Regulation of Angiotensin Type 1 Receptor and Its Gene Expression ...

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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