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Lumbrokinase
attenuates
diabetic
nephropathy
through
regulating
extracellular
matrix
degradation
in
Streptozotocin-induced
diabetic
rats
Huili
Sun
a,1
,
Na
Ge
a,1
,
Mumin
Shao
b
,
Xiaoyan
Cheng
a
,
Yue
Li
c
,
Shunmin
Li
a,
*,
Jiangang
Shen
a,c,
**
a
Department
of
Nephrology,
Shenzhen
Affiliated
Hospital,
Guangzhou
University
of
Traditional
Chinese
Medicine,
Shenzhen,
China
b
Department
of
Pathology,
Shenzhen
Affiliated
Hospital,
Guangzhou
University
of
Traditional
Chinese
Medicine,
Shenzhen,
China
c
School
of
Chinese
Medicine,
The
University
of
Hong
Kong,
10
Sassoon
Road,
Hong
Kong,
China
1.
Introduction
Diabetes
mellitus
is
a
metabolic
disorder
presenting
with
high
blood
glucose.
About
41–60%
of
diabetic
patients
have
diabetic
nephropathy
[1]
which
is
characterized
by
a
progressive
mesangial
expansion
mainly
due
to
the
accumulation
of
the
extracellular
matrix
(ECM)
of
collagen
IV,
laminin,
fibronectin,
proteoglycans,
and
other
matrix
proteins
[2,3].
ECM
is
in
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
5
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
26
September
2012
Received
in
revised
form
17
December
2012
Accepted
7
January
2013
Published
on
line
4
February
2013
Keywords:
Diabetic
nephropathy
Lumbrokinase
Collagen
IV
MMP-2
MMP-9
a
b
s
t
r
a
c
t
Objective:
The
present
study
was
undertaken
to
investigate
the
therapeutic
effect
and
underlying
mechanisms
of
lumbrokinase
on
diabetic
nephropathy.
Methods:
Type
I
diabetes
was
induced
in
male
Sprague-Dawley
rats
via
intraperitoneal
injection
of
Streptozotocin
(STZ).
Lumbrokinase
was
administered
to
the
diabetic
rats
at
a
dose
of
600,000
U/kg
body
weight
by
gavage.
As
a
positive
control,
perindopril,
an
angioten-
sin-converting
enzyme
inhibitor
(ACEI),
was
given
to
diabetic
rats
at
a
dose
of
4
mg/kg
body
weight.
Following
12
weeks
treatment,
we
measured
the
creatinine
clearance
rate
(Ccr),
urinary
albumin
excretion
(UAE)
and
kidney
injury
scores.
In
addition,
the
expression
of
collagen
IV,
MMP-2
and
MMP-9
in
renal
tissue
was
evaluated.
Results:
The
diabetic
rats
developed
proteinuria,
glomerulosclerosis,
tubulointerstitial
fibrosis
and
a
marked
increase
of
renal
cortical
collagen
IV.
In
contrast,
MMP-2
and
MMP-9
were
significantly
reduced
in
the
renal
cortex
of
diabetic
rats.
Interestingly,
lumbrokinase
treat-
ment
markedly
reduced
the
proteinuria
and
improved
the
glomerulosclerosis
and
tubuloin-
terstitial
fibrosis
in
diabetic
rats.
The
induction
of
collagen
IV
and
the
down-regulation
of
MMP-2
and
MMP-9
was
significantly
attenuated
by
lumbrokinase.
All
these
beneficial
effects
of
lumbrokinase
were
comparable
to
the
ACEI
group.
Conclusion:
Lumbrokinase
treatment
attenuated
diabetic
nephropathy
in
rats,
possibly
through
increasing
the
activity
of
MMPs
and
the
subsequent
degradation
of
extracellular
matrix.
#
2013
Elsevier
Ireland
Ltd.
All
rights
reserved.
*
Corresponding
author
at:
Department
of
Nephrology,
Shenzhen
Affiliated
Hospital
of
Guangzhou
University
of
Traditional
Chinese
Medicine,
1
Fuhua
Road,
Futian
District,
Shenzhen
518033,
Guangdong,
China.
Tel.:
+86
755
83002010;
fax:
+86
755
88359333.
**
Corresponding
author
at:
School
of
Chinese
Medicine,
The
University
of
Hong
Kong,
10
Sassoon
Road,
Hong
Kong,
China.
Tel.:
+852
25890429;
fax:
+852
21684259.
E-mail
addresses:
shunminli@126.com
(S.
Li),
shenjg@hkucc.hku.hk
(J.
Shen).
1
Huili
Sun
and
Na
Ge
contributed
equally
to
this
work.
Contents
available
at
Sciverse
ScienceDirect
Diabetes
Research
and
Clinical
Practice
journal
homepage:
www.elsevier.com/locate/diabres
0168-8227/$
–
see
front
matter
#
2013
Elsevier
Ireland
Ltd.
All
rights
reserved.
http://dx.doi.org/10.1016/j.diabres.2013.01.012
dynamic
flux
with
both
synthetic
and
degradative
compo-
nents.
Imbalance
of
synthetic
and
degradative
components
contributes
to
the
progression
of
glomerular
sclerosis
in
diabetic
nephropathy.
Matrix
metalloproteinases
(MMPs)
appear
to
be
critical
ECM-
degrading
enzymes.
MMPs
comprise
a
large
family
of
Zn
2+
-
dependent
enzymes
which
can
degrade
almost
all
extracellular
matrix
components
[4–6].
Among
MMPs,
MMP-2
and
MMP-9
have
been
implicated
in
the
pathogenesis
of
diabetic
nephropa-
thy.
Previous
studies
have
shown
the
reduced
gene
expression
and
activities
of
MMP-2
and
MMP-9
in
the
diabetic
kidney
[7].
High
glucose
decreased
the
degradation
of
mesangium
matrix,
which
is
substantially
mediated
by
reduced
MMPs
activities
[8].
As
shown
by
streptozocin
(STZ)-induced
diabetic
rats
or
glucose
infused
rats,
exposure
to
hyperglycemia
impaired
nephrogen-
esis
in
fetuses
[9].
Importantly,
high
glucose
concentration
in
rat
metanephric
organ
culture
medium
resulted
in
a
dramatic
reduction
of
MMP-2
and
MMP-9
at
both
transcription
and
enzymatic
activity
levels
[10].
In
addition,
the
therapeutic
effects
of
angiotensin-converting
enzyme
inhibitors
(ACEIs)
on
diabetic
nephropathy
are
also
mediated
by
the
modulation
of
matrix
degradation
[8].
Therefore,
MMPs
serve
as
potential
therapeutic
targets
for
drug
development
in
treating
diabetic
nephropathy.
Lumbrokinase,
an
extract
of
lumbricus
rubellus,
was
identified
in
recent
decades
[11].
Lumbricus
rubellus,
as
a
Traditional
Chinese
Medicine,
has
been
used
for
thousands
of
years
in
China.
