from the lung as template. All of the fragments
were subcloned into a Bluescript(KS) vector for in vitro
transcription (15). 32P-Labeled antisense riboprobes were
synthesized by the manufacturer's protocols using a MAXI
scriptTM transcription kit (Ambion Inc., Austin, TX).
Unlabeled sense RNAs were prepared with the same transcription system.
The sense RNAs were gel-purified and stored at
70 °C.
RNase Protection Assay--
The procedure described by Azrolan
and Breslow (16) with minor modifications was used for RNase protection
assay. All reactions were carried out in duplicate for each sample in
all experimental groups. A standard curve was generated using sense RNAs.
Primer Extension Analysis--
Primer preparation and primer
extension analysis for the mouse renin genes were slightly modified
versions of published procedures (17, 18). The primer was a 38-mer
oligonucleotide complementary to Ren-1C mRNA
from positions 1039 through 1076 of the cDNA sequence (19). We
determined that the nucleotide sequence of this region is identical in
the strain B6 Ren-1c gene and the strain 129 Ren-1d and Ren-2 genes.
Autoradiographic bands were quantitated with an NIH image computer
program (version 1.55).
RT-PCR Assay--
Quantitative RT-PCR (20) was used to assess
expression of the gene coding for the type 1A receptor for AngII. Total
RNA from the kidney was reversely transcribed to cDNA (21). A
template plasmid for preparing the internal standard was constructed by making a 120-bp deletion (PvuII/DraI sites) in a
PlmI/SacI fragment from the mouse type 1A
receptor gene. PCR primers specific for the type 1A gene were designed
from sequences of the type 1A and 1B genes (22); they are
5'-ACGAGTCCCGGAATTACACG-3' for the sense primer and
5'-GCGTGCTCATTTTCGTAGACAGG-3' for the antisense primer. Competitive PCR
was performed in the presence of an internal standard, yielding a
320-bp fragment. The RT product corresponding to the type 1A mRNA
is 440 bp long. The amount of the full-length product relative to the
internal standard was determined after hybridization to labeled
full-length fragment as a probe.
Renin Immunocytochemistry in Kidney
Sections--
Immunohistochemical detection of renin was as described
previously (23, 24). Briefly, after deparaffinization, 7-µm kidney sections were incubated with a polyclonal renin antibody (1:10,000, gift from Dr. Tadashi Inagami, Vanderbilt University, Nashville, TN).
The high specificity and characterization of this renin antibody has
been documented previously (25). Immunocytochemistry was done with
kidneys from the Agt one-copy and wild-type mice. Two to
four sections per kidney were examined by direct microscopic visualization. The total number of glomeruli, the number with renin-positive JGA, and the number with renin-positive cells upstream of the JGA were counted in each section. The percentage of
renin-positive JGA was determined as (the number of renin-positive JGA
in all sections) × 100/(the total number of glomeruli observed). The number of cells positive for renin, including those in the JGA itself,
along the afferent arterioles of glomeruli having upstream renin-positive cells was also counted in each section. The figures thus
obtained from each slide were averaged for each animal.
To determine the area of juxtaglomerular apparatuses in the
Agt one-copy and wild-type mice, 10 random fields of each
section were captured with a video camera. Every section was screened using the same magnification (× 400), and only the JGA with a classic
donut-shaped outline were evaluated. All the images were studied with
an image analysis software (MochaTM, version 1.02, Jandel
Scientific). Using the manual measurement mode, the perimeter of each
JGA was outlined, and its area was determined by summing the number of
pixels contained within the outline.
To determine the number of cells in the JGA that had been evaluated for
area, the number of nuclei observed within the outlined perimeter was counted.
