and High Glucose-Induced Alterations in Mesangial Cell Collagen ...

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by Drs. Su Wen. Qian and Anita. Roberts. (Laboratory of Chemoprevention,. National. Cancer Institute,. Bethesda,. MD). Application of Mechanical. Force.

Mechanical Mesangial

Strain- and High Glucose-Induced Cell Collagen Metabolism: Role

BRUCE

L. RISER,

KENICHIRO Division

tion

mesangial

strain

or high

of transforming

cells

growth mechanisms

betic

sclerosis

resulting

renal

cose

interacts

with and

Accordingly, 8 or

mM

stretching,

and

the

MC

were

grown

for

incorporation

to

elevating

‘4C-proline

into

icantly

increased

Of the

causal

greater

In 8 mM MC

factors

glomerulosclerosis,

in the

cose

stimulate

extracellular

largely

mesangiab

expansion

ulosclerosis

(7).

have

demonstrated merular

tures

that The

been

that expansion

involved

outward to which

of

(ECM) In

matrix

to the increased

are

characteristic mechanisms

is translated

into

only

increased

Soc

the

tion

is MC

gluby

these

cells

synthesis

and and

glomer-

identified.

We

have

capillary

pressure compliance

basement are

firmly

results

in gb-

of the

stoic-

causes

the

membrane attached

others

the

of

and

is no

effect high

stretch-

of TGFglucose

is

media,

collagen

importantly,

important

its

implications

net for

syn-

accumuthe

patho-

glomeruloselerosis.

(I

Am

1998)

consequence

of pressure-induced

mechanical

have

strain

shown

strain

to cultured

ecules,

including

IV (8.10). Although

that MC

One

factor-n

TOF-f3l

by

Furthermore.

duction

high

of both

is the

mol-

types

I and

been

bound of

active

synthesis

of

ECM.

whereas

glucose-induced

by MC.

binding

MC pro-

stimulate

collagen,

fully

transforming

and

increased

glomerular

not

cytokine

concentrations

and

of matrix

and

produced

and

mechanical

collagen

have the

We

neutralization

secretion

of collagen

(1 1).

The Accepted October 27, 1997. Bruce L. Riser, Department of Internal Medicine. and Hypertension. CFP-5. Henry Ford Hospital. 2799 Detroit. Ml 48202.

that

glucose

reduces

and

receptor-mediated

MC

TGF-l

production

formation

is both of

cultured

cyclic

of hyperglycemiashown

effect

of the cytokine (9).

the

disten-

of stretch. of

laminin,

(TOF-)

important

form

application

ECM been

glomerular

in the

enhances

mediators

it has

growth

the

fibronectin,

the

identified,

pathogenetie

significantly

increased

spontaneously and

robe

in t’it’o, because

applies

1046-6673/0905-0827$03.00/() Journal of the American Society of Nephrology Copyright tO 1998 by the American Society of Nephrobogy

breakdown

there

In

of diabetic

hypertension-induced

recently

movement of the paramesangial MC cytoplasmic projections

have

In conmedium

breakdown,

glucose

most

no

in collagen aecuplays a positive

of stretch-induced

and,

9: 827-836,

Thus,

deposition

distention

mediator

stretch.

and

TOF-p

and

had

glucose

synthesis,

turnover.

but

but

depo-

gbomerular

collagen

data

Nephrol

that

in low

treatment

by high

increase

collagen

catabolism,

signif-

ECM

marked

and

and

increased

to the

indicate

MC

is a critical

thesis

the

accumulation,

basal

These

gb-

overall

results

in

synthesis

abolished

in

collagen to

MC

TGFsynthe-

glucose,

induced

of

in 35 mM

collagen

in low

changes

However,

lation.

whereby

due

role

hyperten-

of diabetic

pathogenetic

These

regulatory

genesis

medium

situ,

totally

niubation.

limited

the

stretched accumulation.

baseline

stretch-induced

and

those

reduced

treatment

reduced

further

accumulation

(4-6).

