Osteogenic Protein- 1 mRNA Expression Is Selectively Modulated ...

J Am

Osteogenic Modulated

Protein- 1 mRNA Expression after Acute Ischemic Renal MARIA

M. ALMANZAR,*

ANA ANA

I. PIQUERAS,t L. PAREDESt

*Pedjatric.

of Pathology,

Osteogenie

protein-

after

birth

differentiation. repair renal

injury

pediele

was 48

blots.

hydrogenase

ratio signals

ischemie,

Osteogenic is

superfamily isolation

and

a lengthy matrix

opposite,

member

of

growth

of OP-

the

expression

mature

is the

family

significantly

tissues

express

highest

form

before

member

of

occurs

medulla

gbomerular,

including collecting

duet

OP- 1 is expressed

and in the

Northern

Western

blot

(4.0

in

rats.

are

ovary

the

result

cells

results

(5).

and

in

cells rat

(I 1 ,l2).

mal

I046-6673/090

1- l456$03.00/0

may

than

OP-l

mRNA

compared kb

and

with for

the

the

lower

other

identify

returned

to

baseline.

and

after

per

kidneys

is now

to its

molecules,

ment

In

with

OP-l

coronary

with

OP-

size

of

potent

bone

its

an

Recovery

vascular

mechanisms.

to inhibit

bilateral

carotid

infarct

in the

(16).

ability

(13),

neural

cells

neural heart

adherence ( 1 5). ligation

regenera-

model,

treat-

of neutrophils Similarly, in rats

These

cell adhesion

results

to

treatment decreases in

rat

the models

effect. acute

These of

during

function

of neural

in promoting

endothelium

anti-ischemic from

dur-

OP-l

Cultured

ischemia/reperfusion

appears

cerebral

these

molecule

morphogenetic

an effect rat

1 before the

suggest

reversal

the

mesenchy-

understood.

suggesting

(14).

with

have

Overall, of

established,

kidneys

kidneys

signaling relevance

of after

compared

(I 2).

Op- 1 plays a major robe in tissue repair. treated with OP- 1 result in the induction tion

mutant

section

(1 1). Mutant

the

arrest

shortly

morphogenesis,

Although is poorly

nephrogen-

show

in death

epithelialization

injury.

repair.

during

of dysgenic

gbomeruli

In

selectively

for kidney

resulting

by

reper-

renal

mice

1 as an essential

development

are

isehemie

mesenehyme

of branching

nephrogenesis. kidney

protein

acute

analysis

OP-

points.

mRNA

be a key element

and

time after

mRNA

three

all

OP- I protein days

OP- 1-deficient

reduction

at

analyzing Seven

100 in wild-type

In addition

of

in the

animals

reperfusion.

metanephric

condensation,

kidney

an essential

2.4

OP-l

Histologic

less

than

adult

and

lower

in sham

medulla,

transcript

and differentiation,

a dramatic

the

Journal of the American Society of Nephrology Copyright © 1998 by the American Society of Nephrology

and

fivefold

that

when

when

OP-I

the

birth

rat

kidney

Received March 19, 1997. Accepted March 3, 1998. Correspondence to Dr. Ana L. Paredes, Miami Childrens Hospital, Division Nephrology. 3200 SW. 60 Court. Suite 304. Miami, FL 33 155-4078.

of

Homozygous

growth

the

epithe-

was

with

isehemie

in the medulla

renal

ing

(8). has

(10).

Abundant

in renal

and

cells

inducing

findings

BMP

Other

kidney

(6,7), (9)

medium

but

robe esis

more in

kb

significantly

kidney

revealed

to the

the (4).

canine

of

Transfee-

intestine,

tissue

Georgia;

20-fold

obtained

levels

OP- 1 modulation

bone

large

kidney

shown

downregulated

The

1 ).

(2).

increasingly and

conclusion,

factor-n

for

at 24 h after

the

kb)

protein-7

(

fusion,

de-

OP-l

demineralized

renal

kidney

2.4

factors

has been cells

by

The

into

endothelial fetal

and

secretion the

and

determined

is processed

Madin-Darby

mesangial,

and

growth

gonads

of OP- I transcripts lines,

were

that the

in

I , including

content

content

3-phosphate

hamster

expressed OP-

OP-l results

formation

protein

Florida;

time points were comparable. OP- 1 mRNA expression was also affected in the opposite medulla compared with the sham medulla. However, only in the isehemic medulla was the Similar

were

in

Results

relative

1 molecule

bone

Chinese

a 50-kD

only

mRNA

cell

inner

into

of

OP-

cortex.

were

morphogenetie

present new

of the

4.0

In the

kidneys

differentiation

a factor

of inducing

1 eDNA

expression

and

Tifton,

downregubated

and

transforming

of the

for

1 7- to 19-kD OP-I

the

Medical

Miami,

compared

reperfusion.

at 3, 6,

sham-operated

1 ), or bone of

purification

capable

(3).

