Catecholamines Modulate Podocyte Function - Journal of the ...

ARTICLES

Catecholamines

Modulate

TOBIAS

BRUNO

PETER

Podocyte

HUBER,*

JOACHIM

SCHOLLMEYER,*

HERMANN

Function

GLOY,*

RAINER

GREGER,

ANNA

HENGER,*

PETER

MUNDEL,

and

PAVENSTADT*

*Department

of Medicine,

Freiburg,

Germany;

Division

and

of Nephrology

and

tPhysiologisches

ofAnatomy

and

Cell

Department

Institut,

Biology,

Albert-Ludwigs-

Universit#{228}t,

Ruprecht-Karls-Universit#{228}t,

Heidelberg,

Germany.

Abstract.

The aim of this study

of adrenoceptor

agonists

was to investigate

aline

fluorescence and

[Ca2]1 whereas

[Ca2}1.

was

completely

prazosin

The

[Ca2]1

induced channel

phenylephrine

induced

0.1 pM for peak agonist UK 14.304 induced

the

a

increase

of

and plateau), did not influ-

#{176}

by

activity

Noradren-

biphasic

response

inhibited

(10 nM). mM), [Ca2]1

(72.5

by noradrenaline

a1-adrenoceptor

antagonist

In a solution with a high extracellular K was unchanged and the [Ca2]1 increase

by noradrenaline was not inhibited by the L-type Ca2 blocker nicardipine ( 1 jIM). Vm and Gm were examwith

the

configuration.

patch-clamp

podocytes

Catecholamines tions.

and increased

increase

secretion,

and

proximal

tubule

It appears capillaries

renal

(1). Within in the

to increase and decrease

(Kf)

in

Na

and

of many

resistance, water

rats

level, it is known that noradrenaline E2 synthesis and increases the

glomerular

rat mesangial

renal

cells

(4).

represents

the

most

differentiated

cell

type

Journal of the American Society of Nephrology Copyright © 1998 by the American Society of Nephrology

cyclase

a time-

cAMP

in

changed cantly

increased

agonists

crease ization

of

145

to 32

whereas mM).

regulate

of

was

un-

The

signifi-

a reduced

data

Cl

indicate

function:

that

They

and induce depolarization

of a cAMP-dependent

9: 335-345,

solution

it was

with

podocyte

[Ca2]1 via an a1-adrenoceptor via a f32-adrenoceptor. The to an opening

increase

or in an extracellular solution

of

Isoproterenol

isoproterenol

concentration,

in an extracellular

adrenoceptor

the effect

40 nM).

effect

of Na

Ca2 (from

mimicked

Gm (EC50

The

concentration

1 1 8.55 1 (5 to

on Vm. Stimula-

concentration-dependent

in the absence

a reduced

IC!

of isoproterenol

by forskolin

and

podocytes.

with

antagonist

the effect

on Vm and

induced

Recent

in

the

may

ultrafiltration.

(2,3).

On

the

noradrenaline

in the

which

glomer-

forms

studies

stabilizing

in-

a depolaris probably

C1

conductance.

1998)

the outer

indicate

capillary

speculated

that

regulate

Received April 15, 1997. Accepted August 13, 1997. Correspondence to Dr. Hermann Pavenst#{228}dt,Medizinische Universit#{228}tsklinik, Abteilung IV, Labor C4, Hugstetter Strasse 55, 79106 Freiburg, Germany. 1046-6673/0903-0335$03.OO/O

The 2-adrenoceptor inhibited

isoproterenol

ulus,

contracts mesangial cells and thus might decrease Kf by a reduction of the capillary surface area (5,6). The understanding of the influence of catecholamines on cellular functions of glomerular endothelial cells or podocytes is limited. The podocyte

of adenylate

renin

stimulates prosuptake of Ca2 in

Furthermore,

tion

func-

noradrenaline

hypertensive

nM)

(J Am Soc Nephrol

the hydraulic pressure of glomerular the glomerular ultrafiltration coeffi-

spontaneously

pM).

100

1 nM) (EC50

of potency

reabsorption

of glomerular

0.5

due

whole-cell

modulate

the glomerulus,

regulation

slow

and noradrenaline

Gm. The order

regulators

vascular

stimulate

be involved

in the

phenylephrine,

are important

They

cellular taglandin

technique

Isoproterenol,

depolarized

cient

agonist

podocytes.

concentration-dependent

in podocytes (EC50 the a2-adrenoceptor

ence

also

in single

and the a1-adrenoceptor

reversible

med

method

calcium

and ion conductances (Gm) [Ca2]1 was measured by

([Ca2J1), membrane voltage (Vm), in differentiated mouse podocytes. the Fura-2

for the adrenoceptor agonists was isoproterenol (EC50 > noradrenaline (EC50 0.3 .tM) > phenylephrine

the influence

on the intracellular

the

of our

knowledge

cells

is based

on

evaluate obtain studies vivo

podocyte,

not

the

had

or dilation

from

a cobblestone

immunologic properties of pletely fit those of podocytes been

suggested

appearance (14).

that

In a subsequent

borized

podocyte

entiated

podocytes,

better

events

investigate

tions

line,

was

inasmuch

of these

which

in the

did not recently,

podocytes

immortalized markers

line

to study of this on

ar-

of differ-

situation

aim

comit has

a cobblestone

the in vivo The

to

In addition,

cell

catecholamines

to

tried

in these

( 1 5). This

the advantage

cells.

past,

the podocyte

with

expresses

mimic

as it offers of

used

differentiated

a conditionally

cultures,

influence

cells.

podocytes

established

in differentiated the

the cells (10-12).

arborized,

study,

cell

than previous

podocyte ical

into

In the

the cultured cells in vivo (1 3). Very

cultured

convert

may (8,9).

researchers

whereas

architecture

cell,

of glomerular

cells.

