Regulation
Expression:
of Angiotensin
Role in Renal
DONNA
ROBERT
H. WANG,*
C. SPETH,
*Department
of Internal
Branch,
Galveston,
Center,
Jackson,
Physiology,
Low
sodium
late
gene
expression
angiotensin
study
and
II (Ang
presented
has been
of
the
II) receptor
here
tests
the
HUAWEI
DONALD
State
Department
of Physiology
demonstrated
(AT1),
the AT1A
that
subtype.
the
1
The
and treated
for 2 wk with normal
diet plus 3 mg/kg/day
losartan,
diet, or low sodium
were not significantly
renin activity
was significantly
low salt intake,
or a combination
plasma
renin
activity
of
that
renal
AT1
combination
indicated
creased-183%
by
227%
by
the
levels
in
controls.
renin-angiotensin
blood
pressure
has
predominant
strated
that
salt
(RAS)
the
type
subtype
sodium
intake
receptor
in the kidney
(4),
ligand
1 Ang
groups.
Blockade
was
found
was
it
the
rat (6).
the aorta
inhibitory
effects
in the
comitant
hemodynamic
that
expression
salt and
expression
is also
of
the
water
All
homeosta-
Direct
growth
kidney
during
Received
fetal
December
Correspondence
Hypertension
Building,
life compared
with
28,
1995.
Accepted
to Dr.
Donna
H. Wang,
&
Vascular
University
Research
of Texas
Medical
it expression
September
in the
in the adult
Department
of Internal
Laboratories,
8.104
Branch,
Galveston,
(8).
Medical
Medicine,
Research
TX 77555-1065.
Journal
of the American
Copyright
tO 1997
by the
Society
American
Society
of Nephrology
for
lacking
muscle
that
requires
the
All
receptor
by
but
(J Am
arterioles
inhibition
are
to down-
Soc
Nephrol
8:
showed
normal
that
renal
raised
the intriguing
tion
of All
gene
expression
II on
renal
of lufro-McReddie
the
selective
somatic
low
normal
Al
1 blocker,
growth
question
by
in rats.
of whether
activa-
intake
induces
sodium
renal growth.
If the All
gene is linked with
growth,
knowledge
of this link could provide
insight
into
renal
disease
the
relevance
states.
renal
loss
of
For
of All
receptors
example,
it has
growth
may
and
interstitial
functioning
result
abnorfurther
in hypertensive
been
noted
and
that
in the development
fibrosis
nephrons
of
growth-
Ang
and
results
(7). These
report
of
effect
to onemass
wall
of con-
an
the recent
captopril
the
independent
(6,7).
These
versible
of
reduces
changes
until
study
merulosclerosis
of Nephrology
rats
of ACE
inhibits
pensatory
l046-6673/0802-0193$03.00/0
AT 1
mRNA
administration
and skeletal
This
losartan,
abnormal
mal renal
9, 1996.
renal
suggesting
level
of the
in the kidney.
hypertensive
evidence
was
et al.
elevated
of the
II
intake
suggesting
that All
receptor-meare necessary
to sustain
functional
In addition,
both
with
Ang
reported
that the angiotensin
converting
enzyme
(ACE)
inhibitor, captopril,
prevents
the normal
growth
of blood vessels
in
( 1-3). Recently,
we have demonof the AI1A
receptor,
which is the
low
that
sodium
growth,
All
the
losartan
animal,
suggesting
a role for Ang II in renal
growth
and
maturation
(5). In support
of this concept,
we have previously
one-clip
is linked
gene
receptor,
events
with
Low
a normal
The
among
indicating
Blockade
normal
of controls.
expression
to upregulate
All
receptor
expression
193-198, 1997)
role in
growth,
above
but were
different
on renal
factors.
found
that
by
renal
mass
of multiple
kidney,
suggesting
All
in renal
depletion
both
AT 1 receptors
and
an increase
those
significantly
mRNA
revealed
is upregulated
not
normal
is the
kidney
with
no effect
the All
intracellular
method,
compared
to increase
regulate
diated
(All)
with
but to have
and
binding
depletion
treatment
development.
found
to sodium
by sodium
renal
their
and extracellular
Pharmacology
from that in control
rats. Kidney
weight
ratio, and renal DNA and
of renal
normal
with
sis.
