and. *Dep(,rt,,zent of Medicine,. University of Melbourne,. Austin. & Repatriation .... ical Co., Castle. Hill,. NSW,. Australia) in 100. mM Tris-HCI. (pH. 8.0)/SO. mM.
J Am
Soc Nephrol
9: 1464-1473,
Heparin-Binding EGF-Like Growth Factor mRNA Is Upregulated in the Pen-Infarct Region of the Remnant Model: In Vitro Evidence Suggests a Regulatory Role Myofibroblast Transformation GEOFFREY MARINA
KIRKLAND,* KATERELOS,*
*Dep(irt,ne,zts and
of Clinical
*Dep(,rt,,zent
Heidleberg,
Abstract.
(HB-EGF) that
may
study
was
to determine
segmental
renal
hybridization creased
by tubular
At day
3, typical
aetin
the
EGF
mRNA
tocrine
were and,
infarcted
HB-EGF
Recently,
we
described
mitogenie
and
others
factor
HB-EGF
(EGF)
family
transmembrane
precursor
to yield
the
mature
hepanin-binding tamed
within
peptide,
an
which
The
importance transcript
to
factor-nb
duced
expression.
recently
synthesized
lies in (I)
acids
Society Soc
July
work
17.
was
of Nephrology,
Neplirol
Vincent’s 1046-6673/0908-
Copyright
in part
balance
differentiation
February at the
(7).
a
and
The
(5),
41
1996.
Annual
mature
domain
(5,8).
induction
and
published
U
American
1998
the
rethe
in-
1 . Because
TGF-/3
HB-
is usually
HB-EGF
fibrobbast
cells (3)
of cell
American
(J Am
Immunology,
St.
cells,
may
con-
proliferation
and
scarring.
and
chemotaxis
(13),
course
and
of
was
actions,
cell
been
in cutaneous
detected burn
victims
role
(15,16),
and
in this function
(4);
determine
whether
kidney
model,
we the has
wound
and
we
fluids
from
observation
mitogen
study of
cells mitogenesis
in the area surrounding area). Because HB-EGF
this
for this fibroblast
Therefore,
distribution
to
in the remnant
increased staining (the pen-infarct
stress smooth
(14).
designed
upregulated
shear
mesangiab
and
consid-
I! in rat
including
adhesion
studies
noted strikingly infarcted kidney also
including
1 in rat
multiple
cells,
by factors
( 1 1), angiotensin
endothelin-
for
muscle
pathogenesis,
cells
(I 2),
evidence
smooth
regulation
(2)
in disease
endothelial
HB-EGF
form
in abstract
Power,
Victoria
Department
Parade.
Fitzroy
of Clinical 3065.
Victoria,
in tissue
attempted
HB-EGF
in renal
pigs
suggested
an
scarring
and
to determine
the
infarction.
Society
by the American
of
Nephrology
Society
Australia.
Materials
and
Methods
Disease Model and Experimental Protocol In the initial model. the purpose of which was to induce
1464$03.t.X)/() the
of
and
during
(2-4,6,9,10);
important
healing. Meeting
HB-EGF
inhibited
that
myofibroblasts
endothelial
cells
In the
eon-
in several
including
muscle
pro-
6, 1998.
29th
into
transforming
a-SMA, to TGF-J3
between
of
Although
with
strongly
suggests
3,
levels
fibrobbasts.
mitogen
study
high
MRC-5
due
1 and of expres-
1996).
to Dr. David Hospital.
of
Accepted November
1858.
7:
Correspondence
Journal
1997.
presented
a-SMA
days
to myofibroblasts, an HB-EGF contributed
human line
also
fibroblast
to a local
in human
the
the rapid stimuli
of
at
expressing
increased
HB-EGF
tribute
ered as
predominantly
EGF-like
to a C-terminal
by cell
blots
significantly
intensity
apposed whether
(TGF-b)
potent
a
mesangial
epidermal
in
a-SMA
this
types,
epidenmal
region
mRNA of greater
cells
Northern
cells
the
86 amino
is
important Received
West
showed
area
tubular
antiproliferative,
human
This
Australia;
although only
HB-EGF
fibroblast
expression
EGF at
in experimental is
of the
creased
more
endothebial
of the
and
by various
growth
HB-
by an unknown
hydrophilic
numbers
seen
au-
of a smaller
of
also
be
area
to upregulation stimulation
capillaries
study
and
of 75-
of HB-EGF
Centre,
mRNA were directly was made to determine
pen-
to
(HB-EGF)
cationic
N-terminal
is linked
mRNA
is
and
is cleaved
protein
domain
Medical
infarction,
5. Because
at day
the
possible
heparin-binding
(5,6)
zone.
