Impact

of

foliar

herbivory

on

the

development

of

a

root-feeding

insect

and

its

parasitoid

The

majority

of

studies

exploring

interactions

between

above

and

below-ground

biota

have

been

focused

on

the

effects

of

root-associated

organisms

on

foliar

herbivorous

insects

This

study

examined

the

effects

of

foliar

herbivory

by

Pieris

brassicae

L.

-LRB-

Lepidoptera

Pieridae

-RRB-

on

the

performance

of

the

root

herbivore

Delia

radicum

L.

-LRB-

Diptera

Anthomyiidae

-RRB-

and

its

parasitoid

Trybliographa

rapae

-LRB-

Westwood

-RRB-

-LRB-

Hymenoptera

Figitidae

-RRB-

mediated

through

a

shared

host

plant

Brassica

nigra

L.

-LRB-

Brassicaceae

-RRB-

In

the

presence

of

foliar

herbivory

the

survival

of

D.

radicum

and

T.

rapae

decreased

significantly

by

more

than

In

addition

newly

emerged

adults

of

both

root

herbivores

and

parasitoids

were

significantly

smaller

on

plants

that

had

been

exposed

to

foliar

herbivory

than

on

control

plants

To

determine

what

factor

-LRB-

s

-RRB-

may

have

accounted

for

the

observed

results

we

examined

the

effects

of

foliar

herbivory

on

root

quantity

and

quality

No

significant

differences

in

root

biomass

were

found

between

plants

with

and

without

shoot

herbivore

damage

Moreover

concentrations

of

nitrogen

in

root

tissues

were

also

unaffected

by

shoot

damage

by

P.

brassicae

larvae

However

higher

levels

of

indole

glucosinolates

were

measured

in

roots

of

plants

exposed

to

foliar

herbivory

suggesting

that

the

development

of

the

root

herbivore

and

its

parasitoid

may

be

at

least

partly

negatively

affected

by

increased

levels

of

these

allelochemicals

in

root

tissues

Our

results

show

that

foliar

herbivores

can

affect

the

development

not

only

of

root-feeding

insects

but

also

their

natural

enemies

We

argue

that

such

indirect

interactions

between

above

and

below-ground

biota

may

play

an

important

role

in

the

structuring

and

functioning

of

communities

Introduction

Over

the

past

years

studies

exploring

community-level

processes

have

been

based

primarily

on

bi

and

tri-trophic

interactions

in

above-ground

systems

usually

involving

associations

among

plants

insect

herbivores

and

their

natural

enemies

such

as

parasitoids

or

predators

-LRB-

Price

et

al.

Turlings

et

al.

Vet

and

Dicke

Dicke

-RRB-

While

providing

valuable

data

on

a

range

of

selective

forces

shaping

community

structure

and

function

these

studies

have

generally

ignored

biological

processes

that

occur

below-ground

Recently

however

it

has

become

apparent

that

above-ground

trophic

interactions

can

be

strongly

influenced

by

interactions

between

the

host

plant

and

soil-dwelling

organisms

-LRB-

Gange

and

Brown

Masters

and

Brown

Masters

et

al.

Masters

Gange

Van

der

Putten

et

al.

Bardgett

and

Wardle

Bezemer

et

al.

-RRB-

For

instance

recent

studies

have

shown

that

soil-dwelling

organisms

such

as

root-feeding

insects

arbuscular

mycorrhiza

fungi

and

nematodes

can

also

influence

the

growth

development

and

behavior

of

higher

trophic

levels

below-ground

including

parasitoids

-LRB-

Masters

et

al.

Gange

et

al.

Wurst

and

Jones

Bezemer

et

al.

Guerrieri

et

al.

-RRB-

and

even

hyperparasitoids

in

the

fourth

trophic

level

-LRB-

Soler

et

al.

-RRB-

It

is

also

known

that

above-ground

herbivory

can

affect

the

development

of

below-ground

insect

herbivores

-LRB-

reviewed

by

Blossey

and

Hunt-Joshi

-RRB-

when

the

shared

host

plant

has

previously

been

exposed

to

intense

shoot

damage

For

example

Tindall

and

Stout

-LRB-

-RRB-

reported

that

severe

damage

by

insect

foliar

herbivores

was

correlated

with

a

significant

reduction

in

the

body

size

of

an

insect

herbivore

feeding

on

root

tissues

of

the

same

host

plant

compared

with

conspecifics

developing

on

roots

of

plants

without

foliar

herbivores

Other

studies

have

shown

similar

effects

-LRB-

Masters

et

al.

Hunt-Joshi

and

Blossey

-RRB-

However

the

effects

of

foliar

herbivory

on

the

development

of

natural

enemies

in

the

soil

such

as

predators

and

parasitoids

remain

largely

unexplored

Thus

far

the

effects

of

above-ground

herbivory

on

the

performance

of

below-ground

consumers

have

been

explained

mainly

by

quantitative

changes

in

the

amount

of

root

biomass

when

an

extreme

amount

of

leaf

damage

results

in

partial

or

complete

death

of

the

root

system

However

minor

levels

of

shoot

damage

while

not

leading

to

a

significant

reduction

in

root

biomass

may

still

affect

the

quality

of

the

root

tissues

influencing

the

development

of

root-associated

organisms

-LRB-

reviewed

in

Bezemer

and

Van

Dam

-RRB-

Furthermore

interactions

between

above

and

below-ground

herbivores

may

also

be

mediated

by

qualitative

changes

in

the

shared

host

plant

For

example

insect

herbivory

frequently

leads

to

a

systemic

defense

response

within

the

host

plant

resulting

in

enhanced

concentrations

of

secondary

plant

compounds

in

plant

organs

that

have

not

been

damaged

by

herbivory

-LRB-

Karban

and

Baldwin

-RRB-

Secondary

plant

compounds

commonly

have

a

negative

impact

on

herbivore

performance

-LRB-

Karban

and

Baldwin

-RRB-

Several

studies

have

shown

that

root

herbivory

can

result

in

enhanced

concentrations

of

secondary

plant

compounds

in

the

foliage

although

the

reverse

e.g.