Lumbrokinase
consists
of
a
group
of
bioactive
protelytic
enzymes.
Previous
studies
demonstrated
many
beneficial
properties
of
lumbrokinase,
including
anti-inflam-
matory,
anti-oxidative
stress,
anti-fibrotic,
anti-microbial
and
anti-cancer
effects
[12–15].
Lumbrokinase
is
easily
absorbed
in
the
intestinal
tract
without
destruction
of
its
activity
[16]
and
capsules
of
lumbrokinase
are
widely
used
in
China,
Japan,
Korea,
Canada
and
United
States
[17–20].
Similar
to
tissue
plasminogen
activator
(t-PA),
lumbrokinase
dissolves
fibrin
clot
by
converting
plasminogen
to
plasmin
with
the
advantage
of
a
relative
broad
optimal
pH
range
and
good
heat
stability
[16,21–
23].
Lumbrokinase
was
reported
to
protect
ischemic
brains
through
inhibition
of
intercellular
adhesion
molecule-1
(ICAM-
1)
and
the
activation
of
Janus
Kinase1/Signal
transducers
and
activators
of
transcription1
(JAK1/STAT1)
in
experimental
cerebral
ischemia-reperfusion
model
[24].
However,
the
effect
of
lumbrokinase
on
diabetic
nephropathy
is
poorly
understood.
In
the
present
study,
a
diabetic
rat
model
was
established
using
STZ
injection.
Therapeutic
effect
of
lumbrokinase
on
diabetic
nephropathy
was
evaluated
by
determination
of
renal
function,
ECM
deposition
and
the
expression
of
MMP-2
and
MMP-9
in
renal
tissues.
The
findings
from
the
present
study
revealed
an
important
role
of
lumbrokinase
in
amelioration
of
diabetic
nephropathy.
The
regulation
of
collagen
IV,
MMP-2
and
MMP-9
in
kidney
by
lumbrokinase
may
be
involved
in
the
potential
mechanisms
of
renal
protection
in
diabetes.
2.
Materials
and
methods
2.1.
Animals
Adult
male
Sprague-Dawley
rats,
weighting
200–220
g,
were
purchased
from
the
Laboratory
Animal
Center
of
Guangzhou
Medicine.
All
experiments
were
conducted
in
accordance
with
the
NIH
statements
of
‘‘Principles
of
laboratory
animal
care’’.
Rats
were
treated
according
to
the
guidelines
of
the
Institutional
Animal
Care
and
Use
Committees
of
the
Guangzhou
University
of
Traditional
Chinese
Medicine
and
University
of
Hong
Kong.
Animals
were
housed
at
constant
room
temperature
(21
8C)
under
a
controlled
12
h
light
to
12
h
dark
cycle
and
had
free
access
to
water
and
standard
laboratory
diet.
2.2.
Streptozotocin-induced
diabetes
mode
and
drug
treatment
Experimental
diabetes
was
induced
by
a
single
intraperitoneal
injection
of
60
mg/kg
body
weight
Streptozotocin
(STZ,
Sigma
Aldrich,
St.
Louis,
MI,
USA)
in
0.01
mol/l
citrate
buffer
(pH
4.2)
after
a
16
h
overnight
fasting.
Induction
of
the
diabetes
was
confirmed
by
measuring
the
blood
glucose
level
after
3
days
STZ
administration.
The
rats
with
fasting
blood
glucose
concentration
>16.7
mmol/l
were
classified
as
successful
diabetes
model
and
used
in
the
study
[25,26].
The
rats
were
randomly
allocated
into
the
following
experimental
groups:
Normal
control
rats
(non-diabetic
group,
n
=
12),
STZ-induced
diabetic
rats
(diabetic
group,
n
=
12);
lumbrokinase-treated
diabetic
rats
(diabetic
+
lumbrokinase
group,
n
=
12);
ACE
inhibitor
perindopril-treated
diabetic
rats
(diabetic
+
ACEI
group,
n
=
12).
Lumbrokinase
(Lum,
Bokang
Pharmaceutical
Company,
Zhuhai,
China)
were
administered
to
the
rats
at
a
dose
of
600,000
U/kg
body
weight
by
gavage
for
12
weeks.
According
to
previous
studies,
the
suitable
concentration
of
lumbrokinase
was
determined
as
600,000
U/kg
body
weight
in
this
experiment.
As
a
positive
control,
perindopril
(PER,
an
ACE
inhibitor,
Servier
Laboratories,
Neuilly,
France)
was
adminis-
tered
to
the
rats
at
a
dose
of
4
mg/kg
body
weight
by
gavage
for
12
weeks.
The
purification
of
lumbrokinase
from
lumbricus
rubellus
included
5
steps:
1,
hydrolysis
and
autolysis;
2,
centrifugal
separation;
3,
membrane
separation
and
particles
obtainment;
4,
ultrafiltration
and
5,
lyophilization.
One
kilogram
of
the
lumbrokinase
was
obtained
from
the
100
kg
of
fresh
Lum-
bricus
rubellus.
The
specific
activity
of
lumbrokinase
had
20,000
U
pers
1
mg
protein.
2.3.
Physiological
and
metabolic
parameters
Body
weight
was
measured
every
week.
The
kidney
index
was
1000
kidney
weight/body
weight.
The
value
of
serum
creatinine
(Cr)
and
urinary
creatinine
were
determined
by
the
automatic
biochemistry
analyzer
(Olympus
2000,
Tokyo,
Japan).
Ccr
was
calculated
as
urinary
creatinine
urine
volume/serum
creatinine,
and
was
expressed
as
microliters
per
minute
per
gram.
Systolic
blood
pressure
(SBP)
was
measured
by
tail
plethysmography
in
conscious,
preheated
rats
at
the
12th
week
as
described
(IITC
Life
Science,
Woodland
Hills,
CA,
USA)
[26].
In
brief,
rats
were
restrained
and
warmed
with
a
heat
lamp
before
measurement,
then
placed
in
a
holder
with
the
tail
exposed,
allowing
access
to
the
tail-cuff.
An
integrated
sensor-cuff
occluder
operated
to
stop
tail
pulsation
on
inflation
and
to
detect
the
return
of
tail
pulsations
passing
through
the
occluder
cuff
on
each
deflation
cycle
with
the
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
586
photoelectric
sensor.
Three
measurements
were
taken
over
5
min
and
the
mean
value
was
calculated.
2.4.
Urinary
albumin
excretion
(UAE)
Animals
were
placed
into
the
metabolic
cages
(Tecniplast
S.
p.
a,
Buguggiate,
Italy)
for
24
h
urine
collections
at
the
end
of
the
experiment.
Several
drops
of
toluene
were
added
to
the
urine
collection
beaker
to
inhibit
microbial
growth.
UAE
was
measured
using
a
rat
quantitative
ELISA
kit
(Bethyl
Labora-
tories
Inc.,
Montgomery,
TX,
USA)
following
the
instruc-
tions.
