Abstract. Excessive production and deposition of extracellular matrix proteins are characteristic features of diabetic nephrop- athy. This study tests the hypothesis.
Enhanced Collagen from Insulin-Dependent ROBERTO GIANCARLO *
Unit
and
Abstract.
proteins This
patients
study
tests
develop
lism
and
total
(5 mM)
normal
from
14 insulin-dependent
for age,
14 IDDM diabetes
subjects.
7189
(4341
±
collagen
metabo-
subjects
examined fibrobbasts
glucose
patients
incubated
index; in
in
with
nephropathy
mass
serially
concentrations
(IDDM)
without
were
in
cultured
matched
and
the
ne-
presence
terial) was determined radation was determined ing
the
for
8 h with
residual
trations
The
reasons
the
associated
subset
mM),
overall
excess
for
the
ventricular
sion, ney,
arteries,
and
The
heart)
thickening,
and
patients
cells,
We the
have
recently
patients
sodium-hydrogen
poration into with fibroblasts
with
demonstrated nephropathy antiport,
DNA, and obtained
early from
that exhibit
partly
unexplained
increased
[3H1-thymidine
cell differentiation diabetic patients
from synthesis
susceptible
in the sclerotic
from
activity
of
incor-
compared without ne-
1 133$03.O0/O
be
Soc
that
(5
but and
suggest
from diabetic in collagen patients
without
intrinsic
to play
may
that
patients metabo-
The
and 8:
sub-
nephropathy
results
occur
Nephrol
or of
studied,
metabolism.
processes
and cell
in
develop The
control
ne-
increased
those
diabetic
an important
in the kidneys, 1 133-1
function of
subjects
cultured
cells,
may
diabetes
be
and
139,
extraceblular
matrix
complications
is the
major
brotic
processes
arter-
1997)
is evidence
that
extraceblular using
animal
glucose
component
olism
and
models
ofcolbagen
regulation
other
of
in patients
mechanism
particular,
of
with
who
collagen
metabolism
develop
diabetes
the
and
development to explore sclerotic
lesions
compared
with
the
(1-3).
of
shown
that
help provide
of
sclerotic
whether have
cells
(7-11).
about
would
cells
high metab-
and
would
of
studies,
glycoproteins
data
fi-
There
regulation
expression
kinetic
long-
collagen for
heart
have
extraceblular
function
in that
a number
gene
synthesis
it is essential
patients
lines,
who
of the
to a deranged
alter
quantitative
of collagen
and
and
cell
may and
acquisition
lead
biosynthesis,
concentrations
cell
context
responsible
arteries,
may
metabolic
patients
disturbed
in the
results, abnormal-
the
IDDM
is of relevance
kidney,
diabetes
matrix
of
to
between
of diabetes
in the
These that
independent
production
extracelbular
(4-6). suggested
intrinsic
kidney disease. study of the interaction
term
the
healthy
in long-term
production Received August 27, 1996 Accepted December 30, 1996 Correspondence to Dr. Roberto Trevisan, Divisione Malattie del Ricamhio, Policlinico Universitario, Via Giustiniani 2, 35 128 Padova, Italy. 1046-6673/0807-
with
diabetic
collagen
(J Am
groups concentrations
These
to
significant
production
three
patients
control
No
in all groups
unaltered.
without
healthy
protein
glucose
to nephropathy
and
) or
the
lower
labeling
in the patients
P < 0.01).
normal
is likely
ies,
The
Journal of the American Society of Nephrology Copyright © 1997 by the American Society of Nephrology
than
in the
a 24-h
cells;
long-standing
role
heart.
after
0.01
IDDM
intracellular greater
P
served as control subjects. The two diabetic same clinic population and had a similar
duration, subjects
cells
flasks, and at subconfluence, incubation in DMEM with
defined
greater
and concomitant
bong-term
with
nephropathy,
persistently
ofdiabetes
biopsy,
outpatient
and
diabetic
(AER)
duration
on renal
subjects
20 tg/min) from the
with
excretion
urine,
lesions
healthy history
patients
albumin
recruited
concentration
experiment,
Cell Incubation.
and Methods
bosclerotic
medium containing
nondi-
Patients
j.g/min
Eagle’s (FCS)
subjects.
Materials Fourteen
D-glucose
passage, each
Estimation
as a urinary
modified calf serum
concentration of 25 mM. D-Mannitol was medium to achieve equal final osmolality
nitrogen.
complications.
synthesis
in normal-
abetic
that
subjects
on ice and tannic
precipitated
acid.
by
to a counting of radioactivity
for
is a measure
of
collagen. The pellets were then solubilized and counted to represent the radioactivity
tubes [3H]probine
0.5
The
supernaof radio-
by collagenase without
ml
material
determination
solubilized of
with
Acid-insoluble
centrifugation.
vial
radioactivity
0.005 M CaCI,). and without colla-
sub-
collagenase
incorporated
into
with I ml of 0.2 M NaOH incorporated into noncob-
Fibroblast
lagen
protein.
All
of
the
counts
were
corrected
for
cell
number
and
(15-1 7).
of Collagen
Collagen by
was measured
following
the
residual,
experiment
25-mm2
flasks
DMEM
with
of DMEM
and
(18).
in eight
0.4%
FCS
FCS.
l0)
were
quiescent
were
then
100 mg/L
pulse-labeled
L-ascorbic
labeling,
incubation
the radioactive
L-prohne
times with to labeling
was substituted
medium
was
cell
for 15-3H]probine)
was added.
of collagenase-sensitive
determination
of collagen
formula:
tions
minute
per
radioactivity sample
Percentage [dpmj
in the
at
sample
X
time
using
at
l00/(dpm
0, and
-
t0)
of collagen indicates
of
in the
a period
of 2,
was
ANOVA,
individual
experiment,
the mean
of triplicate
calculated.
Statistical
calculation
was
and comparisons
Newman-Keuls determine value
