Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor beta expression in rat glomerular mesangial cells

Angiotensin II stimulates extracellular matrix

protein synthesis through induction of

transforming growth factor-beta expression in

rat glomerular mesangial cells.

S Kagami, … , D E Miller, N A Noble

J Clin Invest.

1994;

93(6)

:2431-2437.

https://doi.org/10.1172/JCI117251

.

Angiotensin II (Ang II) has been implicated in the development of progressive

glomerulosclerosis, but the precise mechanism of this effect remains unclear. In an

experimental model, we have shown previously that TGF-beta plays a key role in

glomerulosclerosis by stimulating extracellular matrix protein synthesis, increasing matrix

protein receptors, and altering protease/protease-inhibitor balance, thereby inhibiting matrix

degradation. We hypothesized that Ang II contributes to glomerulosclerosis through

induction of TGF-beta. Ang II treatment of rat mesangial cells in culture increased TGF-beta

and matrix components biglycan, fibronectin, and collagen type I at both the mRNA and

protein levels in a time- and dose-dependent manner. Saralasin, a competitive inhibitor of

Ang II, prevented the stimulation. Ang II also promoted conversion of latent TGF-beta to the

biologically active form. Coincubation of mesangial cells with Ang II and neutralizing

antibody to TGF-beta blocked the Ang II-induced increases in matrix protein expression.

Continuous in vivo administration of Ang II to normal rats for 7 d resulted in 70% increases

in glomerular mRNA for both TGF-beta and collagen type I. These results indicate that Ang

II induces mesangial cell synthesis of matrix proteins and show that these effects are

mediated by Ang II induction of TGF-beta expression. This mechanism may well contribute

to glomerulosclerosis in vivo.

Research Article

Find the latest version:

Angiotensin 11 Stimulates Extracellular

Matrix Protein

Synthesis through

Induction of Transforming Growth

_Factor-#

Expression in Rat

Glomerular

Mesangial Cells

Shoji Kagami, WayneA.Border,Diane E.Miller,andNancyA. Noble

DivisionofNephrology, Department ofMedicine, University ofUtah SchoolofMedicine,Salt LakeCity, Utah84132

Abstract

AngiotensinII(Ang II) has been implicatedinthedevelopment ofprogressive

glomerulosclerosis,

but the

precise

mechanism of this effect remains unclear. In an

experimental model,

we

have shown previously that

TGF-,6

plays a key role in glo-merulosclerosis by

stimulating

extracellular matrix

protein

synthesis, increasing matrix

protein

receptors, and altering

protease

/protease-inhibitor

balance,thereby

inhibiting

matrix

degradation.

We

hypothesized

thatAngIIcontributesto glo-merulosclerosis through induction of

TGF-fl.

AngII treatment

ofrat

mesangial

cells inculture increased

TGF-ft

and matrix

componentsbiglycan,

fibronectin,

and

collagen

type Iatboth the mRNA and

protein

levels ina time- and

dose-dependent

manner.Saralasin,a

competitive

inhibitor of Ang II,

prevented

the stimulation. Ang II also

promoted

conversion of latent

TGF-,8

tothebiologically active form. Coincubation of

mesan-gial cells with Ang II and

neutralizing antibody

to

TGF-,6

blockedtheAng II-induced increases in matrix

protein

expres-sion. Continuous in vivoadministration of AngIItonormalrats

for7 dresultedin70% increasesinglomerularmRNAforboth

TGF-ft

and collagentypeI.These resultsindicate that AngII

inducesmesangial cell synthesis of matrix proteins and show

that theseeffectsaremediated by AngIIinduction of

TGF-,6

expression. This mechanismmaywell contributeto

glomerulo-sclerosis in vivo.

(J.

Clin. Invest. 1994.

93:2431-2437.) Key

words:fibrosis*collagen*

glomerulosclerosis

*

kidney*

proteo-glycan

Introduction

Marked accumulation of mesangial matrix is a histological

hallmark of progressive glomerular diseasesthat leads to

glo-merulosclerosisandend-stage kidney disease( 1,2).Our

stud-ies inanexperimental model ofglomerulonephritis inducedby

injection

with antithymocyte serumin rats have shownthat

overproduction

ofglomerular TGF-,Bcanunderlie the

develop-ment of glomerulosclerosis (3-6). This work has been

con-firmed by recent studies of Isakaet al. (7) where

liposome-bound

TGF-f31

cDNA wasintroduced intoratkidneysby

in-jection

into the renal artery. Marked glomerular

TGF-flI

Addresscorrespondence to Nancy A. Noble, Ph.D., Division of Ne-phrology, University of Utah School of Medicine, Salt Lake City, UT 84132.

