Tifton,. Georgia; and. Creative. BioMolecules,. Hopkinton,. Massachusetts. Abstract. Osteogenie protein-. 1 (OP- 1 ) is a morphogenetie fac- tor highly expressed.
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,