(8). The

Received July 17, 1997. Correspondence to Dr. Division of Nephrology West Grand Boulevard.

NARINS

Hospital,

collagen

accumulation

on

same

of diabetic

glomerular

and

effect the

TOF-

breakdown,

progression

trast,

induced

increased

stretching

breakdown,

sis,

accumulation. me-

without

and

for mesangial

contributors

hypertension

sition

culture

responsible

are likely

merubar

in

in

cyclic

or protein-asso-

concentrations matrix

(MC)

therefore

0.

Ford

whereas

increased significantly

collagen

secretion,

exposure

and

High

S d of

of collagen

medium,

identified

(1-3).

appear

glucose

hyperglycemia

cells

ROBERT Henri

accumulation,

markedly

greatly

TOF-. plates

fivefold

synthesis

predominate

mesangial

and

not alter

significant

glu-

mRNA levels by 30 combination of these

than

collagen

sion

free

glucose

dia-

collagen by

TOF-l

the dynamics

high

secretion

mRNA.

K. SHARBA,

Medicine,

neutralization

hyperten-

2 to

mRNA,

glucose

whether

influence

to

did

secre-

collagen,

flexible-bottom

TGF-j31 secretion twofold and TGF-l and 45%. respectively. However, the increased

ABDUL

of Internal

cyclic

of

is mediated

on

to assess or

) and

examines

subjected TOF-l

of

to their

intraglomerular

change

media,

ciated hydroxyproline tabolism. Stretching

stimuli

Department

development

strain

this

assayed

the

study

mechanical

glucose

and

YEE,

RODRIGUEZ-BARBERO,

exposed increase

(TOF-l for

This

whether

rat

35

(MC)

from

hyperglycemia.

metabolism

JERRY

Hypertension,

levels

faetor-l

possible

and/or

ALICIA and

glucose

suggesting sion

CORTES,

in

Michigan.

Cultured

mechanical

ASANO,

of Nephrologv

Detroit,

Abstract.

PEDRO

Alterations of TGF-f3

diabetic

streptozotocin-induced

nephropathy, TOFdeficient

a similarly

has also diabetic

of TOF-

renal after

1 to

BB

rat,

TOF-

3 d of

nonobese

diabetic

diabetic

The

antibody

that

are

in the

mouse

In human

treatment

also

levels

hyperglycemia

glomerular

(14). an

mRNA

rat (13).

increased

been reported mice with

in vitro

demonstrated

cortical

( 12),

diabetic

expression

of

of insulinneutralizes

the

828

Journal

of the American

three

mammalian

sion

of collagen

vitro

treatment of glomeruli an antibody to TGF-3l

with gen

isoforms

tion

induced

cyclic tion

and

MC

not been

regarding

strain.

have

that

Although

demonstrated.

shown

that

is both

it might

little degradation.

was

glucose only

(1 8). This occur

tension addressed (21)

influenced suggests

when

are

by

both

the

extracellular

that

We

concentration

net accumulation

hyperglycemia

and

present. The limited number the role of stretch ( 19), TOF-

in MC

matrix

breakdown

have

of ECM

may hyper-

changes

in the

activity of specific metalloproteases or tissue inhibitors of metalloproteases, often yielding conflicting results. This may be due to the complex interplay that occurs among the many proteases and their corresponding inhibitors (7). In the

present

stretch-

and

examining

study,

high

and

We

show

additive

an

glucose,

thesis,

stretch

increases

breakdown,

Materials

glucose

and

two

factors

and

accumulation

and

on

without

altering

increases

act In

break-

collagen

collagen

mediated

syn-

by TGF-.

from

glomeruli. same high

Nu-Serum

glucose. were

a cloned

line

described

by us previously

derived

from

These cells express the Thysensitivity to phorbol-stimulated

outgrowths

(22),

were

of Fisher

1 antigen and demonstrate neutrophib adhesion

lysis, as do early passage MC, indicating continued sion of essential cell surface receptors (23).

and

like

either

subjected

well,

Neutralizing

antibody

and

mRNA

to TGF-l

attempt

to mimic

expres-

( 1 .D I I . I ) that neutralizes Genzyme TGF-13

Corp. blockade

rat TGF-j31

was

iments

were

earned

average

Roberts (Laboratory Bethesda, MD).

on collagen

elongation

(25).

period

provided

by

of Chemoprevention,

A eDNA Drs.

