Genetic Evidence That Lethality in
Angiotensinogen-deficient Mice Is Due to Loss of Systemic but Not Renal
Angiotensinogen*
Yueming
Ding
§,
David E.
Stec¶, and
Curt D.
Sigmund
¶
**
From the
Genetics Interdisciplinary Graduate Program
and the Departments of ¶ Internal Medicine and
Physiology & Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa
52242
Received for publication, May 8, 2000, and in revised form, November 13, 2000
 |
ABSTRACT |
Angiotensinogen (AGT)-deficient mice die shortly
after birth presumably due to renal dysfunction caused by the presence
of severe vascular and tubular lesions in the kidney. Because AGT is
expressed in renal proximal tubule cells, we hypothesized that its loss
may be the primary mediator of the lethal phenotype. We generated two
models to test this hypothesis by breeding transgenic mice expressing
human renin with mice expressing human AGT (hAGT) either systemically
or kidney-specifically. We then bred double transgenic mice with
AGT+/
mice, intercrossed the compound heterozygotes, and examined the
offspring. We previously reported that the presence of the human renin
and systemically expressed hAGT transgene complemented the lethality
observed in AGT
/
mice. On the contrary, we show herein that the
presence of the human renin and kidney-specific hAGT transgene cannot
rescue lethality in AGT
/
mice. An analysis of newborns indicated
that AGT
/
mice were born in normal numbers, and collection of dead
10-day old pups revealed an enrichment in AGT
/
. Importantly, we
demonstrated that angiotensinogen protein and functional angiotensin
II was generated in the kidney, and the kidney-specific
transgene was temporally expressed during renal development similar to
the endogenous AGT gene. These data strongly support the
notion that the loss of systemic AGT, but not intrarenal AGT, is
responsible for death in the AGT
/
mouse model. Taken together with
our previous studies, we conclude that the intrarenal renin-angiotensin
system located in the proximal tubule plays an important role in blood
pressure regulation and may cause hypertension if overexpressed, but
may not be required for continued development of the kidney after birth.
 |
INTRODUCTION |
Regulation of arterial blood pressure, renal hemodynamics, and
fluid and electrolyte homeostasis are well-recognized functions of the
renin-angiotensin system
(RAS).1 The results of recent
studies indicate that the RAS may also regulate renal growth and
development. All the components of the RAS are detectable at the
mRNA and protein level in the developing kidney. Angiotensinogen
can be detected as early as embryonic day 17 (E17) in rodent kidney
where it is expressed primarily in developing tubules (1). Renal
angiotensinogen expression peaks around birth but declines thereafter
(2). Renin can be found in rodents as early as E15 before obvious blood
vessel and nephron formation occurs, and at E17 is expressed in the
renal artery and its main branches. Its expression pattern gradually moves from vascular structures to mature juxtaglomerular cells as
kidney development progresses (1, 3). Similar to angiotensinogen, renin
expression reaches a peak around birth (4). In the rat kidney, ACE can
be detected at E16, and AT-1 and AT-2 receptors are expressed
throughout metanephric development (5-7). Consequently, all the
components of the RAS are expressed during early kidney development in
specific temporal-spatial patterns associated with nephrogenesis,
suggesting the RAS may play an important role during renal development.
There is ample evidence showing that Ang-II has trophic or mitogenic
effects on a variety of target cells and tissues in addition to its
function as a regulator of volume and sodium homeostasis. Ang-II
directly stimulates growth and proliferation of renal mesangial cells,
proximal tubular cells, Henle's loop cells, and vascular smooth muscle
cells in vitro (8-10); Ang-II may also stimulate a cascade
involving growth factors and oncogenes such as transforming growth
factor
, basic fibroblast growth factor, platelet-derived growth
factor, c-Fos, c-Myc, and c-Jun (11). Direct evidence implicating an important role of the RAS in renal development comes
from studies using either pharmacological blockade or targeted disruption of the RAS. ACE inhibitor treatment during pregnancy (even
during pre-eclampsia) is strongly contraindicated, because their
infants have an increased incidence of renal tubular dysplasia, growth
retardation, oligohydramnios, hypotension, hypocalvaria, and death
(12). Similarly, neonatal rats treated with captopril, enalapril, and
losartan exhibit gross histopathologic changes in the kidney, including
tubular atrophy and dilation, papillary atrophy, interstitial
inflammation, and hyperplasia of renal vasculature (13). Similarly,
mice with targeted mutations in the angiotensinogen, ACE, AT-1 receptor
genes, and in both renin genes, have nearly identical phenotypes
characterized by poor survival to weaning, low blood pressure, and
abnormal kidney structure. Their kidneys develop thickened,
hypercellular arterial walls, interstitial fibrosis, inflammation,
papillary atrophy, and tubular dilation (14-17). Given the severity of
the renal lesions, it has been hypothesized, but not proven, that the
lethality is related to renal failure.