Receivedforpublication 29November 1993 and in revisedform 31

January1994.

expression

and

matrix

accumulationwereseen 5dafter injec-tion.

Several lines of evidence suggestthat the intrarenal renin-angiotensinsystem

(RAS)'

playsanimportant rolenotonly in theregulation of glomerular hemodynamics but also in glo-merular growth and

sclerosis (for

reviewsseereferences

8,

9). Angiotensin

converting

enzyme inhibitors and

angiotensin

II

(AngII)receptor

antagonists

attenuated

progressive

glomerulo-sclerosis and interstitial fibrosis in several

disease

models and slowed disease

progression

inseveral human studies( 10-18 ). Elegant studies

showing

that

glomerulosclerosis

candevelop andprogressin the absence of

glomerular hypertension,

and that

angiotensin converting

enzyme inhibition can attenuate

the

progression

of sclerosis without

affecting

the level of glo-merularpressure, raise the

possibility

thatAng II may act

di-rectlyonglomerular cellstoinduce

sclerosis, independent

ofits

hemodynamic

action ( 13, 19). While it has been shown clearly that Ang II induces increased

protein synthesis

without cell

proliferation

inanumberof

kidney

celltypes(9, 20), and Ang

IIhasbeenreportedtostimulatemesangial cell collagen synthe-sis (21, 22),

convincing

dataonwhetherAng IIinduces

extra-cellularmatrix

protein synthesis

byglomerular cellsarescarce

and none exist on the mechanism of such an effect. It was

reported recently that the

addition of

Ang II tocultured

vascu-lar smooth muscle cells stimulates secretion of

TGF-13

(23, 24).

Since

TGF-f

is clearly

apotent

stimulator

of

glomerular

extra-cellular

matrix

production

and

deposition (3,

7,

25-29),

and

since

mesangial

cells are

similar

to vascularsmooth muscle cells in

embryonic origin, histology,

and

functional

properties

(30),

we

hypothesized

thatAng II

induces

TGF-,B

production

andsecretion by

mesangial

cells and that this

TGF-j

inturn

stimulates extracellular matrix production and deposition. Studieswereinitiated using culturedratmesangialcells where

the

confounding effects of

Ang II onglomerularpressurewere not afactor.

Methods

Cell culture.Ratmesangialcellswereobtained from intact glomeruli of 4-6-wk-oldSprague-Dawleyratsaccordingtopublished methods and werecharacterized as described previously (3, 26). Mesangial cells were used between passages 5 and 9 and were maintained in RPMI 1640medium (SigmaChemical Co., St. Louis, MO) supplemented with 18% FBS (Hyclone Laboratories, Logan, UT),100U/ml penicil-lin, 100 ug/ml streptomycin, 0.1 U/ml insupenicil-lin, and 25 mM Hepes bufferat37°C ina5%CO2incubator.

Cellproliferation andproteinsynthesis assays. Confluent mesan-gial cells in 24-well culture plates (Costar Corp., Cambridge, MA) were

1.Abbreviations usedinthispaper:AngII,angiotensin II;GAPDH,

glyceraldehyde-3-phosphate dehydrogenase;MV1 Lu,minklung

epi-thelialcell; RAS, renin-angiotensinsystem.

J.Clin.Invest.

©TheAmerican Society for Clinical Investigation,Inc.

madequiescent by placing them in serum-free medium for 2 d. They were thenstimulated for 24-48 h with10-6_10-12 M Ang II (Sigma

ChemicalCo.). Anderson et al. (20) have shown recently that Ang II is verystablein mesangial cell cultures under serum-free conditions. The

stimulatedcellswerepulsedwith 1MCi/ml [3H]thymidine(Dupont/

NewEngland Nuclear, Boston, MA) during the last 12 h of culture. Cellswerewashed oncewithPBS,twice with ice-cold 5% TCA to re-movetheunincorporated

[3H]thymidine,

and were solubilized in 0.25 NNaOHin0. 1% SDS and neutralized. Aliquots of samples were added

toscintillation fluidand counted(Beckman Instruments, Inc., Fuller-ton,CA). Protein synthesis wasmeasured by incubating cells with

[35S]methionine(100

ACi/ml)

(Dupont/New England Nuclear) for thelast 12 h.Afterlabeling, media were removed, and the cells were washed oncewith PBS, twice with ice-cold 5% TCA, and finally dis-solved in 0.25 N NaOH in0.1%SDS and neutralized. Radioactivity in

10-Mlaliquots was counted.