by infarcted tubules, staining intensity of
is a member
that
Victoria,
5 after
reflecting
HB-EGF attempt
in-
expressed
some
begun
factor
(1-4).
were
appeared
because
factor
growth
disease
growth
cells
have
Fitzroy,
the pen-infarct
expression
perhaps sion
situ
muscle
at
suggesting
tubules
growth
factor-like
kidney
hybridization
in individual
in markedly
infarcted
cells
of the infarct, surrounded HB-EGF mRNA. The
mRNA
growth
these
in
at day
from
increased
of this
a-smooth days,
of
tubules
aim
numbers
succeeding
than
was the
large
Endotheliab
to isehemia
‘s Hospital, & Repatriation
maximal
of tissue
mi-
function
1 postinfaretion,
bordering
Some
by in situ
the periphery also expressed
tease
over area.
The
expressing in
cell
possible
mRNA
cells
present
stimulation.
resistant
day
HB-EGF
epithelial
border
within
At
that
L. WU,t
Vincent
Austin
was
growth
epithelial
healing. and
myofibroblasts
(ct-SMA)
isehemic
in wound
infarction.
St.
of Melbourne,
factor-like
and
its distribution
showed
Nephrology,
University
growth
fibroblast
be important
and
Kidney in
Australia.
epidermab
is a potent
togen
Immunology
of Medicine, Victoria,
Heparin-binding
factor
KATHY PAIZIS,* LEONARD and DAVID A. POWER*
1998
of Nephrology
phy
and
hyperfiltration
injury
in the
remnant
kidney,
45
male
hypertroSprague
J Am Soc Nephrol
Dawley
9:
1464-1473.
rats weighing
studied.
In 24
kidney
was
branches
of
branch
(OP
achieved
by
the
renal
main
was
tobarbitone
group),
the upper
a sham
pole,
the
two
leaving
the
anesthesia
In the control
was
total
that
subcapsular
of
nepen-
I 7 rats
comprised
(SHAM
baparotomy
and manipulation of the renal pediebes. Four rats died intraoperatively, and one each from the SHAM and OP groups died postoperatively. Rats were sacrificed at days I , 3, and S and at weeks 1 , 2, 3, and and the kidneys were harvested. nephreetomy was performed
In an additional group of 10 rats, (i.e. , the contrabateral kidney was
removed). Kidneys were harvested noted for the remnant kidney model. In Situ
was
produced were
HB-EGF
by PCR
selected
protein.
amplification,
to include
This
495-bp
to positions 23 1 to 254 sequence of rat eDNA
as described
the
sequence
eDNA
published
sequence
fragment
eDNA
fragment
(sense
was
probe)
or
synthesized
using
pobymerase
(sense
triphosphate
of rat HB-EGF.
was
linearized
probe)
riboprobe,
transcript
with
incorporating
but
not
on a Northern
pyrocarbonate SSC
the
was
Co.,
blot
Hill,
and
with
rinsed
fixed
in
4%
probe.
(PBS).
paraformaldehyde
Sections
0.02%
in
5X
sodium
for 60 mm
were
prehybridized
SSC,
2%
dodecyl
then
isotype-matched
mAb.
Antibody
layer
comprising
technique, CA)
an-
2.5-kb
sections
Digestion
K (Sigma
mM
of
Chem-
Tris-HCI
with sheep serum mm and incubated
anti-a-smooth muscle aetin (a-SMA) from Sigma (I 8). Negative control binding
rabbit
followed
was
detected
anti-mouse
by
mouse
monowas an
using
Ig diluted
peroxidase
a triple-
antiperoxidase
(Dako) diluted 1:100, each for 30 mm, with washing steps ineubations. Sections were developed using nickel-enhanced nobenzidine ylin.
(Pierce,
Rockford,
IL) and
of Tubular
To determine
counterstained
Staining
Using
of tubular
staining
the location
(Dako,
1 :50
between diami-
with
hematox-
Tubular with
anti-HB-EGF
antibodies, tubular markers were used, as described by Nadasdy ( 19). Sequential sections were stained with fluoreseein-babeled (Sigma)
to
(Arachis
hvpogaea)
identify
distal
convoluted
tubules
and proximal
erythroagglutinin).
Leetin
oxidase-conjugated heim), followed
staining
tubules
(Phaseolus
was
identified
using
anti-FITC Fab by diaminobenzidine
to identify
loop of Henle peroxidase
cortical
(TAL).