the

effects

of

foliar

herbivory

on

changes

in

concentrations

of

root

secondary

plant

compounds

has

rarely

been

reported

-LRB-

reviewed

in

Bezemer

and

Van

Dam

-RRB-

Moreover

herbivory

can

induce

a

stress

response

within

the

host

plant

which

can

lead

to

a

reallocation

of

plant

compounds

such

as

carbohydrates

and

soluble

nitrogen

between

root

and

shoot

tissues

and

the

concentration

of

these

compounds

is

known

to

directly

affect

insect

growth

parameters

-LRB-

Masters

et

al.

Masters

and

Brown

-RRB-

It

is

important

to

stress

that

the

mechanisms

described

above

are

not

necessarily

mutually

exclusive

In

this

study

we

examined

the

effects

of

above-ground

herbivory

on

the

development

and

survival

of

a

below-ground

insect

herbivore

and

its

endoparasitoid

Our

study

is

based

on

interactions

involving

a

naturally

occurring

system

in

Western

Europe

The

black

mustard

Brassica

nigra

L.

-LRB-

Brassicaceae

-RRB-

is

a

widely

distributed

annual

crucifer

that

is

common

along

rivers

and

on

disturbed

sites

in

The

Netherlands

-LRB-

Schaminee

et

al.

-RRB-

It

is

attacked

by

several

adapted

specialist

herbivores

including

the

large

cabbage

white

butterfly

Pieris

brassicae

L.

-LRB-

Lepidoptera

Pieridae

-RRB-

whose

larvae

feed

on

the

shoots

and

flowers

of

this

and

related

species

-LRB-

Harvey

et

al.

-RRB-

Mustard

plants

are

also

attacked

by

specialized

root-feeding

herbivores

including

the

cabbage

root

fly

Delia

radicum

L.

-LRB-

Diptera

Anthomyiidae

-RRB-

-LRB-

Coaker

and

Finch

-RRB-

Trybliographa

rapae

-LRB-

Westwood

-RRB-

-LRB-

Hymenoptera

Figitidae

-RRB-

is

one

of

the

main

parasitoids

of

D.

radicum

in

Europe

-LRB-

Lahmar

-RRB-

The

main

aim

of

this

investigation

was

to

determine

whether

the

growth

development

and

survival

of

D.

radicum

and

its

endoparasitoid

T.

rapae

are

affected

by

foliar

herbivory

by

P.

brassicae

We

also

examined

whether

above-ground

herbivory

leads

to

qualitative

and/or

quantitative

differences

in

root

tissues

We

compared

levels

of

root

biomass

in

plants

with

and

without

shoot

damage

as

well

as

levels

of

nitrogen

in

root

tissues

Finally

we

compared

levels

of

allelochemicals

-LRB-

glucosinolates

-RRB-

in

root

tissues

of

plants

under

the

two

treatments

We

hypothesized

that

above-ground

herbivory

by

P.

brassicae

would

result

in

reduced

root-plant

quality

which

would

negatively

affect

the

performance

of

D.

radicum

Because

T.

rapae

is

a

koinobiont

parasitoid

D.

radicum

larvae

continue

feeding

and

growing

during

much

of

the

parasitoid

host

interaction

-LRB-

reviewed

in

Harvey

-RRB-

As

a

consequence

of

this

close

association

between

host

and

parasitoid

we

hypothesized

that

the

effects

of

P.

brassicae

on

D.

radicum

would

be

carried

over

to

T.

rapae

Since

parasitoids

are

highly

susceptible

to

very

small

changes

in

the

quality

of

the

hosts

the

effect

on

the

parasitoid

should

be

stronger

than

that

on

the

host

Materials

and

methods

Studied

species

Brassica

nigra

like

other

members

of

the

Brassicaceae

possesses

potent

inducible

direct

defenses

via

the

production

of

glucosinolates

and

their

breakdown

products

thus

the

plant

provides

excellent

potential

for

the

study

of

multitrophic

interactions

-LRB-

Van

Dam

et

al.

-RRB-

Pieris

brassicae

is

a

specialist

chewing

foliar

herbivore

that

feeds

on

several

species

of

wild

and

cultivated

crucifers

The

female

butterfly

typically

lays

broods

of

eggs

on

the

underside

of

a

leaf

After

hatching

the

larvae

feed

gregariously

until

the

third

instar

when

they

disperse

on

the

food

plant

Delia

radicum

is

a

specialist

chewing

root

herbivore

that

feeds

on

several

species

of

crucifers

Females

oviposit

near

the

root-stem

interface

The

newly

hatched

maggots

feed

by

boring

galleries

in

the

upper

roots

of

the

plant

Trybliographa

rapae

is

a

fairly

specialized

solitary

koinobiont

endoparasitoid

that

parasitizes

instar

larvae

of

D.

radicum

as

well

as

several

closely

related

species

Adult

parasitoid

females

forage

via

antennation

ovipositor

probing

and

vibrotaxis

in

order

to

find

their

hosts

-LRB-

Vet

and

Van

Alphen

-RRB-

The

wasps

readily

parasitize

larvae

that

are

actively

feeding

in

the

root

galleries

but

refuse

hosts

extracted

from

the

root

-LRB-

Jones

-RRB-

Females

use

their

sharp

but

flexible

ovipositor

to

explore

infested

roots

to

find

hosts

and

evaluate

their

quality

-LRB-

Neveu

et

al.

