CsA treatment decreased serum calcium, increased urine calcium excretion, and decreased calbindin. D28K protein level and immunoreactivity compared with.
J Am Soc Nephrol
Inhibition of Calbindin D28K Expression in Rat Kidney: The Possible Pathogenesis A-Induced Hypercalciuria CHUL
5 1 : 253-258,
SO YOUN MIM,* YOUNG BYUNG KEE BANG*
OK
*Division
of Nephrology, Catholic
study
expression
[VitD]
simultaneously.
of CsA
injury
and tubular known
on renal
most
reabsorbed
in
(mTAL),
the distal
cellular,
and
this
reabsorption
calcium
Received
this
report
the
Society
American
a major renal
wast-
the
limb
the
and
and paracelbular. distal segments
60%)
the connecting and
protein
protein
control
(cabbindin)
with
relative
was
The
New
at the 29th
Orleans,
annual
Louisiana,
meeting in November
1996. Correspondence of
to Dr.
Internal
Medical
Medicine, College.
Copyright
of the 0
Kee
Kangnam
505
1046-6673/0908Journal
Byung
Banpo-Dong,
Bang. St.
Division Mary’s
Seocho-Ku.
of Nephrology, Hospital,
Catholic
Seoul,
137-040,
1998
by the
Society American
of
association
absorption
calbindin
small
segments in the
D28K
wasting.
intestine
(1 1). The
nephron
of
excretion
calbindin
(1 2, 13) of the
process
of calcium
distal
in distal
by Steiner expression
VitD
a role
reg-
reabsorp-
in the
renal
for
and calbindin calbindin
D2SK D28K
et al. (19,20) in
the
demonstrate
kidneys
of
of CsA
a decreased long-term
on segmental
CsAand
sites of calbindin D28K expression and renal calcium are unknown. In this study, we hypothesized that CsA
inhibit
calbindin
this
might
esis,
we
affect
D28K
expression
distal
calcium
examined
the
icity
in
well
known
rats
treated
inducer
in the
with
nephron To
test
of calbindin
or without
eel-
hanmight
and the
that
hypoth-
D,SK
expression
model
of CsA
nephrotox-
exogenous
VitD,
in an acute
of calbindin
distal
reabsorption.
association
and renal calcium handling
in
tubule.
the effects
However,
calcium
is activated
between
suggests
D2SK
of
which
hormone (PTH), and activated of calbindin D28K in the distal
process
strongly
inducer
VitD,
bone
reabsorption,
studies rats.
(16-18).
absorption,
calcium
cascade
is an important
tubule
which
is a
D2SK expression.
Department University Korea.
Materials
and
Methods
Animals
14 16$03.00/0 American
of
D9K is
an
decreased
part
alone.
hormone
calcium
in the proximal
to take
VitD
parathyroid
calcium
D3 (VitD)
absorption
Recent
1 1, 1998.
presented
of Nephrology.
masses
that
protein
D28K protein the
urinary
in renal
distal
calcium
renal
This
lular dling
with
the
calcium
in the
intestinal
calcium
distinct
calbindin
serum
suggests
D28K
the VH group. The calcium, increased
demonstrates
a role
is assumed
D,8K
kidney
Two
of
(14,15).
in
treated
cabbindin
and
in the
and
and
a significant
molecular
described.
March
work
(8-10).
the with
intestinal
tubule.
of calcium
plays
transport
been
Accepted
of this
facilitates
the
and
tDepartment
calbindin
with serum
decreased
concentrations
D28K is expressed
tion,
decreased
proximal tubule by parathyroid VitD induces the transcription
In contrast, calcium is active and trans-
the homeostatic
study
play
in high
ulates is
tubule
This expression
may
reabsorption
of Henle’s
proximal
affect
1 ,25-dihydroxyvitamin
remainder
KIM,* and
compared
not
rats,
expression
about
(approximately
did
levels.
calbindin
and
excretion,
D,8K
rat kidney
tubular
is known
VitD
and
side
and
immunoreactivity
calbindin
have
5, 1997.
A preliminary
little
tubule, in
of calcium
28,000
May
transplantation
excretion,
treatment
present
immu-
magnesium
ascending
convoluted
binding
of
and
and
and
(7).
in the process
subclasses
calcium
thick
is where
occurs
but
tubule
reabsorption
mTAL is largely passive reabsorption in the more
calcium
level
and
500
‘s Hospital,
urine
in CsA-treated
causes
Mars’
rats.
were given for 7 d. calcium, increased
renal
St.
A
Korea.
with
handling.
proximal
medullary
Calcium
CsA
(3-6),
calcium
of the filtered the
in the
segment.
(1,2).
Of these,
Kangnam
YONG
compared decreased
d, subcutaneously)
benefits,
SHIN,*
calcium
calbindin
of organ
its great
SHIN
1998
CHA,t
and immunoreactivity + CsA treatment
separate
important
HO
level VitD
groups, I ,25-dihyCsA groups) were
most
JUNG
Seoul,
CsA
per
of the
Despite
complication
of CsA
reabsorbed
one
dysfunction.