Terminal
deoxynucleotidyl
staining
published
method
treated
tissue
with
labeled method,
Sections
K and,
Binding the ABC were
ascending detected
of Dr.
was
limbs of the using a rabbit
nick
cells and
after
end
labeling
was performed
neutral-buffered
was dewaxed
proteinase
dUTP. using
thick was
gift
Australia),
(Dako).
apoptotie
Briefly,
immunoper-
a generous
Victoria,
transferase-mediated
to detect (20).
affin-embedded
antibody,
Clayton,
binding
assay
duets vulgaris
fragments (Boehringer Mannand hematoxylin as described
medullary
Antibody
antiperoxidase
(TUNEL)
CA).
and
collecting
renal
earlier. Rabbit anti-Tamm-Horsfall H. Y. Lan (Monash Medical Centre, used
and
et a!.
leetins
using
formalin-fixed,
rehydrated.
washing,
Sections
incubated
a
par-
with
were biotin-
was detected by the avidin-biotin-complex Elite kit (Vector Laboratories, Burbingame,
eounterstained
in Harris
hematoxylin.
(pH Northern
immersion Slides
rinsed
Grand Island, NY), using a pro200 homogenizer (Proscientifie, Inc., Monroe, CT), according to the manufacturer’s instructions. Thereafter, I/S vol of chloroform was added to the sample, incubated for S mm at room temperature, then centrifuged at I 2,000 X g, 4#{176}C for IS
block
sulfate
1 A4
blocked for 30
(EDTA) at 37#{176}C for 30 in 2 mg/mI glycine in
for
at 37#{176}C and
a
water.
100
acid were
mm,
15
with 100 mM triethanolamine, to block positively charged
formamide,
The
Paraffin
proteinase
Australia)
phosphate-buffered
saline
in DEPC
20 p.g/ml
NSW,
uridine-
detected
cell RNA.
were RNA
were dewaxed in xybene and dehy0.2 M HCI for 20 mm, rinsed with water twice for S mm, then placed in
8.0)/SO mM ethylenediaminetetra-acetic mm. Digestion was stopped by
equilibrated was added
SacII
enzyme
Germany).
riboprobe,
of mesangial
10 mm
undertaken
Castle
into the
the HB-EGF
restriction
Mannheim,
sense
(DEPC)
at 70#{176}Cfor
sections
subcboned
digoxigenin-labeled
Mannheim,
were cut 4-p.m-thick. Sections drated in alcohol, treated with diethyl
mature
(Promega, Madison, insert was sequenced to be identical to the containing
the
(2).
the
SpeI (antisense probe). Labeled riboprobes T7 RNA polymerase (antisense probe) or SP6
(Boehringer
tisense
pGEM-T
previously
encoding
multiple cloning site of the plasmid pGEM-T WI) between the SP6 and T7 promoters. The over its full length on both strands and found
ical
2/6
Hybridization
Primers
overnight at 4#{176}C with ebonal antibody (mAb)
Localization Markers
eDNA probe corresponding and 707 to 726, respectively, in the published
2X
4,
1465
and dried overnight at 37#{176}C. Sections were dewaxed, and peroxidase activity was inhibited using 3% hydrogen
Carpinteria,
not at some of the same time points as
A rat HB-EGF
(17),
mierotome endogenous
Transformation
peroxide for 10 mm. Sections were then (Life Technologies, Gaithersburg, MD)
posterior
intrapenitoneal
group
performed
left
extrarenal
proximal
with
were
of the
anterior
and a right
under
operation
of two-thirds
of
artery
(0. 1 mb/I 00 g).
group),
infarction
ligation
performed
and Myotibroblast
HB-EGF
150 to 200 g at the start of the experiment
rats
to perfuse
phreetomy
1998
in PBS
rinsed
with
twice
and
PBS,
and
pH 8.0. Acetic anhydride groups that might bind the
in hybridization reagent
[SDS],
buffer
[Boehringer
and
hybridized
0.1%
(50%
Mannheim],
with
mm.