Microvascular Dissection--
To obtain an integrated view of
the distribution of renin within the kidney, the entire renal arterial
tree was dissected as described previously in rats and mice (26, 27)
and stained for renin. The distribution of renin within the kidney was
classified as described previously (28). In a type I distribution,
renin is present along the whole length of the afferent vessel. In type II, renin extends upstream from the glomerulus but does not occupy the
whole length of the vessel. In type III, renin is present as rings
along the afferent vessel. In type IV, renin is restricted to the
classical juxtaglomerular localization. In type V, no renin is found in
the arteriole.
Statistics--
All values are expressed as mean ± S.E.
The two-tailed t test was used for statistical evaluations.
 |
RESULTS |
The Experimental Animals--
The experimental animals used for
investigating long term homeostatic compensations in the RAS have a
single functional copy of the Agt gene and one disrupted by
gene targeting. We refer to them as Agt one-copy mice, and
their blood pressures are about 8 mmHg (approximately 7%) below the
pressures of wild-type mice with two copies of the gene (4). Except
when indicated, the mice studied were F1 progeny-derived from the two
inbred strains 129 and C57BL/6 (B6) and so were genetically identical
except for having different numbers of functional Agt genes.
Because of this genetic uniformity, even small differences between the mice can be ascribed directly to the difference in Agt gene
copy number.
Renin-Angiotensin System Proteins and Peptides--
To determine
what components are homeostatically adjusted in the Agt
one-copy animals, the steady state concentrations of the major RAS
protein components present in plasma were compared in the
Agt one-copy animals and their wild-type controls. The resulting data, Table I and Fig.
1 (below), show two major differences. First, the plasma AGT concentration in the Agt one-copy mice
is markedly reduced, to 33-37% of the AGT concentration in the
controls (p < 0.01), which is also significantly less
than the 50% expected if the amount of protein were directly related
to gene copy number (p < 0.01 versus 50%).
A possible complication affecting this observation is that the
functional Agt gene in the one-copy animals is derived from
mouse strain B6, whereas the wild-type two-copy animals have one copy
from strain B6 and one from strain 129. However, a comparison of AGT
levels in wild-type inbred strain B6 and 129 mice shows that B6 mice
have higher AGT levels (558 ± 30 AngI ng/ml/h, in six
females) than do 129 mice (426 ± 11 AngI ng/ml/h, in six
females), so that if strain differences in Agt gene
expression persist in the F1 hybrids, the AGT concentration in the
Agt one-copy animals should be even more than 50% of the wild-type F1 animals. We conclude that the plasma concentration of AGT
shows no evidence of any compensatory increase in the Agt one-copy animals.
View this table:
[in this window]
[in a new window]
|
Table I
Plasma proteins and peptides in F1 Agt wild-type and F1 Agt one-copy
mice
AGT, renin, and prorenin: ng of AngI/ml/h. ACE activity: units/liter.
AngI, AngII, and bradykinin: pg/ml. Values are means ± S.E.
Values in parentheses are percent relative to wild-type. n,
number of animals; ND, not determined; wt, wild type.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 1.
RAS proteins and peptides in Agt
one-copy mice. A, inter-relationships of the
components measured in the RAS cascade. B, levels of the RAS
components in Agt one-copy mice. The bars show
means ± S.E. as percentages of those in wild-type mice
(Agt two-copy). See Table I and Fig. 3 for details.
|
|
The second major difference is that, in marked contrast to the absence
of any detectable compensation in AGT plasma concentration, the plasma
renin concentration in the Agt one-copy animals is very
significantly higher (240%) than in the wild-type controls (p < 0.001), indicating a marked homeostatic
adjustment in this component of the RAS.
In mice and humans, renin is secreted into the circulation by
renin-producing cells partly as enzymatically inactive prorenin and
partly as enzymatically active renin (29). The observed homeostatic
increase in plasma renin could therefore be partly or wholly the
consequence of a change in the proportion of the secreted protein in
the active versus inactive form of renin. To investigate
this possibility, we determined the plasma concentration of prorenin in
Agt one-copy and in wild-type animals as well as the
concentration of (active) renin. The plasma prorenin concentration in
the Agt one-copy male animals was significantly higher
(234%; p < 0.01) than in the wild-type controls
(Table I). This increase in plasma prorenin is virtually identical to
that of the plasma active renin, so that the same ratio of prorenin and
active renin is observed in Agt one-copy and wild-type mice.