However,

in

been

processes

function

cultured

appearance,

complex

mesangial

of podocytes,

in culture.

has a very

the

(7).

role

it has

of its foot

concerning

barrier

a central

Furthermore,

functions

podocytes

play

(8).

studies

biological

podocytes

diameters

Kf by contraction

Most

of the filtration

part

that

study cellular

might, of the biologwas func-

to

336

Journal

the American

of

Materials

and Methods

Generation

and

Isolated transgenic

mouse

coated

dishes

(Boehringer 100

many),

and

nich,

(16)

RPM!

100

Mannheim, (both

U/ml

Germany)

grown

After 5 d of primary

mouse

synthesis

culture,

fetal

calf

100 U/ml

Technologies,

recombinant

to induce

10%

Germany),

Life

Ger-

(Sigma,

were

replated

and propagated

T-antigen. trypsinized

at 33#{176}C in medium

and

of identical

position. Cells grown at 33#{176}C exhibited an undifferentiated ogy and proliferate. From the initial pool of outgrowing cells, 10 individual cell lines were stained positive for WT-l (Wilrns’ which

in the adult

Only

WT-1

tional

is only clones

After

the

reduced

proliferation

2 rno, to

10 U/mI

or phenotype.

morpholglomerular

in podocytes;

were

selected

reference

and used

concentration without

corn-

of

Subcultivation

of

17).

for addi-

y-interferon

a significant

was

change

confluent

of cell cells

was

in a medium

without grown

these

by culturing

1640

cells

i.e., they

cells,

liferating

ically

RPM!

mM: Seromed,

(2.5

T-antigen

y-interferon

in

exhibit

characterized

fetal

Berlin,

Inactivation

morphology

positive

These serum

and

of the

to the differentiation

10 d. Arborized

for podocyte

calf

Germany).

an arborized

and stained

marker

differentiation.

10%

at 37#{176}Cleads

after approximately

acteristic

to initiate with

were

for WT-l

foot processes

of

and stop

cells

pro-

immunolog-

and pp44,

( 18). Three

a char-

different

cell

clones were used in the study. Cells were used between passages 6 and 30. Only cells that were grown for more than 14 d at 37#{176}C and that

exhibited iment,

an arborized cells

were

Patch-Clamp The

morphology

replaced

scribed

method

( I 9,20).

in detail

on the stage

superfused

with

free

activity,

Ca2’

lished

the and

experiments

In brief,

podocytes

were

from

acid

a standard

(NMDG) filled

with

pipettes

(2 M)

5 D-glucose, had an input

electrode

a patch-clamp Freiburg,

pH 7.2. resistance

equation

Germany)

to estab-

buffers.

The

with

respectively.

1.2 NaH,PO4,

(in 0.73

The

mM):

CaCl,,

95 I .03

ether)-N,N’-tetra-acetic

(Ca2’

activity,

to 3 MIl.

continuously

l0”

M).

A flowing

The

(10 pd/h)

The data were recorded Physiologisches

displayed

was

of the patch-clamp

recorded

continuously

amplifier.

To obtain

using

using

V,

V

in

steps

from the measured

Kirchhoff’s

and Ohm’s

10

of

whole-cell

laws.

many) cence

of intracellular dye

Fura-2

AM

were performed in single microscope setup recently

from a 75-W blocked the

filters

at 340,

mounted

and 380

A dichroic

mirror

activity

([Ca2])

Sigma,

Deisenhofen,

podocytes described

through thermal

(FT

filter

fluoresthe light

filter, which of the three

wheel

( 10 cycles/s).

filters with transmission

Components, Zeiss,

425,

a water damage

maxima

Gr#{246}benzell, Germany).

Oberkochen,

Germany)

Denmark)

could

diaphragm converter

recorded

continuously

Fura-2

150

by means

X

the photocounter

of

fluorescence of the wheel

be chosen to

(50

to-digital

(MPC

64,

on

the

and high

of an adjustable

30

jim).

was

digitized

Zeiss, hard

Ca2

buffers

calculated

from

described

by

complex

of 224

After with

ratio

be

of an AT

as described

performed ionomycin

previously

the fluorescence

Grynkiewicz nmoIIL

60 s after

et

ratio (22).

al.

(37#{176}C)was

and rinsed

were with

the [Ca2]

with

0.5

added

cultured

Germany)

and

computer.

The

in detail

A KD

(21).

for

the

The

Fura-2-Ca2

given

concentra-

removed,

plates.

They

Germany)

To terminate

and cells extraction,

were

cAMP

an enzyme-linked immunosorbent schweig, Germany).

were

rinsed

assay, with

cells

at 37#{176}C preincu-

Biochemiwere

exposed

the supernatants

ice-cold

concentrations assay

After

(Research for S mm,

the

kept

solution.