[

3jS]

Methionineand [35S]sulfate labeling ofcells and

immunopre-cipitation.Confluent mesangialcells were madequiescentasdescribed above.After36 hof incubationwith or without 10-8M Ang II and

10-'

M saralasin (Sigma Chemical Co.), 100 MCi/ml of

[35S]_

methionine or [35S]sulfate (Dupont/New England Nuclear) was

added for another12 h. For bothimmunoprecipitationand

proteogly-can analysis, themedium wascollectedand centrifuged to remove cellulardebris.Immunoprecipitation experimentswereperformed as follows:aliquots (100

_Al)

ofthemediawerediluted with 900

,l

of RIPA

buffer (25 mM Tris-HCI, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5%

Triton X-100,0.5% deoxycholate). Supernatants were treated

sequen-tially withnormalrabbitserum(10Il)to removenonspecific binding, rabbitantibodies (10Al)to ratfibronectin (Telios Pharmaceuticals, Inc., SanDiego, CA),andratcollagentype I(Chemicon International,

Inc.,Temecula,CA) for16 hat4VC. Aftereachantibody incubation,

immune complexeswere removedbytheaddition of protein A-Se-pharose beads(Pharmacia LKB Biotechnology, Inc., Uppsala,

Swe-den).Beadswerewashedfour timeswith RIPAbuffer,and the immun-oprecipitateswereanalyzed byelectrophoresison4-12%SDS-PAGE gelsasdescribedpreviously(3).

Quantitation oflatent andactive TGF-f3 produced by mesangial

cells inthe presenceofAngII. Confluent, quiescent mesangial cells werecultured inserum-free medium containing10-8 M Ang IIfor48 h.Culturesupernatantswerecollected and dialyzed againsta-MEM

(Gibco Laboratory,GrandIsland, NY).Thequantityof

TGF-f#

pres-entin the supernatantswasassayedusingthe minklungepithelialcell

(MVl Lu;American TypeCultureCollection, Rockville,MD)assay, where human recombinantTGF-31 wasusedtogenerateastandard

curve(R&D Systems, Inc.,Minneapolis,MN).Todeterminewhether

mesangialcellsare producinglatent

_TGF-#,

latentTGF-fl was

con-verted to theactiveformbytreatingculture supernatants with 10 N

HCl

(final pH= _{1.5-2.0) for}30 minat roomtemperature,followed by neutralization withequimolar NaOH(29, 31).

MVl Lu cellswerecultured in a-MEMsupplemented with 10% FBS.Bioassayswereperformedasdescribedpreviously(24).Briefly,

MVILucellswereplatedinto 24-well cultureplatesin a-MEM

supple-mentedwith10%FBS. Aftera6-hincubation,mediumwasdiscarded,

andcellswerewashedoncewithserum-freea-MEMand then incu-bated withTGF-Bl orculture supernatant in serum-free medium for 24 h. Thecellswerepulsedwith

[3H]thymidine

(2

MCi/ml)

for the last 6h, andincorporation wasdetermined asdescribed above. Culture supernatantswereserially dilutedtofall within the range of the

stan-dardcurve.Specificityof the observed effectswasprovenby

competi-tion of the

_TGF-(#

activitywitha_purifiedrabbitanti-TGF-,B IgGanda

controlIgG (R&D Systems,Inc.).

RNAanalysis.TotalRNAwasprepared bylysisofmesangialcells inguanidine isothiocyanatesolution followedbyultracentrifugationof thelysatethroughacesium chloridegradientand ethanol

precipitation

(32).RNAconcentrationsweredeterminedusingspectrophotometric

readingsatAbsorbance260.For Northernanalysis,RNAwasdenatured andfractionated by

electrophoresis

through a _{1.2% agarose}

gel

(20

jg/lane)and transferred to a nylon membrane (ICN Biomedicals,

Inc., Costa Mesa, CA).Nucleic acids were immobilized by ultraviolet irradiation (Stratagene, La Jolla, CA). Membranes wereprehybridized with 50%formamide,10% Denhardtssolution,0.1% SDS, 5X SSC, and 200 ug/ml denatured salmon sperm DNA andhybridized with DNA probes labeled with 32P-dCTP by random oligonucleotide priming (Boehringer Mannheim Corp., Indianapolis, IN). The blots were washed in 2x SSC,0.1%SDS at room temperature for 15minand in

0.1X SSC, 0.1% SDS at 550C for 30 min. Inaddition toethidium bromidestaining, autoradiographic signals obtained withthe rat gly-ceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe served as controlsfor equal loading ofthegel.Autoradiographswere scanned on anlaserdensitometer(Ultrascan XL;PharmaciaLKB