We conclude that a change in the ratio of these two products is not
part of the homeostatic adjustment made in the Agt one-copy mice.
To determine the net effect of the observed increase in plasma renin
concentration combined with the observed decrease in plasma AGT
concentration, we compared the steady state concentrations of AngI in
Agt one-copy and wild-type mice. The results show that the
Agt one-copy animals have AngI levels that are 58% and 75% of wild-type in males and females, respectively. Thus the combined effect of the two changes is a partial but not complete restoration of
the AngI concentrations to the wild-type level (p < 0.001 for one-copy versus wild-type), albeit at the expense
of decreasing the steady state concentration of AGT below 50% of
wild-type.
An additional possible means of compensating for the less than normal
AGT and AngI plasma levels in the one-copy animals would be via a
homeostatically induced increase in the level of the converting enzyme
ACE. Measurements of serum ACE activities (Table I), however, show no
significant differences (p = 0.15) between the
Agt one-copy mice and the wild-type controls. An additional indicator of possible changes in ACE function is the plasma bradykinin concentration, since this octapeptide is destroyed by the enzyme. We
found that the bradykinin levels were not different between the
Agt one-copy mice and the wild-type mice (p = 0.87). Thus we conclude that homeostatic compensation has not been
induced at the level of the converting enzyme or of the bradykinin peptide.
The major effector peptide of the RAS is the octapeptide AngII. A
measure of changes in the net status of the circulating arm of the
system can therefore be obtained by comparing the steady state plasma
concentrations of AngII in Agt one-copy and wild-type mice.
The resulting data show that plasma AngII in the Agt
one-copy males and females are, respectively, 50% and 62% of the
levels in the wild-type animals. These levels are significantly less than wild-type (p < 0.01), indicating that homeostasis
is incomplete, as is reflected by the residual differences in blood
pressure between Agt one-copy and wild-type animals.
In summary, (Fig. 1), measurements of the expression of the protein and
peptide components of the endocrine RAS show clear evidence that a
major homeostatic compensation occurs in plasma renin concentrations in
response to a genetic reduction in Agt gene expression.
Other components of the system either show no changes or have changes
that appear to be passive and secondary to the genetic reduction in AGT
levels and the consequent homeostatic increase in renin. The final
result is a steady state concentration of AngII in Agt
one-copy animals that is still significantly less than normal.
Renin-Angiotensin System mRNAs--
To ascertain whether the
changes seen in the circulating protein components of the RAS are
present at the level of transcriptional products, we used an RNase
protection assay to determine the amounts of the relevant mRNAs in
tissues that make the largest contribution to the plasma in
Agt one-copy and wild-type animals. The major site of
synthesis of AGT secreted into blood is the liver (30), which also
contains the highest abundance of AGT mRNA. Fig.
2A presents the data for AGT mRNA in
Agt one-copy and wild-type F1 males and females and in
strain B6 and 129 inbred wild-type males. More mRNA is present in
the female mice than in the males (a disparity also observed in the
plasma AGT). But regardless of gender the liver AGT mRNA levels in
Agt one-copy animals are clearly reduced compared with those
in the wild-type animals (54% in males; 53% in females;
p < 0.001). Recollecting that the single functional Agt gene in the one-copy animals is derived from strain B6
and noting from Fig. 2A that liver AGT mRNA levels in
strain B6 are 1.2 times the corresponding levels in strain 129, we
conclude that the liver AGT mRNA data agree with the plasma protein
data in indicating no homeostatic compensation in the Agt
one-copy mice in the transcription of the remaining functional
Agt gene in the primary tissue of AGT synthesis.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Tissue mRNA levels for primary tissues
synthesizing AGT, renin, and ACE determined by RNase protection
assay. A, AGT mRNA in the liver. n = 10 for each of the four groups of F1 mice; n = 5 for
the wild-type B6 and 129 males. B, renin mRNA in the
kidney. n = 10 for each group of F1 mice;
n = 5 for the wild-type B6 and 129 males and females.