We previously reported that transferring both the human renin (hREN)
and human angiotensinogen (hAGT) genes onto the mouse angiotensinogen
(mAGT) knockout background complemented (rescued) the lethality and
renal lesions associated with the loss of angiotensinogen (18).
However, the hAGT transgene used in that study was driven by its
own endogenous promoter and expressed in multiple tissues such as
liver, kidney, brain, and heart. Therefore, it is unclear whether
restoration of the systemic RAS or intrarenal RAS was responsible for
the rescue. Given that the primary defect in AGT-deficient mice may be
the kidney, the aim of this study was to determine whether the
intrarenal RAS plays a critical role in renal development by
determining if restoring only the intrarenal RAS can rescue the lethal
phenotype in mAGT-deficient mice. The results of this classic genetic
complementation assay are reported herein.
 |
MATERIALS AND METHODS |
Animal Breeding Strategy and Husbandry--
The generation and
characterization of hREN, hAGT, and KAP-hAGT single and double
transgenic mice have been reported previously (19-21). The KAP-hAGT
transgene segregates as an autosomal trait and is kidney-specifically
expressed, whereas the hREN transgene is X-chromosome-linked and
systemically expressed. The generation of mAGT-deficient mice has been
described previously (14). All mice were maintained by backcross
breeding to C57BL/6J, and were fed standard mouse chow (Teklad LM-485)
and water ad libitum unless otherwise indicated. Care of
mice met or exceeded the standards set forth by the National Institutes
of Health in the Guidelines for the Care and Use of Experimental
Animals. All procedures were approved by the University of Iowa Animal
Care and Use Committee. Mice were killed by CO2 asphyxiation.
For the developmental study, KAP-hAGT single transgenic male mice were
crossed with female C57BL/6J, and pregnancy was monitored by daily
examination of vaginal plugs. Mouse fetuses were removed on E7.5
through E18.5. In addition, newborn through 8-week-old mice were also
collected. For the genetic complementation study, hREN/KAP-hAGT double
transgenic females were mated to male mice heterozygous for the mAGT
deletion (+/
) and the compound heterozygotes hREN/KAP-hAGT mAGT+/
were intercrossed. The resulting offspring immediately after birth and
at 10 days and 3 weeks of age were collected. In addition, all
cages were carefully monitored for dead pups, which were immediately
taken for sampling and genotyping.
Genotyping--
Genomic DNA was isolated from tail or placental
biopsies as described previously (20). PCR analysis was performed to
identify the hREN, hAGT, and KAP-hAGT transgenes using specific primer sets as reported (22, 23). To genotype the mAGT locus, different PCR
primer sets were used: Primer a, 5'-GTATACATCCACCCCTTCCA-3'; primer b,
5'-GGAAGTGAACGTAGGTGTTGAA-3'; primer c, 5'-TGCACGGGTTCTGAGGATCCA-3'; and primer d, 5'-TAAAGCGCATGCTCCAGACT-3'. The set of primers a and b is
specific to the wild-type mAGT locus and yields a 750-bp fragment,
whereas primers c and d are specific to the targeted mAGT locus
yielding a 1.3-kb fragment. Southern blot analysis was carried out to
confirm the PCR results as previously described (18). Briefly, tail DNA
was digested with XbaI, separated on 0.8% agarose gel,
transferred to nitrocellulose membranes, and hybridized with a probe
specific for intron 1 and part of exon 2 of the mAGT gene.
After hybridization, the wild-type mAGT locus yields a 5-kb fragment,
whereas the targeted locus yields a 6.5-kb fragment. The sex of fetuses
and newborns was determined by PCR amplification of an sry
gene fragment as described previously (24).