Interestingly,
for
intake,
II receptor
was
to retard
was
of angiotensin
Ii (Ang II) are
receptors.
With the use of a
autoradiographic
that
intake,
losartan
an important
balance,
of Mississippi
losartan
by
four
is required
by low salt intake
treatment.
Renal
plays
electrolyte
The effects
to specific
assays
sodium
in-
two-compared
increased
by losartan
system
All
low
the
binding
renal subtype
gene expression
predominant
by
212%
of
regulation,
shown
significantly
of Texas
Anatomy,
not altered
ratio
activation
were
were
protein/DNA
analysis
levels
significantly
decreased
in vitro
been
mRNA
University
in the rats subjected
lowered
low
receptor
blot
University
Comparative
binding
content
treatment,
with the
Northern
Radioligand
fluid volume
control.
mediated
by binding
competitive
controls.
losartan,
AT1 receptors
were
but were significantly
Ihe
elevated
by losartan
of the two, compared
the
and
sodium
diet,
low sodium
Biophysics,
and
receptor
significantly
Body weight
and MAP
the four groups.
Plasma
diet plus losartan.
different
among
and
Laboratories,
plus losartan
did not differ
weight,
kidney
weight/body
protein
upregulation
of renal
AT1
mRNA
induced
by sodium
depletion
occurs
conjointly
with an elevation
of the AT1 receptor
that modulates
renal growth.
Seven-week-old
male Wistar
rats were divided
into four groups
normal
sodium
P. GRANGER,t
Washington.
AT1
type
Research
of Veterinary
Pullman,
to upregurenal
hypothesis
Vascular
Department
University,
predominant
JOEY
J. DIPETI’E*
and
and
and Its Gene
ZHAO,*
Hypertension
Texas;
Missippi;
Washington
intake
DU,*
Medicine,
Medical
Abstract.
the
YONG
Medical
and
Type 1 Receptor
Growth
of gb-
associated
(9),
and
com-
with
the
irre-
ultimate
194
Journal of the American
outcome
of loss
disease,
e.g.
indicate
that
of nephrons
renal
,
Society of Nephrology
in the
failure.
systemic
kidney
Moreover,
hypertension
subjects
with fewer
Thus,
determination
than 0.8
of the
is end-stage
clinical
is found
glomeruli
mechanism(s)
X
106
cDNA
renal
observations
in virtually
all
per kidney
underlying
(10).
renal
Medicine,
obtain
growth
is important.
In the study
presented
here,
we used
Northern
blot analysis
combined
with radioligand
binding
to
test the hypothesis
that increased
All
gene
expression
by
sodium
restriction
results
in an increase
sity that consequently
modulates
renal
Materials
and
Treatment
Groups
in Al
growth.
1 receptor
Probes
The clone pUC19 containing
a 2.3 kb of the rat AT1 receptor
generous gift of Dr. Tadashi Inagami, Vanderbilt
University
School
den-
Nashville,
a 790-bp
a template
(14)
for
was digested
(- 180 to +610).
making
AT1
cDNA
probes.
The
AT1
cDNA
probes
were labeled with 32P-dCTP using a Multiprime
DNA labeling system
(Amersham
Co., Arlington,
IL) to a specific
activity
of 3 X 108
CPM/jsg.
The labeled
probes were separated
from unincorporated
nucleotide
using Mini-Spin
G-50 DNA purification
spin columns
(Worthington
Methods
TN)
fragment
(a
of
with Kpn I and EcoR I to
This fragment was used as
RNA
Biochemical
Extraction
Co.,
and
Freehold,
Northern
NJ).
Blots
Young 7-wk-old
River Laboratories,
Total RNA of kidney was extracted
using the guanidine
thiocyanate-phenol-chloroform
extraction
protocol
(15). Electrophoresis
of
30 j.g denatured
RNA from each preparation
was performed
in a 1%
into
agarose
four
male Wistar rats weighing
179 ± 4 g (Charles
Inc., Wilmington,
MA) were randomly
divided
(N
13 in each group) and treated for 2 wk with
diet (0.5%), normal sodium diet plus losartan,
low
groups
normal
sodium
sodium
diet
(0.07%),
purchased
from
was
or low
Harlan
sodium
diet
Teklad
plus
Diets
losartan.
