Rat Mesangial Cells - Journal of the American Society of Nephrology

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Materials and Methods. Cells. RMC were obtained from primary glomerular explants and used between passages. 6 and. 10. Using phase-contrast microscopy,.
High

Glucose

Inhibits

Rat Mesangial HOWARD for

accumulation.

high

messenger

directly

stable external

glucose

reduced

nitrite

manner

in this

modulates

and

study

levels

(5.6

high

glucose

in cell

media

was

monophosphate

in rat

The

Hyperglycemia

is a major

complications glucose

plasma

flow

cells

(3).

(ECM)

(2),

oxide

dioxygen

the

by at least

synthase

(NOS)

arteniolar

handling

(6).

tone (10).

possess

response

different participates

and

cells

cytokines

are conflicting

and

(iNOS)

cyclic was

no

on NO

(1).

matrix

L-arginine

renal

yet

April

Correspondence Division

to

of Nephrology.

l()46-6673/0808-

Journal

18,

1996. Dr.

Accepted Howard 269-01

March Trachtman. 76th

Avenue,

17,

(7),

effect

diabetes

increased

I 276$03.00/0

and high

glucose

and

a role

causes

compromises

precursor

reduce

NO

and

other,

production

by

ambient

in the

(J Am Soc

glucose

development

Nephrol

(13,14),

NO

of

8: 1276-1282,

guanosine

meruli

isolated

of NO

in the pathogenesis

and

NO

nitrite,

the effect

diabetic

of high

cells

the

and

monophosphate from

rats

glucose

docu-

NO-dependent generation

by

gb-

(15-17).

To

the

role

clarify

nephropathy, production

measuring

metabolite

indicate

have

(cGMP)

on NO

by

reports

others

decreased

of diabetic

(RMC)

stable

Some

whereas

production

cyclic

mesangial

NO production.

synthesis

reduced

med

cell

in cultured

the

of NO,

we examrat

accumulation

in the

of

incubation

media.

NO

Materials

of experi-

and

Hospital, NY

I 1040.

Methods

Cells

epithelial

Children’s

of the American Society of Nephrology Copyright U 1997 by the American Society of Nephrology

high

of an elevated

play

on renal

NO

in

RMC

were

obtained

passages

identity

from

6 and

of mesangial appearance:

puromycin

aminonucleoside

plated

75-cm2

flasks

analysis ified

index for

of iNOS atmosphere

modified streptomycin,

Eagle’s 100

was

there

was

plates,

25

NO

synthesis of

protein.

Cells

of 10%

C02-90%

medium

were

(DMEM) penicillin,

I0

growth

cells/mb,

10%

and

nitrite

grown Western

fetal

in Dulbecco’s with bovine

of

Cells

at 37#{176}C in a humid-

maintained

supplemented and

were

content

incubated

effect (18).

to assay

RMC

L-arginine

air and

stellate,

no inhibitory

( 19).

the

elongated,

on cell

X

and used

microscopy,

by their

or d-valine of

explants

phase-contrast

confirmed

determination

pg/ml

glomerular

Using

in addition,

in 96-well an

primary

10.

cells

or fusiform

production, Park,

may

mesangial

in normal

cells

on

manner.

1997)

that

1997. Hyde

that

media

glucose

increased

that

to

presence

gbomerulosclerosis.

between

New

indicate

act

high

levels

in the metabolic

in the

a change

mM)

(20

factors

con-

to external

of

in mesangial

Limitation

caused

L-arginine

a dose-dependent

to comparable results

glucose

cell

effect in

(vitamin

anti-transforming

ambient

to 20 mM)

inhibitory

These

by inhibition

tubular

mesangial

tubular

Schneider

(10

of L-arginine

unidentified,

level,

modified of antioxidants

in mesangial

production

content

cells

not

or a pan-specific

of L-arginine

diabetic

were Received

dose

synthesis.

as

was

of affer-

synthesize

the

NO

media.

mented

to mesangial

of the enzyme

and

concerning

University

An elevated

the

cell

glucose

mental

An

nephron

regulation

and

reversed

mesangial

NO

(I 1,12).

data

Washington

addition

of L-arginine

highest

NO in

molecule

(8,9),

of Molecular

the

reduction

Addition

depletion

of target

circulation

balance

NOS

h

(4,5).