Briefly,
plate
wells
were
coated
with
1
mg
of
rat
albumin
in
0.05
mol/l
carbonate–bicarbonate
(pH
9.6)
for
overnight
at
4
8C.
After
blocking
with
0.1
g/l
BSA
solution,
100
ml
of
standard
or
urine
sample
was
incubated
in
microplate
for
60
min
at
37
8C.
After
washing,
100
ml
of
horseradish
peroxidase-conjugated
sheep
anti-rabbit
IgG
was
put
into
the
plate
well
for
1
h
at
37
8C.
Finally,
the
plate
was
incubated
with
100
ml
of
3,30,5,50tetramethylbenzidine
for
10
min
at
room
temperature.
Absorbance
was
measured
at
450
nm.
2.5.
Tissue
preparation
After
administration
of
lumbrokinase
and
PER
for
12
weeks,
rats
were
anesthetized
with
pentobarbital
sodium
(Sigma–
Aldrich,
St.
Louis,
MI,
USA)
at
a
dose
of
50
mg/kg
body
weight
by
i.p.
A
midline
incision
of
the
abdomen
was
cut
and
blood
samples
were
collected
from
the
aorta.
The
kidneys
were
removed
immediately,
weighed
and
rinsed
in
PBS
buffer.
Renal
tissues
were
stored
in
10%
buffered
formaldehyde
for
subsequent
examinations
of
histology
and
immunohis-
tochemistry.
The
rest
of
the
kidney
tissue
was
stored
at
80
8C
for
the
other
analysis.
2.6.
Score
of
glomerulosclerosis
and
tubulointerstitial
fibrosis
Glomerulosclerosis
is
defined
as
the
accumulation
of
ECM
deposits
and
mesangial
expansion.
The
glomerulosclerosis
index
(GSI)
was
assessed
in
periodic
acid-Schiff-stained
sections
in
80
randomly
selected
glomeruli,
and
the
degree
of
sclerosis
was
graded
using
a
semiquantitative
scoring
method
[27].
Tubulointerstitial
fibrosis
refers
to
tubular
atrophy
or
dilatation,
deposition
of
ECM,
and
presence
of
inflammatory
cells.
The
tubulointerstitial
fibrosis
index
(TIFI)
was
assessed
in
Masson’s
trichrome-stained
sections
in
10
randomly
selected
fields
of
view
at
400
magnification
using
a
light
microscope.
The
degree
of
fibrosis
was
graded
using
a
semi-quantitative
scoring
method
[26].
The
degree
of
fibrosis
was
on
a
scale
of
0–3
(grade
0,
normal;
grade
1,
lesion
area
<
25%;
grade
2,
lesion
area
25–50%;
grade
3,
lesion
area
>
50%).
2.7.
Immunohistochemistry
The
rat
kidneys
were
used
for
studies
of
immunohistochem-
istry.
In
brief,
formalin-fixed
kidney
sections
(2
mm
thick)
were
mounted
on
slides,
dewaxed
and
hydrated.
Slides
were
brought
to
a
boil
in
10
mM
sodium
citrate
buffer
(pH
6)
for
10
min
and
cooled
on
bench
top
for
30
min.
After
10
min
incubation
in
3%
hydrogen
peroxide,
sections
were
blocked
with
normal
goat
serum
for
30
min,
and
then
incubated
with
primary
antibodies
Col
IV
(1:500,
Abcam,
Cambridge,
UK),
MMP-9
(1:25,000,
Millipore,
CA,
USA)
and
MMP-2
(1:150,
Millipore,
CA,
USA)
for
overnight
at
4
8C,
respectively.
After
washing
with
rinse
buffer,
sections
were
incubated
with
biotinylated
anti-rabbit
and
anti-mouse
IgG
(Vector
Labora-
tories,
Burlingame,
CA,
USA)
for
10
min,
followed
by
incubation
with
avidin-biotin
HRP
complex
for
5
min.
Localization
of
peroxidase
conjugates
was
revealed
by
using
diaminobenzidine
tetrahydrochloride
solution
as
chromo-
gen
and
hematoxylin
for
counterstaining.
The
immunoper-
oxidase
staining
of
Col
IV,
MMP-2
and
MMP-9
was
quantified
by
gray
scale
analysis
(Image-Pro
Plus
6.0).
The
multi-
plications
of
positive
area
and
the
gray
intensity
in
10
randomly
selected
fields
of
view
at
a
magnification
of
200
were
calculated.
2.8.
Western
blot
analysis
Snap-frozen
kidneys
were
homogenized
in
a
buffer
with
20
mmol/l
Tris–HCl
(pH
7.5),
150
mmol/l
NaCl,
1
mmol/l
Na
2
EDTA,
1
mmol/l
EGTA,
1%
NP-40,
1%
sodium
deoxycho-
late,
2.5
mmol/l
sodium
pyrophosphate,
1
mmol/l
b-glycer-
ophosphate,
1
mmol/l
Na
3
VO4,
1
mg/ml
leupeptin
and
1
mM
PMSF.
Homogenates
were
centrifuged
at
12,000
rpm
for
1
h
at
4
8C.
Then
the
supernatant
was
collected
and
the
protein
Table
1
–
Physical
and
biochemical
parameters
in
various
groups
of
rats
at
12
weeks.
Non-diabetic
Diabetic
Diabetic
+
lumbrokinase
Diabetic
+
ACEI
Body
weight
(g)
576
30
269
29
a
278
57
a
278
43
a
Kidney
index
2.74
0.15
5.95
0.43
a
5.99
0.77
a
6.06
0.47
a
Urine
(ml/24
h)
31
11
285
41
a
269
32
a
309
55
a
Water
(ml/24
h)
50
13
342
37
a
328
50
a
348
47
a
Food
(g/24
h)
25
12
63
4
a
63
6
a
61
4
a
UAE
(mg/24
h)
0.61
0.51
20.09
21.18
a
6.14
4.62
b
2.57
1.43
b
SBP
(mmHg)
117
12
105
9
a
101
13
a
98
9
b
BG
(mmol/l)
5.99
1.97
28.24
3.35
a
28.12
2.76
a
25.23
4.18
a
Ccr
(ml/min/g)
2.35
0.97
8.82
3.13
a
8.00
2.63
a
7.74
2.22
a
Data
are
shown
as
means
SEM.
a
P
<
0.05
vs.
non-diabetic
rats.
b
P
<
0.05
vs.
untreated
diabetic
rats.
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
5
87
Fig.
1
–
(A–C)
Effects
of
lumbrokinase
on
the
formation
of
glomerulosclerosis
and
tubulointerstitial
fibrosis
in
the
renal
tissues
of
STZ-induced
diabetic
rats.