Gene Expression Analysis--
RNase protection assay was used to
compare the time course of expression of hAGT transgene with that of
the endogenous mAGT gene during development. The hAGT probe
was derived from exon 2 at nucleotides 302-819 relative to the
transcription start site as described previously (21). The mAGT probe
was derived by cloning a reverse transcriptase-PCR product from
nucleotides 305-739. The oligonucleotides used to clone the mAGT
cDNA probe were 5'-GCCGCCGAGAAGCTAGAGGATGAG-3' and
5'-TGGGAAGAGGGCAGGGGTAAAGAG-3'. This cDNA fragment was first cloned to pCR2.1 vector (Invitrogen) and then subcloned into
pBluescript SK
to obtain the desired antisense orientation relative
to the T7 promoter. The mouse
-actin cDNA (mAct) probe was
provided by Ambion (Austin, TX). Antisense RNA probe was prepared using an in vitro transcription reaction containing
[
-32P]UTP. Total RNA was isolated from whole fetuses
from E7.5 to E13.5, and kidneys from E16.5 to 8-week-old mice as
described previously (22). Total tissue RNA (20 µg) was hybridized to the probes and were treated according to the manufacturer's protocol for Hyb-Speed RNase protection assay kit (Ambion). The length of
the full-length probe for hAGT, mAGT, and mAct are 630, 591, and 330 nucleotides, respectively, and the expected protected fragment are 518, 425, and 250 nucleotides, respectively.
Physiological Analysis and Statistical
Analysis--
Immunohistochemistry and measurements of urinary hAGT
and blood pressure were described in detail previously (20, 21). Chi-square analysis was performed to compare the genotype frequencies with expected Mendelian ratios using the SigmaStat software package.
 |
RESULTS |
The purpose of this study is to genetically determine whether the
intrarenal renin-angiotensin system alone can rescue the postnatal
lethality observed in mAGT knockout mice. To accomplish this, we
performed a complementation assay employing one transgenic mouse
expressing hREN and two transgenic models expressing hAGT
one expressing hAGT systemically (hAGT or A+) and one expressing hAGT specifically in proximal tubule cells of the kidney (KAP-hAGT or KA+).
In the systemic model, hAGT mRNA is abundant in the liver, kidney,
heart, and brain, and elevated levels of hAGT protein are present in
the systemic circulation (23). Double transgenic mice expressing both
hREN and the systemic hAGT transgene exhibit a 4-fold elevation in
plasma Ang-II and are chronically hypertensive (19). In the KAP-hAGT
mice, hAGT mRNA and protein are restricted to the kidney as a
consequence of being driven by the kidney-specific KAP promoter (20).
Double transgenic mice containing both the hREN and
KAP-hAGT genes do not have circulating hAGT protein, have
normal plasma Ang-II levels, but are nevertheless hypertensive (20).
To perform the genetic analysis we first bred hREN mice with either
hAGT or KAP-hAGT mice to generate systemic (R+/A+) and kidney-specific
(R+/KA+) double transgenic mice (Fig. 1).
Double transgenic females were then bred with male mice heterozygous for the mAGT deletion (+/
), and the compound heterozygotes (R+/A+ mAGT+/
or R+/KA+ mAGT+/
) were identified. These compound
heterozygotes were then intercrossed, and the resulting offspring were
genotyped by PCR and Southern blot analysis (Fig.
2). According to Mendelian genetics, the
offspring would exhibit one of 12 different genotypes with different
combinations of R+, A+, or KA+ transgenes and mAGT alleles (Table
I). Our previous data indicated that
single transgenic mice (R+/A
or R
/A+) have the same level of plasma
renin activity, plasma Ang-II, and blood pressure as normal mice
(R
/A
) and therefore can be stratified together as a single group
(18, 19). This is because the enzymatic reaction between renin and
angiotensinogen is species-specific, such that human renin cannot
cleave mouse angiotensinogen and vice versa (25). However,
Ang-I generated from the enzymatic reaction between human renin and
human angiotensinogen can be cleaved to form Ang-II by mouse ACE,
because Ang-I is evolutionary conserved in rodents and humans.
Therefore, we stratified offspring from the third round of breeding
into a double transgenic group (RA+ or RKA+), and a nondouble
transgenic group (RA
or RKA
). The nondouble transgenic group
consisted of mice that were either nontransgenic, single transgenic,
but not doubly transgenic. This simplified the 12 original genotypes
into 6 relevant genotypes (mAGT+/+, mAGT+/
, and mAGT
/
for
each).

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Fig. 1.