(Madison,
The
WI).
rat food
Losartan
(3
mg/kg/day)
was dissolved
in 0.5-I
mL of water and given by oral
gavage. This dose of losartan
was chosen because
it has previously
been demonstrated
to provide
angiotensin
II antagonism
without
lowering blood pressure (personal
communication
with Dr. Pancras C.
Wong, DuPont Merck Pharmaceutical
Company,
Wilmington,
DE).
An equal amount of water was given by oral gavage
to both the NS
and LS rats. All animal procedures
were in accordance
with the
National
ratory
Institute
of Health
guidelines
for the care
and use of labo-
animals.
gel containing
Renin
with
a single
intraperitoneal
all the rats were anesthetized
injection
of
100
mg/kg
citrate
saline
of thiopental
placed
Plasma
renin
activity
(PRA)
was
available
radioimmunoassay
ter, MN).
Tissue
Protein
OH) coupled
to a Gould
2400S
were heparmnized
(1 unitlg),
and
(3 mL) were collected
ethylenediamine-tetraacetic
from the carotid artery and
acid (EDTA)
tubes.
determined
using
kit for angiotensin
(Eastman
Kodak
correct
the differences
Preparation
Content
and
Measurement
Rochester,
NY)
dodecyl
with
in RNA loading,
sulfate
intensifying
Northern
as the ratio
Binding
Assay
Tissues
were
homogenized
phosphate,
pH
7.1-7.2).
g for 20 mm
of AT1
mRNA
to GAPDH
in hypotonic
Homogenates
at 4#{176}C.
Cell
mRNA.
buffer
were
membranes
(20
at
were
resuspended
in
phosphate,
150 mM NaC1,
and recentrifuged.
Bacitracin
1 mM EDTA,
and EDTA
are included
in the assay buffer to inhibit peptidase
activity in the
tissue preparation.
After resuspension
in assay buffer, an aliquot of the
cell membrane
suspension
was used for protein assay using modified
Bradford
dye-binding
procedure
(Bio-Rad,
Hercules,
CA) (13). To
and
to 2 nM
mono
by
Speth
et al.
0.1%
(16)
in a final
bovine
serum
DNA
of the angiotensin
receptors,
and it is resistant
to peptidase
virtue of the lack of a free amino tenninal
amino group.
content
of solid
cells
(1 1). Protein
Therefore,
Hoechst
DNA
33258
tissues
is an indirect
content
measurement
each
species,
DNA
of the number
of
is an accepted
indicator
of cell size (1 1).
with the fluorescent
compound
content
was assayed
(Sigma,
St. Louis,
MO)
using
a fluorescence
sodium
centrifuged
total
from
mM
then
p.m.
cells
To
(Tris)-
62.5
in euploid
,
screen.
measure
is constant
and
blots were incubated
To obtain tissue for Northern blot analysis, a midline incision in the
abdomen
was performed.
The left kidney was promptly
removed,
weighed,
frozen in liquid nitrogen,
and processed
for RNA extraction.
To obtain tissue for the binding study and the measurement
of DNA
and protein content,
the right kidney was divided
into three pieces,
weighed,
and stored at -70#{176}
until processed.
Because the amount of
per cell
(SDS),
in 20 tris(hydroxymethyl)-aminomethane
assay buffer (50 mM sodium
0. 1 mM bacitracin,
pH 7.2)
Co., Stillwa-
of DNA
Co.,
10 mm
expressed
48,000
a commercially
I (Instar
sodium
,
Ligand
of plasma
in chilled
0.5%
pg/mL
transducer
recorder
samples
(SSC),
of denatured
salmon sperm DNA. The membrane
was
hybridized
with the 32P-labeled
probes in the hybridization
buffer for
18-20
h at 42#{176}C.
Then it was washed
successively
in 2X
1 X and
0.5 X SSC (two times, 10 mm each) containing
0.1% SDS. Stringencies of washes were 65#{176}C.
Blots were exposed to XAR-5 X-ray film
200
was
Cleveland,
The rats
was transferred
standard
sodium. The right carotid artery was catheterized
for the measurement
of mean arterial pressure
(MAP)
with a Statharn
231 D pressure
(Gould
Inc.,
(Gould
Inc.).
RNA
HC1 (pH 8.0) to strip off the cDNA probes and rehybridized
with
32P-labeled
glyceraldehyde
3 phosphate
dehydrogenase
(GAPDH)
cDNA probes.