-RRB-

Seeds

of

B.

nigra

were

collected

from

a

single

population

in

the

Northwest

of

Wageningen

The

Netherlands

Seeds

were

surface

sterilized

and

germinated

on

a

bed

of

glass

pearls

-LRB-

pearls

of

mm

diameter

-RRB-

One

week

after

germination

seedlings

were

transplanted

into

pots

filled

with

soil

collected

from

a

restoration

area

that

was

abandoned

in

at

De

Mossel

in

Ede

The

Netherlands

The

sandy

loam

soil

was

sieved

-LRB-

cm

-RRB-

and

mixed

with

white

sand

-LRB-

to

improve

the

drainage

of

the

soil

-RRB-

and

sterilized

using

gamma

radiation

-LRB-

KGray

-RRB-

to

eliminate

all

soil

organisms

The

plants

were

grown

in

a

greenhouse

at

relative

humidity

at

°C

-LRB-

day

-RRB-

and

°C

-LRB-

night

-RRB-

on

a

day

night

cycle

Natural

daylight

was

provided

by

metal

halide

lamps

-LRB-

μmol

s

m

PAR

lamp

per

-RRB-

Plants

were

watered

daily

and

supplemented

with

nutrients

-LRB-

Hoagland

solution

-RRB-

once

when

the

plants

were

weeks

old

Pieris

brassicae

was

obtained

from

the

insect

culture

maintained

at

the

Laboratory

of

Entomology

of

Wageningen

University

The

Netherlands

D.

radicum

and

T.rapae

were

obtained

from

insect

cultures

maintained

at

the

Laboratory

of

Ecobiology

of

Insect

Parasitoids

of

Rennes

University

France

Experimental

design

and

insect

measurements

To

test

the

effect

of

above-ground

herbivores

on

the

performance

of

soil

insects

we

reared

D.

radicum

and

its

parasitoid

T.

rapae

on

roots

of

plants

previously

exposed

-LRB-

treatment

-RRB-

and

not

exposed

-LRB-

control

-RRB-

to

P.

brassicae

larvae

Twenty-five

days

after

transplanting

three

newly

hatched

larvae

of

P.

brassicae

were

carefully

placed

with

a

small

brush

on

the

youngest

fully

developed

leaf

of

each

plant

and

enclosed

in

a

cylindrical

clip

cage

-LRB-

cm

diameter

-RRB-

in

order

to

ensure

that

the

damage

was

localized

After

days

the

larvae

were

moved

to

another

new

leaf

on

the

plant

The

larvae

were

allowed

to

feed

for

a

further

days

after

which

each

larva

and

clip

cage

was

removed

Thus

each

foliar-damaged

plant

was

exposed

to

feeding

by

three

P.

brassicae

larvae

for

a

total

of

days

A

second

set

of

plants

was

kept

undamaged

and

served

as

control

To

standardize

the

possible

effects

of

the

clip

cages

on

plant

shoots

they

were

also

placed

on

control

plants

following

the

same

methods

as

for

the

foliar-damaged

plants

A

total

of

plants

were

used

in

the

experiment

-LRB-

plants

with

and

without

foliar

herbivore

damage

-RRB-

One

day

after

removing

the

P.

brassicae

larvae

four

D.

radicum

larvae

were

introduced

to

each

plant

by

carefully

placing

the

larvae

with

a

brush

onto

the

soil

surface

immediately

adjacent

to

the

stem

Using

a

magnifying

glass

each

plant

was

carefully

checked

min

after

inoculation

to

ensure

that

all

the

root

fly

larvae

had

successfully

entered

into

the

soil

For

each

plant

two

of

the

larvae

were

healthy

and

two

had

been

parasitized

by

T.

rapae

within

approximately

the

prior

h

In

a

previous

study

we

reported

that

there

was

no

significant

difference

in

final

root

biomass

in

B.

nigra

plants

with

initial

densities

of

five

unparasitized

D.

radicum

larvae

per

plant

when

compared

with

undamaged

plants

-LRB-

Soler

at

al.

-RRB-

Furthermore

parasitized

larvae

consume

less

plant

material

and

attain

smaller

final

masses

than

healthy

hosts

-LRB-

Grandgirard

-RRB-

thus

we

assumed

that

plant

root

biomass

would

not

be

a

limiting

factor

for

root

herbivore/parasitoid

development

It

is

also

important

to

stress

that

larvae

of

D.

radicum

are

not

cannibalistic

-LRB-

A.M.

Cortesero

unpublished

observations

-RRB-

making

it

possible

for

unparasitized

and

parasitized

larvae

to

share

a

host

plant

To

obtain

parasitized

D.

radicum

larvae

approximately

first

instar

larvae

were

offered

to

mated

T.

rapae

females

over

the

course

of

h.

To

facilitate

and

ensure

high

rates

of

parasitism

a

slice

of

B.

napus

-LRB-

turnip

-RRB-

root

of

cm

in

height

-LRB-

cm

diameter

-RRB-

that

had

previously

been

infested

with

the

larvae

of

D.

radicum

was

offered

to

the

parasitoid

females

The

larvae

were

collected

from

the

root

after

h

and

larvae

were

then

transferred

to

the

experimental

B.

nigra

plants

Because

direct

observations

of

parasitism

are

almost

impossible

in

T.

rapae

additional

larvae

from

the

infested

turnip

slice

were

removed

and

placed

on

a

new

turnip

where

parasitoid

emergence

was

monitored

From

these

larvae

T.

rapae

adults

emerged

and

produced

D.

radicum

adults

Following

the

below-ground

inoculation

of

unparasitized

and

parasitized

D.