Bio-technology, Inc.). Changes in mRNA levels weredetermined byfirst correctingfor thedensitometricintensity ofGAPDHforeachsample. Forcomparison,this ratio was set at unity for normal control samples, orsamplesatzerotime and other lanes on the same gel were expressed asfold increasesoverthisvalue.

cDNAprobes.Amouse

_TGF-#31

cDNA probe (plasmidMUI1 ) was provided kindly by R. Derynck (33). Plasmid P16, which contains a humanbiglycan insert,was a kind giftof L. W. Fisher (34). A rat procollagen

_a,

cDNA(plasmidpalRl) was a gift from D.Rowe (35), and a rat GAPDH cDNA probe was agift fromM. B.Sporn (36). A

24-bp oligonucleotide probe was prepared from a ratfibronectin se-quence asfollows.Total RNAfromratglomeruli was reversely

tran-scribed, and thesubsequent cDNA wasamplified using

fibronectin-specific primers.Thespecificity ofthefibronectin cDNA was verified

bySouthern blotanalysis.

In vivo administration ofAng IL Miniosmotic pumps (model 2ML4; Alza Corp., Palo Alto, CA) were implantedsubcutaneously into eight normal Sprague-Dawley rats. Four pumps contained Ang II dissolved in0.01Nacetic acid for in vivo delivery of100ng/min. Four pumpscontained 0.01Naceticacid alone. This dose ofAng II has been shown toproducesubstantialglomerulosclerosisby 12 wk oftreatment

(37).After Iwk,RNAwasobtainedasdescribed above from glomeruli

isolatedbypreviously publishedmethods (3).

Statisticalanalysis.Results arepresentedasmean±SD.Statistical significancewasdeterminedusing Studentt tests.Triplicatewells were

analyzed foreachexperiment, and each experimentwasperformed independentlyaminimum of threetimes.

Results

Effect

ofAngII

on mRNA

levelsfor TGF-3 and

matrix

compo-nents.Incubation of

quiescent mesangial

cellswith 10-6MAng

IIand subsequent Northern blot analysis revealed increased

mRNA levels for

TGF-,3

and extracellular matrix

proteins

(Fig. 1). Expression ofTGF-,3 wasweaklydetected in

quies-centmesangialcells(time 0). However,4 hafterAngII

treat-ment,TGF-,3mRNA waselevatedsignificantly bymorethan threefold(P<0.01).This increasepeakedat8hwithasixfold

increase,

and at 48 h

TGF-,B

message was stillthreefold

ele-vatedcompared with control

(P

<0.01

). Message

for the three extracellularmatrixproteins measured,whichare knowntobe

inducedby

TGF-fl,

allshowed

progressive

increases with time

fromthefirst timepointof 4 h until 48h when theexperiment

wasterminated.

Biglycan,

fibronectin,

and

collagen

type I

tran-scripts

wereincreased

significantly by

8 h

(P

<

_0.05)

and

re-mainedelevated,

showing 4.9-, 4.3-,

and2.8-fold

increases,

re-spectively,

at48 h

(Fig.

1; P<

0.05).

When the response of

these

transcripts

toAngIIdosewas

investigated

after 24 h of

incubation,

itwas seenthat each

responded

ina

dose-depen-dentmannerbetween 10-12 and 10-6 M

(Fig. 2).

At 10-6M

AngII, all

transcripts

wereelevated

significantly

overcontrol values

_(P

<0.01

).

Theresults oftreatmentof cellswith

kb

B

B y -2.6

-8.0

D

-7.2

E

* -1.3

0 4 8 24 48

Hours

Figure 1. Northern blot

ofmRNAfrom

quies-centmesangialcells treated with 10-6M Ang II forincreasing

times. A,

TGF-0;

B, biglycan; C, fibronectin;

D,collagen typeI;and E, GAPDH. Resultsare

representative of

tripli-cateexperiments. Mo-lecularsizemarkersare

shownontheright.

binding, are showninFig. 3. While

l0-'

M saralasin had no

significanteffecton mRNA levels in the absence ofAngII,it

dramaticallydecreased theAng II-inducedincreases.With the

E

kb

O*

**qpqp -2.5

II.

-2.6

C

-8.0

D

Figure 3. Northern blot

-7.2

of_Angeffect of saralasin_{II-inducedin-}on

creasesinmesangialcell mRNA. A,

TGF-f3;

B, biglycan; C, fibronectin;

D,collagentypeI; and E, GAPDH. Resultsare

representative of

tripli--1.3

_-1~ cate_lecularexperiments._sizemarkersMo-_are

shownontheright.