In general,
9,000
mg/kg
is nephrotoxicity
is a well
the effect
role
+
in the management
disease.
effect
The
CsA
two
KIM,t
YOUNG Medicine,
by Cyclosporin of Cyclosporin
urine
D28K
handling rats,
VitD
(25
remains
drugs
autoimmune
loop
CsA-treated versus
CsA
A (CsA)
nosuppressive
ing
calcium
HEE
of Korea,
Pharmacol calbindin
A (CsA)-treated
renal
versus
College
(Biochem
(0.5 tg&g per d, subcutaneously) group showed decreased serum
Cycbosporin
is
[VH]
D3
and VitD The CsA
and
in
(vehicle
droxyvitamin
et al.
a decreased
of
of Internal
Medical
of cyclosporin
association
D2SK
experiments
by Steiner
YOUNG KIM,*
Department
University
demonstrated
kidneys
the
cabbindin
YANG,*
Anatomy,
in the
evaluate
done
KIM,t
1 996)
expression
To
uN
A recent
Abstract.
WOO
9: 1416-1426,
Adult
of Nephrology Society
of Nephrology
housed
male
Sprague
in individual
Dawley cages
rats
weighing
in a temperature-
200
to 225
and
light-controlled
g were
J Am Soc
Nephrol
environment. Madison,
WI),
magnify
9: 1416-1426,
1998
Inhibition
All rats received a low-salt diet (0.05% which is known to accelerate the
the structural
and
functional
effects
incubation
sodium; Teklad, time course and
of CsA
in rats
with
incubated
CsA,
provided
diluted
by
Yu Han
sterile
Sandoz
in olive
(Bonkey,
water
oil
Research
to a final
to a final
Institute
(East
concentration
Pharmaceutical
Experimental
Co.,
Seoul,
concentration
Hanover,
NJ),
of 25 mg/mI.
VitD
Korea)
was dissolved
I :00
in
of I mg/mb.
Groups
subcutaneous group,
injection
overnight
a daily
7).
=
subcutaneous (n 7).
In
Experimental
25 mg/kg,
for 7 d (n
subcutaneous
the
injection
search
Expression
X-bOO
diluted
by CsA
in PBS
and,
1 : 10 in PBS
mouse
1417
subsequently, for
antiserum
15 mm,
against
and
calbindin
VitD
of CsA,
injection
CsA
+
In the
of VitD,
group,
25 mg/kg,
7).
=
rats
and VitD,
pairs
of rats
were
randomly
a daily
assigned
After
for 7 d
to the dif-
containing chloride,
sulfate,
0.02%
10 p.M
NY).
The
blood
next
day
sample
abdomen
and
through
bated
retrogradeby
With
the aorta
veins
opened
preserved
were
a midline below
were
incision, the
occluded by
anesthetized
specimens
with
After
the abdominal
renal
arteries
by ligation
above
with
maldehyde
first perfused
away
briefly
all blood
needle.
arteries
and renal
the kidneys the abdominal
phosphate-buffered
subsequently,
with
a the
was cannu-
I 8-gauge
the renal
with
and,
and
opening
aorta an
a small incision for outflow, perfusion fixation through
were
to rinse
ketamine,
obtained.
by in vivo
kidneys
(PBS)
animals tissue
Intact for intact
was
by an immunoradiometric
(Nichols
Institute,
coefficients was
were
extracted
VitD
using
calf
was
measured
level
thymus
from
and
plasma
source
Serum
phosphorus
8.4
with
Kodak,
Rochester,
measured
by RIA
(Incstar,
kidneys
sion
in the same
were
dehydrated and
sections incubation
azide, and
1 mM
1 mM
the with
IgG
sections
were
bight microscopy.
was homogenized
on sodium
dodecyl
the membranes were at room temperature,
affinity-purified
150 mM dodecyl
ethylenediaminetetra-acetic
acid, fluoride.
X g for 20 mm in
were then To reduce
The
at 4#{176}C. After
the supernatant by the samples were ebectropho-
IL),
subfate-polyacrylamide
condition. Proteins by electrobbotting.
in lysis
Triton X-bOO, 0. 1 % sodium
phenylmethybsulfonyl
concentration Rockford,
gel
ebectrophoresis
transferred nonspecific
to nitrocellubose antibody bind-
blocked with 5% nonfat and then incubated for
24 h at 4#{176}C with
anti-cabbindin
D,SK
( 1:50.000).
dried
The
milk
for 30
membranes
were
then washed in several changes of blotting buffer containing 0.01 M PBS, pH 7.4, and 0. 1 % Tween 20, and incubated for I h with peroxidase-babeled donkey anti-mouse IgG (I : 1000, Jackson ImmunoResearch Laboratories). Visualization was made after a 10- to
Densitometry
30-mm
exposure
to enhanced
chemibuminescence
Buckinghamshire,
for
United
analysis
EYETMII
of Eagle
ether,
Reinhardt
Stillwater,
and
Plasma
were
ex(22).
assay
was
performed
Still Video
densities (mean each band.
at room
using
System
± SD)
(Amersham
Kingdom)
the Zero-Dscan
(Stratagene,
were
obtained
software
La Jolla, after
Life
temperature.
three
CA). The determina-
mounted
with were with
were
fixative
removed
400
concentrations
solution
and embedded on
and
treated
with
primary
for 2 h at 4#{176}C. Slices
in polyester
gelatin-coated
xybene
glass
ethanol,
and,
methanobic
antibody,
additionally wax,
slides.
after H,O,
device
were
the sections
by immer-
of kidney
and sections The
sections
tissue
were were
rinsing in tap water, for 30 mm. Before were
calculated
6. 1 . 1 for unpaired
as mean
with
Statistical
Macintosh. t test.
The
SEM,
±
Comparisons
bevel
and all statistical
Package
for Social
between
of statistical
groups
significance
analyses
Sciences
version
were
was
done
by
as P
chosen