Su

Wen

National

probe

of study.

concentration a total

Qian

Cancer

studied,

in which

cells

were

of the In an

during

possible

(24),

all exper-

of 10 s of stretch

the

sur-

purified

over

effect

an

culture

of 20 tg/ml serum

exposed

and

set to provide

the entire

of high

to 35 mM

glucose

glucose

for

of 1 1 to 12 d.

mRNA

Analyses

RNA

was

Stat-60,

isolated

Tel-Test

analysis.

by

Inc.,

glyoxal/DMSO

guanidinium

Ebeetrophoresis % agarose

using

mRNA

that

32P-labeled

carried

were were

England methods

individual were

Gels

Gels

New standard

TX).

I h, and

was

gel.

photographed.

(Dupont

and

Friendswood,

at 55#{176}Cfor

then

isoforms,

out

sodium

in

10 mM with

onto

the

Hudson, NH), and of Health Image,

Spring, MD). The assayed (185 and 285) to correct yielded

values

that

glyeeraldehyde-3-phosphate

phos-

membranes Boston, MA), and probed for

corresponding

priming

image analysis version 1 .59,

for

bromide

GeneSereen

hexamer

in

used

ethidium

Products, hybridized

using

by random

(RNA

denatured was

stained

blotted

extraction were

20 j.tg of RNA

Nuclear Research (26). Blots were

TGF-f3

phenol Samples

eDNA

(Sigma

kit, Sigma Chemical, St. Louis, MO). Autoradiograms tatively analyzed by scanning densitometry (Howtek,

TGF-3

Prime-i

were quantiSeanmaster 3

was performed Twilight Clone

TGF-j3 mRNA were normalized for sample loading differences. were

similar

to those

dehydrogenase

to This

normalized

(GAPDH)

+

(NaBBS,

to

mRNA.

Bioassay

The

biological 24 h after were

seeded

After

hours

for Anita

Institute,

later,

Bedford, counter index,

medium

last (17,27).

into

I h, test

96-well

cells

were

were

exposed

onto

filter

MA),

in the filters (Beckman expressed

(1.0

l0

X

containing

or TGF-

standards,

introduced

into

to 1 .0 p.Ci

(MultiScreenlM

and

was

dishes

fixed

measured

Instruments, as inhibitory

with

medium

was

a modification

of

(mink

lung) cells

per fetal

with

in

bovine

or without

[3H]thymidine/well were

Assay ethanol.

Millipore,

radioactivity

Irvine,

The

reflects

harvested,

System,

The

CA).

in a

then

Beckman

(IU),

well)

Twenty-two

in an LS 350 units

epithelial

0.2% cultures.

for 4 h at 37#{176}C. Cells

wells

lysed,

culture

using

CCL-64

medium

samples

antibody,

in the change,

In brief,

Eagle’s

of 200 p.1 of medium

washed tamed

of TGF-

the

modified

TGF-l

total

activity

method

serum.

and

mouse

negative

stretch

the entire

additions

Strain

timed,

was

daily

In experiments

(Flexercell

pressure

intensity

or control

iden-

the bottom to its surface.

8% over

received

antibody

was

added

specific

wells

TGF-3

the

by controlled flexible-bottom

cycles

Vacuum

and

mediated of the

cell

alternating

mHz).

