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INTRODUCTION |
The renin-angiotensin system
(RAS)1 plays a critical role
in the regulation of blood pressure and volume homeostasis in mammals. The main effector of the RAS is the octapeptide hormone angiotensin II
(Ang II) produced by the successive enzymatic cleavage of the hepatic
glycoprotein angiotensinogen (AGT) by kidney-derived renin and
angiotensin-converting enzyme (ACE), a metalloprotease located at the
surface of endothelial cells (1). Although Ang II acts through two
receptors, type I and type II (AT1 and AT2, respectively), all of the
known cardiovascular effects of Ang II are mediated by the AT1
receptor. In addition to this circulating RAS, the mRNA and protein
for all of the RAS components have been identified in different tissues
as brain, heart, adrenal gland, reproductive organs, and kidney
(reviewed in Ref. 2), leading to the suggestion that tissue RAS (tRAS)
might mediate cardiovascular and other functions within tissue sites.
Inactivation of the RAS during development either by pharmacological
inhibition or gene ablation also causes severe anomalies in the
neonatal kidney, leading to hydronephrosis and impaired urine
concentration (3). In humans, inhibition of the RAS during pregnancy is
associated by a high rate of spontaneous abortion and in some strains
of mice with a high rate of mortality in the first 3-5 weeks of life
(4-8). In an effort to understand the mechanisms that implicate the
RAS in renal development, all of the genes coding for the components of
the RAS from AGT to the angiotensin receptors have been disrupted in
mice (9-15). Although these results confirm that Ang II acting through
the AT1 receptor is required for normal renal development, renal
defects in mouse pups are only observed when both of the two AT1
isoforms present in mice (AT1A and AT1B (16-18)) are inactivated,
leading to the suggestion that each isoform can compensate in the
absence of the other in the target tissue (19). Because only limited
tissues, including the brain, adrenal gland, and testis, express both
the AT1 receptor isoforms in mice, it is possible that the effects of
the RAS on renal development are mediated through one of these non-renal tissues. Supporting this hypothesis, specific restoration of
AGT in the circulation by targeting adipocytes (20) or multi-tissue restoration of AGT by using the ubiquitously expressed metallothionein promoter (21) or the native angiotensinogen promoter (22) prevents
kidney abnormalities seen in AGT
/
mice, whereas
targeted restoration of AGT only in the kidney of these animals does
not prevent the renal anomalies (8). In the current study, we have used
a novel transgenic strategy to directly address the ability of brain
Ang II to correct the renal anomalies in AGT
/
mice and
have compared these results to those obtained when Ang II is
specifically restored in the circulation of these mice.
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MATERIALS AND METHODS |
Generation of Transgenic and Angiotensinogen Knockout
Mice--
The expression vector used for releasing Ang II is shown in
Fig. 1, and the details of its construction have been described previously (23). Briefly, the signal peptide of human prorenin was
linked to a fragment of the heavy chain constant region of IgG2b mouse
immunoglobulin lacking the BIP binding, hinge, and intermolecular disulfide bridge regions. This domain is followed by a
portion of the human prorenin prosegment, which includes a consensus
cleavage site (RVRTKR) for furin, a ubiquitous protease, immediately
linked to the human Ang II peptide sequence. This fusion protein coding
sequence was placed downstream of a 2.2-kb fragment of the human glial
fibrillary acidic protein promoter (hGFAP), which targets expression to
astrocytes of transgenic mice (24). The transgene was excised from the
plasmid vector and co-injected with a tyrosinase gene for pigmentation
marking in the pronucleus of fertilized eggs from FVB/N mice as
previously described (25). The resulting transgenic founder lines were called GFAP-AngII. Generation and characterization of transgenic mice
with cardiac-specific expression of frog skin Ang II (fsAng II 2C) has
been described previously (26).