Sh-i

-8.0

Figure 2. Northern blot of dose dependency of

__a mRNA levels in

re-_ W _ -7.2 sponse toincubationof

quiescentmesangial cellstoAngII.A, TGF-,B;B,biglycan; C, fibro-nectin; D, collagen type I; and E, GAPDH. Re-sultsare

representative

of triplicate

experi-ments. Molecular size

C

1f71210.1010.8

i7

10i markers are shown on

Ang II (M) theright.

exception ofbiglycan mRNA, which remained significantly elevatedovercontrols,mRNAlevelsinthe presence of sarala-sinwere not

statistically

elevatedovercontrols. These results indicate that the observedAngTI effectsarereceptormediated

(Fig.

3).

Effect ofAng

II

on

TGF-f activity ofmesangial

cell cultures.

Toestablish that the rise inTGF-f mRNA inmesangialcell

culturewas

accompanied

by thesynthesis of active or latent

forms of

TGF-f

protein,

TGF-j3

activitywasmeasured by

us-ing

thestandardMV 1Lucell bioassay,whereTGF-f secreted

by mesangial cells into culture medium is measuredbyits abil-ity to inhibitcell growth. Fig. 4 shows that the mean active

TGF-f

presentin sixcontrol mesangial cell cultures was 0.24

ng/ml(Fig.4, openbar).Whenstimulated with10 -8 M Ang

IT,

a 12-foldincreaseto2.9ng/ml was seen(Fig.4, P<0.001). This increasedactivitywas completely abrogated by neutraliz-ing anti-TGF-4 antibody (Fig.4,black bar),but not by

non-immunerabbit IgG(data not shown). Acid treatmentof

con-ditioned medium resulted in significant increases in active

TGF-f

in both control and Ang II-treated cultures (P < 0.05). However, acidtreatment ofcontrol conditioned medium

re-A

O

to 01

_0

Co_{_}

0

_

0c

-CL..L. -0 X _cD

o 0 C

ocon

C

C ._a

co

7n0₀L..

CO

kb

-2.5

A

ll.

to

4MA:

B

A

B

C

D

E

-.vw.s. _{IM IM} fm

4&#&

2.6

AMwLAwMAftmm&

IRMW

40

-2.5

.."

F. _4"i

..- 0

_A;;: 1.

Control

£

so

4 0

co

0

-00..

7- 2

c.-000 C

0 0 c

0

CO <

TGF-antibody

Figure 4. Effect of10-8M AngII onTGF-#bioactivitybefore and

afteracidification ofculturemedium as measuredbythe standard

MVl

Lubioassay.Open barsrepresent mean±SDof

TGF-ft

in cul-turesupernatantfrom six mesangial cell cultures stimulated withand without10-8M AngII.Hatched bars represent mean±SD of

_TGF-#

activities ofthe same culture supernatantsaftertransient acidification

toactivatelatent

_TGF-fl.

The black bar represents the mean±SDof

TGF-#

activitiesin the presenceofboth Ang II andneutralizing

anti-body to

TGF-fl.

sulted in 2.1 ng/ml

active

TGF-f3,

while active TGF-f3

inAng

II-treated

cultured media increased to 6.35 ng/ml (Fig. 4,

hatched bars).

Thus,an averageof only11%

of

the total

TGF-3

in control cultureswas

active,

while 45%of the total

TGF-f3

in

AngII-treated cultureswasactive(P <0.05).Thisresult

indi-catesthatAng II

increased

notonly the

production

of both latent and

active

TGF-3

but also stimulated the conversion of latentto

active

_TGF-#

(Fig.

4).

Effect ofAngII

onmesangial

cellsynthesis

ofmatrixcompo-nents.

Equal

amounts

of [35S] methionine-labeled conditioned

medium

were

immunoprecipitated with specific antibodies

to

fibronectin

and

collagen

typeI.

_{[3H]Thymidine}

incorporation

did

not

differ

between Ang

TI-treated

and control

cultures,

in-dicating

that

this represented medium from

an

equal

number ofcells

(see Fig.

6B

citation below). Consistent with

theresults

of

Northern blot

analysis

with or

without saralasin (Fig.

3),

immunoprecipitated fibronectin

and collagentype I were

signif-icantly increased

by

I0-1

M AngII4.1-and

3.5-fold

over

con-trols,

respectively (Fig.

5,AandB;P<

0.01). Similarly,

when

conditioned medium

from

[35S]

sulfate-labeled

mesangial

cell cultureswas

subjected

to

SDS-PAGE

and

fluorography,

a

2.4-fold increase

in

sulfate-labeled

proteins

was seenwith 10-8M AngII treatment

for 48

h

(Fig.