C, ACE mRNA in the lung. n = 5 for each
group of F1 mice. The bars show means ± S.E.
Asterisks indicate Agt one-copy means
significantly different from wild-type means (p < 0.001).
|
|
The high steady state plasma renin (and prorenin) concentrations
observed in Agt one-copy animals suggest a substantially increased level of renin gene transcription. Fig. 2B
presents the relevant data and shows that the steady state renin
mRNA contents of the kidneys of the Agt one-copy males
and females are respectively 182 and 165% of the wild-type two-copy
controls (p < 0.001). Thus a major part of the
homeostatic adjustment in renin production is a consequence of an
increase in amount of renin mRNA.
Since the steady state serum ACE activities of the experimental and
control mice do not differ significantly, we expected to see no
differences in the ACE mRNA levels in tissues in which ACE is
synthesized. The lungs are a major site of ACE synthesis in both sexes
(31). In addition, a truncated form of ACE is synthesized in the testis
from a testis-specific promoter (32). Fig. 2C presents data
showing that the ACE mRNA contents of the lungs of Agt
one-copy males and females are slightly increased (108 and 105%)
relative to their two-copy controls, but the difference is not
statistically significant (p = 0.09). The ACE mRNA
level in the testis was also slightly higher (data not shown), but
again the difference was not significant (108% wild-type,
p = 0.65). Thus there is no evidence for significant
homeostatic compensation at the level of ACE mRNA.
In the mouse, three receptors (types 1A, 1B, and 2) control the
cellular and physiologic actions of AngII (22, 33). The results of
administering receptor antagonists that specifically block the actions
of either the type 1 or the type 2 receptors establish that blood
pressure changes are chiefly executed by the type 1 receptors (34).
Genetic experiments disrupting the genes coding for the type 1A
receptor gene (35, 36) or the type 1B receptor gene (37, 53) show that
approximately 90% of the endocrine pressor effects of AngII are via by
the type 1A receptor. Another possible means of homeostatic adjustments in the face of a chronic decrease in blood pressure would therefore be
to increase expression of the type 1A receptor. However, comparison of
the type 1A receptor gene expression by RT-PCR (Fig.
3) in Agt wild-type mice
(lane 1) and the Agt one-copy mice (lane
2) revealed no detectable differences, although the same assay
readily detected the decreased level of type 1A receptor mRNA in
animals having only one copy of the 1A receptor gene (lane
4) in place of the normal two copies (lane 3), and
showed no product in 1A receptor gene zero-copy mice (lane
5). Thus chronic homeostatic changes in expression of the type 1A
angiotensin II receptor gene do not occur in the Agt
one-copy animals.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 3.
Comparisons by RT-PCR of type 1A angiotensin
II receptor mRNA in Agt one-copy and wild type
mice. The products of 25 cycles of PCR with reverse transcripts
from the kidneys of five mice are visualized. The 440-bp band is the
product from the mouse type 1A receptor mRNA; the 320-bp product is
from a PCR internal standard. The genotypes of the mice were:
Agt wild-type, 1A receptor wild-type (lane
1); Agt one-copy, 1A receptor wild-type (lane
2); Agt wild-type, 1A receptor wild-type (two-copy)
(lane 3); Agt wild-type, 1A receptor one-copy
(lane 4); Agt wild-type, 1A receptor zero-copy
(lane 5).
|
|
Other Tissues--
A great deal of work by many investigators has
been directed toward assessing the possible autocrine/paracrine
contributions to blood pressure homeostasis by RAS components
synthesized in tissues other than those directly involved in the
endocrine/circulatory aspects of the system (38, 39). Homeostatic
changes in other tissues were therefore investigated in the
Agt one-copy and wild-type animals. The data for AGT
mRNA are summarized in Fig.