radicum

larvae

experimental

plants

were

placed

individually

into

large

meshed

cylindrical

cages

-LRB-

height

m

diameter

cm

-RRB-

to

ensure

that

newly

emerged

adult

flies

and

wasps

did

not

escape

and

could

be

immediately

captured

Plants

from

the

two

treatments

were

randomly

distributed

in

the

greenhouse

and

were

moved

around

the

room

every

days

to

avoid

any

potential

effects

of

positioning

on

insect

development

The

cages

were

checked

twice

daily

-LRB-

once

in

the

morning

and

once

in

the

afternoon

-RRB-

to

record

the

emergence

of

adult

flies

and

parasitoids

Newly

emerged

adults

of

D.

radicum

and

T.

rapae

were

collected

with

an

aspirator

At

emergence

the

following

data

were

recorded

-LRB-

-RRB-

development

time

between

inoculation

and

adult

eclosion

in

days

and

-LRB-

-RRB-

adult

body

size

based

on

the

measurement

of

hind

tibia

length

in

both

the

herbivore

and

parasitoid

Hind

tibia

length

has

been

commonly

used

as

a

measure

of

adult

size

in

many

insects

especially

parasitoids

-LRB-

Godfray

Harvey

et

al.

Bezemer

and

Mills

-RRB-

The

hind

tibia

lengths

of

D.

radicum

and

T.

rapae

were

measured

using

a

calibrated

slide

under

a

stereomicroscope

Adult

survival

-LRB-

calculated

as

the

proportion

of

insects

that

emerged

relative

to

the

number

of

larvae

introduced

in

each

pot

-RRB-

was

also

recorded

Plant

responses

Because

the

measurement

of

glucosinolate

and

nitrogen

concentrations

requires

the

destructive

sampling

of

plants

root

samples

were

taken

from

an

additional

set

of

plants

-LRB-

nine

plants

per

treatment

-RRB-

The

plants

in

the

additional

set

were

treated

in

exactly

the

same

way

as

the

foliar-damaged

and

control

plants

described

above

Root

material

was

collected

h

before

the

other

set

of

experimental

plants

was

inoculated

with

parasitized

and

unparasitized

D.

radicum

larvae

to

evaluate

the

quality

of

the

control

and

foliar-damaged

plants

that

larvae

encountered

after

inoculation

After

carefully

washing

the

roots

of

each

plant

the

fine

roots

were

separated

from

the

main

root

Roots

less

than

mm

thick

were

considered

fine

-LRB-

with

a

hair-like

appearance

-RRB-

and

those

thicker

than

mm

were

considered

to

be

main

roots

The

roots

were

immediately

frozen

at

°C

then

freeze-dried

and

ground

Glucosinolate

contents

were

determined

using

high

performance

liquid

chromatography

-LRB-

HPLC

-RRB-

as

described

by

Van

Dam

et

al.

-LRB-

-RRB-

Nitrogen

content

was

determined

using

a

Carlo

Erba/Fisons

type

CHN

analyzer

The

total

root

biomass

of

the

experimental

plants

was

determined

at

the

end

of

the

experiment

The

roots

of

each

plant

were

harvested

oven-dried

at

°C

and

subsequently

weighed

on

a

microbalance

Statistical

analysis

The

effects

of

foliar

herbivory

-LRB-

with

or

without

-RRB-

and

larval

status

-LRB-

parasitized

or

unparasitized

-RRB-

on

survival

of

D.

radicum

and

T.

rapae

were

tested

using

a

generalized

linear

mixed

model

-LRB-

with

binomial

distribution

and

logit

link

function

including

plant

as

random

term

-RRB-

The

effects

of

foliar

herbivory

on

developmental

time

and

hind

tibia

length

of

D.

radicum

and

T.

rapae

were

tested

using

a

generalized

linear

model

-LRB-

with

normal

distribution

and

identity

link

function

-RRB-

The

effects

of

foliar

herbivory

and

root

type

-LRB-

primary

or

secondary

roots

-RRB-

on

plant

quality

-LRB-

nitrogen

and

glucosinolate

levels

-RRB-

were

tested

using

a

linear

mixed

model

including

plant

as

random

term

to

consider

that

the

fine

and

main

roots

were

sampled

from

the

same

plants

To

test

the

effect

of

foliar

herbivory

on

plant

quantity

-LRB-

root

biomass

-RRB-

we

used

analysis

of

variance

Normality

independence

and

homogeneity

of

variance

were

checked

by

inspection

of

the

residuals

after

model

fitting

Analyses

were

carried

out

in

Statistica

and

Genstat

Results

Foliar

herbivory

significantly

affected

the

survival

of

the

root

herbivore

D.

radicum

and

its

parasitoid

T.

rapae

-LRB-

Wald

P

-RRB-

Relative

to

control

plants

the

survival

of

D.

radicum

and

T.

rapae

decreased

by

more

than

when

the

host

plants

were

previously

exposed

to

foliar

herbivory

-LRB-

Fig.

-RRB-

and

the

effect

was

qualitatively

similar

for

root

herbivore

and

parasitoid

responses

indicated

by

a

non-significant

interaction

term

-LRB-

Wald

P

-RRB-

There

was

a

significant

effect

of

foliar

herbivory

on

D.

radicum

and

T.

rapae

adult

size

measured

as

hind

tibia

length

-LRB-

Wald

P

-RRB-

Both

herbivore

and

parasitoid

adult

sizes

were

reduced

in

the

presence

of

foliar

herbivores

on

the

shared

host

plant

-LRB-

Fig.

-RRB-

Again

the

negative

effect

of

foliar

herbivory

did

not

differ

between

host

and

parasitoid

-LRB-

interaction

term

Wald

P

-RRB-

Development

times

of

D.

radicum

and

T.

rapae

were

not

significantly

affected

by

foliar

herbivory

-LRB-

Wald

P

-RRB-

Fig.