(A)
Representative
periodic
acid-Schiff
staining
for
detecting
glomerulosclerosis
periodic
acid-Schiff
staining
in
kidneys
from
formalin-fixed
kidney
sections
taken
from
the
rats
of
each
group
(n
=
8
rats/
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
588
concentration
was
measured
using
the
BCA
protein
assay
kit
(Pierce
Biotechnology,
Rockford,
IL,
USA.)
according
to
the
manufacturer’s
specifications.
For
Western
blotting
analysis,
10
mg
of
each
sample
was
subjected
to
10%
sodium
dodecylsulfate–polyacrylamide
gel
electrophore-
sis.
Proteins
were
transferred
to
nitrocellulose
membrane
(Bio-Rad
Laboratories,
Hercules,
CA,
USA).
Non-specific
binding
sites
were
blocked
at
room
temperature
for
1
h
with
5%
non-fat
milk
in
Tris-buffered
saline/Tween-20,
then
incubated
overnight
at
4
8C
with
the
primary
anti-
bodies:
a
rabbit
anti-collagen
IV
polyclonal
antibody
(1:2500,
Abcam,
Cambridge,
UK),
a
rabbit
anti-MMP-9
polyclonal
antibody
(1:750,
Millipore,
CA,
USA).
Next,
the
blots
were
incubated
with
goat
anti-rabbit
IgG
(H
+
L)
HRP
(Promega
Corporation,
WI,
USA),
an
affinity
purified
HRP-
conjugated
secondary
antibody.
Blots
were
visualized
using
an
enhanced
chemiluminescence
(Pierce
Biotechnol-
ogy,
Rockford,
IL,
USA.).
A
mouse
anti-GAPDH
monoclonal
antibody
(1:2000,
Proteintech
Group,
Chicago,
USA)
was
used
as
a
control
for
equal
loading
of
proteins.
For
quantitative
evaluation
of
Western
blot
results,
the
films
were
scanned
and
the
optical
densities
were
quantified
by
using
VisionWorks
LS
Imagine
Acquisition
and
Analysis
Software
(UVP
Inc.,
Upland,
CA,
USA).
2.9.
Statistical
analysis
Data
were
expressed
as
mean
SEM.
The
differences
were
analyzed
with
one-way
ANOVA
with
correction
for
multiple
comparisons
using
the
least-significant
difference
post
hoc
test
for
multiple
comparisons.
Statistical
significance
was
set
at
p
<
0.05
level.
3.
Results
3.1.
Physiological
and
metabolic
parameters
During
the
12
weeks
study
period,
3
rats
died
in
all.
One
rat
died
in
normal
group
due
to
the
fighting
accident.
For
diabetic
and
lumbrokinase
treated
groups,
one
animal
died
in
each
of
these
two
groups
possibly
due
to
the
diabetic
complications.
No
animal
death
was
found
in
ACEI
treated
group.
We
detected
physiological
and
metabolic
parameters
in
all
the
study
groups.
As
shown
in
Table
1,
the
STZ-induced
diabetic
rats
had
the
clinical
characteristics
of
polyuria,
polydipsia,
polyphagia,
reduced
body
weight,
increased
the
blood
glucose
(BG),
kidney
injury
and
proteinuria,
indicating
a
successful
rat
model
with
diabetic
nephropathy.
Treat-
ment
of
lumbrokinase
resulted
in
a
remarkable
decrease
of
UAE.
The
effects
of
lumbrokinase
on
the
UAE
were
similar
to
that
of
ACEI.
Meanwhile,
the
STZ-induced
diabetic
rats
showed
lower
systolic
blood
pressure
(SBP)
than
non-
diabetic
control
rats.
Treatment
of
ACEI
further
decreased
SBP
in
rats.
However,
lumbrokinase
did
not
affect
the
level
of
SBP
in
diabetic
rats.
In
addition,
both
lumbrokinase
and
ACEI
had
no
effect
on
body
weight
change,
kidney
index,
24
h
urine
volume,
water
or
food
intake,
BG
and
Ccr
in
diabetic
rats.
These
results
suggested
that
lumbrokinase
could
reduce
urinary
albumin
excretion
in
the
STZ-induced
diabetic
rats.
3.2.
Glomerulosclerosis
and
tubulointerstitial
fibrosis
We
further
evaluated
the
effects
of
lumbrokinase
on
the
glomerulosclerosis
and
tubulointerstitial
fibrosis
in
the
STZ-
induced
diabetic
rats.
As
showed
in
Fig.
1A
and
B,
the
diabetic
rats
had
more
glycogen
deposits
and
collagen
fibers
in
glomerular
mesangium
and
basement
membrane
than
non-
diabetic
rats.
There
were
typical
histological
changes
of
glomerulosclerosis
and
tubulointerstitial
fibrosis
in
the
dia-
betic
rats
as
evidenced
by
mesangial
expansion
and
deposi-
tion
of
extracellular
matrix
and
the
tubulointerstitial
injury.
Quantitative
analyses
showed
that
the
diabetic
rats
had
remarkably
increased
glomerulosclerosis
index
(GSI)
[non-
diabetic,
0.11
0.02
arbitrary
units
(AU);
diabetic,
0.72
0.05
AU,
P
<
0.05]
and
tubulointerstitial
fibrosis
index
(TIFI)
(non-diabetic,
0.05
0.02
AU;
diabetic,
0.83
0.07
AU,
P
<
0.05).
Supplementation
of
lumbrokinase
remarkably
at-
tenuated
the
severity
in
lesions
and
reduced
the
scores
of
GSI
and
TIFI
(GSI,
0.29
0.01
AU;
TIFI,
0.27
0.04,
P
<
0.05),
whose
effects
were
similar
to
ACEI
(Fig.
1C).
These
findings
suggested
that
lumbrokinase
could
reduce
the
glomerulosclerosis
and
tubulointerstitial
fibrosis
in
the
STZ-induced
diabetic
rats.
3.3.
Expressions
of
collagen
IV
To
elucidate
the
potential
mechanisms
of
lumbrokinase
in
prevention
of
diabetic
nephropathy,
we
investigated
the
effects
of
lumbrokinase
on
the
expression
of
collagen
IV
in
the
renal
tissues
of
the
diabetic
rats.
Representative
results
are
shown
in
Fig.
2.
In
non-diabetic
control
group,
the
group)
at
12
weeks.
Imaging
was
obtained
from
kidney
samples
of
following
group:
(A)
non-diabetic;
(B)
diabetic;
(C)
diabetic
+
lumbrokinase;
(D)
diabetic
+
ACEI.
The
diabetic
kidneys
are
characterized
by
moderate
glomerulosclerosis
with
thickening
of
glomerular
basement
membrane
and
mesangium
expansion
(as
showed
with
arrow).
(B)
Representative
Masson’s
trichrome
staining
for
detecting
tubulointerstitial
fibrosis.
Masson’s
trichrome
staining
in
kidneys
from
formalin-
fixed
renal
sections
was
taken
from
representative
rats
from
each
group
(n
=
8
rats/group)
at
12
weeks.