Breeding scheme. A schematic of the
breeding strategy utilized to generate mice containing combinations of
the hREN and either the hAGT or KAP-hAGT transgenes and gene-targeted
disruptions of the mAGT gene. Male mice are represented by
squares and females by circles. Brother/sister
intercrosses of double transgenic mAgt+/ mice yielded offspring that
were genotyped. The individual hREN, hAGT, and KAP-hAGT transgenics
utilized to generate the double transgenic mice in the first breeding
are heterozygous for each of the transgenes. The hREN transgene is
present on the X-chromosome and exhibits a sex-linked mode of
inheritance. The hAGT and KAP-hAGT genes are
autosomal.
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Fig. 2.
Sample genotype data. A, a
representative Southern blot of XbaI-digested tail DNA from
offspring of the breeding shown in Fig. 1 is shown. Hybridizing bands
corresponding to the wild-type and targeted loci are indicated. The
deduced genotype for each animal is shown at the top of each
lane. B, a representative gel of tail DNA PCR reactions
using primers specific for the targeted and wild-type allele of mAGT is
shown. Bands corresponding to the wild-type and targeted loci are
indicated. The deduced genotype for each animal is shown at the
top of each lane. C, a representative gel of tail
DNA PCR reactions using primers specific for the hREN
(R) and KAP-hAGT (K) genes is shown.
Positions of the PCR products are indicated. The deduced genotype for
each animal is shown at the top of each lane. D,
a representative gel of tail DNA PCR reactions using primers specific
for the sry locus is shown.
|
|
We previously genotyped 131 offspring from an intercross of R+/A+
mAGT+/
mice that survived to 3 weeks of age (18). In the absence of
lethality, we expect 25% of offspring to be mAGT
/
. Only three of
the nondouble transgenic mice that inherited both targeted alleles of
mAGT survived to weaning. However, all double transgenic mice
inheriting both targeted alleles of mAGT survived to weaning,
demonstrating that systemic expression of hREN and hAGT can complement
the lethality observed in AGT
/
mice. In fact, all R+/A+ mAGT
/
mice survived to at least 6 months of age, and the kidneys appeared
histologically normal. The mice also exhibited slightly elevated
blood pressure. Although we did not repeat this test breeding, we
have since generated a fully homozygous inbred
strain of R+/A+ mAGT
/
(R+/+A+/+mAGT
/
) mice,
which we have been able to continuously maintain for the past 2 years.
We genotyped 128 offspring 3 weeks of age from the R+/KA+ mAGT+/
intercross (Table I) and then stratified the data into a nondouble
transgenic and a double transgenic group (Table
II). As evident from these data, no RKA
mAGT
/
or RKA+ mAGT
/
mice survived to 3 weeks of age.
2 analysis showed a highly significant difference
between the numbers of mice observed and those expected based only on
Mendelian inheritance assuming no lethal effects of the mAGT deficiency
(p < 0.0001). To determine whether the mAGT
/
mice
were born at the appropriate ratio (25%) or died in utero,
we performed a second set of breeding between R+/KA+ mAGT+/
parents and collected newborns from 27 litters. Genotype analysis of
159 offspring revealed appropriate proportions of all six genotypes as
predicted by Mendelian segregation, including RKA
mAGT
/
and RKA+
mAGT
/
(Table III). To further determine when mAGT
/
mice died, we carefully examined offspring from newborn to 10 days of age and collected dead pups as they appeared. Similar to the findings at 3 week of age, of a total of 52 live 10-day-old offspring, none of them genotyped as mAGT
/
(data
not shown), suggesting all homozygous mAGT
/
mice died before 10 days of age (generally between 4 and 5 days of age). Indeed, when we
genotyped the dead pups, we found that 20 of 27 pups (12 of 16 RKA+ and
8 of 11 RKA
) were also mAGT
/
, a clear enrichment in that genotype
(Table IV).
Because the expression of hAGT in the KAP-hAGT model is sexually
dimorphic and androgen-responsive, we considered the possibility that
females would have increased lethality as compared with males. We
therefore genotyped mice at the sry locus located on the Y chromosome to distinguish between males from females (Fig. 2). There
was no significant difference between the number of males and females
in any group of mice, including those that died between birth and 1 week of age (Table V).
To ensure that the lack of complementation was not due to the absence
of hAGT protein, we performed immunohistochemistry on kidney sections
from nontransgenic and transgenic mice (Fig.