5

C;

P<

0.01

). Concomitant

incubation with 10-5M

saralasin

and

o0-8

MAngII

markedly

reduced the levels

of

Ang

IT-stimulated synthesis

of these

pro-teins and

proteoglycans

sothatthe

quantity

immunoprecipi-tatedor

electrophoresed

didnot

differ

from that obtained

from

control conditioned medium

(Fig. 5).

Effect

ofcoincubation with TGF-3 antibody

in

AngII-stim-ulated

mesangial

cell culture. Toassesswhether secreted TGF-,B

protein

mediated the

effects of

Ang IIonthe

expression

of matrixproteins, Ang

TI-treated

mesangial

cellswere

coincu-batedwith

TGF-# neutralizing antibody

( 100

zg/ml)

or

con-trol

IgG

(100

Atg/ml).

As

reported previously (20), mesangial

celltreatmentwith10 -8 MAng II stimulates

protein

synthesis

but not cell

replication (Fig. 6).

Treatment of cultures with

neutralizing antibody significantly,

but not

completely,

B

wa

i_

Wm

* 0:.-.b..

C

CO)

+

C

kd

Figure5.Effect ofAng

-200

IIonmesangial cell

synthesis ofextracellular

matrixcomponents. -_{1 16}

Mesangial

cells cultures

incubatedwith saralasin

alone,Ang11,or Ang

IIand saralasin were

ra-2200

diolabeled

with

[35jS]

methionine. Culture

su-116 pernatants were used

for immunoprecipita-tion of(A)fibronectin

and (B) collagen type I. C shows SDS-PAGE

of[

"S]sulfate-labeled

proteins

(proteogly-0200

cans)

from similar cul-tureslabeled with

[35S]-sulfate. Molecular

-1 1

6 weight markersare

shown on theright.

blocked the

stimulation

of protein synthesisobserved at 48 h

(P < 0.05)

(Fig.

6 A). In contrast, addition of neutralizing

antibody significantly increased

_{[3HIthymidine}

incorporation

of

mesangial

cells

incubated

in the presence ofAng II (P

<

0.05),

indicating increased

cell

proliferation.

Control IgG hadno

effect

(Fig. 6 B). The factthatsufficient

TGF-,3

neutral-izing antibody

had been addedwasconfirmedby thefinding

that the

conditioned medium from

mesangialcellsincubated with Ang II and

TGF-j3

neutralizingantibody for 48 h

con-tained control levels of

TGF-3

activity (0.15ng/ml) as

mea-suredby

bioassay.

When

antibody-treated

mesangial cellcultures were radio-labeled with

[355]methionine

or

[355]sulfate,

and culture su-pernatant was

either

immunoprecipitated

or

subjected

to

SDS-PAGE,

the resultsseenin

Fig.

7were

obtained. While addition

of

neutralizing antibody

to

TGF-,3 (Fig.

7,lane 2) and control IgG (Fig. 7, lane 3)had noeffecton the synthesisoffibronectin

(Fig.

7A),collagentype I(Fig.7B),or proteoglycans(Fig.7

C), neutralizing antibody

to

TGF-f3

(Fig. 7, lane 5) but not

control

antibody

(Fig.

7, lane6) greatly reduced theAng II-in-duced increases

(Fig.

7,

lane

4)

in

production

of these extracel-lular matrix components to levels thatdidnotdiffer

statisti-cally

from controls.

Thus, autocrine TGF-(3 secretion

appears

tomediate alarge

portion

of the Ang II-induced increases in

production of

matrix

proteins by mesangial

cell cultures.

Ang IIadministrationin vivo.

Fig.

8 shows the results ofin

vivoadministration of Ang IIonthemRNAlevels from iso-latedglomeruli for

TGF-#

Iandcollagentype I.After

only

1 wk

oftreatment,a

significant

increase inmRNAfor both

proteins

V..

A

;-fl::::

A

TGF-P Control Ang 11 antibody antibody

0

0

cO

D

_ 0

0~~~

- I

-o oc +_

°

< << kd

I__*__I~i~u -220

0

T 0

0.=

o c

e-_CO_,I 00

iEE

x1

>la

20-

15-10

-il

B

-I

5

0-Control Control antibody

T1

Ang AngIl+ AngIf+ TGF- control antibody antibody

Figure 6. Effect of coincubationofAngIIandneutralizing antibody

toTGF-3 onprotein synthesisand cellproliferation. Radioactivity

precipitated by TCA from mesangial cells incubated under conditions describedinMethods.A, [35S]Methionine radioactivity; B, [3H]-thymidine.