4A and show that, as in the
liver, the AGT mRNA levels in the kidney, submandibular gland and
testis of the Agt one-copy animals are close to the levels
expected for animals having only a B6-derived Agt gene. Thus
there is no evidence of significant homeostatic compensation at the
level of Agt gene transcription in these tissues. In the brains of both males and females, the Agt one-copy animals
likewise have lower amounts of AGT mRNA than wild-type animals, but
the difference in the brain is less than in all other tissues. Further studies will be required to determine whether this is due to a compensatory change in Agt gene expression in the brain or
to some other factors.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 4.
Tissue mRNA levels for secondary tissues
synthesizing AGT and renin determined by RNase protection assay.
A, AGT mRNA levels in the kidney, brain, submandibular
gland, and testis; n = 10 for each group. B,
renin mRNA levels in the adrenal gland and submandibular gland;
n = 10 for each group. The bars show
means ± S.E. Asterisks indicate Agt
one-copy means significantly different from wild-type
(p < 0.001).
|
|
Fig. 4B presents the data for renin mRNA levels in
various extra-renal tissues. With one exception, renin mRNA in
extra-renal tissues does not differ between the Agt one-copy
and wild-type animals. The exception is the adrenal gland of males,
where the level of adrenal renin mRNA in Agt one-copy
males is four times that of wild-type males (p < 0.001). However, no difference in adrenal renin mRNA is seen
between the Agt one-copy and wild-type females
(p = 0.48). Note also that Agt wild-type
males have approximately four times the adrenal renin mRNA of
wild-type females, so that gender-related differences in renin
expression are seen in the adrenal glands irrespective of their
Agt genotypes.
Gender-related Effects--
Some strains of mice including 129 have a duplicated renin locus that includes a closely linked
androgen-responsive renin gene (Ren-2) in addition to a
gender-indifferent gene (Ren-1); male mice carrying the
Ren-2 gene synthesize large amounts of mRNA in their
submandibular glands and secrete renin into their saliva (40, 41).
Other strains, including B6 (and humans), have only the
Ren-1 gene, and so have much lower salivary gland renin
mRNA levels, comparable with the levels seen in females. The
Ren-2 gene has not been described in any species other than mouse. The F1 mice used in the present study have the single strain B6-derived Ren-1c gene and the two closely
linked strain 129-derived Ren-2 and Ren-1d genes. We therefore carried out
experiments to determine to what extent the homeostatic changes in
renin expression seen in the Agt one-copy animals differed
by locus as well as by gender.
The relative contribution to kidney renin mRNA of the
Ren-1 and Ren-2 genes was determined by a primer
extension analysis. We found that in the Agt one-copy
females 20% of the renin mRNA was derived from the
Ren-2 gene, which does not differ significantly from the
percentage in their wild-type sisters or in a 50/50 mixture of the two
female parental mRNAs. In the Agt one-copy males 40% of
the mRNA was from the Ren-2 gene, which is greater than
that in their wild-type brothers (10%) or in a 50/50 mixture of the two male parental mRNAs (10%). Thus, in Agt one-copy
females the homeostatic increase in renin expression is mediated by the
two renin genes in the same proportion as they are expressed in their wild-type sisters, but in males a greater proportion of the increase is
mediated by the androgen-sensitive Ren-2 gene.
Cellular Responses--
A great preponderance of evidence supports
the view that the kidney is the chief source of circulating active
renin and its enzymatically inactive precursor prorenin in humans and
mice (42). In the normal kidney, renin is produced by modified smooth
muscle cells that are associated mainly with the juxtaglomerular
apparatus (JGA) and to a much lesser extent with the afferent upstream
(proximal) portion of the glomerular arterioles. Several, not
necessarily mutually exclusive, mechanisms could therefore mediate the
chronic homeostatic elevation of kidney renin mRNA that we observe.