M 3-isobutyl-I-methylxanthine

agents.

of the fluores-

cAMP Ringer’s-type

Cologne,

[Ca2]

to the equation

peak.

in 6-well

a physiologic

International,

rapidly

analog-

at the end of the (5 jmol/L) and low

according

assumed.

of Intracellular

Podocytes

to the

the

a 12-bit

Oberkochen, disc

rectan-

amplification,

tions for the [Ca2’] peak refer to the highest magnitude fluorescence ratio. The given [Ca2] plateaus refer to the

cals

a

values for the three excitation wavelengths for each turn were computed. In 20 experiments, the calibration of the

fluorescence signal could using the Ca2’’ ionophore

protocol,

bation

and

filter

of the phototube

cence

the Ger-

with an inverted (21). In brief,

in a motor-driven

nm (Laser

with

(500 to 530 nm; Lys and Optisk Laboratories, Lyngby, were used in the emission light pass. The fluorescence field

bandpass

gular

calcium

filters were bandpass

360,

Activity

(5 pM;

xenon lamp was directed infrared light, to avoid

The excitation

was

Calcium

ethanol

were (70 gIL).

were measured

(Amersham

Buchler,

with Braun-

Denmark). In were replaced

patch

the current

recorder.

the whole-cell

clamp

The

following

agents

were

used:

noradrenaline,

phenylephrine,

isoproterenol, prazosin, metoprolol, IC! I 18.551, UK 14.303, forskolin, (all Research Biochemicals International), nicardipine, A 23187 (Sigma,

Deisenhofen,

Germany).

using Institut,

on a pen

Chemicals

KC1

The access conductance (Ga) was monitored in most of the experiments by the method recently described (20). Membrane voltage (V5) of the cells

Measurements Ca2’-sensitive

After an ethanol

Ca2

and determined

containing

(Fr#{246}beand Busche, and

To vary

according

Copenhagen, Na’ or Cl

was used as a reference. amplifier

in a bath

solution.

or gluconate,

of2

de-

ether)-N.N’-

as Ca2

a solution

been

at 37#{176}C,and

ethyleneglycol-bis(f3-aminoethyl

K-gluconate, 30 KC1, 4.8 Na2HPO4, MgCl2, I ethyleneglycol-bis(/3-aminoethyl and

prepared

electrode (Radiometer, studies, the extracellular were

kept

Ringer-like were

has

mounted

microscope,

nitrilotriacetic

was calculated

pipettes

in these

solutions

by N-methyl-D-glucamine

acid,

After each exper-

examined.

used

of an inverted

with

acid

a Ca2’4-selective ion replacement patch

were

or hyperpolarizing

was calculated

of Intracellular

Measurement

a phosphate-buffered

techniques

tetra-acetic activity

cells

Experiments

patch-clamp

chamber

were examined.

and new

Measurement

photocurrent

performed with trypsin at 37#{176}C after approximately 7 d ( 1 :5 dilution). After trypsinization, cells were thermoshifted to 37#{176}C and maintained cells were L-glutamine

G

(I), Ga, and

excitation

by limiting dilution. Cells antigen, a nuclear protein,

found

protein-positive

studies.

gradually

kidney

cloned tumor

current

Mu-

passed through a 33-nm pore size sieve to remove remaining glomerular cores consisting mainly of mesangial and endothelial cells. Cells were

by depolarizing

mV.

penicillin,

of the immortalizing cells

measured

mV to ±40

serum

Eggenstein,

-y-interferon

outgrowing

was

H-2K”-tsA58

at 37#{176}C on collagen-I-

containing

the voltage of the respective cell was clamped in the voltagemode (Vu) to V,,1. Starting at this value, the whole-cell current

clamp

Lines

heterozygous

were

1640

streptomycin

Cell

1O-wk-old

kidneys

Mannheim,

tg/ml

of Podocte

from

with

of Nephrology

(G5),

Culture

glomeruli

Society

mode

conductance

Statistical

Analyses

The data are given of experiments.

The

as mean average

± SEM, where n refers to the number of the effect of the agonist before and

after the experiment was taken as control. Paired compare mean values within one experimental 0.05 was considered statistically significant.

t test series.

was used to A P value

and

Adrenoceptors

The Effect Is Mediated

Results Noradrenaline and the a, Agonist Increase [Ca2±] in Podocytes Addition 24)

to single

transient teau. n

of noradrenaline

5) increased ±

25

[Ca2’111

to 294

agonists

and

[Ca2]1

returned

podocytes

and

19 nM,

±

removal

The

phenylephrine

was

of approximately

of Noradrenaline on [Ca2 by an a1-Adrenoceptor

nanomolar

inhibited

of the

completely

[Ca2}1

increase

(n

ing of the effect

=

5).

Figure

[Ca2]1

increase.

In contrast,

14.304

(0.1

n

proterenol cytes.

Figure

2A

2B

n

prazosin

shows

an original

the a2-adrenoceptor -adrenoceptor

5) did not influence

summarizes

record-

on noradrenaline-mediated

5) and the

(1 jtM,

antagonist

the noradrenaline-induced

of 10 nM prazosin

j.tM,

in Podocytes

a1-adrenoceptor

and reversibly

337

the

agonist

UK

agonist

iso-

[Ca2]1

in podo-

data.

a typical

a single biphasic

and

of the solution,

1A shows

from

pM).

pla(1 M,

Ringer-like

Figure

obtained (1

an EC50

(Figure

After

=

in a fast

by a [Ca2]1

1 1 to 262

±

with

values.

measurement

with

agonists

49

resulted

(n

and phenylephrine

respectively.

to baseline

to noradrenaline

dependent,

Fura-2

followed

35)

=

from

to noradrenaline

sponse

was n

nM,

54

rinsing

fluorescence exposed

±

with

which

(1 tM,

Ten

or phenylephrine

56)

=

loaded

peak,

Noradrenaline

=

76

podocytes

[Ca2i1

(n

Phenylephrine

Podocytes

podocyte [Ca2]1

re-

concentration-

0. 1 jM

for both

1B).