3). Along with our previous report
showing expression of hAGT mRNA in proximal tubule cells (21),
these results clearly demonstrate the production of hAGT protein in the
kidney. Moreover, consistent with apical secretion of hAGT in proximal
tubule cells (26), we detected hAGT protein in the urine of transgenic
mice (5.01 ± 0.69 pmol/ml) but not nontransgenic mice (0.34 ± 0.14 pmol/ml, representing the background of the assay). Although we
could not accurately measure intrarenal Ang-II in the KAP-hAGT model,
double transgenic mice containing the hREN and KAP-hAGT transgene
exhibit hypertension (143 ± 5 versus 113 ± 3 mmHg in control mice) due to the production of Ang-II in kidney (20).
Studies using the Ang-II AT-1 receptor antagonist losartan
confirmed that the blood pressure elevation was due to an intrarenal
Ang-II-dependent mechanism (20).

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Fig. 3.
Cell-specific expression of the KAP-hAGT
transgene. Confocal immunofluorescence images of kidney
from nontransgenic (A-C) and transgenic mice
(D-E) are shown. A and D are low
power micrographs (magnification, × 4), B and
E are moderate power micrographs (× 10), and C
and F are high power (× 20) micrographs. Staining is in
bright orange.
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|
Finally, we considered the possibility that, because hAGT is driven by
the KAP promoter in KAP-hAGT transgenic mice, the inability to rescue
the lethality may be due to the absence of transgene expression during
late development. To examine this, we performed timed breeding and used
RNase protection to compare the time course of hAGT transgene
expression during gestation and neonatal life with that of the
endogenous mAGT and KAP genes. As anticipated, the KAP-hAGT transgene was expressed at precisely the same
developmental stages as endogenous KAP (Fig.
4B). Expression was evident in whole fetuses at E11.5 and in kidney beginning at E16.5. Transgene expression in kidney was evident in newborns and in mice 1 and 2 weeks
of age. When compared with endogenous mAGT expression, it was evident
that expression of the transgene tended to precede and was higher than
expression of endogenous mAGT (Fig. 4A), although fetal
expression of mAGT was evident on a longer exposure (data not shown).
It is therefore unlikely that the inability of the kidney-specific
transgene to rescue the lethal phenotype was related to the lack of
transgene expression.

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Fig. 4.
Developmental expression of the KAP-hAGT
transgene. Representative RNase protection assays showing
developmental expression of KAP-hAGT, endogenous KAP (B),
and endogenous mAGT (A) are shown. The position of the
protected products is indicated. RNA was isolated from whole E11.5 and
E13.5 fetuses (labeled 11 and 13) and from kidney
of E16.5 (labeled 16) and E18.5 (labeled 18),
newborn (N), 1 week of age (1), and 2 weeks of
age (2). Expression of actin was used as an internal
control.
|
|
 |
DISCUSSION |
The major finding of the present study is that transfer of both
KAP-hAGT (localized to proximal tubule) and hREN transgenes onto the
mouse angiotensinogen knockout (mAGT
/
) background is not sufficient
to rescue the lethality associated with this model. Consistent with
previous reports, mouse AGT knockout mice were born in predicted
Mendelian ratios, but died soon after birth (14, 18). In the present
study, no mAGT
/
mice survived past 10 days of age, regardless of
the presence or absence of the KAP-hAGT and hREN transgenes.
One possibility for the inability to rescue is that the transgenes were
not active during a critical developmental period or perhaps they were
expressed in inappropriate locations. However, an examination of
temporal and spatial expression of the transgenes suggests that this
explanation is unlikely. According to our data, the KAP-hAGT transgene
was expressed in a similar temporal window as the endogenous gene.
Moreover, we previously reported that the transgene is expressed in
proximal tubule cells, the same cells that express mAGT endogenously in
the kidney (21). Furthermore, unlike our observation in adult mice,
hAGT is similarly expressed in both males and females during early
stages of development. It is not until after 4 weeks of age that
expression in males becomes much higher than in females.
Although all studies report significant mortality and renal
abnormalities associated with deletion of RAS function, the severity (or penetrance) appears to be highly variable among models and laboratories. The perinatal mortality rate of mAGT
/
mice in this
study (essentially 100%) is much higher than previously reported (60-70%). One explanation is that genetic background may contribute to the high mortality rate we observed. The heterozygous mAGT+/
mice
used in our study were maintained by six generations of
backcross breeding with inbred C57BL/6 mice. Similarly, the KAP-hAGT
and hREN mice used in this study were maintained by 5 and 10 generations of backcross breeding with C57BL/6, respectively.
