is a recipient of a Established. Investigator. Award from the American. Heart Association. We thank. Dr. Richard ... S. Chaki. 5, Konishi. F, Bardhan. S. Tibbetts. C,.
Regulation Expression:
of Angiotensin Role in Renal
DONNA ROBERT
H. WANG,* C. SPETH,
*Department
of Internal
Medical
Branch,
Medical
Center,
and
Abstract.
Low
late
gene
the
angiotensin study
sodium
intake
II (Ang
Galveston,
the
tests
the
HUAWEI
DONALD
Texas;
State
demonstrated
(AT1),
the AT1A that
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
indicated
activity
of
that
renal
combination
creased-183%
AT1
by
227%
by
the
levels
in
controls.
pressure
has
shown
predominant strated that
All
assays
(RAS)
type
intake
receptor
in the kidney
(4),
ligand
and
regulate diated
role in
binding
method, (All)
it
is the
in the
kidney
suggesting
that
is linked
is upregulated
expression
with
by
of
salt and
the
water
low
sis.
kidney
All
during
fetal
gene
expression
life compared
is also
with
elevated
it expression
in the
in the adult
December
Correspondence Hypertension
Building,
28, to Dr.
&
1995. Donna
Vascular
University
Accepted
H. Wang,
Research
of Texas
September
Medical
Department
of Internal
Laboratories,
8.104
Branch,
Galveston,
the
rat (6).
kidney,
Copyright
of the American tO 1997
Medicine, Research
TX 77555-1065.
by the
Society American
Society
of Nephrology
of the
growth,
renal
AT 1
that
level
requires
the
All
receptor
by
of the
All
II
intake
suggesting
a normal
Blockade
Ang
sodium
mRNA
but
to down-
suggesting that All receptor-meare necessary to sustain functional in the kidney.
both
In addition,
(J Am
the aorta
inhibitory Direct
(8).
This
losartan,
results
study
Soc
Nephrol
8:
gene
are
captopril
to one-
the
mass
wall
(7). These independent
of con-
effect
of
Ang
showed
that
renal
the
and
the intriguing expression
II on
report
selective
somatic
low
Al
renal
1 blocker,
growth
question by
normal
of lufro-McReddie 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
growth
loss
and of
of All
For
receptors
example, may
interstitial
functioning
of
growth-
(6,7).
the recent
normal
raised
of All
inhibition
an
until
inhibits
These
versible
for
lacking
of reduces
arterioles
changes
evidence
was
muscle
of ACE
hemodynamic
growth
rats
and skeletal
effects
comitant
administration
hypertensive
merulosclerosis
of Nephrology
factors.
the
losartan
that
Low
expression
above
receptor, events
one-clip
pensatory
l046-6673/0802-0193$03.00/0
Journal
Medical
mass
with
indicating
on renal
to upregulate
the All intracellular
abnormal mal renal
9, 1996.
and
The
among
reported that the angiotensin converting enzyme (ACE) inhibitor, captopril, prevents the normal growth of blood vessels in
tion Received
growth,
normal
of controls.
different
development.
mRNA
of multiple
those
but were both
AT 1 receptors
renal
no effect
found
with
with
significantly
renal
in renal was
not
normal
to increase
depletion
depletion
treatment
of renal
normal
but to have
et al.
Interestingly,
was
to sodium
animal, suggesting a role for Ang II in renal growth and maturation (5). In support of this concept, we have previously
All
homeosta-
Pharmacology
by sodium
compared
All receptor expression 193-198, 1997)
that
of Mississippi
from that in control rats. Kidney weight ratio, and renal DNA and losartan
intake,
for
an increase
their
and extracellular
II receptor
by
to retard
found
with
( 1-3). Recently, we have demonof the AI1A receptor, which is the
subtype
sodium
was
of angiotensin Ii (Ang II) are receptors. With the use of a 1 Ang
found
is required
intake,
an important
balance,
sodium
groups.
losartan
revealed
of Texas
Anatomy,
not altered
Blockade
was
by low salt intake treatment. Renal
plays
autoradiographic the
salt
increased by losartan
The effects to specific
that
low
by
were
four
in-
two-compared
electrolyte
renal subtype gene expression
predominant
the
binding
system
regulation,
in vitro
been
212% of
University
in the rats subjected
ratio
activation
significantly
binding
protein/DNA
analysis
were
University
Comparative
lowered
low
receptor
blot
Biophysics,
and
content
treatment, with the
Northern
levels
significantly decreased
fluid volume control. mediated by binding competitive
mRNA
Radioligand
renin-angiotensin
blood
controls.