Autoradiographic
signals were scanned
with a laser
densitometer
(Ultrascan
XL, Pharmacia).
Relative
gene expression
Activity
At the end of the 2-wk diet treatments,
formaldehyde.
charged nylon membrane
(Fisher Co., Houston,
TX).
was baked at 80#{176}C
for 2 h in a vacuum oven (Fisher
TX) and prehybridized
for 5 h at 42#{176}C
in hybridization
deionized
formamide,
5 X Denhardt’s
solution,
5 X
at 90#{176}C
for
Plasma
2.2 mollL
to a positively
The membrane
Co., Houston,
buffer-50%
spectro-
photometer
(F-4500, Hitachi Co., Tokyo, Japan) (12). Protein content
was measured
using a modified
Bradford
dye-binding
procedure
(Bio-Rad,
Hercules,
CA) (13). The results were normalized
to whole
kidney
by weight
and expressed
as DNA
(mg)Ikidney
or protein
(mg)Ikidney.
containing
used
Ang
presence
because
affinity
the AT1 receptor
100
‘25I-(Sar,
as the radioligand
in its binding
the
II receptors,
to high-
density,
or absence
g
Ile)
protein
Ang
volume
was
of 200
‘25I-(Sar,
albumin.
incubated
II prepared
with
as described
L
assay buffer
Ile)
Ang
II was
it is an antagonist
that does not vary
and low-affinity
agonist
conformations
1 nmolfL
of the
‘25I-(Sar,
specific
AT!
activity
by
To measure
Ile) Ang II was used in
antagonist
losartan
(1
mol/L).
Nonspecific
binding
was measured
in the presence
of 1
rnol/L
unlabeled
Ang II. The binding assay was performed
for 120
mm at room temperature
and followed by immediate
filtration through
glass-fiber
filters
radioactivity
was
3801,
CA).
Irvine,
(Whatman
GF/C,
counted
in a gamma
Receptor
affinity
Hillsboro,
OR).
spectrometer
and
concentration
The
filter-bound
(Beckman,
LS
were
calcu-
Angiotensin
lated
by Scatchard
gram
Ligand
analysis
using
the computerized
curve-fitting
pro-
(17).
0.05),
or the
combination
with
the ratio
found
Figure
Statistical
Analyses
Results
one-way
were
renal
expressed
analysis
as mean
of variance
± SEM.
followed
Comparison
test. Differences
at the P < 0.05 level.
was analyzed
were
no significant
changes
(Table
1). The
of
treatment
confirmed
statistically
significant
in MAP
among
the four
to modulate
the renin angiotensin
by observation
of elevated
PRA
system
in the
(RAS)
was
three treat-
nificant.
compared
with
that
1).
body
was
not significantly
weight
and kidney
groups,
ratio were
conjunction
Table
2),
in rats receiving
a low salt intake,
indicating
or kidney
weight/body
activation
of the
that
weight
endogenous
ulate renal growth.
To determine
whether
control
(P
group
different
blockade
of
ratio
(Table
RAS
does
altered
losartan-treated
2). DNA
groups
0.05),
indicating
renal cell number.
that losartan
Furthermore,
determined
altered
(Table
by
losartan
<
among
weight/body
(Bmax)
and
groups
alone
rats
the
or in
(P <
binding
renal
that
necessarily
stim-
not
in the
compared
losartan-
in decreased
ratio was not
with
rats,
indicat-
ing that losartan
DNA and protein
does not affect
cell size. In contrast,
renal
content,
and the protein/DNA
ratio were not
altered
by a low
does not change
salt intake,
either cell
indicating
that
number
or cell
sodium
deficiency
size (Table
2).
All
mRNA
content
in each of the four
was determined
groups
of rats
by Northern
(Figure
iA).
Renal
analysis
sitometric
analysis
mRNA-to-GADPH
sartan
treatment
Table
1. MAP
(Figure
mRNA
(0.94
iB) indicated
that
was significantly
±
and PRA
0.07),
sodium
in four
groups
restriction
=
+
115
DUP
6
57
121±3
LS
LS
+
a
MAP,
mean
arterial
pressure;
PRA,
treated
with
a normal
sodium
diet;
1 13 ±
DUP
treated with a low sodium
b p
< 0.05 versus
NS.
p < 0.05 versus LS.
±
67
diet. Values
plasma
DUP,
renin
±
±
LS,
SE.