Therefore, all the mice described herein are essentially C57BL/6
congenic. It is interesting to note that homozygous AT-1A
receptor-deficient mice containing a mixed C57BL/6 and 129 background
survive normally and exhibit normal kidney morphology, whereas
homozygous AT-1A receptor-deficient mice maintained on a congenic
C57BL/6 or inbred 129 background exhibit substantially reduced survival
and abnormal kidney structure (27).
We reported that transfer of both the hREN and systemically expressed
hAGT transgenes onto the mAGT knockout background restores survival and
corrects the renal abnormalities caused by the mAGT deletion (18). In
that study, human angiotensinogen was expressed not only in proximal
tubule cells but also in other tissues such as liver, brain, and heart,
etc., all normal sites for AGT expression. Because hAGT was expressed
in the liver, plasma AGT and angiotensin II levels were elevated. This
increase in plasma Ang-II was sufficient to not only rescue lethality
but to reverse the hypotension and correct the renal lesions normally
associated with mAGT
/
mice. This stands in contrast to the KAP-hAGT
model used in the present study, in which circulating hAGT levels
remain undetectable and will not have any circulating Ang-II when bred
onto the mAGT
/
genetic background (20, 21). Unfortunately, the very
early lethality of mAGT
/
observed in our study precluded any
detailed renal histological analysis. Nevertheless, examination of
several mAGT
/
kidneys from mice, which died before 10 days of age,
did not show any clear evidence of renal lesions. This is not
particularly surprising, because most of the lesions previously
reported were in those rare mice that survived well past 3 weeks of
age. This raises the intriguing possibility that circulating, rather
than proximal tubule-derived Ang-II, is required for continued
development of the kidney after birth. In agreement with this
hypothesis, Kang et al. (28) reported that mAGT-deficient
mice expressing the rat angiotensinogen (rAGT) only in the brain and
liver did not exhibit renal lesions. These findings suggest that
normalization of circulating Ang-II alone is sufficient to rescue the
renal lesions in mAGT-deficient mice and provide additional evidence of
the crucial importance of circulating Ang-II in renal development.
In the present study, the use of the KAP promoter allowed for precise
targeting of hAGT to the proximal tubule, the predominant site for AGT
production in the kidney. The exact function of proximal tubule AGT
production remains unclear; however, multiple lines of evidence suggest
an important role in electrolyte balance and blood pressure regulation.
The localization of AGT in proximal tubule, its polarized secretion
into the proximal tubular lumen, and the presence of high levels of
Ang-II in proximal tubular fluid have been clearly demonstrated (26,
29, 30). Ang-II has been reported to stimulate the sodium/hydrogen
exchanger, an important regulator of proximal tubule sodium
reabsorption, independently of systemic angiotensin, and may also
indirectly affect proximal reabsorption by increasing the sensitivity
of the tubuloglomerular feedback system (31-33). Importantly, our previous study showed that overexpression of the intrarenal RAS in
transgenic mice chronically increased blood pressure independent of
circulating Ang-II, providing strong evidence for the possibility that
the intrarenal RAS per se plays a significant role in blood pressure regulation (20).
In conclusion, our results suggest that the renin-angiotensin system in
the proximal tubule may not necessary for normal renal growth and
development, and its loss is unlikely to be the cause of neonatal death
in mAGT-deficient mice. Based on all available data, we conclude that
the intrarenal renin-angiotensin system located in the proximal tubule
plays an important role in blood pressure regulation and may cause
hypertension if overexpressed but may not be required for continued
development of the kidney after birth.
 |
ACKNOWLEDGEMENTS |
We acknowledge the outstanding technical
assistance of Kelly Andringa, Patricia Lovell, Lucy Robbins, and Norma
Sinclair for generation and genotyping of hREN, hAGT, and KAP-hAGT
transgenic mice, Deborah Davis for assistance with timed breeding, and
Henry Keen and Robin Davisson for reviewing the dissertation (written by Y. D.) leading to this manuscript. We would like to thank Dr. Oliver Smithies for the gift of angiotensinogen-deficient mice.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL55006 and HL48058. The transgenic mice were generated and maintained at the University of Iowa Transgenic Animal Facility, which
is supported in part by the College of Medicine and the Diabetes and
Endocrinology Research Center. DNA sequencing was performed at the
University of Iowa DNA Core Facility.