Results
shown
are
representative
images
from
kidney
samples
of
following
groups:
(A)
non-diabetic;
(B)
diabetic;
(C)
diabetic
+
lumbrokinase;
(D)
diabetic
+
ACEI.
The
collagen
fibers
were
stained
blue
(arrow)
and
the
cytoplasm
was
red
and
(C)
Quantitative
analysis
on
glomerulosclerosis
and
tubulointerstitial
fibrosis.
Data
were
showed
as
the
glomerulosclerosis
index
(GSI)
and
tubulointerstitial
fibrosis
index
(TIFI).
*p
<
0.05
when
compared
to
non-diabetic
group.
**p
<
0.05
when
compared
to
diabetic
group.
The
results
suggest
that
both
lumbrokinase
and
ACEI
could
prevent
the
formation
of
glomerulosclerosis
and
decrease
the
accumulation
of
collagen
fibers
in
renal
tissues
of
diabetic
rats.
(For
interpretation
of
the
references
to
color
in
this
figure
legend,
the
reader
is
referred
to
the
web
version
of
the
article.)
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
5
89
Fig.
2
–
(A–D)
Immunohistochemical
staining
and
Western
blot
analysis
for
detecting
the
expressions
of
collagen
IV
in
renal
tissues.
(A)
Formalin-fixed
kidney
sections
taken
from
representative
rats
from
each
group
(n
=
8
rats/group)
at
12
weeks
were
mounted
on
slides
and
stained
with
anti-collagen
Iv
polyclonal
antibody.
Representative
immunohistochemical
staining
of
collagen
IV.
Results
shown
are
representative
images
from
the
kidney
samples
of
following
groups:
(A)
non-
diabetic;
(B)
diabetic;
(C)
diabetic
+
lumbrokinase;
(D)
diabetic
+
ACEI.
(B)
Densitometric
analysis
of
immunohistochemical
staining
for
renal
collagen
IV
protein
from
8
rats
in
every
group.
*p
<
0.05
when
compared
to
non-diabetic
group.
**p
<
0.05
when
compared
to
diabetic
group.
The
results
suggest
that
both
lumbrokinase
and
ACEI
could
decrease
the
expression
of
collagen
IV
in
renal
tissues
of
diabetic
rats.
(C)
Representative
immunoblot
results
obtained
from
non-diabetic,
diabetic
and
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
590
immunostaining
of
collagen
IV
was
localized
at
basement
membranes
of
proximal
and
distal
tubules
and
collecting
ducts,
as
well
in
the
mesangial
areas
in
the
glomeruli.
In
diabetic
group,
an
apparent
increase
in
the
intensity
of
collagen
IV
immunostaining
was
found
in
these
areas,
and
the
expanded
mesangial
areas
had
higher
level
of
collagen
IV
expression
than
other
areas.
Treatment
of
lumbrokinase
significantly
decreased
the
expression
of
collagen
IV
in
the
same
areas
of
the
diabetic
kidney.
Western
blot
analysis
revealed
similar
results
as
immunohistochemical
studies.
Fig.
3
–
Immunohistochemical
staining
for
detecting
the
expression
of
MMP-2
in
renal
tissues.
(A)
Formalin-fixed
kidney
sections
taken
from
representative
rats
from
each
group
(n
=
8
rats/group)
at
12
weeks
were
mounted
on
slides
and
stained
with
anti-MMP-2
monoclonal
antibody.
Representative
immunohistochemical
staining
for
MMP-2.
Results
shown
are
representative
images
from
different
kidney
samples
of
following
groups:
(A)
non-diabetic;
(B)
diabetic;
(C)
diabetic
+
lumbrokinase;
(D)
diabetic
+
ACEI.
(B)
Densitometric
analysis
for
immunohistochemical
staining
of
MMP-2
protein
from
8
rats
in
each
group.
*p
<
0.05
when
compared
to
non-diabetics.
**p
<
0.05
when
compared
to
diabetics.
The
results
suggest
that
both
lumbrokinase
and
ACEI
could
promote
the
expression
of
MMP-2
in
renal
tissues
of
diabetic
rats.
diabetic
+
lumbrokinase
rats
are
shown.
(D)
Densitometric
analysis
for
the
expression
of
collagen
IV
protein
in
extracts
prepared
from
6
rats
in
each
group.
*p
<
0.05
when
compared
to
non-diabetic
group.
**p
<
0.05
when
compared
to
diabetic
group.
The
results
suggest
that
lumbrokinase
could
decrease
the
expression
of
collagen
IV
in
renal
tissues
of
diabetic
rats.
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
5
91
Fig.
4
–
(A–D)
Immunohistochemical
staining
and
Western
blot
analysis
for
detecting
the
expression
of
MMP-9
in
renal
tissues.
(A)
Formalin-fixed
kidney
sections
taken
from
representative
rats
from
each
group
(n
=
8
rats/group)
at
12
weeks
were
mounted
on
slides
and
stained
with
anti-MMP-9
polyclonal
antibody.
Representative
immunohistochemical
staining
for
MMP-9
expression.
Results
shown
are
representative
images
from
kidney
samples
of
following
group:
(A)
non-diabetic;
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
592
Those
results
suggested
that
lumbrokinase
could
inhibit
the
expression
of
collagen
IV
in
the
renal
tissues
of
diabetic
rats.
3.4.
Expressions
of
MMP-2
and
MMP-9
To
understand
whether
the
beneficial
effects
of
lumbrokinase
on
diabetic
nephropathy
are
related
to
MMPs,
we
investigated
the
expressions
of
MMP-2
and
MMP-9
in
the
kidney.
As
shown
in
Figs.
3
and
4,
the
positive
immunostaining
of
MMP-2
and
MMP-9
located
in
the
proximal
tubules,
distal
tubules,
collecting
ducts
and
the
mesangial
areas
of
the
glomerulus
in
non-diabetic
group.
In
agreement
with
previous
reports
[8,10],
an
apparent
reduction
of
immunostaining
of
MMP-2
and
MMP-9
was
found
in
the
renal
tissues
of
diabetic
group.
Treatment
of
lumbrokinase
remarkably
reversed
this
reduc-
tion
of
MMP-2
and
MMP-9
in
the
kidneys.
The
effects
of
lumbrokinase
on
the
expressions
of
MMP-2
and
MMP-9
were
similar
to
that
of
ACEI.
Western
blot
analysis
further
confirmed
immunohistochemical
results.
These
findings
highly
suggested
that
the
reversed
MMP-2
and
MMP-9
activities
may
be
involved
in
the
protective
actions
of
lumbrokinase.
4.
Discussion
The
present
study
provided
the
first
evidence
that
lumbroki-
nase
could
ameliorate
glomerulosclerosis,
tubulointerstitial
fibrosis
and
decrease
urine
albumin
excretion
via
reducing
collagen
IV
deposition
and
up-regulating
MMP-2
and
MMP-9
in
the
kidneys
of
STZ-induced
diabetes.