Knockout mice for angiotensinogen (AGT
/
, a gift
from Drs. Kim and Smithies, University of North Carolina, Chapel Hill,
NC) were crossed from the original C57BL/6 strain onto the FVB/N
background for 10 generations to generate AGT
/
(FVB)
mice. GFAP-Ang II or fsAngII 2C transgenic mice were crossed into the
AGT
/
(FVB) background. All mice were used at 12-16
weeks of age unless otherwise specified. The animal protection
committee of the Clinical Research Institute of Montreal approved all
animal protocols.
Transgene Expression Analysis in GFAP-Ang II Mice--
Total RNA
from brain or other tissues was prepared by the
guanidinium-thiocyanate-phenol-chloroform method (25), and RNase protection assays were performed as described previously (27) with
minor modifications. Labeled RNA probes corresponding to the transgene
or to histone H4 (an internal control for RNA loading) were synthesized
in presence of the [
-32P]CTP (Amersham
Biosciences). Total RNA (10 µg) from tissue samples was hybridized
with a mixture of the labeled probes (6 × 103
counts·min
1 ml
1).
The distribution of transgene expression in brain was assessed by
in situ hybridization as described previously (28). Briefly, mice were killed by decapitation and brains were removed and quickly embedded in OCT and frozen in isopentane at
30 °C. Serial
frozen sections (10-µm thick) were cut by cryostat at
20 °C and
thaw-mounted on Superfross slides (Fisher). Tissue sections were fixed
for 1 h in 4% paraformaldehyde in 0.1 M
phosphate-buffered saline and followed by a 10-min wash in a buffer
containing 0.1 M triethanolamine, 0.1 M acetic
acid glacial, and 0.02 M acetic anhydride. Ribonucleotide probes corresponding to the transgene were synthesized by in
vitro transcription in the presence of [35S]UTP and
[35S]-CTP. The labeled probes (8 × 107
counts·min
1 ml
1) were hybridized
overnight at 55 °C. Non-hybridized probes were digested by
incubation in RNase A solution (200 µg/ml of RNase A in 1 × SSC), and the slides were extensively washed and dipped in photographic
emulsion (Kodak NTB-2). Slides were exposed for 6 days and developed in
Kodak D19 solution.
Characterization of GFAP-Ang II Mice--
The levels of Ang II
in brain, tissues, and plasma were determined by radioimmunoassay (RIA)
(29). Ang II in tissue or plasma was obtained by acid/alcohol
extraction (80% ethanol, 0.1 M HCl) and purified on
Sep-Pak hydrophobic C18 cartridges (Waters, Milford, MA). The peptides
were eluted with ethanol, and lyophilized peptides were quantified by
RIA using a polyclonal rabbit antibody, which has 100%
cross-reactivity with Ang II (angiotensin 1-8), Ang III (angiotensin
2-8), and Ang IV (angiotensin 3-8) but no reactivity with Ang I
(angiotensin 1-10) (29). To determine the form of angiotensin peptides
released by the fusion protein in brain, whole brain acid/alcohol
extracts from three mice were pooled and fractionated by
reverse-phase HPLC and elution fractions were lyophilized and subjected
to RIA using an antibody specific for Ang II and its metabolites (26,
29).
Plasma renin activity was determined as described previously (26).
Briefly, blood (5 drops) was obtained by orbital puncture of mice under
light ether anesthesia in cold tubes containing 25 µl of 0.5 M EDTA (pH 8.0). Blood was immediately centrifuged, and the
plasma was frozen in liquid nitrogen and stored at
20 °C until the
assay was performed. Plasma renin activity was determined by an Ang I
generation assay in presence of excess angiotensinogen substrate from
sheep (26). The resulting Ang I was measured by RIA using antibody
specific for Ang I.