3). However,
losartan
treat-
number
restriction
(fmol/mg
(P <
protein)
in the
0.05).
Sodium
increase
control
total
receptor
number,
was not statistically
sig-
was
a significant
decrease
fmoL/mg
protein,
the
possibility
‘251-(Sar,
lie)
N
in losartan5), and a signif-
=
5).
=
that
previously
Ang
II binding
resuspended
5) to dissociate
tissues
bosartan.
and
=
in All
sites
bound
to All
losartan
receptors,
we
in 50 mmol
sodium
We found
that, when
rats (8. 1 and
still substantially
(23.2
and
lower
20.8
than
fmollmg
10.0 fmol/mg
protein,
unlikely
that the reduction
from the previously
bound
protein,
N
that of rats fed normal
N
2),
=
in the All
losartan.
2) was
sodium
diet
that
it is
indicating
receptor
any
in
density
results
sodium
intake
A receptor
in the
Discussion
have
kidney
(4).
recently
demonstrated
the gene
expression
of the
The
presented
here
hypothesis
that
depletion-induced
All
receptor
study
that
low
All
was
designed
to test
the
AT1
receptor
activation
resulting
from sodium
increases
in Ang II leads to an increase
in
density
that consequently
modulates
renal
growth.
We found that the blockade
All
receptors
by losartan decreases
report
that Ang II mediates
receptors
(8). Surprisingly,
of the binding
of Ang
renal mass, confirming
normal
however,
restriction,
both renal
II to
the
renal growth
through
the renal mass was
despite
the
All
mRNA
fact that low
and receptor
8b
4b,c
somatic
growth.
Reduced
renal growth
in the
rats does
not appear
to be caused
by tissue
losartan-treated
hypoperfusion
because
losartan
lufro-McReddie
MAP in the
that although
present
study.
27 mg/kg/day
MAP,
was
activity;
losartan;
are means
±
of
above a certain
factors.
renal
but not
46±4b
3
(Kd)
density,
suggesting
that increases
in renal mass
normal
level requires
the activation
of multiple
Losartan
at a dose of 3 mg/kg/day
reduced
34±2
±
constants
rats receiving
a low sodium
diet (29.7
± 1.6
N = 5) compared
with controls
(18.4
± 0.4
N = 5). The increase
in All
receptor
binding
sodium
restriction
was prevented
by losartan
not altered
by sodium
sodium
intake
increases
ng/mL/h
(N - 6 rats/group)
113±4
NS
1.6
losartan-treated
prior
All
PRA
13 rats/group)
±
eliminate
We
of All
by lo(1 .08
To
upregulates
blot
Den-
of rats”
M AP mm Hg
(N
the ratio
elevated
II to
calculated
the tissue was exposed
to a mildly
acidic wash to eliminate
residual
ligand,
the specific
‘251-(Sar,
Ile) Ang II binding
content
were lower in
in the control
rats (P <
of control
II. There
increase
in
fmollmg
protein,
fmol/mg
protein,
sites induced
by
homogenized
acetate
(pH
treatment
resulted
the protein/DNA
that
Ile) Ang
inhibits
or in cell size,
ratio were
to
from
of Ang
The
3. There
is no signifamong
renal prepara-
(1 pM losartan
displaceable)
binding
rats (6.2 ± 0.5 fmollmg
protein,
N
icant
(15.4
2), indicating
growth
and protein
of rats than
of
renal
weight
(Table
receptor
of salt
P < 0.05).
groups.
dissociation
compared
0.05)
of the binding
summarized
in the Table
in the binding
constants
the four
±
195
Figure
3 shows
the All
receptor
density
in the kidneys
of
four groups
of rats measured
in the presence
of 1 nmolIL
receptor
treated
weight
losartan,
either
than in control
treated
rats resulted
from changes
in cell number
renal DNA and protein
content
and protein-to-DNA
C
binding
alone
tended
the difference
‘25I-(Sar,
weight
kidney
lower
with
of the
endogenous
Ang II to Al 1 receptors
retards
normal
growth.