-LRB-

SE

-RRB-

survival

of

D.

radicum

-LRB-

herbivore

-RRB-

and

T.

rapae

-LRB-

parasitoid

-RRB-

on

B.

nigra

plants

that

had

been

exposed

to

foliar

herbivory

-LRB-

by

P.

brassicae

-RRB-

-LRB-

shaded

bars

-RRB-

and

on

control

plants

-LRB-

open

bars

-RRB-

Within

herbivore

and

parasitoid

significant

differences

-LRB-

P

-RRB-

between

treatments

are

indicated

by

different

lettersFig

-LRB-

SE

-RRB-

hind

tibia

length

of

D.

radicum

-LRB-

herbivore

-RRB-

and

T.

rapae

-LRB-

parasitoid

-RRB-

on

B.

nigra

plants

that

had

been

exposed

to

foliar

herbivory

-LRB-

by

P.

brassicae

-RRB-

-LRB-

shaded

bars

-RRB-

and

on

control

plants

-LRB-

open

bars

-RRB-

Within

herbivore

and

parasitoid

significant

differences

-LRB-

P

-RRB-

between

treatments

are

indicated

by

different

letters

Plants

under

both

treatments

demonstrated

similar

root

biomass

-LRB-

P

Fig.

-RRB-

Root-nitrogen

levels

also

did

not

differ

significantly

between

control

plants

and

plants

exposed

to

foliar

herbivory

-LRB-

Table

-RRB-

Independent

of

the

treatment

nitrogen

levels

were

significantly

lower

in

secondary

than

in

primary

roots

-LRB-

Table

-RRB-

Foliar

herbivory

significantly

affected

the

levels

of

indole

glucosinolates

-LRB-

neoglucobrassicin

and

glucobrassicin

-RRB-

in

the

roots

-LRB-

Table

-RRB-

In

the

presence

of

foliar

herbivory

the

levels

of

neoglucobrassicin

-LRB-

Fig.

-RRB-

and

glucobrassicin

-LRB-

Fig.

-RRB-

significantly

increased

in

both

primary

and

secondary

roots

Independent

of

the

treatment

levels

of

neoglucobrassicin

and

glucobrassicin

were

significantly

higher

in

secondary

than

in

primary

roots

Concentrations

of

gluconasturtin

-LRB-

Fig.

-RRB-

and

sinigrin

-LRB-

Fig.

-RRB-

did

not

differ

significantly

between

the

roots

of

control

plants

and

plants

that

had

been

exposed

to

foliar

herbivory

-LRB-

Table

-RRB-

Independent

of

the

treatment

gluconasturtin

levels

were

significantly

higher

in

primary

than

in

secondary

roots

Sinigrin

levels

did

not

differ

significantly

between

primary

and

secondary

roots

Fig.

-LRB-

SE

-RRB-

root

biomass

-LRB-

a

-RRB-

and

percentage

of

nitrogen

in

primary

and

secondary

roots

-LRB-

b

-RRB-

of

Brassica

nigra

plants

that

had

been

exposed

to

foliar

herbivory

-LRB-

by

P.

brassicae

-RRB-

-LRB-

shaded

bars

-RRB-

and

in

those

of

control

plants

-LRB-

open

bars

-RRB-

Table

F-test

-LRB-

F

and

P

values

-RRB-

for

the

fixed

effects

and

variance

component

estimates

from

REML

-LRB-

restricted

maximum

likelihood

-RRB-

analysis

of

the

effect

of

foliar

herbivory

-LRB-

by

P.

brassicae

-RRB-

and

root-type

on

nitrogen

and

glucosinolate

levels

of

B.

nigra

rootsFactorsNitrogenNeoglucobrassicinGlucobrassicinGluconasturtinSinigrinFPFPFPFPFPFoliar

herbivory

-LRB-

-RRB-

-LRB-

-RRB-

Interaction

-LRB-

-RRB-

Fig.

-LRB-

SE

-RRB-

levels

of

glucosinolates

-LRB-

μmol

g

dry

mass

-RRB-

-LRB-

a

neoglucobrassicin

b

glucobrassicin

c

gluconasturtin

d

sinigrin

-RRB-

in

primary

and

secondary

roots

of

Brassica

nigra

plants

exposed

to

foliar

herbivory

-LRB-

by

P.

brassicae

-RRB-

-LRB-

shaded

bars

-RRB-

and

in

those

of

control

plants

-LRB-

open

bars

-RRB-

Within

each

root

type

significant

differences

-LRB-

P

-RRB-

between

treatments

are

indicated

by

different

letters

Discussion

This

study

provides

evidence

that

shoot-feeding

insects

via

systemic

changes

in

the

root

quality

of

the

shared

host

plant

can

influence

the

development

of

root-feeding

insects

sharing

the

host

plant

Moreover

the

effects

can

be

carried

over

at

least

one

trophic

level

higher

affecting

the

performance

of

a

parasitoid

We

found

that

foliar

herbivory

was

significantly

correlated

with

a

reduction

in

successful

development

of

Deliaradicum

and

Trybliographarapae

Survival

of

the

herbivore

and

its

parasitoid

was

two

to

three

times

higher

on

plants

that

were

undamaged

above-ground

than

on

plants

that

were

previously

exposed

to

foliar

herbivory

In

addition

when

compared

with

those

on

control

plants

newly

emerged

adult

D.

radicum

and

T.

rapae

were

significantly

smaller

when

the

insects

developed

on

plants

that

had

been

initially

damaged

by

foliar

herbivores

Several

authors

have

argued

that

body

size

is

the

main

target

of

selection

in

parasitoids

and

other

insects

because

it

is

correlated

with

life-history

and

demographic

characters

such

as

searching

and

mating

efficiency

longevity

and

fecundity

-LRB-

Godfray

Harvey

et

al.