A

400’

350 ‘‘300

A

0 400

.

250 C

,

350

‘

300

200 C

0

0

.

0

150

E

250

.100

(is

a)

0

(3 0 0 150 E :

100

0

50

50 0 5 mm

B

0

L

Nor

25O C

1mm

I

300

I

C

B

200

: (is

300

C

C C 0

-.-

Noradrenaline

-A---

Phenylephrine

C 0

150

0

E 100

a)

C.) (is

5-

C

0

C 0

0

0

E C.)

I

Nor

Nor

Nor

]

so

UK 14.314J

P-i

50

a)

Figure

0 0

of the

10

iO

Agonist Noradrenaline

Figure

1.

calcium

activity

effect of response

2. The

noradrenaline-induced by an a1-adrenoceptor.

mediated

1 ,M curve

([Ca2’]1)

and

[M]

phenylephrine

in podocytes.

increase

(A) Original

the

cytosolic

recording

of the

noradrenaline (Nor) on [Ca2]1. (B) Concentrationof the agonists. Averaged [Ca2]1 peak responses

(mean ± SEM) to noradrenaline the number of experiments).

and phenylephrine

are shown

(n

ce1-adrenoceptor

antagonist

noradrenaline

(Nor;

of the

effect

of prazosin

crease

and the effects in podocytes

on

the

agonist control.

before

and

[Ca2]

(both after

agonist a

=

5).

c

(Prazo;

10 nM)

increase. agonist

the

the

(Iso;

[Ca2]1

in-

14.314

(0.1

I MM)

on

statistical

the average of

on

(B) Summary UK

isoproterenol indicates

experiments, addition

increase in podocytes is recording of the effect

noradrenaline-induced

of the a2-adrenoceptor

In this and in subsequent =

prazosin

1 j.tM)-mediated

p.M) and the -adrenoceptor [Ca2’]1

[Ca2’]1 (A) Original

significance. of the effect of the

antagonist

was

taken

as

338

Journal

of the American

Society

of Nephrology

Effect of a Low Extracellular Ca2 the L-tvpe Ca2 Channel Antagonist the Noradrenaline-Induced [Ca2 ], In a solution

with

a reduced

MM), the noradrenaline reduced,

whereas

Figure

3A

[Ca211

peak

an original

influx through of the L-type

pM) by

did

not

of the

noradrenaline

whether two [Ca2 with

(ii

additional

increase

(Figure

in culture sets

4).

were

extracellular

K’

To

were

measured

was 6).

(ii

induced investigate

Ca2’

performed:

separately

concentration

channels, V,

and

in a solution

of 72 mM,

and

on 1Ca2] an inhibitor

was investigated of the endoplasmic

reticulum

An increase

of the extracellular

(23).

[Ca2I1

effect

in

220).

=

C

-

a,

nM)

depolarized

-41

± 2 mV

After

did

not

and

significant

6).

=

cause

Figure

presence

an addidecrease

4B

shows

the

of thapsigargin.

capacitance of 67

and

reached, of 34

(ii

significantly

outward

1 to

lasted

±

as

2 nS,

long

Figure

5 shows

an original

( 10 nM)

on V

noradrenaline

as

from

the

caused

(‘i

a reversible

(n

of

isopro-

phenyleph-

and

3 1 to 77).

=

The

present.

effect

isoproterenol,

I5 ±

27).

=

were

the

to

increase

1 1 ± 2 to

agonists of

(10 2 mV

±

significant

respectively

recording

depolarization

-58

I

an input

Isoproterenol

and

G1. Like

and

± 2 pF and

from

conductance

13

podocytes

139).

=

led to a simultaneous

and

9 ±

and

(Cs) 5 nS

±

podocytes

inward and

V1 was

a stable

terenol

E

100

for

cytes =

was

0.3

the 150-

the

adrenoceptor

isoproterenol

pM)

agonists

(ECS()

concentration-

The

rank

order

(EC50

of

and

G55 (Panel

B), respectively.

Isoproterenol Adrenoceptor

Depolarizes

SM).

for

the

phenylephrine

podo-

noradrenaline

>

0.5

=

curves

noradrenaline,

in depolarizing

I nM)

phenylephrine

>

concentration-response

enol,

0

(is

plateau (ii

in the

(Ga)

potency

0 0

±

was

-

0 C

plateau

a small

6 nmollL

±

membrane

dependent

200

[Ca2j

had

rine

-

panel).