The
equal mm,
N-bauroylsareosine)
overnight
Kidneys
denatured
was
Blot
Analysis
were
homogenized
upper
volume and
phase
of isopropanol,
then
washed
aqueous
in Trizob
centrifuged
twice
with
at
75%
was
reagent
removed
(Life
and
Technologies,
precipitated
with
an
incubated
at room temperature for 5 X g, 4#{176}C for 10 mm. The pellet and centrifuged at 8000 X g, 4#{176}C
12,000
ethanol
digoxigenin-babebed
sense and antisense riboprobe at a concentration for 5 mm. The pellet was then air-dried and dissolved in DEPC-treated of ISO ng/mb in a humidified chamber at 42#{176}C. After treatment of the water, and the RNA was used for Northern analysis. RNA samples slides with DNase-free RNase A (5 p.g/mb) for 30 mm at 37#{176}C, were ebeetrophoresed on I % agarose gels containing 7.2% formaldecoborimetric detection of mRNA after hybridization was aecomhyde and transferred to nylon membranes (GeneSereen Plus, Dupont). plished with the nonradioaetive nucleic acid detection kit (Boehringer RNA was fixed using a Stratalinker (Stratagene, La Jolla, CA), and Mannheim) according to the manufacturer’s instructions. Sections the membranes were prehybridized in hybridization solution (5 X
were each
counterstained of three
rats
with was
Immunohistochemical Muscle Tissue
embedded
Nuclear
stained
Fast
and
Red.
examined
Staining
Tissue at each
to Detect
obtained time
from
point.
ct-Smooth
Actin blocks
were
in paraffin.
immersion-fixed
Paraffin
sections
in 4%
paraformaldehyde
4 p.m thick
were
and
cut on a
saline-sodium
phosphate-EDTA,
5X
Denhardt’s,
added and incubated overnight. Membranes were then washed with 2X SSC/O.l% SDS, I X SSC/0.l% SDS, SSC/O. 1% SDS for 15 mm at 42#{176}C. Filters were then
sequentially and 0. 1 X exposed
to x-ray
film
at -70#{176}C. Membranes
DNA)
50%
insert
denatured
sperm
SDS,
and 100 p.g/mb sheared which 2 X 106 counts/mI
was
herring
0.5%
formamide, 42#{176}C, after
32P-Iabeled
were
then
for 4 h at HB-EGF
hybridized
1466
Figure
Journal
1. (a through
(a) Normal is also
some
the
of
American
h) In situ
kidney
with
staining
Society
Nephrology
of
hybridization
using
almost
no staining.
in necrotic
tubules
J Am
antisense
(b) Infarcted (thin
arrow)
heparin-binding
kidney and
epidermal
at 24 h, showing
gbomerubi
(thick
growth
prominent
arrow).
factor-like
staining
(c) Higher
Nephrol
Soc
growth
factor
(HB-EGF)
in the pericapsular
power
magnification
9: 1464-1473,
area,
of the perinecrotic
necrotic
area
is marked
together
with
intense
staining
3 postinfaretion, tubules
within
there the
is a generalized
infarcted
area
increase (thick
arrow).
in tubular Between
staining necrotic
abutting tubules,
the some
infarcted positive
area cells
(*),
are
seen
area
(thin
riboprobe.
to the left.
day 1 , showing upregulation of HB-EGF mRNA by most of the viable tubules. The middle of the necrotic from the papibla at day 1 , showing expression by urothelium (thin arrow) and tubules (thick arrow). The
is marked
arrow),
(d)
(*).
which
may
1998
Section (*).
(e)
of a few be
There area
at
taken At day
viable
endothelial
J Am
Soc Nephrol
with
GAPDH
9: 1464-1473,
as above.
densitometry
Quantification
(Molecular
of signals
Dynamics
300A)
using
ImageQuant
sample
were
expressed
was
Computing
Software,
version
as the ratio
performed
by
Densitometer,
3.0.
Values
of HB-EGF
HB-EGF
model
signal
for
Vitro
Stimulation
of Fibroblasts
and
each
branches
to GAPDH
Detection
titled
of
experiments
Gabbiani
and
were
coworkers
based
on studies
(21).
The
fetal
reported lung
previously
fibroblast
from American Type Culture Collection between passages S and 7 after thawing.
were plated
tissue
RPMI 24
in 6-well
containing
h later
and
15% the
culture
fetal
calf
following
plates
recombinant
The
medium
was
human
growth
factors
line
phenybmethybsulfonyl
fluoride,
and
Initially,
the marked
occurred
urothelium
bd)
and clumping
of
of HB-EGF
mRNA
1 , b through
(Figure
tubular
and
pole iden-
hybrid-
cells
in the papibla
(Figure
positive
at day
necrotic
increasing
3, and
tubules
bordering
(Figure
overlying
the
Id),
as well
capsular
fibro-
were
expression
probably
5, the peri-infarct
cells
intense (Figure and venubes
was
were (Figure
of spindle-shaped which was not
These
there
that
capillaries
at day
large areas (Figure 2b), 2a).
in most
were
of the in situ
expression
those
bower
cells
Ib).