Bezemer

and

Mills

Harvey

-RRB-

Consequently

our

results

reveal

not

only

that

D.

radicum

and

T.

rapae

suffer

from

increased

mortality

on

plants

previously

exposed

to

foliar

herbivory

compared

with

control

plants

but

that

fitness

of

the

surviving

adults

is

also

compromised

In

a

recent

study

based

on

the

same

multitrophic

system

we

reported

that

root

herbivory

by

D.

radicum

reduced

the

performance

of

the

foliar

feeder

P.

brassicae

-LRB-

Soler

et

al.

-RRB-

However

several

others

found

no

significant

relationship

between

root

herbivory

and

the

performance

of

above-ground

herbivores

-LRB-

Moran

and

Whitham

Salt

et

al.

Hunt-Joshi

and

Blossey

-RRB-

and

yet

others

found

a

positive

relationship

-LRB-

Masters

and

Brown

-RRB-

These

results

clearly

show

that

the

effects

of

below-ground

herbivores

on

their

above-ground

counterparts

are

likely

to

be

association-specific

although

this

contrasts

with

the

rather

consistent

negative

effect

thus

far

observed

in

most

reciprocal

studies

of

above-ground

herbivory

on

below-ground

insects

-LRB-

this

study

Moran

and

Whitham

Masters

and

Brown

Salt

et

al.

Tindall

and

Stout

Hunt-Joshi

and

Blossey

-RRB-

More

work

is

clearly

required

to

elucidate

possible

patterns

in

interactions

between

above

and

below-ground

systems

and

to

determine

whether

there

are

any

clear

patterns

emerging

from

them

We

also

found

that

the

amount

of

root

biomass

did

not

differ

significantly

between

the

foliar-damaged

and

control

plants

suggesting

that

the

negative

effects

of

above-ground

herbivory

by

P.

brassicae

on

the

development

of

D.

radicum

and

T.

rapae

were

based

on

qualitative

rather

than

quantitative

changes

in

the

root

system

Because

only

three

young

caterpillars

were

allowed

to

feed

from

the

foliage

for

a

limited

period

of

time

the

physical

amount

of

shoot

tissue

removed

was

negligible

-LRB-

R.

Soler

personal

observation

-RRB-

Changes

in

root

biomass

as

a

result

of

source

sink

relationships

within

the

plant

are

likely

to

occur

when

above-ground

herbivore

damage

is

much

more

extensive

-LRB-

Hunt-Joshi

and

Blossey

-RRB-

Root

nitrogen

levels

were

also

not

significantly

affected

by

foliar

herbivory

and

thus

it

is

highly

unlikely

that

changes

in

this

important

-LRB-

and

often

limiting

-RRB-

primary

plant

compound

were

responsible

for

the

observed

effects

However

levels

of

indole

glucosinolates

were

significantly

increased

by

about

in

plants

that

had

been

exposed

to

foliar

herbivores

Glucosinolates

and

their

breakdown

products

including

indoles

have

been

shown

to

be

involved

in

reducing

plant

quality

for

both

generalist

and

specialist

insect

herbivores

and

nematodes

-LRB-

Li

et

al.

Van

Dam

et

al.

-RRB-

Although

indoles

constituted

only

a

small

fraction

of

total

glucosinolates

in

the

plant

-LRB-

with

sinigrin

being

by

far

the

most

prevalent

-RRB-

studies

with

other

secondary

compounds

have

shown

that

only

trace

amounts

in

the

diet

are

required

to

exert

strong

effects

on

the

development

of

herbivores

and

their

parasitoids

-LRB-

Barbosa

et

al.

-RRB-

Consequently

the

negative

relationship

between

above-ground

herbivory

and

the

development

of

D.

radicum

may

be

at

least

partially

correlated

with

increased

levels

of

indole

glucosinolates

in

the

roots

of

the

shared

host

plant

that

were

induced

by

feeding

damage

from

P.

brassicae

larvae

above

ground

It

has

been

well

established

that

allelochemicals

in

the

herbivore

's

diet

can

exhibit

negative

effects

on

the

morphology

development

size

and

survival

of

their

predators

and

parasitoids

-LRB-

reviewed

in

Harvey

Ode

-RRB-

T.

rapae

is

a

koinobiont

parasitoid

of

D.

radicum

and

therefore

after

being

parasitized

Delia

larvae

continue

feeding

and

growing

until

the

final

larval

instar

has

been

reached

At

this

time

the

developing

parasitoid

larvae

begin

destructive

feeding

and

exponential

growth

-LRB-

Harvey

et

al.

-RRB-

At

the

end

of

the

host

's

larval

development

allelochemicals

that

may

be

present

in

the

hemolymph

and

other

tissues

such

as

fat

body

are

rapidly

consumed

by

the

koinobiont

parasitoid

-LRB-

Harvey

-RRB-

Due

to

the

tight

linkage

between

host

and

koinobiont

parasitoid

development

in

our

study

increased

mortality

and

reduced

adult

size

observed

in

the

T.

rapae

adults

on

plants

that

had

been

exposed

to

foliar

herbivory

could

probably

be

attributed

to

a

corresponding

increased

mortality

and

reduced

size

of

its

host

D.

radicum

Parasitoids

in

particular

are

highly

susceptible

to

very

small

changes

in

the

quality

of

the

host

's

internal

biochemical

environment

-LRB-

Harvey

-RRB-

Other

studies

have

shown

that

parasitoids

can

be

more

affected

than

their

hosts

by

the

quality

of

the

host

plant

-LRB-

Harvey

et

al.

Soler

et

al.