isoforms

a time-dependent

mesangial

glucose

single

of

antibody.

partially

synthesis

injury

dismutase),

tent.

to 72

nephropathy

in the

island

Department

cells

C (H-7),

factor-a

cell L-arginine

of extracellular

glomerular salt

Mesangial

inducible

to various

There

pathogenesis

nitrogen

NO

Long

Center,

and

Laboratory,

growth

The

glucose

cell-signaling

three in the

of sodium

proliferation

in the

guanidino

York;

E or superoxide

the

inducible

of high

cells

L. CRIMMINS

Medical

by mesangial

the

there

of

oxidative

New

kinase

nitrite,

24

however,

production

DAN

Jewish

Chemistry

Raising

decreased

effect

is a volatile,

from

by

enhances

by renal (NO)

synthesized

for

in NO

including

causes

the

components

Nitric

cells

GFR

enhances

matrix

in normal

decline

concentration

and

and

factor

of diabetes

in

whether

of

media.

expression

suppressive

oxide

in a time-dependent

generation;

cell

of protein

tested

mM

paralleled

mesangial

protein.

elevated

The

production

by rat mesangial

of 14 ± 3% of the amount

alteration

organ

33.3

Park,

Acid

concenpromotes

mesangial

it was

supernatants

(P < 0.01).

mM)

guanosine synthase

to

Island

Hyde

Nucleic

participates

NO synthesis

concentration

to a nadir

media

ent

GFR,

New

develop-

Nitric

that

in vitro by measuring the accumulation metabolite of NO, in the incubation

cells

is

flow,

the

kidney.

molecule

blood

Therefore,

glucose

to

glucose and

in the

and

and

Long

‘s Hospital,

of Medicine, Protein

contributes

matrix

of renal

in Cultured

Missouri.

A high-serum hemodynamics

of extracellular

the regulation

and

St. Louis,

directly

is a short-lived

Production

FUTTERWEIT,

Children

College

Pharmacology.

Hyperglycemia

(NO)

Einstein

of Medicine,

ment of diabetic nephropathy. tration alters intraglomerular deposition

STEPHEN

Schneider

Albert

and

School

Abstract.

TRACHTMAN, of Nephrology,

Biology

Oxide

Cells

Division

Campus

Nitric

1(X) g/ml serum.

in

High

Experimental

Conditions

At 24 h after reached

plating

confluence

randomly (1)

tions:

glucose:

to one

Control: DMEM

concentration

Western RMC

into 96-well

in 75-cm2

assigned

flasks

of the

plates

(usually following

DMEM containing supplemented with

or when

within two

5.6 27.8

of 33.3 mM. Lipopolysacchanide (IFN)-y (50 U/mI) were added

interferon

I wk),

the cells cells

experimental

mM mM

were

(2) High to a final

of the cytokine combiconsisted of DMEM

netetra-acetic

of 125

tl

was added

and

Nitrite

production

was of

naphthylethylene

measured

a solution

diamine

cGMP

10 mm,

and

the

containing

1%

dihydnochloride, was

assay

Samples

were

measured

0.1%

phosphoric

acid

incubated

at 550

nm.

at

and say

glucose

concentration.

RMC

cGMP in the supennatants after acetylation. using

Chemical

Co.,

Ann

were

grown

was measured a commercially

Arbor,

1% Triton

0.05%

Lexington,

sham,

Arlington

in 25-cm2

ex-

Protein a

flasks,

protein

RMC fate

in these was

used

to determine

the number

Twenty-five

of viable

A total of 100 jd of phenazine

microliters

in 96-well

plates. 37#{176}C in the 10% 490

freeze-

dodecyl for 45 mm,

sulfate gel. Gels and protein was

20, the membrane

was

to iNOS

followed

exposed

to a

(Transduction

by a secondary

Labo-

antibody

(horse-

of the iNOS (Amen-

IL).

of the cell

blue

reagent

homogenates

(BioRad,

were

Richmond,

determined

CA).

polyclonal

(TGF)-

antibody,

and TGF-5, MN).