8 mM

under

deforming attached

of approximately

Selected

monoclonal

recom-

, -2, and -3 was a generous gift of MA) and was used in the studies of

metabolism.

(50

and

of the plates

group),

precisely

of mesangial

me-

environment

in intraglomerular

out using

10 s of relaxation

(Minneapantibody

TGF-/31

(Cambridge, kindly

and

conditions

MA)

system

provides

magnitude, the cells

oscillations

Dulbecco’s

antihuman)

system

growth

one-half

(experimental

a computer-assisted This

I plates,

streptomycin,

Bedford,

in a static

well)

(Flex

penicillin,

incubation,

stretching

cycles of known well and stretching

assayed

from R&D Systems assay. A monocbonab

or 48-h

diameter

rat the and

Probes

(chicken

binant human TGF-)31 were obtained ohs, MN) and were used in a biological

contained

maintained

pressure culture

cells

Standards,

were

eells/25-mm

Research,

a 24-

using

Danielpour’s

Antibodies,

and 1640,

to cyclic

plates

correction

and

PA) RPMI

(Collaborative

After

Silver rRNA

Methods

characterized

obtained

in 1 mb/well

MeKeesport, medium,

The

Densitometer, tional Institutes

Cells

Cells,

cultured

Flexereell, dium.

and

stretch and

bottoms

phate/l

TGF-3

production. synthesis

accumulation,

in by

of

levels

stretch

TGF-/3

effects

TGF-3l collagen

TOF-p net

these the

increase

glucose,

and

and

Mesangial

high

of

metabolism

breakdown,

that

of

of

analyzing

to

In high

role

collagen

influence by

manner

independent

accumulation.

the

MC

on the synthesis,

of collagen. normal down

the

production

neutralization

investigate

glucose-induced

separately

TGF-f3

in

we

elastin-coated

face

of studies that have (20), or high glucose

examined

(5 X l0

flexible

low-frequency of

glomerular

seeded

with

Unit#{174},Flexercell).

recently reported that cyclic strain stimulates collagen breakdown as well as synthesis (18). The net difference, i.e., accumulation,

were

culture

is known

Cells

cells

tical conditions (control group). Stretch was cycles of vacuum applied to the underside

antici-

ECM metabolism, ECM production

in matrix

to Cultured

plates

remaining

cyto-

be

Force

Mesangial

20%

the secre-

In addition,

cytokine

collaproduc-

augments

change would dictate altered of TGF-f3 to stretch-induced of this

total ECM

a response

rats

of Mechanical

into six-well

in

diabetic

We

significantly (17).

action

Similarly,

in the increased

of TGF-1,

clearly

the

(15).

the increased

mechanical

Application

overexpres-

insulin-deficient

corrects

isoform-specific

pated that such the contribution has

by

renal

mRNA

from

a role

of cultured

activation

and

kine-

reduces

fibronectin

play

in MC

strain

of Nephrobogy

of TOF-f3

IV and

synthesis (16). TGFmay also

Society

TGF-3 the

con-

scintillation activity

activity that

is

Stretch-

specifically

attributable

to TGF-f3.

This

index

is calculated

from

difference in [3H]thymidine incorporation of a sample assayed and without antibody divided by radioactivity incorporated presence

of antibody,

TGF-

standard

multiplied by 100. In this to 2.4 IU of TGF-l

then

was equivalent

diluted to provide activities dose-response curves with

growth were

vation

lN HCI,

of the latent TGF-

TGF-

activity

assay, I pg of Samples were

in the linear range as determined TGFstandards. The small amount

TGF-3 activity detected in the MC from the determined values. Samples total

.