I (OP-

a

search

tion

and

1

transcript

kidney

strikingly

isehemic

OP-

for

killed

transcript

Administration

‘s Hospital,

of Georgia,

were

histology

OP-l

h after

was

ischemic

clamping

Rats

1/glycerabdehyde

each

kidney

unilateral

24

and several

Acute

reperfusion, by

OP-

for

protein-

(BMP-7),

by

growth

involves

isehemia.

in OP- 1 mRNA of

renal

whether

after

analyzed

Changes

the

examined

7 d after

and

measuring

content

isehemic

of

and

Veterans

Children

University

whole

injury

in rats

Miami

Miami

Medicine,

H. DUBE,t

F. CHARETTE,

and

mRNA

by reperfusion.

h and

mierodissected

from

achieved

followed

and

Western

study

of Miami

fac-

arrest

1998

Massachusetts.

1 ) is a morphogenetie

to

is modulated

MARC

of Nephrology,

of Veterinary

PHILIP

and involved in tissue repair OP- 1-deficient mice die

postisehemie

this

expression

I 2, 24,

mainly

Because

renal

hal

due

mechanisms,

mRNA

College

Hopkinton,

1 (OP-

in the kidney Homozygous

University

tDivision

9: 1456-1463,

Is Selectively Injury

S. FRAZIER,

K. JONES,t

Medicine,

Florida;

BioMolecules,

tor highly expressed and development. shortly

and

Miami,

Departnient

Creative

Abstract.

WILLIAM

Nephrologv

Center/GRECC,

KENDALL

Soc Nephrol

nephrogenesis

renal

injury

mechanisms (

occurs

include 1 7),

progression

through a possible through

several stepwise the

cell

I Am Soc Nephrol

9: 1456-1463,

cycle

cells

of viable

factors

(1 9).

been

A

shown

growth,

and

repair

after

renal

failure

factors

have

but also Ischemic

acute

and

shown

modulation kidney

and

Experimental Young to 200

g were

MA). cycle

Rats and

nation

and

was

Charles

was induced incision,

anesthesia,

core

removed

body

and

in

temperature. return

(Wilmington,

The

the

was

the

of 150

an intraperitoneab kidney

was

pediebe

was

on a warm

1 h of isehemia,

kidney

was

blanket the clamp

assessed.

Animals

that did not reperfuse completely were excluded from the experiment. The wound was sutured and the animals were returned to their cages after

their

complete

recovery

12, and 24 h with the

thoracie and

flushed

a lethal

and

incision,

dose

abdominal

the

with

from

heart

anesthesia.

was

30 cc of cold

punctured

normal

saline.

A fraction

in liquid

nitrogen

for RNA

labeled

isehemie

and

were

Another

group

kidneys

were

separated freezing.

by free-hand The medulla

tan section) papilla

visible

(white

opposite animals

of animals

was

removed,

and

used

killed

the

section).

We

medulla

labeled

this

kidney).

48 h. Both were

medulla

group

was

The

quickly

a no. 1 1 scalpel before the outer stripe medulla

eye and the inner

also

cortex and medulla. for all time points

removed

analysis.

(contralateral

and

and

the rest

protein

at 3, 6, 24, and

cortex

dissection using section included

to the naked

were

for histology;

opposite

died,

a midline syringe

kidneys

and

at 3, 6,

animal

through

a 21-gauge

Both

snap-frozen

killed

the

exposed

with

was

were

Before

were

and cut longitudinally. kidneys

Rats

of anesthesia.

cavities

snap (redl

including

as isehemic

The control group consisted that either underwent surgery

and

of paired without

clamping of the renal pedicle (sham rats) or had no surgery (control rats). A third group of animals was killed 7 d after reperfusion to

evaluate mRNA and histologic per time point studied. RNA

Preparation

Total

RNA

assay), RNA

using was

from

stripped

La Jolla,

(150 Sacehi

to 200 (23).

using

the

Each

sample

CA).

denaturing

(Stratagene,

and

cellulose

San Diego,

ratio

using

CA)

Co.

(BioRad

optical was

a

density

was

GS

670,

units.

The

determined

for each

sample.