-RRB-

This

appears

not

to

be

the

case

in

our

studied

trophic

chain

since

we

found

comparable

effects

of

foliar

herbivory

on

the

root

herbivore

and

its

parasitoid

In

summary

this

study

has

reported

that

herbivore

damage

of

plant

shoots

can

have

significant

negative

effects

on

the

development

of

below-ground

herbivores

and

their

natural

enemies

Combined

with

the

results

of

a

previous

study

based

on

the

same

system

we

have

demonstrated

that

these

effects

are

largely

reciprocal

-LRB-

Soler

et

al.

-RRB-

Our

research

further

highlights

the

importance

of

integrating

the

below-ground

domain

with

above-ground

systems

in

order

to

better

understand

the

myriad

of

factors

that

shape

the

evolution

assembly

and

functioning

of

communities

and

ecosystems

-LRB-

Wardle

et

al.

-RRB-

Studies

over

larger

spatial

scales

have

already

shown

that

above-ground

herbivores

can

influence

the

community

structure

of

the

below-ground

system

and

vice-versa

-LRB-

De

Deyn

and

Van

der

Putten

Wardle

et

al.

-RRB-

Further

studies

combining

interactions

between

above-ground

and

below-ground

communities

in

a

multitrophic

framework

and

which

seek

to

explore

both

processes

and

mechanisms

are

required

if

we

are

to

develop

a

better

understanding

of

the

many

complex

factors

that

influence

community

structure

and

function

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

delia radicum
glucosinolates
trybliographa rapae

above-/below-ground interactions
plant–insect interactions


Molecular mechanism of edema formation in nephrotic syndrome : therapeutic implications Sodium retention and edema are common features of nephrotic syndrome that are classically attributed to hypovolemia and activation of the renin -- angiotensin -- aldosterone system . However , numbers of clinical and experimental findings argue against this underfill theory . In this review we analyze data from the literature in both nephrotic patients and experimental models of nephrotic syndrome that converge to demonstrate that sodium retention is not related to the renin -- angiotensin -- aldosterone status and that fluid leakage from capillary to the interstitium does not result from an imbalance of Starling forces , but from changes of the intrinsic properties of the capillary endothelial filtration barrier . We also discuss how most recent findings on the cellular and molecular mechanisms of sodium retention has allowed the development of an efficient treatment of edema in nephrotic patients . Introduction Interstitial edema is a common clinical feature of nephrotic syndrome -LRB- NS -RRB- . It is often massive -LRB- up to 30 % of body weight -RRB- and constitutes a functional constraint , owing to locomotive restriction and eyelid shutting . Expansion of the interstitial compartment volume results from the combination of abnormal renal sodium retention and alterations of fluid transfer across capillary walls . Renal retention of sodium in nephrotic syndrome Site of sodium retention Most of our current knowledge on the site and mechanism of renal sodium retention in NS comes from experimental models of the disease , in particular the puromycin aminonucleoside -LRB- PAN -RRB- rat model . Following a single injection of PAN , rats develop massive proteinuria within 4 -- 5 days and sodium retention within 2 -- 3 days . Sodium excretion resumes after 9 -- 10 days , while proteinuria lasts for 2 -- 3 weeks -LSB- 1 , 2 -RSB- . Unilateral NS can be induced by the injection of PAN into one of the renal arteries -LSB- 3 -RSB- . Early in vivo micropuncture studies in rats with unilateral PAN nephrosis demonstrated that sodium retention in the nephrotic kidney originates beyond the last nephron segment accessible to micropuncture , i.e. , between the late distal convoluted tubule and the tip of the collecting duct -LSB- 3 -RSB- . This part of the nephron encompasses the connecting tubule and the cortical and outer medullary collecting ducts , which reabsorb sodium and are the major sites of the adjustment of sodium balance under the homeostatic control of aldosterone , and the inner medullary collecting duct , which is able to secrete an overload of sodium in response to atrial natriuretic peptide -LSB- 4 -RSB- . Methods allowing in vitro analysis of isolated sub-segments of the distal nephron demonstrated a marked stimulation of sodium reabsorption in the cortical collecting duct -LRB- CCD -RRB- of PAN nephrotic rats -LSB- 1 , 5 -RSB- . This stimulation of sodium transport likely extends upstream to the connecting duct but not downstream to the outer medullary collecting duct . In addition , in NS , the inner medullary collecting duct becomes insensitive to the natriuretic action of atrial natriuretic peptide -LSB- 6 -- 8 -RSB- , thereby preventing any compensation of sodium retention in upstream nephron segments . Cellular mechanism of sodium retention in the collecting duct In vitro microperfusion of isolated CCDs confirmed that the rate of sodium reabsorption and the trans-epithelial voltage are negligible in CCDs from control rats , whereas both are high in CCDs from PAN nephrotic rats -LSB- 5 -RSB- . CCDs are made of principal and intercalated cells , which account for sodium and water reabsorption and potassium secretion for principal cells , and proton , bicarbonate and likely chloride transport for intercalated cells -LSB- 4 -RSB- . In principal cells , sodium reabsorption proceeds along a two-step mechanism -LRB- Fig. 1 -RRB- : Na,K-ATPase , exclusively present in the basolateral membrane , energizes the active extrusion of sodium at the expense of ATP hydrolysis . The electrochemical gradient for sodium generated by this primary process drives passive entry of sodium through the apical membrane , which contains selective sodium channels -LRB- epithelial sodium channels , ENaCs -RRB- . Both