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
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L:J Static
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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.