Accumulation
of
glomerular
mesangial
matrix
is
a
pivotal
event
in
the
pathogenesis
of
renal
diseases
[28].
Plasmin/
MMPs
cascades
affect
glomerular
ECM
accumulation
in
diabetic
nephropathy
[29–33].
Decreased
degradation
of
the
glomerular
ECM,
due
to
reduced
glomerular
proteolytic
activity,
contributes
to
the
progression
of
glomerulosclerosis
in
diabetic
nephropathy
[34,35].
Collagen
IV
is
a
normal
constituent
of
the
mesangium
and
glomerular
basement
membrane
[36,37].
Accelerated
matrix
deposition
(collagen
IV)
is
present
in
the
microalbuminuria
stage,
an
early
stage
of
diabetic
nephropathy
[38].
MMP-2
(gelatinase
A)
and
-9
(gelatinase
B)
are
two
major
proteinases
responsible
for
breaking
down
type
IV
collagen
and
laminin.
MMPs
are
secreted
in
an
inactive
zymogen
form,
which
is
subsequently
activated
by
the
cleavage
of
the
pro-enzyme
[39].
In
the
present
study,
the
STZ-induced
diabetic
rats
had
decreased
expression
of
MMP-2
and
MMP-9
in
the
renal
tissues.
These
results
are
consistent
with
a
previous
report
[10].
Moreover,
typical
diabetic
nephropathy
injuries,
including
glomerulo-
sclerosis,
tubulointerstitial
fibrosis,
increased
urinary
albu-
min
excretion
and
collagen
IV
accumulation,
were
shown
in
this
STZ-induced
diabetic
rats.
The
results
from
present
study
indicated
that
the
down-regulation
of
MMP-2
and
MMP-9
was
associated
with
decreased
degradation
of
the
ECM
and
the
development
and
progression
of
diabetic
nephropathy.
Lumbrokinase
is
a
group
of
proteolytic
enzymes
with
molecular
weight
from
25
to
32
kDa
[40],
which
includes
plasminogen
activator
and
plasmin
[25].
The
plasminogen
activator
(e-PA)
in
lumbrokinase
is
similar
to
t-PA
and
urokinase
(UK).
Lumbrokiase
cleaves
plasminogen
at
four
hydrolytic
sites:
Lys77–Arg78,
Arg342–Met343,
Ala444–Ala445
and
Arg557–Ile558
[41].
The
site
Arg557–Ile558
is
also
recog-
nized
and
cleaved
by
t-PA
and
UK.
Lumbrokinase
contains
the
plasminogen
activator
(e-PA),
which
is
one
of
the
important
components
in
ECM
protease
systems.
Thus,
we
hypothesized
that
lumbrokinase
may
attenuate
the
development
and
progression
of
glomerulosclerosis
and
tubulointerstitial
fibro-
sis
by
activating
the
PA/plasmin/MMPs
cascade
in
diabetic
nephropathy.
The
PA/plasmin/MMPs
cascade
is
initiated
by
single
chain
t-PA
(sc-tPA)
[42].
The
plasmin
produced
by
sc-tPA
could
convert
sc-tPA
and
sc-uPA
to
tc-tPA
and
tc-uPA,
respectively
[43,44],
making
them
more
active
in
the
conver-
sion
actions
of
plasminogen
to
plasmin.
Plasmin
can
degrade
the
non-collagenous
components
of
the
ECM,
whereas
tc-uPA
activates
latent
MMP
and
degrades
type
IV
collagen
[45].
A
previous
study
reported
that
type
IV
collagen
degradation
by
gelatinase
A
in
the
mesangial
cell
required
the
presence
of
plasmin
[45],
whereas
MMPs
can
increase
serine
protease
activity
by
inactivating
serine
protease
inhibitors
[46].
Thus,
the
interactions
between
PAs
and
MMPs
could
enhance
the
effects
of
the
ECM
proteases.
In
our
study,
treatment
of
lumbrokinase
significantly
ameliorated
glomerulosclerosis
and
tubulointerstitial
fibrosis
and
reduced
urinary
albumin
excretion
in
the
STZ-induced
diabetic
rats.
Treatment
with
lumbrokinase
also
reversed
the
down-regulation
of
MMP-2
and
MMP-9
and
reduced
collagen
IV
deposition
in
the
STZ-
induced
diabetic
rats.
These
protective
effects
of
lumbroki-
nase
were
similar
to
the
effects
of
ACEI.
Given
that
the
reduction
of
MMP-2
and
MMP-9
can
delay
the
ECM
degradation
and
prompt
the
development
and
progress
of
diabetic
nephropathy,
and
ACEI
can
up-regulate
MMP-2
and
MMP-9
expressions
and
improve
matrix
degradations
[8,10],
it
is
reasonable
that
the
therapeutic
effects
of
lumbrokinase
are
associated
with
the
regulations
of
the
PA/plasmin/MMPs
cascade
in
the
STZ-induced
diabetic
nephropathy
model.
Nevertheless,
our
experiments
were
performed
in
a
STZ-
induced
diabetic
model
which
is
considered
a
model
of
type
1
diabetes.
The
current
experimental
evidence
might
not
be
suitable
for
understanding
the
potential
effects
of
lumbroki-
nase
on
diabetic
nephropathy
in
type
2
diabetes.
Recent
studies
reported
that
increased
MMP-2
and
MMP-9
levels
are
associated
with
arterial
stiffening
and
angiogenesis
in
with
(B)
diabetic;
(C)
diabetic
+
lumbrokinase;
(D)
diabetic
+
ACEI.
(B)
Densitometric
analysis
of
immunohistochemical
staining
for
renal
MMP-9
protein
from
8
rats
in
each
group.
*p
<
0.05
when
compared
to
non-diabetics.
**p
<
0.05
when
compared
to
diabetics.
The
results
suggest
that
both
lumbrokinase
and
ACEI
could
promote
the
expression
of
MMP-9
in
renal
tissues
of
diabetic
rats.
(C)
Representative
immunoblot
result
obtained
from
non-diabetic,
diabetic
and
diabetic
+
lumbrokinase
rats
are
shown
and
(D)
Densitometric
analysis
for
Western
blot
results
of
MMP-9
protein
in
extracts
prepared
from
6
rats
in
each
group.
*p
<
0.05
when
compared
to
non-diabetics.
**p
<
0.05
when
compared
to
diabetics.
The
results
suggest
that
both
lumbrokinase
could
promote
the
expression
of
MMP-9
in
renal
tissues
of
diabetic
rats.
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
5
93
type
2
diabetes
[47,48].
Therefore,
it
is
still
necessary
to
further
investigate
the
therapeutic
values
of
lumbrokinase
in
diabetic
nephropathy
of
type
2
diabetic
model.
In
summary,
the
present
study
demonstrated
the
thera-
peutic
potential
of
lumbrokinase
in
the
treatment
of
diabetic
nephropathy
as
shown
by
the
significant
attenuation
of
glomerulosclerosis,
tubulointerstitial
fibrosis
and
proteinuria.