§
A predoctoral fellow of the American Heart Association Iowa
(Heartland) Affiliate.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
An Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Depts. of Internal Medicine
and Physiology & Biophysics, 2191 Medical Laboratory, The University of
Iowa College of Medicine, Iowa City, IA 52242. Tel.: 319-335-7604; Fax:
319-353-5350; E-mail: curt-sigmund@uiowa.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M003892200
 |
ABBREVIATIONS |
The abbreviations used are:
RAS, renin-angiotensin system;
E, embryonic day;
ACE, angiotensin converting
enzyme;
Ang-I and -II, angiotensin I and II;
hREN, human renin;
hAGT, human angiotensinogen;
mAGT, mouse angiotensinogen;
rAGT, rat
angiotensinogen;
PCR, polymerase chain reaction;
bp, base pair(s);
kb, kilobase(s).
 |
REFERENCES |
1.
|
Gomez, R. A.,
and Norwood, V. F.
(1995)
Am. J. Kidney Dis.
26,
409-431[Medline]
[Order article via Infotrieve]
|
2.
|
Darby, I. A.,
and Sernia, C.
(1995)
Cell Tissue Res.
281,
197-206[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Gomez, R. A.
(1998)
Kidney Int.
67,
S12-S16
|
4.
|
Gomez, R. A.
(1990)
Pediatr. Nephrol.
4,
421-423[Medline]
[Order article via Infotrieve]
|
5.
|
Jung, F. F.,
Bouyounes, B.,
Barrio, R.,
Tang, S. S.,
Diamant, D.,
and Ingelfinger, J. R.
(1993)
Pediatr. Nephrol.
7,
834-840[Medline]
[Order article via Infotrieve]
|
6.
|
Tufro-McReddie, A.,
Harrison, J. K.,
Everett, A. D.,
and Gomez, R. A.
(1993)
J. Clin. Invest.
91,
530-537[Medline]
[Order article via Infotrieve]
|
7.
|
Kakuchi, J.,
Ichiki, T.,
Kiyama, S.,
Hogan, B. L. M.,
Fogo, A.,
Inagami, T.,
and Ichikawa, I.
(1995)
Kidney Int.
47,
140-147[Medline]
[Order article via Infotrieve]
|
8.
|
Anderson, P. W.,
Do, Y. S.,
and Hsueh, W. A.
(1993)
Hypertension
21,
29-35[Abstract]
|
9.
|
Dubey, R. K.,
Roy, A.,
and Overbeck, H. W.
(1992)
Circ. Res.
71,
1143-1152[Abstract]
|
10.
|
Wolf, G.,
and Neilson, E. G.
(1990)
Am. J. Physiol.
259,
F768-F777[Abstract/Free Full Text]
|
11.
|
Wolf, G.
(1995)
Adv. Exp. Med. Biol.
377,
225-236[Medline]
[Order article via Infotrieve]
|
12.
|
Barr, M. J.
(1994)
Teratology
50,
399-409[Medline]
[Order article via Infotrieve]
|
13.
|
Friberg, P.,
Sundelin, B.,
Bohman, S. O.,
Bobik, A.,
Nilson, H.,
Wickman, A.,
Gustafsson, H.,
Petersen, J.,
and Adams, M. A.
(1994)
Kidney Int.
45,
485-492[Medline]
[Order article via Infotrieve]
|
14.
|
Kim, H. S.,
Krege, J. H.,
Kluckman, K. D.,
Hagaman, J. R.,
Hodgin, J. B.,
Best, C. F.,
Jennette, J. C.,
Coffman, T. M.,
Maeda, N.,
and Smithies, O.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
2735-2739[Abstract]
|
15.
|
Krege, J. H.,
John, S. W. M.,
Langenbach, L. L.,
Hodgin, J. B.,
Hagaman, J. R.,
Bachman, E. S.,
Jennette, J. C.,
O'Brien, D. A.,
and Smithies, O.
(1995)
Nature
375,
146-148[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Oliverio, M. I.,
Kim, H. S.,
Ito, M.,
Le, T.,
Audoly, L.,
Best, C. F.,
Hiller, S.,
Kluckman, K.,
Maeda, N.,
Smithies, O.,
and Coffman, T. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15496-15501[Abstract/Free Full Text]
|
17.
|
Tsuchida, S.,
Matsusaka, T.,
Chen, X.,
Okubo, S.,
Niimura, F.,
Nishimura, H.,
Fogo, A.,
Utsunomiya, H.,
Inagami, T.,
and Ichikawa, I.