Western

Blot

Tissue

Analysis

samples

minced,

from

collected,

and

protein

The

protein

assay

from each phoresis

using

bibon-P

membranes

CA), (Millipore,

0. 1% Coomassie

Blue

apparatus

Sheep

polycbonab

antibody

(PBST).

The

tion ofthe then

membranes

primary

incubated

(Pierce) analyzed

with

gel CA)

and

per

Poly(A)

mRNA

Micro-

was run on

transferred

membranes

mg

Histopathologic The kidney

Assessment fractions were

to Du-

by capillary

PAS

embedding

from and

Specimens

were

point

and

renal

injury:

mation; each

chemicals The

were

membranes

lesion

each

three

involving

pH

for immunobbotagainst

mature

mature 0.01%

overnight

with

PBST.

Membranes

rabbit

Sciences,

OP-I, Tween this

anti-sheep

dilu-

were antibody

(HE)

and

six

dilation

and

was

necrosis;

as follows:

of the field;

3+, lesion involving >60% of the field. were

2

+ ,

also

evaluated The

were

(2)

interstitial

damage.

lesion

(three studied.

at each with

0, minimal

41 to 60% Sham-operated

present.

sections

fields

consistent

and (4) glomerular

lesions

acid-Sehiff

for histologic

for lesions

lesions

before

cut at 5 p.m

periodic

six paraffin

microscopically for

were

was included

section,

and ECL

IL).

in formabin

Sections

point

each

Arlington,

and fixed

we analyzed

graded

(1) tubular

kidneys

with

removed

From

examined

20%

(PBS) produced

incubated

Life

kidney,

HE).

subjectively

of these

718),

and processing.

(3) hemorrhage;

one

with

NaPO4,

at room temperature, washed in PBST, to x-ray film (Lightning Plus) using (Amersham

paraffin

with

using biotinylated in PBS containing

and washed

ehemilumineseenee

routine

stained

or blocked

in 50 mM

peroxidase-conjugated

for 30 mm by exposure

Instruto Immo-

and either

saline

(no.

were

antibody

Scientific

of loading

dissolved

OP-b domain and affinity-purified was diluted (1: 100 dilution, 5 p.g/ml) 20

MA),

uniformity

5% bovine serum albumin (Sigma) 7.4, 100 mM NaCI phosphate-buffered ting.

of protein

gel ebeetro-

by electrophoresis

Bedford,

to assess

to

was

by bicinehoninie

(Hoefer

transferred

heated

micrograms

by SDS-polyaerylamide

Small

Francisco.

Forty

dissected,

and

supernatant

determined

IL).

was separated

were

centrifuged,

was

Rockford,

medulla

( 1 : I dilution),

were

concentration

a Mighty

San

and

buffer

samples

(Pierce,

sample

ments,

cortex

in Laemmli

100#{176}C for S mm. acid

kidney

homogenized

MO).

Chemical

memwith

OP- I or GPDH

as arbitrary

(St. Louis,

Sigma

for either

densitometer

of OP-I/GPDH

ing renal pathology was source of the specimen.

from

scan

signal

contralateral

obtained

lane,

hybridized

dehydrogenase (GPDH) eDNA and exposed to film fi.r 6 h. The

expressed

all

DNA

otherwise

in each

water,

and

specified,

sperm

Unless

content

of the integrated

for 2 h at 45#{176}Cin a rotating

salmon

at

-80#{176}C using

for 48 h (Lightning

mRNA

signal

a laser

tured

oven.

at

After

SDS

screens

in deionized

3-phosphate CA), washed,

the field; involving

sonicated

OP-I

by boiling

blotting with lOX SSC. After cross-linking at 1200 milliJoules, membranes were prehybridized with 50% formamide, 10% dextran sulfate, 1% sodium dodecyb sulfate (SDS), 1 M NaC1, and 100 p.g/ml denahybridizing

of 32P-labeled

mouse eDNA). 0. 1 X SSC/0. 1 C/

Message

of the autoradiographie

quantified

analysis;

tissue

of Chomezynski

formaldehyde

ralon-ultraviobet

Blotting kidney

1457

NY).

and stained with hematoxylin/eosin (PAS). A total of four rats per time

by oligo(dT)

kit (Invitrogen,

agarose

A total of four rats was used

Northern

isolated

the method selected

FastTraek a 1%

and was

changes.