A low salt intake
alone did not alter the kidney
rats
experimental
from
restriction
although
low
± 0.06,
the four
tions
or losartan
ability
(0.51
from
group are
difference
Growth
(1 .16
plots
each
icant
intake
groups
the four
NS
maximal
in controls
and Renal
two
Scatchard
tissues
salt
0.05, Table
Although
both
by
Multiple
of the
2 shows
ment decreased
total
presence
or absence
groups
0.05,
data
were considered
Results
There
ment
The
by the Tukey-Kramer
Receptor
rats
NS,
of losartan
did not decrease
et al. have shown
significantly
the range
of renal
of renal
blood
flow
decreased
perfusion
can
be
MAP
pressures
in which
achieved
(8).
still within
autoregulation
Therefore,
they
also
196
Journal
Table
of the
BW,
2.
KW,
American
Society
of Nephrology
KW/BW
ratio,
renal
N
NS
NS
+
DUP
LS
LS
+
DUP
a
BW (g)
ATI
*
Protein/DNA
(mg/mg)
120
± 0.02
4.03
± 0.05
7.2
± 0.3
±
002b
3.78
±
004b
6.0
±
03b
13
265
±
5
1.08
±
0.02
4.07
±
0.05
7.4
±
0.4
13
262
±
6
0.99
±
0#{149}03b,c
3.80
±
003b.c
5.8
±
03b.c
6
7
8
_
9 10 1112
3b
124
9
NS, rats treated
5
±
4b,c
with a normal
sodium
40
60
16.7
± 0.4
16.6
± 0.7
17.0
± 0.8
16.7
±
diet;
DUP,
1.1
of rats.
131415
16 1718
a
NS
NS+DUP
LS
V
LS+DUP
S
LS+ DUP
S
1920
;T;;:;
up**
± 5
30
Ls
20
0
.-
-
Protein
1.05
number
of rats”
(mg/kidney)
0.98
weight;
groups
DNA
± 5
KWIBW,
kidney weight to body
diet. Values are means ± SE. N
in four
(mg/kidney)
± 7
NS +DUP
*
i0)
264
NS
4 5
KWIBW
(X
ratio
259
A
3
and protein/DNA
13
body weight; KW, kidney weight;
LS, rats treated with a low sodium
< o.osversus NS.
p < 0.05 versus
LS.
2
content,
13
BW,
I
and protein
K W (g)
losartan;
L, p
C
DNA
a
GA.PDH
U-
B
0
I .5
10
*
#{163}1.2
*
*
Q-
0
0.9
0
z
20
Bound(fmoI/mg
E
p0.6
Figure
2. Scatchard
treated
with
N = 4), low
0
Co
0.3
the mean
NS
Figure
1. Northern
blot
panel)
to hybridize
RNA
normal
sodium
(NS,
diet
(GAPDH)
cDNA
of Northern
blot
mean
lane
labeled
from
AT1
kidneys
diet (LS,
cDNA
of rats
lane
+
McReddie
growth
those
from
The blots
were
glyceraldehyde
stripped
and
3
phosphate
rehybridized
with
dehydrogenase
involved
SE.
*
for
0.05
P <
B is densitometric
GAPDH
in each
blot.
Results
Our
role
it is unlikely
in determining
that
renal
hypoperfusion
played
impairment
observed
growth
sartan-treated
weanling
rats
(8).
observations,
indicate
that
Ang
II
during
development
via
the
All
receptor.
and
content,
but
not
that
normal
both
DNA
ratio,
were
tan
decreases
cell
proliferation
results
are
in
These
lower
protein
in losartan-treated
agreement
Their
results,
along
stimulates
renal
a major
with
DUP (N
results
(DUP,
constants
(Kd) from four
3. Each symbol
represents
animals
induces
receptor
mechanism
in each
groups
is
negative
that
losar-
cell
size.
Tufro-
suggests
a dissociation
It
feedback
has
All
mechanism
well
as
with
Ang
II
Il-induced
All
is
growth
mRNA
and
chronic
the
expression
reported
kidney
suggesting
modulate
Ang
that
upregulates
been
rat
as
that
between
the
losartan
in the
coarctation,
affecting
is unexpected
and
(8),
hyper-
(18-20).
density
mRNA
II levels
that
increases
unknown.
Ang
and
receptor
protein
aortic
kidney
decreases
demonstrating
losartan
by which
found
indicating
All
losartan
that
observation
receptor
protein-to-DNA
the
This
growth
We
from
by
ment.
sion
rats,
reports
processes
receptor
our
developing
in growth
All
in lo-
without
with
+
to four
that
laboratories
creases
All
that
demonstrating
in the
finding
losartan
suggested
of three
other
is mediated
analysis
NS.
versus
of rats
losartan
3). The
+
II concentration.
et al.
plastic
in A corrected
tissues
4), NS
with
losartan
one rat (30 pg of
panel).