Transcription of one or more of the renin genes could be up-regulated
in the usual renin-producing cells of the kidney, or the number of
these or other cells capable of synthesizing renin could be increased. To help distinguish between these various possibilities we determined by immunohistochemistry the number and distribution of renin-containing cells in the kidneys of Agt one-copy animals and wild-type controls.
The immunohistochemical results are illustrated and diagrammed in Fig.
5. In wild-type animals the majority of
renin-producing cells are confined to the juxtaglomerular end of the
arterioles in the manner typical of a classical JGA (Fig. 5,
A and C). This distribution of expression
corresponds to the type IV pattern (28) shown diagrammatically in the
bottom panel of Fig. 5. A much smaller proportion of
wild-type glomeruli have additional renin-staining cells extending
along the afferent arterioles as well as being present in the JGA
(types II and III). Some wild-type glomeruli have no renin-staining
cells (type V). In the Agt one-copy animals (Fig. 5,
B and D), there is a considerable increase in the
proportion of glomeruli having renin-staining cells extending along the
afferent arteriole as well as being present in the JGA (types II, III,
and mixed types II/III and III/IV).

View larger version (140K):
[in this window]
[in a new window]
|
Fig. 5.
Renin immunolocalization in the kidneys.
A, kidney tissue section from a wild-type mouse. Renin
staining (brown) in this type IV glomerulus is confined to
the JGA area. B, kidney tissue section from an
Agt one-copy mouse. Renin immunostaining in these type II
glomeruli includes the JGA area but also extends along the afferent
arterioles in the direction of the interlobular artery. C,
renin immunolocalization within the renal arteriole tree microdissected
from a wild-type mouse. Renin is present mainly in the JGA area (type
IV). Renin is not present in interlobular arteries. D, renin
immunolocalization in the renal arteriole tree microdissected from an
Agt one-copy mouse. In addition to renin staining in the
juxtaglomerular region (type IV), renin is present in cells along the
afferent vessels in distributions corresponding to types II, III, and
mixed types (types II/III and III/IV). Renin is present in interlobular
arteries. The diagram below A-D illustrates the types of
distribution I through V.
|
|
A summary of a statistical analysis of these immunochemical data is
presented in the upper portion of Table
II, using combined data from 12-week-old
F1 Agt one-copy (n = 5) and wild-type mice (n = 5) and from 10-week-old F2 litter mates having the
same genotypes (n = 5 and 3, respectively); the F1 and
F2 data were essentially indistinguishable. This analysis shows: (i)
that the proportion of glomeruli having renin-staining cells in the
classical JGA region is somewhat (about 25%) greater in the
Agt one-copy mice than in wild-type (p < 0.05);
View this table:
[in this window]
[in a new window]
|
Table II
Renin-staining cells in kidneys of Agt wild-type and Agt one-copy mice
Values are means ± S.E. Areas are pixels × 103.
Values in parentheses are percent relative to wild-type. Counts are
averages from two to four sections from each kidney. n,
number of animals; wt, wild-type.
|
|
(ii) that in the Agt one-copy mice the number of afferent
arterioles with renin-staining cells upstream from the glomerulus is
three times that of wild-type mice, and this is highly significant (p < 0.0001); (iii) that the number of renin-staining
cells along the individual afferent arterioles of the Agt
one-copy mice is also increased significantly (1.4 times wild-type;
p < 0.05); (iv) that the total number of
renin-staining cells along the afferent arterioles of the
Agt one-copy mice is more than 4 times wild-type (p < 0.0001).
