Inhibition of Calbindin D28K Expression by Cyclosporin A in ... - JASN

0 downloads 0 Views 2MB Size Report
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