II to renal
=
and dissociation
in the Table
II binding
Ang
lane represents
(A, lower
of Ang
(A, upper
treated
(DUP, lane
1 1, 12, 13, 14, 15), LS +
1 , 2, 3, 4, 5), NS
of the Ang
at a particular
LS+DUP
Each
probes
±
LS
32P-dCTP
isolated
16, 17, 18, 19, 20).
total renal
RNA).
32P-dCTP-labeled
represent
NS+DUP
using
6, 7, 8, 9, 10), low sodium
(lane
of binding
of maximal
binding
(Bmax)
groups
of rats are summarized
0.0
DUP
plot
protein)
sodium
diet (NS, N
diet (LS, N = 4), LS
normal
sodium
80
that
but
deserves
treatment
with
expression
of
All
its
decom-
of the
gene.
mRNA
The
expres-
previously
that
is downregulated
All
following
high
local
receptor
gene
expression
by
It is possible
that
blockade
(21).
and/or
circulating
of the binding
of Ang II to its receptor
by losartan
eliminates
a negative
feedback
signal and increases
All
mRNA
expression. However,
it has also been reported
that intrarenal
All
a
Angiotensin
Table
3.
losartan
Changes
in Ang
and/or
sodium
II receptor
+ DUP
LS
LS
a
+ DUP
Ang
in response
for
to
restrictiona
Kd
Bmax
N
NS
NS
density
(fmol/mg
reasons:
ligand
did
Ang
II binding,
1.5 ±0.2
1.9 ± 0.2
bound
1.5 ±0.2
upregulates
1.7 ± 0.3
dium
depletion
binding;
sion
in the
59±5c
3
32 ±
II; Bmax,
calculated
6d
maximal
to the All
Consistent
Kd,
C
that
gene
All
expression
mRNA
receptor
mRNA
upregulates
the
gland
elevated
finding
NS
NS+DUP
LS
LS+DUP
Ang
region
It is possible,
induced
by low
mRNA
expression
and
Figure 3. Bar graph showing AT1 receptor density in kidneys of four
groups of rats measured
in the presence
of 1 nmol/L
‘251-(Sar, Ile)
Ang II. There are five rats in each group. MS. rats treated with normal
sodium diet; DUP, losartan;
LS, rats treated with low sodium diet.
* p < 0.05
versus
MS. + P < 0.05 versus
MS + DUP. # P < 0.05
versus LS.
loading
increased
(27
rats
mg/kg/day)
for
did
not
change
3 wk
(8).
The
after
to distinguish
The
may
among
these
possibili-
AT!
or other consequences
of
on the genomic
structure
of Al!
elements
have
exist
shown
that
in the 5’-
tubule
Ang
weight/body
by sodium
weight
ratio,
not
altered.
This
local
RASs
proximal
tubule
It is well
established
stimulator
of
in the
early
All
Micropuncture
of proximal
indicates
of Ang
II binding
density
that
steps,
e.g., posttranscriptional
events,
needed
to transcribe
the
distinct
ologic
increase
All
mRNA
into
receptor
protein.
Regulation
of
actions
has
demonstrated
events
posttranscriptional
by
our
laboratory,
contribute
to
expression
whereby
in
losartan
All
functional
receptor
as
regulation
is that blockade
interfere
with
by agonists
well
as
by
of
the
AT!
the
kidney.
Another
could
decrease
All
occupancy.
This
does
others
been
of the renal
intermediary
(22,23),
development
and
may
its
gene
receptor
and
possible
receptor
mechanism
binding
is by
not appear
to be the case
Ang
II and
is Ang
other
renal
circulating
of
One
may
to
the
roles in normal
renal development
and altered
physiconditions.