To assess the possible occurrence of a hyperplastic response in the
renin-containing cells of the JGA of the Agt one-copy mice,
the number of renin-staining cells in a sampling of glomeruli having
classic donut-shaped JGA was determined by counting nuclei within the
renin-staining areas in Agt one-copy mice, and this was
compared with wild type using the 10-week-old F2 litter mates. No
difference was observed (lower part of Table II, p > 0.7). To assess the possible occurrence of a hypertrophic response, the
area of renin staining was determined for each JGA. Again no difference
between the two genotypes was observed (Table II, p > 0.3). The sizes of the individual renin-producing cells, as judged by
the area per cell, are indistinguishable in the classic JGA of the two
genotypes. The intensity of renin-staining per cell was likewise not
observably different in the two genotypes.
Overall these data show that the higher plasma renin levels and the
greater amount of total kidney renin mRNA in the Agt
one-copy animals relative to wild type is mediated by their having a
somewhat greater proportion of glomeruli with renin-expressing cells in the JGA region and a severalfold greater number of renin-expressing cells along the afferent arterioles of their renal glomeruli rather than by hyperplasia or hypertrophy of JGA cells already committed to
renin synthesis or by up-regulation of renin gene expression in these cells.
 |
DISCUSSION |
The main purpose of the present study was to determine the major
long term adjustments directed toward homeostasis that occur in mice
inheriting a precisely determined genetic variation with a blood
pressure lowering tendency, namely inactivation of one copy of the
Agt gene. The first finding is that in the liver, the prime
site of AGT synthesis, no homeostatic up-regulation of the remaining
functional Agt gene can be detected at the mRNA level.
Thus the single copy of the Agt gene in the experimental animals yields essentially half the amount of mRNA achieved by the
two copies in the wild-type controls. This absence of autoregulation of
a normal functional gene to compensate for some unusual behavior in its
homologous allele appears to be very widespread and probably universal
in genes that do not code for products that act directly with their own
regulatory machinery. There is a considerable body of data from
previous studies showing that the expression of many genes is directly
and precisely proportional to gene copy number. Epstein (43), for
example, compiled from previous studies convincing evidence in support
of this proportionality in humans and mice having trisomies,
monosomies, and deletions involving over 40 different loci mainly
coding for enzymes and plasma proteins. And we, in "gene titration"
experiments with mice, have demonstrated a direct proportionality
between gene copy number and expression with the genes coding for AGT
(3, 4), for ACE (44) and for the natriuretic peptide receptor A (45).
In none of these instances is the normal gene up-regulated in the
absence of a functional homologue, while three copies of a gene produce
very close to 1.5 times the amount of immediate gene product resulting from two copies, indicating a similar absence of down-regulation. These
observations do not exclude the existence of mechanisms to up-regulate
or down-regulate genes via less direct and more complex pathways, but
they do exclude the general occurrence of autoregulation.
The implication is that most genetic variants which affect expression
will not be corrected by adjusting the transcription of either the
variant gene or its nonvariant homologue.
The second finding is that the major route whereby homeostasis in the
endocrine side of the RAS is attempted in the Agt one-copy animals is through an increase in plasma renin concentration mediated by a modest increase in the proportion of glomeruli with
renin-producing cells in their JGA and by the presence of considerably
greater numbers of renin-producing cells along the afferent glomerular arterioles in the Agt one-copy animals than in the wild-type
animals, rather than by hyperplasia or hypertrophy of JGA cells already committed to renin synthesis or by an increase in their renin content.
No significant changes were detected in expression of the
Ace gene or of the gene coding for the type 1A AngII
receptor, which mediates most of the blood pressure-related functions
of the system.
