Heparin-Binding EGF-Like Growth Factor mRNA Is Upregulated in the ...

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