The
therapeutic
effects
of
lumbrokinase
might
be
associated
with
an
increase
in
MMPs
and
subsequent
degradation
of
glomerular
ECM.
Conflict
of
interest
The
authors
declare
that
they
have
no
conflict
of
interest.
r
e
f
e
r
e
n
c
e
s
[1]
The
Diabetes
Control
and
Complications
Trial
Research
Group.
The
effect
of
intensive
treatment
of
diabetes
on
the
development
and
progression
of
long-term
complications
in
insulin-dependent
diabetes
mellitus.
N
Engl
J
Med
1993;329(14):977–86.
[2]
Adler
S.
Structure–function
relationships
associated
with
extracellular
matrix
alterations
in
diabetic
glomerulopathy.
J
Am
Soc
Nephrol
1994;5(5):1165–72.
[3]
Miner
JH.
Renal
basement
membrane
components.
Kidney
Int
1999;56(6):2016–24.
[4]
Matrisian
LM.
Metalloproteinases
and
their
inhibitors
in
matrix
remodeling.
Trends
Genet
1990;6(4):121–5.
[5]
Davies
M,
Coles
GA,
Thomas
GJ,
Martin
J,
Lovett
DH.
Proteinases
and
the
glomerulus:
their
role
in
glomerular
diseases.
Klin
Wochenschr
1990;68(22):
1145–9.
[6]
Nyska
M,
McCabe
C,
Linge
K,
Laing
P,
Klenerman
L.
Effect
of
the
shoe
on
plantar
foot
pressures.
Acta
Orthop
Scand
1995;66(1):53–6.
[7]
McLennan
SV,
Fisher
EJ,
Yue
DK,
Turtle
JR.
High
glucose
concentration
causes
a
decrease
in
mesangium
degradation.
A
factor
in
the
pathogenesis
of
diabetic
nephropathy.
Diabetes
1994;43(8):1041–5.
[8]
McLennan
SV,
Kelly
DJ,
Cox
AJ,
Cao
Z,
Lyons
JG,
Yue
DK,
et
al.
Decreased
matrix
degradation
in
diabetic
nephropathy:
effects
of
ACE
inhibition
on
the
expression
and
activities
of
matrix
metalloproteinases.
Diabetologia
2002;45(2):268–75.
[9]
Amri
K,
Freund
N,
Vilar
J,
Merlet-Benichou
C,
Lelievre-
Pegorier
M.
Adverse
effects
of
hyperglycemia
on
kidney
development
in
rats:
in
vivo
and
in
vitro
studies.
Diabetes
1999;48(11):2240–5.
[10]
Duong
Van
Huyen
JP,
Viltard
M,
Nehiri
T,
Freund
N,
Belair
MF,
Martinerie
C,
et
al.
Expression
of
matrix
metalloproteinases
MMP-2
and
MMP-9
is
altered
during
nephrogenesis
in
fetuses
from
diabetic
rats.
Lab
Invest
2007;87(7):680–9.
[11]
Mihara
H,
Sumi
H,
Akazawa
K,
Yoneta
T,
Mizumoto
H.
Fibrinolytic
enzyme
extracted
from
earthworm.
Thromb
Haemost
1983;50(1):258.
[12]
Cooper
EL.
CAM,
eCAM,
bioprospecting:
the
21st
century
pyramid.
Evid
Based
Complement
Altern
Med
2005;2(2):125–7.
[13]
Balamurugan
M,
Parthasarathi
K,
Cooper
EL,
Ranganathan
LS.
Earthworm
paste
(Lampito
mauritii,
Kinberg)
alters
inflammatory,
oxidative,
haematological
and
serum
biochemical
indices
of
inflamed
rat.
Eur
Rev
Med
Pharmacol
Sci
2007;11(2):77–90.
[14]
Prakash
M,
Balamurugan
M,
Parthasarathi
K,
Gunasekaran
G,
Cooper
EL,
Ranganathan
LS.
Anti-ulceral
and
anti-
oxidative
properties
of
‘‘earthworm
paste’’
of
Lampito
mauritii
(Kinberg)
on
Rattus
Norvegicus.
Eur
Rev
Med
Pharmacol
Sci
2007;11(1):9–15.
[15]
Ismail
SA,
Pulandiran
K,
yegnanarayan
R.
Anti-
inflammatory
activity
of
earthworm
extracts.
Soil
Biol
Biochem
1992;24(12):1253–4.
[16]
Hu
R,
Zhang
S,
Liang
H,
Li
N,
Tu
C.
Codon
optimization,
expression,
and
characterization
of
recombinant
lumbrokinase
in
goat
milk.
Protein
Expr
Purif
2004;37(1):
83–8.
[17]
Hwang
CM,
Kim
DI,
Huh
SH,
Min
BG,
Park
JH,
Han
JS,
et
al.
In
vivo
evaluation
of
lumbrokinase,
a
fibrinolytic
enzyme
extracted
from
Lumbricus
rubellus,
in
a
prosthetic
vascular
graft.
J
Cardiovasc
Surg
(Torino)
2002;43(6):891–4.
[18]
Huang
ZD,
Li
ZW,
Zhang
WX.
Lumbrokinase
in
treating
cerebral
infarction.
Chin
J
New
Drugs
Clin
Remedies
2000;6(19):453–5.
[19]
Liao
RH.
Analytical
report
of
treating
30
patients
of
ischemic
cerebrovascular
disease
with
anford
lumbrokinase
and
nimodipine.
Strait
Pharm
J
1997;9(3):
25–6.
[20]
Pang
SQ,
Ding
MC,
Xie
SP.
A
clinical
study
of
therapeutic
effectiveness
in
treating
ischemic
cerebrovascular
disease
with
Lumbrokinase
(Boluoke).
Chin
J
Neurol
Psychiatry
1993;26(4):229–32.
[21]
Nakajima
N,
Sugimoto
M,
Ishihara
K,
Nakamura
K,
Hamada
H.
Further
characterization
of
earthworm
serine
proteases:
cleavage
specificity
against
peptide
substrates
and
on
autolysis.
Biosci
Biotechnol
Biochem
1999;63(11):2031–3.
[22]
Park
Y,
Ryu
E,
Kim
H,
Jeong
J,
Kim
J,
Shim
J,
et
al.
Characterization
of
antithrombotic
activity
of
lumbrokinase-immobilized
polyurethane
valves
in
the
total
artificial
heart.
Artif
Organs
1999;23(2):210–4.
[23]
Mihara
H,
Yineta
T,
Sumi
H,
Soeda
M,
Maruyama
M.
A
possibility
of
earthworm
powder
as
therapeutic
agent
for
thrombosis.
Thromb
Haemost
1989;62(1):545–9.
[24]
Ji
H,
Wang
L,
Bi
H,
Sun
L,
Cai
B,
Wang
Y,
et
al.