106 cpm/ml

5 X

intensifying

Rochester,

the relative

were

tissue

combi-

left

vascular

placed

After to

Injury

with a I 2: 1 2 lightidark bibitum throughout the

sodium. and

Renal

to autoradiography

of RNA

Hercules, weight

Laboratories

using

the animal

blood

Kodak,

To quantify branes

then

an average

River

a dorsolateral

During

subjected

film with

Eastman

Quantification

here

Preparation

rats with

pentobarbital

clamped. to maintain

Tissue

and

by

is mod-

We report downregulated

in an animal facility laboratory diet ad

Anesthesia

of ketamine

were

X-OMAT

Plus;

Ischemic

for 20 h at 45#{176}C with

they

intensity

Dawley from

Kodak

after

probe (680 bp Hindlll-Ba,nHb membranes for 30 mm with

human glyceraldehyde (Cbontech, Palo Alto,

Sprague

obtained

exposed

growth

65#{176}C(7),

Expression

we sought

expression

(AIRI).

of

Methods

were housed fed standard

experiment.

of these

is selectively

Animals female

of

models

observations,

injury

that OP-b mRNA expression the ischemie kidney.

Materials

of several

renal

have

in mechanisms

OP- 1 mRNA

isehemic

OP-b eDNA washing the

development,

nephrotoxic

of these

were hybridized

growth

factors

in kidney

to participate

injury.

whether

after

of many growth

a role

On the basis

to determine

intervention

polypeptide

to play

function, renal

OP-I

the

of

only

(20-22).

ulated

(1 8), and

number

not

1998

time

isehemie inflam-

The severity or no lesion;

involving

of I

+,

2 1 to 40%

of

of the field; and 4+, lesion kidneys and nonsurgical to confirm

pathologist

that was

no underly-

blinded

to the

1458

Journal

Statistical

American

Society

J Am Soc Nephrol

of Nephrology

ischemic

Analyses

Data and

of the

from each

expressed

animal

as the

significance

was

test and defined

(n

mean

established

as P

4 animals/time

=

±

SEM

by

ANOVA

point)

for

each

were

time

and

point.

Statistical

Bonferroni

the

post

total

resulted

hoc

from

kidney

is present

(4.0 kidney

transcripts

opposite

in a fivefold

ischemic

downregulation

0.05.


60%

and

necrosis,

per time point of

the

field;

of the field.

is shown.

2+,

lesion

Ise,

isehemie;

at

J Am Soc

Figure time

Nephrol

9: 1456-1463,

1. (A) Light point.

microscopy

(B) Light

(C) Light microscopy

24

h after

19-kD

viously also

1998

microscopy

(3).

reperfusion.

recognized

Higher

of a rat kidney of a rat kidney

of rat kidney

Antibody

band corresponding molecular

by antibody

OP-I

section

6 h postreperfusion

section

24 h postreperfusion.

7 d postreperfusion.

7 1 8 recognized

to mature weight 718

section

the

OP- 1 as described OP- 1 protein species

(approximately

There

17-

to

preare

50 to 41 kD

There There

are numerous

23

tubular

kD).

previously cies

(50

casts

are necrotic

is minimal

and

Expression

and evidence

and dilated

dilatation;

These

to 4 1 kD) that

and band

Renal

bands

with

have

early

or casts

are observed.

been

cells

as

secreted

forms

(23

kD,

kD

at this hyabine

ovary 19

1459

multiple

hamster 17 to

Injury

of hemorrhage

tubules

no inflammation

protein

in Chinese

OP- 1 . Note

after bschemic

is present

casts.

characterized prodomain I 9 to in

spe1 7 kD)

cortex

of and

1460

of the American

Journal

Society

of Nephrology

Soc Nephrol

J Am

#{149}NU

____

-

_.

..

,

1998

4.0kb

.

2.0

9: 1456-1463,

#{149}dI2.4kb1

;w=,

GPDH

1.5 -INonnal

.1.

Sham

x

0 0

0..

E:J

I

1.0

0

oe schenic

-

0.5

HOURS 2. (Top

Figure

Panel)

Representative

or isehemia/reperfusion

osteogenie protein1 (OP-b) and in total kidney for OP-I transcript

are expressed versus

as arbitrary

sham

and

medulla

of the

However, medulla.

the

These to l9-kD the

with

blots of total kidneys

normal

animals.

glyceraldehyde 4.0 kb. Values

optical

density

Each

3-phosphate are means

units for OP-b

opposite

kidney

and

in the

is significantly

kidney.

in the

represents

normalized

to GPDH.

*P

the

ischemie

reduced

in the

The

ischemie 5).

17- to

l9-kD

medulla

band

compared

adaptation

cortex.

one

animal

0.01,