Physiological Measurements--
Systolic blood pressure (BP) was
measured by tail-cuff plethysmography (BP-200 system, Visitech Systems,
Apex, NC). Mice were trained for 7 days and BP was recorded for an
additional 3 days. After the initial 10 days of BP recording, some mice
were treated with an ACE inhibitor (Captopril, Sigma) at 100 mg/kg/day
by intraperitoneal injection in 0.9% NaCl or an angiotensin
AT1 receptor antagonist (Candesartan, a gift from
Astra-Zeneca) 15 mg/kg/day by gavage for a further 5 or 3 days while
measuring BP during the treatment.
For determination of water intake and urine output, animals were placed
in metabolic cages with free access to water and food. After a 24-h
adaptation period, water intake and urine output volume were measured
for three consecutive days. Urine and plasma osmolality were determined
by the freezing-point depression method (µOsmette, Precision
Systems). Water bottles were removed for 24 h, and urine volume
and osmolality were again measured to evaluate the capacity of the mice
to concentrate urine when faced with dehydration.
Histology--
Animals were killed by CO2
inhalation, and the kidneys were fixed in Bouin's fixative and
embedded in paraffin. Serial cuts (5 µm) were prepared and stained
with either Sirius Red or hematoxylin/eosin for morphological analysis
by standard techniques.
Statistical Analysis--
Results are expressed as mean ± S.E. One-way ANOVA with Dunnett's post test or unpaired t
test was performed using GraphPad Prism version 3.00 for Windows (Graph
Pad Software, San Diego, CA).
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RESULTS |
Transgene Expression--
A fusion protein capable of releasing
Ang II into the secretory pathway of expressing cells was placed under
the control of the human GFAP promoter (Fig.
1) to drive over-production of Ang II
specifically in the brain of transgenic mice. Six founder lines were
obtained, and the tissue distribution of transgene expression in the
brain was determined by RNase protection and in situ
hybridization. Three lines (GFAP58.8, GFAP58.5, and GFAP58.2) were
found to express the transgene (Fig.
2A) at different levels in
brain. Line GFAP58.8 (which had the highest expression level in the
brain and was used for most of the experiments in this study) was found
to express the transgene exclusively in the brain (Fig. 2B)
and in situ hybridization demonstrated that this expression
was evenly distributed throughout the entire brain (Fig.
2C). Notably, expression of the fusion protein had no effect
on the pattern of expression of the endogenous GFAP gene
(data not shown).

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Fig. 1.
Diagram of the vector used to release Ang II
in the brains of transgenic mice. A 2.2-Kb fragment of the human
GFAP (hGFAP) promoter was used to drive expression of a
fusion protein comprised of a signal peptide sequence (Pre),
a fragment of the heavy chain constant region of mouse immunoglobulin
IgG2b (Ig), and a human prorenin prosegment as molecular
spacer (Pro) containing the furin cleavage site (RVRTKR)
followed by the sequence encoding Ang II peptide (Ang II).
The coding sequence for Ang II is followed by an intron and
polyadenylation signal from rabbit -globin.
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Fig. 2.
Transgene expression analysis in GFAP-Ang II
mice. An RNase protection assay was performed on total RNA from
brain (A) or non-brain tissues from line GFAP58.8
(B). TG, expected size of the protected transgene
mRNA. Histone H4, expected size for the histone internal
control mRNA; tRNA, negative control;
C+, mRNA from GH4 cells transfected with the
vector in Fig. 1 (10 µg RNA by lane). In situ
hybridization on brain sections from line GFAP58.8 (C) or
non-transgenic control mice (D) with an antisense transgene
probe (AS).
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Dosage and Characterization of Ang II in Brain of GFAP-Ang II
Transgenic Mice--
Angiotensin peptides were extracted from various
tissues and plasma from line GFAP58.8 by acid/ethanol extraction and
quantitated by radioimmunoassay. The results show that brain Ang II was
increased by 3-7-fold in the transgene-expressing lines as compared
with non-transgenic littermates (Fig.