losartan,
AT1 receptors were but were significantly
Ihe
elevated by losartan of the two, compared
the
and
Body weight and MAP the four groups. Plasma
diet plus losartan. different among
receptor
significantly
sodium diet, low sodium
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
and
of Veterinary
Pullman,
subtype.
the
JOEY
Research
of Physiology
to upregurenal
ZHAO,* Vascular
Department
University,
predominant
hypothesis
and
Department
and
and Its Gene
J. DIPETI’E*
Hypertension
Missippi;
has been of
II) receptor
here
and
DU,*
Medicine,
Washington
expression
presented
YONG
Jackson,
Physiology,
Type 1 Receptor Growth
result
it has
abnorfurther
in hypertensive
been
noted
and
that
in the development fibrosis
nephrons
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
in virtually
glomeruli mechanism(s)
X
per kidney underlying
106
cDNA
renal
observations all
Medicine,
(10). renal
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
to a positively The membrane Co., Houston, buffer-50% standard
saline
Renin
with
a single
all the rats were anesthetized
injection
of
100
mg/kg
(Eastman
Kodak
of thiopental
transducer recorder
Ligand
placed
of plasma in chilled
Plasma
renin
samples
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
Preparation Content
and
Measurement
Rochester,
dodecyl
sulfate
NY)
with
intensifying
Northern
as the ratio
Binding
Tissues
were
phosphate,
pH
of AT1
mRNA
to GAPDH
mRNA.
Assay homogenized
in hypotonic
7.1-7.2).
g for 20 mm
buffer
Homogenates
at 4#{176}C. Cell
were
membranes
(20
mM
content cells
of solid (1 1). Protein
Therefore, Hoechst
tissues
DNA 33258
is an indirect
content
content (Sigma,
from
measurement
each
centrifuged
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
species,
DNA
of the number
of
is an accepted indicator of cell size (1 1). with the fluorescent compound
was assayed St. Louis,
MO)
using
a fluorescence
sodium
then
of the angiotensin receptors, and it is resistant to peptidase virtue of the lack of a free amino tenninal amino group.
cells
To
(Tris)-
DNA
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
sodium
in RNA loading,
10 mm
expressed
48,000
a commercially
I (Instar
Co.,
the differences
was
Cleveland, The rats
0.5%
,
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.).
(SSC),
was transferred
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 intraperitoneal
citrate
RNA
(Fisher Co., Houston, TX). h in a vacuum oven (Fisher 5 h at 42#{176}C in hybridization Denhardt’s solution, 5 X
pg/mL
correct
At the end of the 2-wk diet treatments,
formaldehyde.
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
at 90#{176}C for
Plasma
2.2 mollL
charged nylon membrane was baked at 80#{176}C for 2 TX) and prehybridized for deionized formamide, 5X
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.
62.5
by
total
p.m.
Speth
et al.
containing
used
Ang
0.1%
mono
(16)
presence
serum
density,
or absence
protein Ang
was
low-affinity
1 nmolfL of the
‘25I-(Sar,
specific
AT!
assay buffer
Ile) that
agonist
with
as described
L
of 200 ‘25I-(Sar,
it is an antagonist and
incubated
II prepared
volume albumin.
because to high-
g Ile)
in a final
bovine
affinity
the AT1 receptor
100
‘25I-(Sar,
as the radioligand
in its binding
the
II receptors,
to 2 nM
Ang does
II was not vary
conformations 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 counted Receptor
GF/C,
Hillsboro,
in a gamma affinity
OR).
spectrometer and
concentration
The
filter-bound
(Beckman, were
LS calcu-
Angiotensin
lated
by Scatchard
gram
Ligand
analysis
using
the computerized
curve-fitting
pro-
(17).
0.05),
or the
with
the ratio
Figure Statistical
Analyses
Results one-way
were
renal
expressed
analysis
as mean
of variance
Comparison test. Differences at the P < 0.05 level.
were
groups
data
were considered
no significant
(Table
treatment confirmed
was analyzed
1). The
groups
the four
changes
ability
compared 1). body
groups,
ratio were conjunction
in MAP
of
low
2),
that
maximal
salt
of the
among
the four or losartan
restriction although
system in the
(RAS) was three treat-
nificant.
not significantly
weight
(P
group different
and kidney
in rats receiving a low salt intake, indicating
weight/body
activation
of the
that
weight
blockade
of
or in
(P