the

with in the

form

(active

with

plus

medium analyzed and

by of

was subtracted after acid actitherefore

ity in the supernatants radiobabeled nase-resistant

treated

and nontreated

Immediately scraped

Methods radiobabeling

previously

medium

represent

and

dard,

briefly

period

used

to a medium

with

lacking

proline

72 h in an identical 210

mCi/mmol). proline

medium,

M

but containing

ascorbic

Previous

acid,

and

experiments

incorporation

into

by

collagen

us

period

of radiolabeling

(8). All tissue

culture

24 h to provide

140 p.M fresh

ascorbic

period, medium and the cell layer

and consecutively

[3H]-hydroxyproline

acids

total

in 75%

protein

ethanol

in the medium

at -5#{176}C.After

the addition

rated

by

medium Danvers,

solid-phase

supernatants MA), and

extraction, Laboratories,

(Poly-Prep#{174}, Bio-Rad

altered

ments

in which plus

in probine 25.3

nmob/mg

DNA/24h,

n

a 72-h

differences

supplemented

aspirated, the plate with 2 to 6 ml of of six wells

samples

was

of 89 pCi

the

precipitated as

in Centricon”’-3 acids were sepa-

using AG5OW-X8 Richmond, CA).

acid dansylated

derivative

two-tailed

t test.

ANOVA.

Significant

by Fisher’s

protein

[3H]-hydroxyprobine

subsequently

(H) columns Purified amino

(8).

hydroxyproline

precipitate,

tion

on

the

digestible

obtained

beast

Effects

of Glucose

TGF-1

Production

were

(8). In

as described

40 .tmol

and the pellet precipitate

fluoride,

mM two

amounts solution. equal

whereas

The

second

per

method

into

1 ml of medium

(providing

were

of ‘4C incorpora-

incorporated

0. 1 mmol

was

the other

37#{176}C, the undigested acid

and

enzymatic and was

140 used

protein and

0.5%

CaCI,

was

digestion, units/mb

tannic

SEM.

±

no differ-

hydroxyprobine mannitol:

Unless

26.1

1.6

±

otherwise

evaluated

comparisons

among

using were

groups

significance

noted,

the

first

were

unpaired

analyzed

by

then evaluated

test.

colbagenase

removed acid.

After

was was

the

a final divided

added

incubation

by precipitation Finally,

activity,

compared

at the

5

n

to one, with

due

10%

TOF-/31 48

determine

to changes

indicated

had

that

is higher concen-

fivefold an

=

Direct

of

counting that

lower

cell

6; 35 mM: 4.93 ± 0.52 stretch did not significantly

whether

increased

of the specific

increase

in TGF-3l

exposure

to

number

(8

X 106, n = 6, alter the cell

secretion transcript, RNA was mechanical mRNA

stretch

of cells

or high glucose cultures (stretch, 6; stretch, 35 mM: 4.27 ± 0.33

in the level

a 30%

TGF-f31

cultured

effect

demonstrated

as described above, and their . In cells cultured in low glucose,

h induced

to 35 mM in

controls

additive

in-

glucose

to a similar

increase

in a significantly

in low

High

exposed

static

cells in cultures

twofold

secretion

secretion.

experiments

resulted

in either low x 106, n

6). To next

2 h at

‘4C radioactiv-

of these

± 0.9

cultured

MC

cultured 1).

of cells

nonstretched,

on TGF-3l

mM: 7.48 ± 0.63, ,i P < 0.009). However,

into

for

This

increased

than

with

glucose

glucose

6.47

on

than

(Figure

strain

a greater

glucose.

end

of stretch,

In cells

a greater

TOF-J3l

mechanical

elicited

phe-

of

absence

strain.

induced

secretion

glucose

with

to provide

sample

in the

However,

number

concentration,

Stretch

glucose” concentration (8 mM) level (5 mM), it is the minimum

stretch

the

level.

3 .tmol

phenylin the

Cyclic

8 mM glucose medium were medium (1 1 to 12 d total).

to 48 h of cyclic

levels,

was mixed

the pH to 7.5, were added

added

in

high

and

of glucose

grown in mM glucose

medium,

and high

ethylenediaminetetra-acetie

the

as a control. was

or S mM

showed

SEMI).