3A), and Ang II production in the brain of each line was roughly proportional to the level of transgene expression (Fig. 2A). Measurement of the levels of
Ang II in various tissues of line GFAP58.8 confirmed that Ang II
production was only increased in the brain of this line as we could not
detect any increase of Ang II content in non-brain tissues tested (Fig. 3B). Importantly, we were also unable to detect any increase
in circulating Ang II in the plasma of GFAP-Ang II mice as compared with control littermates, suggesting that the Ang II being produced in
the brain of the transgenic animals is not spilling over into their
circulation (Fig. 3C). To confirm this finding, we measured plasma renin activity, a sensitive indicator of circulating Ang II
levels, because renin secretion from the kidney is potently suppressed
by increases in circulating Ang II. Notably, GFAP58.8 mice actually
show a slight increase in plasma renin activity (GFAP58.8, 1721 ± 404; non-transgenic 1193, ± 185 ng Ang I/ml/h; p < 0.05), supporting the conclusion that these animals do not liberate
brain-derived Ang II into the circulation. Because the brain is
rich in aminopeptidases that can break down Ang II to less active
metabolites (30, 31), we investigated the form of Ang II contained in
the brain of line GFAP58.8 by RIA-coupled HPLC of whole brain
acid/alcohol extracts. The results clearly demonstrate that the
majority of the peptide extracted from the brain of GFAP58.8 mice
migrates as bona fide Ang II (Fig.
4).

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Fig. 3.
Level of Ang II in tissues and plasma of
GFAP-Ang II mice. Angiotensin peptides were extracted from brain
of the three founder lines of mice (A), tissues
(B), or plasma (C) from line GFAP58.8 mice
(solid bars) or control (open bars) and subjected
to RIA using an antibody specific to Ang II and its metabolites;
n 4. Values are expressed as means ± S.E.;
***, p < 0.001 compared with control.
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Fig. 4.
Characterization of angiotensin peptides
present in brain of GFAP-Ang II mice. Tic marks
indicate the migration of angiotensin peptide standards for Ang II
(angiotensin 1-8), Ang III (angiotensin 2-8), and Ang IV (angiotensin
3-8). Peptides extracted from the brains of three transgenic
(closed circles) or three control (open diamonds)
mice were separated by HPLC, and fractions were subjected to RIA with
an antibody specific for Ang II and its metabolites.
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Blood Pressure Measurement of GFAP-Ang II Mice--
To determine
the effect of increases in brain Ang II on cardiovascular
functions, systolic BP was measured by the non-invasive tail-cuff
method (32). We found that the BP was significantly increased in the
transgenic mice (GFAP58.8, 150 ± 12; control, 123 ± 6 mmHg; p < 0.001). Despite the differences
observed in the levels of Ang II in brain of the three transgenic lines
we characterized, there was no significant difference in the degree of
hypertension seen in mice from these lines (data not shown). Treatment
of line GFAP58.8 mice with a specific angiotensin II AT1 receptor
antagonist (Candesartan) at a dose of 15 mg/kg/day by gavage normalized
BP after 3 days of treatment (Fig. 5),
confirming that the hypertension seen in these mice was due to the
action of Ang II on the AT1 receptor. In contrast, treatment of these mice with the angiotensin-converting enzyme inhibitor Captopril at a
dose of 100 mg/kg/day intraperitoneally for 4 days did not reduce the
BP of transgenic mice to a greater extent than seen in non-transgenic
littermates (Fig. 5). This dose of Captopril was effective, however, in
correcting RAS-dependent hypertension in mice, which
over-express human renin in the liver under the control of
transthyretin promoter (TTRhRen-A3 before treatment 169 ± 10 and
after treatment 112 ± 9; p < 0.001 (29)). These results clearly demonstrate that the hypertension seen in line GFAP58.8
is due to the direct action of Ang II on the AT1 receptor and not due
to activation of the endogenous mouse RAS, which would have responded
to Captopril treatment.

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Fig. 5.
Blood pressure of GFAP-AngII mice.