B

-190

of

__

140

-200116

Figure 7. Effect of coin-cubation ofAngII and

neutralizing antibodyto

TGF-f3 onsynthesisof

extracellular matrix proteins found in

cul-ture supernatants. Con-ditionsare asdescribed

in Methods. Immuno-precipitated [35S]-methionine-labeled (A) fibronectin and (B) col-lagentypeI.C shows SDS-PAGE of[35S]_

sulfate-labeled

proteo-glycans. Molecular weight markersare

shownontheright.

is seen(Fig. 8, A and B; P< 0.01). When corrected for the

housekeepingenzyme GAPDH (Fig. 8 C) and plotted, itis

seenthat TGF-31messageis 1.7-foldandcollagentype I mes-sageis 1.8-fold increasedby AngII(Fig. 8 D).

Discussion

Ourlaboratory has shownthat inaratmodel of acute glomeru-lonephritis, TGF-3 isacritical determinant ofmatrixsynthesis,

matrixdegradation, and glomerular expressionofintegrin

re-ceptorswhich mediate matrixdeposition (3-6). Arecent

re-port,where TGF-31 wastransiently expressed in normal rat

glomeruli using in vivo gene transfection, has dramatically confirmed thatTGF-,B alonecanproduce marked glomerulo-sclerosis(7). Recent human studies have shown that the level ofglomerular TGF-f expression correlates with the degree of mesangial matrix expansion in IgA nephropathy and in other glomerular diseases characterized by accumulation ofpatholog-ical matrix (38, 39).

AngIIhasalso beenimplicatedinpathological matrix

ac-cumulation inseveral differentanimal models ofprogressive glomerulosclerosis and in patients with chronic renal diseases. Angiotensin convertingenzymeinhibitors, which block Ang II

generation, have been shownto attenuateor evenreversethe development of sclerosis (10-18). The question of whether Ang II leadsto increased matrixprotein synthesis directlyor

whether theseeffectsaresolely secondarytoincreased intraglo-merularpressure wasaddressedusing mesangial cells in culture

where theeffects of increased intraglomerularpressureare

ab-sent(40). Our hypothesiswasthatAngIIhasadirect effecton

TGF-,3 production. This hypothesisisconsistent with recent

datashowingthatAngIIstimulates theexpressionof

TGF-#

1

invascular smooth muscle cell cultures(23, 24). Wefurther

hypothesized that the TGF-f producedinresponse toAngII

treatmentstimulatesproductionof matrixcomponentswhich

lead to pathological matrix accumulation. This is consistent

with several briefreportsthatAng II stimulates collagen

pro-ductioninculturedratmesangialcells(21, 22).Weevaluated

the matrixcomponentsfibronectin, collagentype I and

bigly-canwhichareknowntobe increasedbyTGF-f3 and whichare

associated with theprocessof sclerosis(34, 35, 41, 42). The data presented support our hypotheses. Ang II

in-creasesthe level ofTGF-,3 mRNAandincreasesproduction of

both latent and activeTGF-f3.Similartothefindings ofWolfet

al.(43) with proximal tubular cells, Gibbonsetal.(44) using vascular smooth musclecells, and Andersonetal.(20) using mesangial cells,we found that Ang II-treated mesangial cells

showhypertrophy butnothyperplasia(Fig. 6).When

neutraliz-ing antibodyto

TGF-#

and Ang IIwereadded together,a signif-icantincrease in cellproliferationwas seenconsistent with the

idea that AngIImayinfluence thebiological action of TGF-3

invivo.

Our data also showthat AngII treatmentleadsto an

in-creaseinthe conversion of latenttoactiveTGF-f asit doesin

vascular smooth muscle cells (44). Itwasshownrecently that

plasmin generated by the action ofplasminogen activator is required for the activation of latentTGF-f3 in cell culture stud-ies(45). AngIIis knowntoincreasecell-associated

plasmino-genactivatorinvascular smooth musclecell cultures(46) and similarly maystimulatemesangial cell synthesis of

plasmino-A

c

7-.2

coL. 6

0 clb =

0 _AD: 5

C-.. to

3

4

C- -I 0 X

-c E 3

-E 0

2 co

-c

0

Control

kb

-2.5

-7.2

Figure8. Effect of

con-tinuousin vivo

admin-istrationof 100ngAng II/mintonormal rats

for 1wk onglomerular

mRNA for TGF-3 and

collagen type I(Col 1).