methosul-

with

anti-transforming

reagents

were

growth

I , TGF-2,

TGF-

was purchased

All other

Co. (St. Louis,

neutralizing

reactive

from

TGFI .2, Systems (Minne-

R&D

purchased

from

Sigma

Chemical

MO).

to 2 ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-

methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

dark.

was

(25 pg of protein)

Reagents

apolis,

experiments.

added

suspension lysates

anti-mouse IgG). Immunoblots with enhanced chemibuminescence

contents

a Coomassie

TGF-/33,

was

ether)phenylmethylsul-

by enzyme immunoasavailable kit (Cayman

MI).

method

buffer

ethylenediami-

Assay

The using

Assay

A colonimetnic

The ofthe

antibody

Heights.

A pan-specific.

Proliferation

of lysis

mM

a nitroceblulose membrane electromembrane with buffer containing

Tween

KY)

peroxidase-binked were visualized

2

I mM

X-l00). aliquots

monoclonal

radish protein

factor

Cell

NaCl,

Nitrite

production was normalized to the number of viable cells and pressed as a percentage of the value in control media containing normal

mM

at 1000 rpm for 5 mm in 100 l

dithiothreitol,

and equal

and

murine

ratonies,

(19).

sulfanilamide,

and 2.5%

media.

absorbance

Griess

1277

ethyleneglycol-bis(3-aminoethyI

1 mM

and times,

gelatin

primary

Production

using

to I 25 j.tl of conditioned

25#{176}C for

2 mM

dissolved

loaded onto a 7.5% acrylamide-sodium run at 200 V and 70 mA (milliamps)

0.25%

Briefly,

was 100

then transferred from the gel onto phonetically. After blocking the

Measurement

RMC

in Cultured

in PBS and centrifuged

acid,

three

were were

mM mannitol.

Nitrite

acid,

fluoride,

thawed

+

Production

The pellet pH 7.6,

N,N’-tetra-acetic fonyl

NO

were harvested

to sediment cells. (50 mM Tnis/HC1,

condi-

glucose; glucose

Inhibits

Analysis

RMC

(LPS) ( 10 pg/ml) and to both test media because

NO production was negligible in the absence nation. A hyperosmolality control medium 27.8

Glucose

in the

Statistical

to each well

Results

inner

of this mixture

salt

was added

Plates were wrapped in foil and incubated for I h at CO,-90% air atmosphere. Absorbance was read at

nm in samples

and

solution

Analyses are

experimental group

presented groups

comparisons

as mean were

were

± SEM.

compared made

The

using

using

means

ANOVA;

between post

the Bonferroni

hoc

the inter-

correction.

blanks.

Results Production

NO

HPLC

Assay

for

After contain

incubation LPS and

L-Argimne in normal IFN-y, cell

phosphate-buffered pension were

was

saline spun

Vacuum-dried hydroxysuccinimidyl

and high monolayers

(PBS).

at 10(X) rpm

resuspended

methanol was and the cleared

There

in 500

l

RMC

glucose were were

for 5 mm of

PBS,

media rinsed

removed,

and

to sediment and

then

derivatized with using the Waters

the

sus-

the cells. 1000

added. Samples were spun at 10,000 supernatants were analyzed by HPLC. extracts were carbamate,

that did not twice with

X

l

They

of

g for

100% 10 mm,

6-aminoquinolyl-Nkit (Millipore,

Mil-

by

IFN-y exposure inhibition

exposure

media

10 pJ of 20 mM

maximum

HPLC

of 20 M1 of the denivatized

column

without

obliterating

the chromatogram. Therefore, the sample to be analyzed. were

HCI,

30 l

of borate

adapted

Calibration was (Pierce Chemical

from

Liu

this Mobile

(2 1) for

performed with Co., Rockford.

the internal standard. ible to

U, -J

w

neuronal activity

75

L

> U U I-

I

25

z 0

E

%E.o 24H Figure

1. Effect

levels

of high

(nmol/unit

percentage point). versus

(50

normal

glucose

paired

that

synthesis

per

control

cell

Results

media

(RMC)

nitrite

are expressed

(n

10 for

=

glucose

RMC

media,

Escher#{252}hia

two The

media

before

media.

values represents ratio of NO,/NO,

high

glucose

with

20 mM

The

as a

each