Systolic blood pressure of male GFAP58.8 (solid bars) and
non-transgenic or control (open bars) mice at 10-12 weeks
of age (BP) was recorded by the tail cuff method. Shown is the pressure
difference after treatment with the ACE inhibitor Captopril or the
angiotensin II AT1 receptor antagonist Candesartan. Values are
expressed as means ± S.E., n 6; ***,
p < 0.001 compared with control relative.
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Effect of Brain or Circulating Ang II on AGT
/
(FVB)
Renal Defects--
Inactivation of the AGT gene in
C57BL/6 mice leads to multiple renal defects including hydronephrosis,
hypertrophy of renal arteries, increased thirst, and an inability to
concentrate urine when faced with dehydration and results in a high
mortality rate by weaning (6-8). To test for the effect of
complementation of Ang II on these phenotypes, we first bred the
AGT
/
mice onto the FVB/N background (hereafter referred
to as AGT
/
(FVB)). After 10 generations,
AGT
/
(FVB) mice had all of the expected phenotypes but
did not exhibit any increased mortality at weaning (data not
shown). These mice were bred to GFAP58.8 transgenic mice or to
transgenic mice over-expressing frog skin Ang II in the heart (line 2C;
Ref. 26). The 2C line has an approximate 1,000-fold increase in cardiac
Ang II and spills this peptide into the circulation as evidenced by a
suppression of plasma renin content (line 2C; Ref. 26). Blood pressure
in line 2C mice is not increased at 5 weeks, but is slightly elevated by 10-12 weeks as compared with non-transgenic littermates (26). The
GFAP58.8 and line 2C transgenic mice were bred into the
AGT
/
(FVB) background, and the resulting mice were
characterized. Selective restoration of Ang II either in brain or in
plasma normalizes BP of the AGT
/
(FVB) mice
(*AGT
/
(FVB) 106 ± 7; GFAP-Ang
II/AGT
/
(FVB) 110 ± 9;
2C/AGT
/
(FVB) 112 ± 12 and control 120 ± 5 mmHg; *, p < 0.01 versus control).
The kidneys of AGT
/
(FVB) mice present hydronephrosis,
increased perivascular, glomerular and interstitial fibrosis, medullary cysts, and hypertrophy of interlobular arteries (Fig.
6, C and D). All
(22/22) kidneys examined from GFAP-Ang II/AGT
/
(FVB)
mice were free of hydronephrosis (Fig. 6E), whereas the vast
majority (8/10) of kidneys from 2C/ AGT
/
(FVB) were
hydronephrosis-free (Fig. 6G). Interestingly, some glomerular atrophy and hypertrophy of interlobular arteries persists despite restoration of Ang II in brain or the circulation of
AGT
/
animals (Fig. 6, F and H).
Small cysts were also present in the renal cortex of animals restoring
Ang II only in the circulation of the AGT-deficient animals (Fig.
6H).

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Fig. 6.
Histopathology of the kidneys (Sirius Red
staining). Kidney sections from control (A and
B); AGT / (FVB) (C and
D); 2C/ AGT / (FVB) (E and
F); and GFAP-Ang II/AGT / (FVB) (G
and H) mice were stained for extracellular matrix with
Sirius Red. Original magnification was 2× (A, C,
E, and G) and 20× (B, D,
F, and H) AGT / (FVB) mice present
hydronephrosis (C, open arrows) cortical cysts
(C, arrowheads), glomerular fibrosis
(D, F, and H; note increased dark
staining), and hypertrophy of the interlobular arteries (D,
note thickening of arterial walls as denoted by open
arrows) as compared with control FVB mice (A and
B). Hydronephrosis was corrected in GFAP-Ang
II/AGT / (FVB) (E) and
2C/AGT / (FVB) (G) mice while cortical cysts
(E and G, arrowheads) and hypertrophy
of interlobular arteries (F and H, note
thickening of arterial walls, arrowheads) persist.