± 4 to 150

±

(ii

of the

350

250

right

40

3

not

mV

conductance

400

300

4A,

from

±

did

Isoproterenol, Phenylephrine, and Noradrenaline Depolarize the V, and Increase the G,,, of Podocvtes The resting V1 value obtained in podocytes was -56

depolarization

C C 0 a)

cause

of noradrenaline

3 nS

A

did

18

15, Figure

=

-48

but

in a solution with a reduced bath Ca2 (I Addition of 1 jM noradrenaline into the

but

by

from

shown),

[Ca211

[Ca2]1

increase,

not

thapsigargin-induced

thapsigargin-induced of

(ii

increased

inhibited data not shown).

tional

podocytes

data

[Ca2I1

nM)

6). The

=

a resting

the

effect of 10 M noradrenaline the presence of thapsigargin, Ca2-ATPase

(ii

28,

=

increase.

[Ca211

further

the (1

(20

depolarized

(ii

completely

pM,

noradrenaline-

[Ca211

L-type

I mV increase

17 nM

we tested nicardipine

of the

possess

of experiments

j of podocytes a high

5)

=

podocytes

the

±

Thapsigargin

with normal and resix paired experiplateau was due to a

( 1 jtM)-induced

inhibit

17

-

to 1

plateau

an L-type Ca2’ channel, Ca2 channel antagonist

on the noradrenaline

Nicardipine

1 mM

unchanged

recording

from 3.6 to 72 mM

to

significantly

(from

[Ca2]

was

induced increase of the [Ca2]1 plateau duced bath Ca2. Figure 3B summarizes ments. To examine whether the [Ca2]1 Ca2 effect

K

and on

increase Ca2’4

(I pM)-mediated

the

shows

bath

Concentration Nicardipine

effect

6 shows

of isoproter-

V

on

(ECS()

Figure (Panel

A)

and

50-

0 0’ , 5 mm

B :

I C

,

The

300-

ited

specific

the

32-adrenoceptor

I

ited

the

1 M not of

the

to 1 0 nM

Figure 1 1 8.55

(0.1

with

jtM)

ICS()

(ii

8),

=

by

whereas inhibited

I 6 ± 3%

of

the

7, data

=

(ii

recording

inhibition

of

inhib-

metoprolol

an original

1 -induced

an

completely

isoproterenol

on V1 only

7 shows

on V1 (Panel

1 1 8.55 1 inhib-

IC!

antagonist

of isoproterenol

IC!

terenol

I 18.551

f31-adrenoceptor

shown).

a

depolarization

IC!

response

Vm

of the

the effect 200150-

8 nM.

Via

antagonist

isoproterenol-induced

approximately

250-

Podocvtes

of the effect

effect

of

isopro-

A) and the concentration-response

curve

T

50-

Hl

p)at.

peak

I

peak

NorJ

plat.

prazosin

peak

I

Nor

(Panel B). Pretreatment ization

plat.

The

NOr

Ca2,

Figure

3. Original

noradrenaline extracellular *

indicates

recording

(Nor: I pM) Ca24 (Ca2 statistical

presence

(A) and summary (B) of the effect of on [Ca2]1 in a bath solution with reduced

:

1 cM)

significance.

solution

(ii

=

6).

Mean

±

SEM.

induced

nM)

75

by

effect

effect

of podocytes

(10

with

inhibited

± 5%

(ii

=

of

ICI

depolarization.

14.304 (0.1 shown).

jIM)

was

antagonists 1 1 8.55

I 1 8.55

5) or 36 ± 7%.

of phenylephrine of both

IC!

1 (0. 1 pM)

phenylephrine-mediated

(ii

1 and

=

prazosin

The

did not depolarize

7), respectively.

(ii

completely 5).

inhibited

Figure on

in the

8 summarizes the

the

phenylephrine-

a-,-adrenoceptor podocytes

or

depolar-

agonist (ii

=

7, data

UK not

and Podocytes

Adrenoceptors

339

250

A

250-

2001

200’

I

C

_Q

I

1501

0

150

-

a)

C-) (

I

lC

C

II 1oo

(

100

E

E

I

i_x cn

5O

.

50

C)

C)

I




=

0.4

phenylephrine, a

time-

6)(Figure

=

the

Forskolin

and

and

forskolin

I 1, A and B). The rank order cAMP

(EC5()

#{176}

0.2

was jtM)

isoprotere>

phenyl-

jtM).

( I jtM)-induced ±

1

±

5 mV) 5 mV,

was ii

=

5,

Discussion Stimulation cy-dependent

of renal fall

sympathetic

in GFR

that

to

concentration-dependent

in increasing

forskolin

increase

nerves is due

produces

a frequen-

to an increase

in glomer-

340

Journal

of the American

5’

Society

of Nephrology

0

E

I

-20

-40-

>

a, C (is

-60-

.0

E a)

-80-

I

20

‘so

-

(I,

E.

10

C)

C a)

0-

0 V

C 0 0

Figure

.

-=

-20

.

.

5. Isoproterenol

(Iso) causes a sustained depolarization of the respective cell was clamped to the resting

the voltage by depolarizing

or hyperpolarizing

and G,, (bottom

panel).

ular

resistance,

arteriolar

pressure

gradient,

(24,25).

stimulation

glomeruli

been

a decrease

in glomerular

have

the

mV.

that

onist

of the

that it plays

an active

of Kf

role in the regulation

(8,9). By

using

new

culture

techniques,

it has

been

possible

to

antigen.