Comment is required on our observation of differences between the
relative expression of the Ren-1 and Ren-2 in the
male but not the female Agt one-copy and wild-type mice. In
assessing the relevance of these findings to the overall problem of the
homeostasis of blood pressure, it is important to recollect that the
androgen-responsive Ren-2 locus has been reported only in
the mouse and then not in all strains. Past work by others (17, 18) has
shown that the Ren-1:Ren-2 expression ratio
differs markedly between different cell types in males but not in
females, ranging from around 1:1 in the male kidney to 1:100 in the
male salivary glands. The relative increase in Ren-2
expression seen in the kidneys of male but not the female
Agt one-copy animals suggests that the additional
renin-positive cells along the afferent arterioles have some features
like cells in the salivary glands and so show gender differences in our
F1 mice.
Comment is also required on the 4-fold greater amounts of renin
mRNA observed in the adrenals of the male Agt one-copy
animals relative to the expression in their wild-type brothers. At
least two arguments suggest that this increase in renin expression does not represent a general mechanism for achieving homeostasis in the
circulatory arm of the RAS. First, the effect is male-limited and is
therefore again likely a consequence of the mouse strain-specific androgen-responsive Ren-2 gene. Second, the plasma active
renin concentration in the Agt one-copy males is actually
not as much increased (2.2 times wild type) as in the one-copy females
(2.6 times wild type). However, we do not exclude the possibility that the increased adrenal expression of renin in the males can act on
a local arm of the RAS (46), and the phenomenon merits further investigation.
Our finding that homeostasis in the genetically modified animals is
accompanied by considerably greater numbers of renin-producing cells in
the kidney alters the nature of subsequent questions. In place of
asking how up-regulation of the renin genes in cells already producing
renin is induced and executed in the Agt one-copy animals,
the question becomes what mechanism leads to the observed presence of
more renin-producing cells. No definite answer is available. However,
the changes seen in the normal rat kidney during the period from late
gestation to early maturity and in adult kidneys exposed to
experimentally lowered blood pressures suggest some interesting
possibilities. Initially in the developing rat kidney (28) the afferent
glomerular arterioles stain positively for renin along the whole of
their lengths (type I, as illustrated in Fig. 5). Shortly after birth,
type I-staining glomeruli are much less frequent, but glomeruli can be
seen in which the renin staining extends less far along the arterioles
from the glomerulus (type II). These in turn partly disappear, and
glomeruli are seen that show the classic adult pattern, with renin
staining confined to the JGA (type IV), together with glomeruli showing
no renin staining (type V). The type of renin distribution that is
severalfold more frequent in the Agt one-copy mice than in
wild type has a staining pattern very similar to the type II pattern in
the rat. This similarity suggests the possibility that in the
Agt one-copy mice more of the renin-producing cells
initially present along the afferent arterioles persist into adult life
and fewer glomeruli cease to produce renin in their JGA.
A second possibility is that additional renin-producing cells are
formed in the Agt one-copy mice from non-renin-producing smooth muscle cells. This type of metaplastic/metamorphic
conversion/recruitment has been described in rat kidneys exposed to
blood pressures reduced by partial arterial ligation (47, 48) and in
the kidneys of rats treated with high doses of ACE inhibitor (23, 49).
Cell recruitment has also been demonstrated in other systems, for
example during estrogen-induced synthesis of apolipoprotein II in avian hepatocytes (50), during glucose-induced synthesis of pro-insulin in
purified pancreatic beta cells (51), and during thyrotropin-induced formation of intracellular colloid droplets in thyroid follicular cells
(52).
In conclusion, it is clear that much remains to be done if we are to
understand how homeostasis is achieved in the face of quantitative
genetic variations that are likely to be common in the genes which
control our internal environment. The present results suggest that
lifelong genetically determined disturbances may be corrected by
homeostatic adjustments in the extent to which different cell
populations are retained (or discarded) during development while still
allowing disappearance (or reappearance) of the same cells in maturity
if circumstances change. Attempts to determine the signals and
mechanisms involved in genetic homeostasis should not only look for
chronic up-regulation or down-regulation of immediately relevant genes,
but should also look for possible shifts in the relative frequencies of
the different types of cells that participate in the system.