±

Concentration

the effect

subjected

glucose

in 8 mM

colbagenase-

milliliter:

HCI. After adjusting and N-ethylmabeimide

as before, For

portions

triehloroacetie

‘4C

acids

N-ethylmaleimide). Medium protein was precipitated, 5 times with cold 10% triehboroacetie acid. was then resuspended in 1N NaOH, and the solution with lN fluoride

as a experi-

required to sustain long-term growth of these To examine the effect of stretch, half of the

crease

was washed

was neutralized methybsulfonyl same

total

solution

Amino

for measurement

In this method,

inhibitor

nylmethylsulfonyl

This

of

(8).

standard.

hydroxyproline.

amount

protein

a proteinase acid,

by HPLC

protein-associated

into

based

as an internal

analyzed

mannitol

preliminary

plus

were

differences

protected

To examine

above, was hydrolyzed with 6N HC1 at 1 10#{176}C, and amino acids were separated as above by solid-phase extraction after the addition of 3.32

pCi

mean groups

Multiple-group

been continuously transferred to 35

was estimated by two was obtained according

into protein-associated

medium

10 [mean

two

tration culture.

method,

with

because

were

[3H]-probine

were filtered the free amino

Net collagen accumulation in the medium independent methods. The first measurement this

for the

hydroxyproline.

10; glucose

as

between

HPLC

‘4C incorporation

=

stan-

separated

protein-associated

=

of as

Analyses

Although this “low than the physiologic

to the

and acid

as an internal

medium

into

n

were were

for 5 d in 5 mM glucose

incubated

are expressed

acids were resuspended in 0.1N HCI, and [‘4C]-hydroxyproline, total proline, and proline specific activity was determined by reverse-phase

of the amino

the

the separation was hydrolyzed

incubated

mannitol-containing

± 4.9,

layers

precipitates

to alkaline and

added

into

cultures

incorporation

(glucose:

was

cell

The

subjected

osmolality

were

15 mM

acid,

cold.

and DNA precipitate

incorporation

medium

MC

and

and collagebetween the

Results

contained

internal standard, filters (Amicon,

measured,

chromatographically

did not include

glucose

Data

At the termination

and cell layers

were

of [“C]

Statistical

[‘4C]-

over

were

acid.

(82.3

that

linearly

was rapidly was covered

cold 0.2N perchboric acid. The media pooled as one sample for analysis. The

demonstrated wells

829

3-aminopropi-

[‘4C]-probine

increases

every

of the radiolabeling was placed on ice,

mM

confor

was

perchloric

in the

amino

for

ences

the exception

a final proline out by incubation

0. 15 mM

183

modiflea-

the start of the

(with

of that contained in Nu-Serum, which produced centration of 40 .tM). Radiolabeling was earned onitribe,

minor

24 h before

of 0.2N

homogenized

above,

and

control (8) were

addition

then lipid-extracted

described

latent).

was changed

precipitate

hydrolysis for extraction of RNA protein (8). The cell layer protein

Studies

described

The culture

tions.

protein

Metabolism

samples.

after

Experiments

Metabolism

and

Collagen

incorporation into collagenase-digestible protein was determined from the difference

quantification

Collagen

and Glucose-Altered

8 mM: X

was

,

10

likely

MC

were

probed strain

for for

(Figure

2).

830

Journal

of the American

Society

of Nephrology

30

-

Static

S

L:J Static

Stretch

Static

Stretch

Stretch

(1)

ci)

.

0

I

0

0 1

20

C

-

0

C-) V

S

*

\,

I

ZC)

C,)

C

‘-

10-

0 F-

-I-

-I



ci)

T

0

0

F-

a)

0_

8 mM Figure

1. Secretion

glucose

of

35 mM

transforming

growth

mesangiab cells (MC): effect of stretch sent TGF-/3l accumulated during the conditions.

Significance

in all groups).

ap

0.005.

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