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Effect of Brain or Circulating Ang II on Kidney Function of
AGT
/
(FVB) Mice--
To test for the effect of brain or
circulatory Ang II on kidney function in AGT
/
(FVB)
mice, animals were placed individually in metabolic cages with free
access to water and food, and urine output and water consumption were
measured. As expected, AGT
/
(FVB) mice show an increased
water intake and urine output (Fig. 7,
A and B), but both were significantly attenuated
in GFAP-Ang II/AGT
/
(FVB) and
2C/AGT
/
(FVB) mice. Urine osmolality of
AGT
/
(FVB) and GFAP-Ang II/AGT
/
(FVB) was
significant lower than that of control mice (Fig. 7C). In
contrast, 2C/AGT
/
(FVB) mice correct this decrease in
urine osmolality (Fig. 7C), consistent with the known
importance of circulating Ang II for kidney function (8). As previously
reported, AGT
/
mice are unable to concentrate urine
when deprived of water (Fig. 7C). After a 24-h period of
water deprivation, GFAP-Ang II/AGT
/
(FVB) mice show the
same capacity as control mice to concentrate urine (an approximate
2-fold increase) even though the starting osmolality is lower than that
of control mice (Fig. 7C). The
2C/ AGT
/
(FVB) as well AGT
/
(FVB)
cannot concentrate their urine after being water-deprived as compared
with control littermates (Fig. 7C). These results demonstrate that brain Ang II plays an important role in regulating fluid balance in conditions of dehydration.

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Fig. 7.
Effect of restoring brain or circulating Ang
II on renal function in AGT / (FVB) mice. Water
intake (A) was measured in mice with free access to food and
water. Values are the mean of 3 days' measurements; ***,
p < 0.001 compared with control; , p < 0.05 compared with AGT / (FVB). Urine output
(B) was measured in mice before (open bars) or
after (solid bars) water deprivation for 24 h; ***,
p < 0.001 compared with euhydrated relative.
C, urine osmolality before (open bars) and after
(solid bars) water deprivation; ***, p < 0.001, *, p < 0.05 compared with euhydrated
relative.
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DISCUSSION |
We have used a novel technique to generate transgenic mice with a
chronic over-production of Ang II peptide in the brain. This locally
produced Ang II does not spill over into the circulation as evidenced
by an absence of transgene mRNA or Ang II peptide increases in the
non-brain tissues tested, a lack of detectable increases in circulating
Ang II, and a lack of suppression of circulating renin, a very
sensitive indicator of circulating Ang II. Our results demonstrate that
restoration of brain Ang II in mice deficient for an endogenous
renin-angiotensin system prevents the hydronephrosis seen in the
non-complemented AGT
/
mice and restores their ability
to concentrate urine when challenged by dehydration. In contrast,
although restoration of circulating Ang II in the AGT
/
mice also corrects hydronephrosis, it is unable to correct their ability to concentrate urine when challenged with dehydration, suggesting differences in the site of action of Ang II in these two
models. Although both complementation strategies partially corrected
the increased drinking and urine output seen in AGT
/
mice, neither improved the observed hypertrophy of the interlobular renal arteries. Taken together, these results suggest that the panoply
of renal phenotypes described in RAS-deficient mice may be due to the
action of Ang II on multiple target tissues.