For example,
one can speculate
that an
in renal mass above
a normal
level needs
synergistic
need to be considered.
receptor
by losartan
stimulate
of
ties
All
has
us
kidney
and/or
finding
not
site (28,29).
in the
Although
currently,
the mechanism
by which
losartan
decreases
the All
receptor
density
is unclear,
several
possibili-
triguing
led
S 1 seg-
kidney
weight,
kidney
and protein
content
were
activation
does
Na-H
studies
have
tubule
also con-
receptor
restriction,
and DNA
is
sodium
diet,
to an
potent
concentration
in its
it has
density
with
reabsorption
tubule
(28 -30).
these segments
sodium
a low sodium
Ang II binds
to modulate
retention.
to
suggest-
II receptor
decreased
is given
produced
extremely
by
levels,
lead to an increase
In support
of this,
and
receptors
in AT!
remains
induced
mRNA
levels
receptors.
an animal
or locally
increased
mechanism
density
depletion
although
for the increase
in All
mRNA
release
This
receptor
Interestingly,
was
modulate
the binding
of Ang II to Al!
losartan
has a direct
effect
on
expression
or that other mech-
kidney.
bicarbonate
the highest
also
mechanism.
However,
sodium
diet are also
of aldosterone
in sodium
ments of proximal
demonstrated
that
this
accounts
proximal
is an
for
intake
of
II
mechanism
PRA by sodium
depletion
is activated.
Therefore,
it
by aldosterone
number
and
soexpres-
mRNA
the
resulting
Ang
intake
stimulation
functions
tamed
inconsis-
tency could be the result of differences
in the strain of the rat
studied,
or the duration
and dose of losartan
given. At present,
no information
receptor
effects
studies
increased
that
sodium
Similarly,
that
sodium
(27). When
circulating
exchange
in Sprague-Dawley
All
feedback
fed a low
the changes
that
low
for
in the
with
in
differ-
account
in All
shown
decrease
therefore,
sodium
parallel
increased
induced
no significant
(4),
II levels
aldosterone
Previous
(25,26).
been
previously
expression.
(14,24).
by a positive
levels
in rats
ing that increased
All
translational
products-Al!
0
we have
ties.
All
regulatory
depletion
to remove
receptors.
our previous
be explored.
The changes
*
197
Growth
in losartan
treated
versus
if a competing
ligand
was
and the promotor
region
sequence
several
glucocorticoid-responsive
+
Renal
was
affinity
occur
with
exist, e.g.,
depletion.
sodium
*
treatment
losartan
and (2) there
adrenal
appears
anisms
+
levels
the
alter
elevated
when losartan
blocks
receptor,
indicating
that either
the regulation
of All
mRNA
40
losartan
wash
upregulation
is unclear.
Increased
indicates
that the circulating
RAS
dissociation
constants;
NS, rats treated with a normal sodium diet;
DUP, losartan;
LS, rats treated with a low sodium diet. Values are
means
± SE. N
number of rats.
b p <
0.05 versus NS.
p < 0.05 versus MS + DUP.
d p
< 0.05 versus
LS.
mRNA
not
and
acidic
ence in 125151
Ang II binding
control
kidneys,
which
would
4b
4
mild
‘25ISI
(X l0)
45±2
22 ±
(1)
bound
protein)
4
4
II, angiotensin
two
Receptor
propose
factors,
that
growth.
the
whereas
This
RAS
normal
inplays
renal
Il-dependent.
In conclusion,
we have demonstrated
that blockade
of endogenous
Ang II by losartan results in retardation
of the normal
renal
growth.
Although
sodium
restriction
AT1 mRNA
and receptor
protein,
by sodium
depletion.
These
results
increases
both
renal
renal mass was not altered
suggest
that Ang II modu-
198
Journal of the American
lates
renal
growth
tor. However,
the activation
Ang
Society of Nephrology
normal
during
development
via the All
increases
in renal mass above normal
of multiple
factors.
An understanding
II in growth
information
regulation
regarding
pathogenesis
of the
the
kidney
regulation
of hypertension
may
of blood
kidney
and
14.
recep-
Iwai
N, Yarnano
levels require
of the role of
Sasaki
provide
tion.
pressure
II receptor:
new
the
and
15.
Ann
16.
Acknowledgments
This
study
was
Grant
HL-52279
Donna
H.
supported
and a grant
from
Dr. Donald
J. DiPette
Wang.
Investigator
Award
from
Merck
D. Bukoski
Wilma
for her expert
Institutes
Research
Heart
for his critical
review
secretarial
skills.
of Health
Laboratories
is a recipient
the American
Dr. Richard
Frye
by National
in part
to Dr.
of a Established
Association.
17.
and
18.
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