Podocytes

differentiated

from

and

this

mouse

arborized

grown

In addition,

express specific immunologic markers ( 14, 15). Only cells with a characteristic resembling that of the in vivo The present results indicate [Ca2411

in podocytes.

adrenaline

was

which

followed

was

the peak,

was

solution,

indicating

tracellular extracellular

smooth induced

The

biphasic,

stores,

[Ca2]1

with

plateau.

in an extracellular, that noradrenaline

but

space.

muscle cells Ca2 uptake

also

Similar

stimulated observations

(27). In mesangial has been observed,

induced

transient The

plateau,

nominally released Ca2’ have

by

[Ca2’j1

Ca2 influx been

but not Ca2 tfree

which

but decreased

reported

in

other

agonists

not

only

inhibit

the

an

Ca2’

Ca2

(top

panel)

Ca2

or a

channel

influx

increase

antag-

mediated

by

in extracellular

Vm and should

cell

Na

K

therefore

exert water

multiple

and

their

balance,

subtypes

vasculature,

lead

cytes.

line was completely

reported

in

In addition,

antagonist

The

is the studies.

on

Compared

the

renal

induce

case

in the

hemodynam-

secretion

by

activating

(29).

adrenoceptors

with

noradrenaline,

elicited

the

the a2-agonist UK 14.304 did not influence [Ca2]1

[Ca2]1

inhibited

increase

induced

by 10 nM of the

by

a1-

in-

(3 1) and in podonoradrena-

-adrenoceptor

prazosin.

direct

renal

of acute

In

are of

an equipotent

influence

of noradrenaline

on podocyte

function

not only may play a role under physiologic conditions, may participate in the pathogenesis of renal diseases acute

that

also

(30). The present data indicate on [Ca2411 was in fact mediated

phenylephrine

crease of [Ca2]1, whereas the /3-agonist isoproterenol

in-

this

renin

of

been

suggested but

in future

the predominating

agonist

effects

have

of a- and J3-adrenoceptors

an a1-adrenoceptor.

adrenoceptor

been

increase,

influence and

the a1-adrenoceptor subtype that the effect of noradrenaline by

it has

Whether

to be clarified

Similar

of thapsigargin

a [Ca2’]1

(28).

needs

and

in podocytes.

types,

stimulate

Catecholamines ics,

[Ca2]1

in the presence

of [Ca2]1

cells

the

anisms of the Ca2’4 influx are not known (4). There is a great body of evidence that the Ca2 influx in podocytes is probably not mediated by an L-type Ca2 channel, but may be due to the

L-type

depolarized

on [Ca2’]

from

cells, a noradrenalinebut the precise mech-

not

The

In addition,

agonists

the renal

nor-

from

did

( 10 nM) on V,

or store-controlled

channel:

increase,

distinct

peak,

of isoproterenol

To obtain G0, was measured

to an influx ofCa2’ via L-type Ca2 channels, did not increase [Ca2]1. In the presence of thapsigargin, noradrenaline did not

a

they

of podocytes in vivo arborized phenotype,

increase

a fast

nicardipine

noradrenaline.

present

situation, were used in this study. that noradrenaline increases the

by a [Ca2i1

absent

at 37#{176}Cexhibit

morphology.

cation

an export

culture differentiated podocytes from a mouse carrying a transgene for a thermosensible variant of the simian virus 40-T-

effect

of a receptor-operated

concentration,

by sym-

nerve stimulation may be due at least partially to a of glomerular surface area (26). The podocyte conto the regulation of capillary wall tension, and it has

assumed

ofthe

nonselective

sympathetic

of Kf induced

decrease

recording

opening

(Kf)

of the diameter

of the whole-cell conductance (Ga,) in podocytes. (Vm). Starting at this value, the whole-cell current

Original

hydraulic

coefficient

shown

in a decrease

Therefore,

and an increase membrane voltage

of 10 mV to ±40

ultrafiltration

studies

results

(26).

pathetic decrease tributes

in steps

V,

and glomerular

Morphologic

nerve

:

-

failure.

renal

failure

The

precise

pathophysiologic

are not clearly

understood,

but also such as

mechanisms but the fol-

Adrenoceptors

A

A -U-

Isoproterenol

-..--

Noradrenaline

and

Podocytes

341

0 -10

5’ -20 a)

5

C)

C

> a) C

-30 .2 N

-40

.0

E a)

C (5

_5

-50

0. ‘I)

-60

0

DD

-70

i0-

10-10

Agonist

B

B

i0-

10-8

i0-

10.8

[M]

_._

6-

-.-

Noradrenaimne

-A--

Phenylephrmne

C,,

C a) 0 C

it (21)

0-

(5

0 V C 0 0

I

iO

510-

7. The

iO

10-10

iO

108

Agonist

isoproterenol-induced

sponse

10.6

[M]

to

isoproterenol

of the

curve

inhibitory

(depolarization) 6. Concentration-response

nol,

noradrenaline,

and

of

depolarization

curves

and phenylephrine

G0, (ZGm

B). Mean

of the effect

on the change

SEM (n

±

of isoprotere-

number

of Vm (LVm

number

induced

(Iso;

10

nM).

effect

of

ICI

by

10 nM

is mediated

by a

lowing events have been (2) tubular obstruction; in glomerular

latter

mechanism,

artery

of dogs

infusion

of

acute

renal

caused

vasoconstriction, adrenaline-induced filtration

tubular acute barrier

cytes.

Flattening

occur

(33).