Newborn mice deficient for a RAS have histologically normal
kidneys at birth and develop frank hydronephrosis in the first 3-5
weeks of life (8, 33), a period when the newborn kidney must adapt to a much higher flux in fluid than it was
exposed to in utero. Although both transgenic models reduce
drinking behavior and urine output in the AGT
/
background, these values do not return to levels seen in control mice,
and it seems unlikely that this partial reduction in urine flux could
account for the complete disappearance of the hydronephrosis. Taniguchi
et al. (33) have suggested that the hydronephrosis seen in RAS-deficient C57bl/6 mouse pups is due to an incomplete development of a smooth muscle layer around the upper ureter, which
results in a loss of ureteral peristalsis and hydronephrosis due to
reflux pressure. Surprisingly, in the FVB background we see no
deficiency in the formation of the smooth muscle cell layer around the
upper ureter of the AGT
/
animals (data not shown) even
though all of these animals develop hydronephrosis, raising the
possibility that the hydronephrosis is not directly linked to this
developmental phenomenon. Correction of hydronephrosis in RAS-deficient
mice has also been reported by other groups who have targeted
components of the RAS to different tissues. Double transgenic mice
expressing both human renin and angiotensinogen in numerous tissues
correct all of the renal abnormalities seen in AGT
/
mice (22). Similar results were reported with expression of AGT in
AGT
/
mice using the metallothionein promoter that
results in expression in multiple tissues including brain, kidney, and
liver (21). Somewhat more surprisingly, rescue of the hydronephrosis in
AGT
/
mice has been reported with complementation of
angiotensinogen expression selectively in adipocytes (20). Notably all
of these approaches would result in a partial or complete restoration
of circulating Ang II. One possible explanation for the ability of both
brain and circulating Ang II to correct hydronephrosis might be their
parallel effects on blood pressure, which might counter reflux pressure
in the ureter by increasing the filtration pressure in the
kidney. Indeed, whereas AGT
/
mice are mildly
hypotensive, GFAP-Ang II/AGT
/
(FVB) and
2C/AGT
/
(FVB) mice had a normal BP. However, Kessler
et al. (34) recently reported that replacement of a soluble
form of ACE in ACE
/
knockout mice resulted in the
rescue of kidney anomalies even though these mice remain hypotensive
(34), making blood pressure an unlikely reason for the correction
of hydronephrosis.
Another possible explanation for the ability of circulating Ang II to
correct renal defects in AGT
/
mice would be its uptake
by the kidney itself. Even though the kidney can indeed take up Ang II
from the circulation via the AT1 receptor (35), restoration of Ang II
production only in the kidney is not sufficient to rescue anomalies in
kidneys of AGT
/
mice (8), suggesting that the effect of
Ang II on hydronephrosis is mediated by a non-renal tissue.
Alternatively, Ang II produced in the circulation might act on the
brain to influence the development of hydronephrosis, which would
explain why both of our transgenic mouse models correct this phenotype.
Although the blood-brain barrier is impermeable to peptides such as
angiotensin, the circumventricular organs, such as subfornical organ
(SFO), paraventricular nuclei, and organum vasculosum of the lamina
terminalis (OVLT), allow peptides and hormones to leave the brain
without disrupting blood-brain barrier and permit substances in plasma
that do not normally cross this barrier to trigger changes in brain
function (reviewed in Ref. 36). These organs are rich in AT1 receptors
(37), and injection of 125I-Ang II into the circulation
results in Ang II binding in SFO, OVLT, median eminence, and area
postrema (38-40) to affect both blood pressure and drinking behavior.
These findings raise the possibility that both brain and circulating
Ang II act at the same sites in the brain to influence
either post-natal renal development or function in newborn mice.
Although the actual mechanisms need further investigation, both central
and circulating Ang II can influence renal sympathetic nerve activity,
which in turn could have a direct effect on kidney function by
stimulating the peristalsis of the ureter and in this way prevent the
urinary reflux (41). Interestingly we found that the high rate of
neonatal mortality that has been associated with renin-angiotensin
system deficiency depends on genetic background use. By using
AGT
/
(FVB) it seems that the kidney anomalies seen in
mice with RAS disruption are an adaptation problem instead of a
developmental problem, because obvious renal defects are not seen until
4 to 5 weeks of age.
In conclusion, we have demonstrated that chronic elevations of Ang II
specifically restricted to the brain can prevent the hydronephrosis and
urine concentration deficit seen in RAS-deficient mice. These
findings may have important implications for understanding the
fetotoxic effects of RAS inhibitors seen in humans (4, 5).