Molecularsize markers

Ang 11 areshownonthe_right.

genactivator, generationofplasmin,and conversion of latent tobiologicallyactive

_TGF-f3.

Inadditiontoconfirmingthat thegrowthresponse toAng II is mediatedbyincreased

_TGF-f3

productionandthatAngII

treatment leads to increased conversion of latent to active

TGF-f3,

these studiesmake theoriginalcontributionthat links

Ang II to production ofpathological matrixproteinsat both

the mRNA and protein level through induction of

TGF-#l.

These molecularresponsestoAngII aredosedependent and

sincetheycanbe blockedbytheAngIIreceptor blocker,

sara-lasin, they are specific. The Ang II-induced levels of active

TGF-#

are well within the range needed to increase matrix

protein synthesis (25, 26).The increasein mRNA for matrix

components is somewhatdelayed comparedwith inductionof

TGF-3 mRNA(Fig. 1),consistentwith thehypothesisthat the

AngIIeffectson matrixprotein synthesisareindirect and

de-pend uponautocrine secretion of TGF-(3 by mesangial cells.

Thishypothesiswasconfirmedbyaddition ofneutralizing

an-tibodyto

_TGF-f#

(Fig. 7).These resultssupportthe existenceof

a molecular cascade where Ang II stimulates theproduction

and secretion of active

TGF-f3

and this

_TGF-0

in turn

stimu-latessynthesisanddepositionofmatrixcomponents.

Although AngII concentrationsin normalratglomerular filtrate have beenreportedtobe - lO-8_{Mandthereforewell}

withinthe rangewe usedin theseexperiments (47),until

re-cently

tools were not

available

to determine the role of the intrarenal RAS in

disease

and under various

therapeutic

strate-gies.

Studies

havenowshown that all

components ofthe

intrare-nal RASarepresent in human

kidney

(48),

and

studies

using

animal models

are now

_{appearing. Rosenberg}

etal.

(49)

re-ported

that

low

_protein

diet

butnot

_{uninephrectomy}

decreases renal

renin

mRNA

but

does not

_change

either

renalor

liver

angiotensinogen

mRNA

_expression.

Anderson

etal.

₍₅₀₎

dem-onstrated increases

in

intrarenal

renin

_protein

contentand

ex-pression

of

mRNAs

for

renin

and

_{angiotensin converting}

en-zyme in

streptozotocin-induced

diabetic rats,

suggesting

acti-vation of

intrarenal RAS in

diabetes.

Total renal

_angiotensin

converting

enzyme

activity,

however,

was

_reduced,

but

immu-nohistochemical

staining

revealed

redistribution of

angioten-sin

converting

enzyme

protein,

with

increased

_staining

inten-sity

in

_glomeruli.

Wehave shown

previously

that low

protein

diet

suppresses

TGF-,3

expression

and extracellular

matrix

de-position

in

experimental glomerulonephritis

(51).

We have

further

shown that

increases

in

_{TGF-# expression}

are

corre-lated

with matrix accumulation

in

streptozotocin

diabetes

(38).

Therefore,

our

_{previous studies,}

the data

presented here,

and those

of

Rosenberg

etal.

(49)

and Andersonetal.

₍₅₀₎

are

all

consistent with

a role

for

glomerular Ang

II-induced

and

TGF-#l-mediated

alterations

in extracellular

matrix

produc-tion

and

deposition.

In

conclusion, by

studying

the response

of

_mesangial

cells in culture to

exogenously

added

Ang

II,

we have shown that

Ang

II may

play

an

_important

role in the

sclerotic

_changes

within

the

glomerulus

independent

of

_changes

brought

about

by

increased

_glomerular

pressure. We have

further

shown that these

effects

are

mediated

through

_Ang

II

induction of

TGF-#

synthesis

and

secretion.

A

_{large body}

of

worknow

exists

show-ing

that

TGF-,B

isa

_key

_fibrogenic

cytokine

in

diseases of

liver,

lung,

and centralnervous

_system

aswellasin the

kidney (27).

The data

presented

raise

the

possibility

that,

when the

_systemic

and/or

intrarenal RAS is once

activated

under

pathological

conditions such as

_systemic

hypertension

and

chronic

renal

disease,

both

intraglomerular

hypertension

and

activation of

TGF-,B

exert

additive

effectson

accumulation

of extracellular

matrix

and

progression

of

_glomerular

_injury.

Acknowledgments

We wishtothank Markus Kettelerfor his

helpful

comments.

This

study

was

_{supported by}

_grantDK-43609toW. A. Borderfrom

theNational Institute of

Diabetes,

Digestive

and

Kidney

Diseases.

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