Mechanisms
of
lumbrokinase
in
protection
of
cerebral
ischemia.
Eur
J
Pharmacol
2008;590(1–3):281–9.
[25]
Matsuba
S.
Complementary
and
alternative
approaches
to
biomedicine.
Evid
Based
Complement
Altern
Med
2004;1:345–8.
[26]
Sun
HL,
Sun
L,
Li
YY,
Shao
MM,
Cheng
XY,
Ge
N,
et
al.
ACE-
inhibitor
suppresses
the
apoptosis
induced
by
endoplasmic
reticulum
stress
in
renal
tubular
in
experimental
diabetic
rats.
Exp
Clin
Endocrinol
Diab
2009;117(7):336–44.
[27]
Saito
T,
Sumithran
E,
Glasgow
EF,
Atkins
RC.
The
enhancement
of
aminonucleoside
nephrosis
by
the
co-
administration
of
protamine.
Kidney
Int
1987;32(5):
691–9.
[28]
Klahr
S,
Schreiner
G,
Ichikawa
I.
The
progression
of
renal
disease.
N
Engl
J
Med
1988;318(25):1657–66.
[29]
Liotta
LA,
Goldfarb
RH,
Terranova
VP.
Cleavage
of
laminin
by
thrombin
and
plasmin:
alpha
thrombin
selectively
cleaves
the
beta
chain
of
laminin.
Thromb
Res
1981;21(6):663–73.
[30]
Catania
JM,
Chen
G,
Parrish
AR.
Role
of
matrix
metalloproteinases
in
renal
pathophysiologies.
Am
J
Physiol
Renal
Physiol
2007;292(3):F905–11.
[31]
Nagase
H,
Visse
R,
Murphy
G.
Structure
and
function
of
matrix
metalloproteinases
and
TIMPs.
Cardiovasc
Res
2006;69(3):562–73.
[32]
McLennan
SV,
Fisher
E,
Martell
SY,
Death
AK,
Williams
PF,
Lyons
JG,
et
al.
Effects
of
glucose
on
matrix
metalloproteinase
and
plasmin
activities
in
mesangial
d
i
a
b
e
t
e
s
r
e
s
e
a
r
c
h
a
n
d
c
l
i
n
i
c
a
l
p
r
a
c
t
i
c
e
1
0
0
(
2
0
1
3
)
8
5
–
9
594
cells:
possible
role
in
diabetic
nephropathy.
Kidney
Int
2000;58(Suppl.
77):S81–7.
[33]
Han
SY,
Jee
YH,
Han
KH,
Kang
YS,
Kim
HK,
Han
JY,
et
al.
An
imbalance
between
matrix
metalloproteinase-2
and
tissue
inhibitor
of
matrix
metalloproteinase-2
contributes
to
the
development
of
early
diabetic
nephropathy.
Nephrol
Dial
Transplant
2006;21(9):2406–16.
[34]
Liu
Y.
Renal
fibrosis:
new
insights
into
the
pathogenesis
and
therapeutics.
Kidney
Int
2006;69(2):213–7.
[35]
Nath
KA.
The
tubulointerstitium
in
progressive
renal
disease.
Kidney
Int
1998;54(3):992–4.
[36]
Kim
Y,
Kleppel
MM,
Butkowski
R,
Mauer
SM,
Wieslander
J,
Michael
AF.
Differential
expression
of
basement
membrane
collagen
chains
in
diabetic
nephropathy.
Am
J
Pathol
1991;138(2):413–20.
[37]
Zhu
D,
Kim
Y,
Steffes
MW,
Groppoli
TJ,
Butkowski
RJ,
Mauer
SM.
Glomerular
distribution
of
type
IV
collagen
in
diabetes
by
high
resolution
quantitative
immunochemistry.
Kidney
Int
1994;45(2):425–33.
[38]
Liu
X,
Luo
F,
Pan
K,
Wu
W,
Chen
H.
High
glucose
upregulates
connective
tissue
growth
factor
expression
in
human
vascular
smooth
muscle
cells.
BMC
Cell
Biol
2007;8:1.
[39]
Sato
H,
Seiki
M.
Membrane-type
matrix
metalloproteinases
(MT-MMPs)
in
tumor
metastasis.
J
Biochem
1996;119(2):209–15.
[40]
Cho
IH,
Choi
ES,
Lim
HG,
Lee
HH.
Purification
and
characterization
of
six
fibrinolytic
serine-proteases
from
earthworm
Lumbricus
rubellus.
J
Biochem
Mol
Biol
2004;37(2):199–205.
[41]
Zhao
J,
Li
L,
Wu
C,
He
RQ.
Hydrolysis
of
fibrinogen
and
plasminogen
by
immobilized
earthworm
fibrinolytic
enzyme
II
from
Eisenia
fetida.
Int
J
Biol
Macromol
2003;32(3–5):165–71.
[42]
Hoylaerts
M,
Rijken
DC,
Lijnen
HR,
Collen
D.
Kinetics
of
the
activation
of
plasminogen
by
human
tissue
plasminogen
activator.
Role
of
fibrin.
J
Biol
Chem
1982;257(6):2912–9.
[43]
Ichinose
A,
Fujikawa
K,
Suyama
T.
The
activation
of
pro-
urokinase
by
plasma
kallikrein
and
its
inactivation
by
thrombin.
J
Biol
Chem
1986;261(8):3486–9.
[44]
Ichinose
A,
Kisiel
W,
Fujikawa
K.
Proteolytic
activation
of
tissue
plasminogen
activator
by
plasma
and
tissue
enzymes.
FEBS
Lett
1984;175(2):412–8.
[45]
Baricos
WH,
Cortez
SL,
el-Dahr
SS,
Schnaper
HW.
ECM
degradation
by
cultured
human
mesangial
cells
is
mediated
by
a
PA/plasmin/MMP-2
cascade.
Kidney
Int
1995;47(4):1039–47.
[46]
Desrochers
PE,
Jeffrey
JJ,
Weiss
SJ.
Interstitial
collagenase
(matrix
metalloproteinase-1)
expresses
serpinase
activity.
J
Clin
Invest
1991;87(6):2258–65.
[47]
van
der
Zijl
NJ,
Hanemaaijer
R,
Tushuizen
ME,
Schindhelm
RK,
Boerop
J,
Rustemeijer
C,
et
al.
Urinary
matrix
metalloproteinase-8
and
-9
activities
in
type
2
diabetic
subjects:
a
marker
of
incipient
diabetic
nephropathy?
Clin
Biochem
2010;43(7–8):635–9.
[48]
Chung
AW,
Yang
HH,
Sigrist
MK,
Brin
G,
Chum
E,
Gourlay
WA,
et
al.
Matrix
metalloproteinase-2
and
-9
exacerbate
arterial
stiffening
and
angiogenesis
in
diabetes
and
chronic
kidney
disease.
Cardiovasc
Res
2009;84(3):494–504.
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0
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)
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–
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