of the peritubular
apro-
feature
3, tubular expression was more cells of some small arteries
bordering
1467
and
tubular
or loss of nuclei
striking
including
(Figure
strongly
(R&D
nuclei most
of the upper
necrotic
area from day 1 onward
this
area,
At day Endothelial
changed
10 p.g/ml
was
studies
blasts
Minneapolis, MN) were used: 10 ng/mb HB-EGF, 5 ng/mb TGF-, and 10 ng/ml platelet-derived growth faetor-BB (PDGF-BB). All experiments were performed six times and each was analyzed separately. After S d, the cells were washed twice with normal saline and lysed with 100 p.1 of lysis buffer per well (SO mM Tris-HCI, pH 7.5, 150 mM NaC1, S mM EDTA, 0.5% Triton X-b0O, 10 p.g/ml 1.5 mM
ization
ligation
artery,
pyknotic
Transformation
Infarction
after
renal The
as the
(RockCells
Systems,
beupeptin,
by their
necrotic
at 2 X iO cells per well in
serum.
of the
the cytoplasm.
d).
by
cell
MRC-S was obtained ville, MD) and used
in Renal hours
in the peri-infarct
ct-SMA
These
mRNA
Twenty-four
signal.
In
and Myofibroblast
HB-EGF
1998
area
by cells
endothebiab
be).
At
was
this
cells
time,
found
to distinguish
and
to contain
cells that expressed present in normal kidney
difficult
be). were
from
a-SMA (Figure
endothebiab
tinin) for 30 mm at 4#{176}C. Lysed cells were then removed using a cell scraper and centrifuged at 15,000 rpm at 4#{176}C for S mm to remove
cells except by staining with a-SMA antibody. Electron microscopy of sections taken at day 5, together with their spindle
nuclei. Protein tein Standard
that
concentrations Assay
were
(BioRad
lysates
the BioRad
Minigeb
reducing
Aus-
The
Bio-Rad
with
buffer,
apparatus.
shape
Pro-
NSW,
at -70#{176}C.For Western blots, 20 p.g of protein was run on a SDS-pobyacrylamide gel eleetrophoresis gel and transferred to the
mixed
Park,
stored I 2.5%
using
aliquoted,
using Regents
trabia).
nitrocellubose
were
measured
Laboratories,
and
Blots
were
with 5% skim milk powder in Trts-buffered saline, then for 60 mm with a monocbonal anti-a-SMA antibody (I 8), by Sigma, and diluted 1 :4000. After washing with 0.5% 20 in Iris-buffered saline, the blots were probed with horseperoxidase-conjugated rabbit anti-mouse antibody (Dako) di1 :2000 for 30 mm. Antibody binding was detected using the Super-Signal System (Pierce). For Coomassie blue-stained 10 i.g of lysate was added per lane. Molecular sizes were
Tween radish luted Pierce gels,
calculated
sham
using
PLC,
High
Bucks,
densitometry
using
and ImageQuant Statistical
analyses
version
test
Molecular
Rainbow
Signals
Dynamics
Markers
were
Computing
as described
were
2.01,
(for
from
performed
using ANOVA, test (for equal unequal
software
pack-
comparisons
Results
cells.
undetectable
(0
expression S within
necrotic
Maximal
by in situ
staining
by isolated a predominantly
tubules
in Normal HB-EGF
HB-EGF
mRNA cells
hybridization
intensity
tubular
of tubules
elements
necrotic
area.
(*) at day 5. Magnification:
(thin (h)
(Figure
bordering arrow),
cells
Higher
power
X25
were
area
surrounding
necrotic f; X50
cells
expressing
a-SMA
apposed (Figure were
stage,
too,
necrotic
area
(Figure
mainly
in visceral 2, g and
(presumed
areas
that
endothelial
and
were
2, c and d). myofibroblasts cells
It in
e and f) as well
2,
cells were strongly or within the infarcted
(Figure
very
to tubules that 2, e and d). Some
in
At this
by
mRNA
for HB-EGF mRNA (Figure however, to identify individual the
HB-EGF
surrounded
HB-EGF
directly mRNA
myofibrobbasts
for
were
positive (Figure area expressed
parietal
gbomerular
h).
of infarction poles of the
the necrotic
in a, b, e, and
strongly
tubules
levels seen in normal was a distortion of and
(*)
some
kidneys. The only tissue architecture continued
3).
One-way between
at day 5. tubules
showing
in d; X75
(g)
(thick
ANOVA treatment
Higher
expression in e and
by g;
and
showed (P
X200
in these
cells in h.
of
a highly