Therefore,

cyte

function

may

permeability

marked

body

and

noradrenaline-induced be the

key

event

of acute renal failure. Our noradrenaline might directly

In many

cell

known

to increase

events,

which

types [Ca2]1

are known

was

the

de-

transient

the

phenylephrine-induced that a part of the

renal

not due

(e.g.

,

in this

of the of foot

to

podo-

changes

of podo-

experimental

model

to be mediated

the

Mean

Vm. These

Phenylephrine,

cellular

by the a1-adrenocep-

The

It has

and

V, ±

response SEM

(ii

=

Ca2

ionophore

of the effect

the

f32-adrenoceptor

selective that

ephrine 14.304

response, did

not

that

[Ca2]1-mobilizing

also

apparently because depolarize

acted

was

a2-adrenoceptor

podocytes.

conduc-

inhibited

by

1 1 8.55 1 (36),

a /32-adrenoceptor.

is not involved the

by

agonists

a Cl

IC! via

the

podocytes

by activating

antagonist

of

indicating by an a1-

that the depolarby an increase of

of phenylephrine

phenylephrine

An a2-adrenoceptor

depolarized

podocytes

tance (35). Two-thirds

one-third

was inhibited, was mediated

This suggests is mediated

shown

human

noradrenaline, podocytes. In the prazosin, the Ca2

approximately

depolarization depolarization

been

depolarize

indicating

is

abolished

depolarized antagonist

activating a Cl conductance. izing effect of phenylephrine [Ca2i1.

that

was

adrenoceptor.

also

processes

results support the concept influence podocyte function. muscle cells), noradrenaline

and to depolarize

(34).

and the a-agonist isoproterenol presence of the a1-adrenoceptor

Regarding

into

which

abnormalities obliteration

isoproterenol.

A)

leakage, or tubular obstruction. Norrenal failure causes an alteration of

and

of cell

(32).

noradrenaline failure,

Concentration-response

of experiments).

implicated: (1) renal vasoconstriction; (3) back leak of filtrate; and (4)

capillary

(B)

1 18.55 1 on

of experiments).

tor, lead to cell contraction

crease

[MI

f32-adrenoceptor. (A) Original recording of the effect of the 2adrenoceptor antagonist ICI 1 18.551 (ICI; 0.1 pM) on the V,,1 re-

cm

Figure

io-

10-8

CI 118.551 Figure

the

Smmn

-

Compared

in the phenylagonist with

UK phenyl-

342

Journal of the American

Society

of Nephrology

*

I 00

0-

-I-

-

5’

*

a) C

80

-

CC

60

-

.CO

40

-

0 >

* I..-.

OD 0

-20

0)

l)

-

[11_H

C (5 5-.

20

-40

-

+

E

-

(5)

*

(47)

0

(7)

(8)

0-

Jlici

I Figure

Summary

8.

prazosin

(Prazo;

Prazo

lici

I

118.5511

of ICI

on the phenylephrine

I col I Co IICI2 I ICa2’lI

1 1 8.55 1 (0. 1 M) and (Phen; 1 or 10 SM)-

I

the -adrenoceptor

agonist

IONa’i

iso

Figure 9. Summary of the effect of a reduction of the extracellular Cl’ (from 145 to 32 mM, replacement by gluconate), Ca2’ (Ca2’ from

1 mM

to

I j.tM),

and

N-methyl-D-glucamine)

response.

Mean

indicates

statistical

under

and noradrenaline,

-

1

induced depolarization. The figure represents five to seven experiments per series. Mean values ± SEM. indicates statistical significance when compared with the depolarization induced by phenylephrine.

ephrine

(220)

-60

Phen

of the effect

1 pM)

I

118.5511

Prazo

control

values

Na

isoproterenol

±

SEM

(Iso;

(n

significance

conditions

145 to 0 mM,

(from

on

10

number

=

between

the

and isoproterenol

replacement

by

nM)-induced

Vm

of experiments). effect

of

+

isoproterenol

in the presence

of 32 mM

Cr.

isopro-

terenol was the most potent depolarizing agent. To characterize the f3-adrenoceptor involved in the isoproterenol-induced depolarization,

have

the

effect

of

f3-

and 2-adrenoceptor

blockers

isoproterenol-induced

been

depolarization

was

significantly

in-

examined. Low concentrations of the f37-adrenoceptor blocker ICI 1 18.55 1 inhibited the response to isoproterenol, whereas high concentrations of the f31-adrenoceptor antagonist

creased. activation

metoprolol

u-intercalated cells (37). As with isoproterenol, the effect of forskolin on Vrn was augmented in a solution with a lowered extracellular Cl concentration, indicating that the C1 con-

only

had a small

depolarization.

The

data

effect

strongly

tion induced by isoproterenol ceptor. An isoproterenol-induced ported

in

whereas

epithelial

in muscle

tion (38). Stimulation adenylate skolin, effect

of

cyclase a direct

cells

the

activator

concentration-response depolarization

were

by

f3-intercalated

induces

2-adrenoceptor of the adenylate curves

very

tion,

with whereas

depolariza-

cells

to

of cAMP cyclase,

similar.

j32-adrenoceptor

for cAMP These

data

agonists

(37),

a hyperpolariza-

is known

in an increase

mediated by an increase of cAMP. The depolarizing effect of isoproterenol solution

the

of

a Cl

activate For-

mimicked

the

is activated

observed

in epithelial

been

performed

production

and

de-

the kidney

activates

indicate

that

the

was not altered

in a

a lowered extracellular Ca2 or Na in the presence of a low extracellular

concentraC1, the

cAMP. (40),

secretion,

which

different

and

cAMP

antagonize

nists,

Spontaneously

increase but

not

in response to

reported

results

have

in mesangial

forskolin

However,

diabetic

these

infusion

hormone

enol on podocyte function. Under changes of glomerular responses reported:

Similar whereas

been

in

been cells,

a

has been reported (41). of the f3,-adrenoceptor-medi-

(42). in rats,

might

also

in podocytes are not yet clear. Direct into the renal artery did not change

hemodynamics

have

is

by cells

ated conductance changes infusion of isoproterenol glomerular

has

hyperpolarization consequences

and the

in podocytes

conductance

ductance

cAMP-mediated The functional

(39).

on Vm and on ion conductances,

of isoproterenol

polarization

as

isoproterenol

resulting

that

was mediated by a 2-adrenodepolarization has been re-

such

cells

activation

on isoproterenol-induced

indicate

This indicates that the depolarization was due to the of a Cl conductance. An isoproterenol-induced

systems,

a direct

such

effect

rats

show

Thus,

conditions, have been an

attenuated

j3-adrenergic the

into

as renin

of isoproter-

pathophysiologic to isoproterenol

to glomerular (43).

experiments

of isoproterenol

podocyte,

agowhich

Adrenoceptors

and

343

Podocytes

A

A 20

100

-

a)

Cl) (5

a)

16-

0 C

E C

0.

60

12-

0

(is

(5 N 5-

.xE 8-

(5

40’

Isoproterenol

-.--

Forskolin

-A--

Phenylephnne

(5

0

E

0.

I.-

a)

0

-U-

20’

0

4-

0 0

0-

io-

10.6

io-

2

1

Time

i0-

[mm]

B

[M]

Forskolin

1/2

Isoproterenot

16-

B

. 6-

Forskolmn Phenylephnne

-A-

0

E 0.

12

0. Cl) C

0

a)

0

C) C

(5

8-

a) U)

0-

(5

a)

C)

4-

0

C

V C 0

0 I

I

10-10

I

I

io-

iO

I

1

io-

io-

io-

Agonist EM] I

-

I

i0-

I

io-

10.6

Forskolin Figure

10. Concentration-response

Vm (A; tance

LVm,

curves

(B; Gm)

podocytes.

fl

I

I o-

I 0-5

EM] of forskolin

of whole-cell ±

Figure cAMP

11. Effect of isoproterenol, accumulation in podocytes. induced

increase

of the effect

and the change Mean values

depolarization)

I

SEM (n

=

on

conduc-

number

of

tLM forskolin preincubated

by 10 nM isoproterenol,

cAMP

data point).

to be

involved

in the

pathogenesis

of diabetic

pathy, may participate in the defective response nol occurring in diabetes mellitus (44).

In summary,

the effect

tions of podocytes nologic properties increase

[Ca2]1

adrenoceptor, an activation tion

of catecholamines

cAMP

in podocytes

on cellular

via an a1- and

Insel

/32-

Acknowledgments also

thank

was

supported

483/1-3).

Dr. Ivar

by the

Kugelgen Deutsche

for helpful

technical comments.

Forschungsgemeinschaft

assistance. This (DFG

2.

and forskolin.

data

of

After preincuto the time

point).

Snavely

Myers

BD,

MD:

function.

Deen

WM,

Catecholamines

Annu

Rev Physiol

Brenner

BM:

and the kidney:

Recep-

43: 625-636,

1989

Effects

of norepinephrine

and angiotensin II on the determinants of glomerular ultrafiltra. tion and proximal tubule fluid reabsorption in the rat. Circ Res 37: 101-110, 1975

We

Uchino

K, Nishikimi

blockade

work Pa

PA,

tors and renal

3. von

6 for each

6 for

=

of the increase

M IBMX for 5 mm, podocytes were exposed and the assay was terminated after an incubation

=

were for 5

References

of the cells.

von Hofer for excellent

(n

time

curves

I

func-

respectively. Stimulation of the receptors led to of a Cl conductance, resulting in a depolariza-

We thank Monika

Podocytes (IBMX)

for the indicated

phenylephrine,

and

to isoprotere-

with characteristic morphologic and immuwas examined in this study. Catecholamines and

the agents

by isoproterenol,

of 5 mm (n

nephro-

with

on

of the cAMP

1 p.M phenylephrine,

(B) Concentration-response

bation with 0.5 indicated agent, seems

and forskolin

in podocytes. Mean values ± SEM. with 0.5 M 3-isobutyl-I-methylxanthine

mm and stimulated each

experiments).

phenylephrine,

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Pavenst#{228}dt H,

Wanner

C, Schollmeyer

calcium

activity

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Yaoita

13.

inositol

epithelial

Kidney

epithelial

markers

107:

when

cultured

in vitro.

15.

Mundel

P, Reiser

W, Zeller

contacts ditionally

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L, Kioussis lines 17.

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proved

patch-clamp cells

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mouse.

Proc

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85-90,

1981

BJ:

Adrenergic

II.

Kidney

angiotensin Miner

Brayden

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calcium

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Rev

JA, Summers

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Electrolyte

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distribution

Turner

JT,

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in the human

Phar?nacol

cell

Bylund

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JD, Briner

VA,

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JH, Ferris

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44

TF: Studies

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