(1998)
J. Clin. Investig.
101,
755-760[Abstract/Free Full Text]
|
18.
|
Davisson, R. L.,
Kim, H. S.,
Krege, J. H.,
Lager, D. J.,
Smithies, O.,
and Sigmund, C. D.
(1997)
J. Clin. Invest.
99,
1258-1264[Abstract/Free Full Text]
|
19.
|
Merrill, D. C.,
Thompson, M. W.,
Carney, C.,
Schlager, G.,
Robillard, J. E.,
and Sigmund, C. D.
(1996)
J. Clin. Invest.
97,
1047-1055[Abstract/Free Full Text]
|
20.
|
Davisson, R. L.,
Ding, Y.,
Stec, D. E.,
Catterall, J. F.,
and Sigmund, C. D.
(1999)
Physiol. Genomics
1,
3-9[Medline]
[Order article via Infotrieve]
|
21.
|
Ding, Y.,
Davisson, R. L.,
Hardy, D. O.,
Zhu, L.-J.,
Merrill, D. C.,
Catterall, J. F.,
and Sigmund, C. D.
(1997)
J. Biol. Chem.
272,
28142-28148[Abstract/Free Full Text]
|
22.
|
Sigmund, C. D.,
Jones, C. A.,
Kane, C. M.,
Wu, C.,
Lang, J. A.,
and Gross, K. W.
(1992)
Circ. Res.
70,
1070-1079[Abstract]
|
23.
|
Yang, G.,
Merrill, D. C.,
Thompson, M. W.,
Robillard, J. E.,
and Sigmund, C. D.
(1994)
J. Biol. Chem.
269,
32497-32502[Abstract/Free Full Text]
|
24.
|
Yang, G.,
and Sigmund, C. D.
(1998)
Am. J. Physiol.
274,
F932-F939[Abstract/Free Full Text]
|
25.
|
Hatae, T.,
Takimoto, E.,
Murakami, K.,
and Fukamizu, A.
(1994)
Mol. Cell. Biochem.
131,
43-47[Medline]
[Order article via Infotrieve]
|
26.
|
Rohrwasser, A.,
Morgan, T.,
Dillon, H. F.,
Zhao, L.,
Callaway, C. W.,
Hillas, E.,
Zhang, S.,
Cheng, T.,
Inagami, T.,
Ward, K.,
Terreros, D. A.,
and Lalouel, J. M.
(1999)
Hypertension
34,
1265-1274[Abstract/Free Full Text]
|
27.
|
Le, T. H.,
Fogo, A.,
Oliverio, M. I.,
Peterson, A. S.,
Smithies, O.,
and Coffman, T. M.
(1999)
Genetic modifiers in type 1A (AT1A) angiotensin II receptor deficiency.
J. Am. Soc. Nephrol.
10,
396A(Abstract)
|
28.
|
Kang, N.,
Walther, T.,
Tian, X.,
Lippoldt, A.,
Ganten, D.,
and Bader, M.
(1999)
Local angiotensin synthesis is crucial in hypertension-induced end-organ damage.
Hypertens.
34,
344 (abstr.)
|
29.
|
Ingelfinger, J. R.,
Zuo, W. M.,
Fon, E. A.,
Ellison, K. E.,
and Dzau, V. J.
(1990)
J. Clin. Invest.
85,
417-423[Medline]
[Order article via Infotrieve]
|
30.
|
Navar, L. G.,
Imig, J. D.,
and Wang, C. T.
(1997)
Semin. Nephrol.
17,
412-422[Medline]
[Order article via Infotrieve]
|
31.
|
Saccomani, G.,
Mitchell, K. D.,
and Navar, L. G.
(1990)
Am. J. Physiol.
258,
F1188-F1195[Abstract/Free Full Text]
|
32.
|
Quan, A.,
and Baum, M.
(1996)
J. Clin. Invest.
97,
2878-2882[Abstract/Free Full Text]
|
33.
|
Ploth, D. W.,
Rudulph, J.,
LaGrange, R.,
and Navar, L. G.
(1979)
J. Clin. Invest.
64,
1325-1335[Medline]
[Order article via Infotrieve]
|
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