Na,K-ATPase and ENaCs are targets for multiple , and often coordinated , regulations in CCDs -LSB- 4 -RSB- . Increased sodium reabsorption along the CCDs of PAN nephrotic rats , as well as sodium retention and edema formation , are associated with stimulation of both basolateral Na,K-ATPase -LSB- 1 , 9 , 10 -RSB- and apical ENaC -LSB- 11 -- 13 -RSB- -LRB- Fig. 1 -RRB- , but the latter is dispensable -LRB- see below -RRB- . Stimulation of Na , KATPase is fully accounted for by transcriptional induction of its α and β subunits and targeting of newly synthesized pumps to the basolateral membrane of principal cells -LSB- 14 -RSB- . In contrast , stimulation of ENaC mainly results from the targeting of a pre-existing pool of latent intracellular channels to the apical membrane of principal cells and slightly from transcriptional induction of the α and β subunits -LSB- 11 -- 13 -RSB- . The patch-clamp technique shows that the intrinsic properties of ENaC -LRB- open probability and unitary conductance -RRB- are not altered in the CCD of nephrotic animals -LSB- 13 -RSB- . Fig. 1Cellular mechanism of sodium reabsorption in principal cells of collecting ducts from normal rats and nephrotic rats . Sodium reabsorption in principal cells proceeds along a two-step mechanism that includes active extrusion of intracellular sodium ions by the basolateral Na,K-ATPase and passive apical entry of sodium via the amiloride-sensitive epithelial sodium channel -LRB- ENaC -RRB- . In CCDs from normal rats -LRB- top panels -RRB- , most ENaCs are sequestered in the intracellular compartment of principal cells -LRB- left panel -RRB- , and basolateral expression of Na,K-ATPase in collecting ducts -LRB- asterisk -RRB- principal cells is very weak , in comparison with that in thick ascending limbs -LRB- T -RRB- and even proximal tubules -LRB- P -RRB- -LRB- right panel -RRB- . Accordingly , the rate of sodium reabsorption is very low . In CCDs from PAN nephrotic rats -LRB- bottom panels -RRB- , ENaC is expressed at the apical border of principal cells -LRB- left panel -RRB- , and expression of basolateral Na,K-ATPase is drastically increased in collecting ducts -LRB- asterisk -RRB- . Polarized increases in expression of ENaC and Na,K-ATPase in principal cells account for increased sodium reabsorption in CCDs . In both normal and nephrotic rats expression of Na,K-ATPase is undetectable in the glomerulus -LRB- G -RRB- ; in CCDs , unlabeled cells for both ENaC and Na,K-ATPase are intercalated cells -LRB- redrawn from -LSB- 13 , 14 -RSB- -RRB- Aldosterone and activation of mineralo-corticoid receptors are not involved in sodium retention in nephrotic syndrome Historically , it is acknowledged that sodium retention and edema formation result from hypovolemia-induced stimulation of the renin -- angiotensin -- aldosterone system . Hypovolemia is supposed to proceed as follows : proteinuria induces hypoalbuminemia and reduces plasma oncotic pressure , which generates an imbalance of Starling 's forces across capillary walls , leading to interstitial leakage of fluid and decreased efficient volume -LSB- 15 -RSB- . Although this mechanism is consistent with the renal site of sodium retention , with the activation of ENaC and the induction of Na,K-ATPase in the collecting duct , and with the hyperaldosteronemia observed in PAN nephrotic rats -LSB- 13 -RSB- , a large body of clinical evidence argues against this theory . Analbuminemic patients display no sodium retention and no , or only modest , edema , despite low plasma oncotic pressure -LSB- 16 -RSB- . In children with steroid-sensitive minimal change disease , natriuresis resumes at the same time as proteinuria stops , before normalization of albuminemia -LSB- 17 -RSB- . Blood volume is not correlated with plasma oncotic pressure in nephrotic patients -LSB- 18 -RSB- . Among children with relapse idiopathic nephrotic syndrome , -LRB- a -RRB- 21 % display hypertension but only 4 % exhibit collapse -LSB- 19 -RSB- , and -LRB- b -RRB- only 1 % display low blood volume , whereas 17 % present hypervolemia -LSB- 20 -RSB- . Intravenous injection of albumin induces volume expansion but promotes only mild natriuresis -LRB- reviewed in -LSB- 21 -RSB- -RRB- . Blockade of mineralo-corticoid receptor or inhibition of angiotensin-converting enzyme has no effect on natriuresis in most patients -LSB- 22 , 23 -RSB- . The lack of a role for hyperaldosteronemia in sodium retention in NS was directly demonstrated in PAN nephrotic rats . In this model it is possible to blunt PAN-induced hyperaldosteronemia through bilateral adrenalectomy and corticosteroid replacement through implanted mini-pumps delivering constant physiological level of aldosterone and glucocorticoids . Administration of PAN to these corticosteroid-clamped rats reduces sodium excretion , establishes sodium balance and promotes ascites formation with similar time course and intensity as in adrenal-intact rats -LSB- 11 , 13 -RSB- . These findings also exclude a possible role in sodium retention of promiscuous activation of the mineralo-corticoid receptor -LRB- MR -RRB- by glucocorticoid brought about by decreased 11β-hydroxysteroid dehydrogenase type 2 -LRB- 11β-HSD2 -RRB- . Indeed , use of dexamethasone instead of corticosterone for glucocorticoid replacement prevents MR activation , even in the presence of 11β-HSD2 inhibition . The lack of involvement of MR activation in sodium retention in nephrotic syndrome is consistent with the fact that nephrotic syndrome does not induce potassium secretion . None of the known factors that control sodium reabsorption in the collecting duct accounts for sodium retention in PAN-nephrotic rats Since aldosterone , the major factor that controls sodium reabsorption in collecting ducts , is not involved in sodium retention , several hormones or paracrine factors that modulate this process have been considered as putative candidates : Vasopressin -LRB- AVP -RRB- , through activation of its V2 receptors coupled to the cAMP pathway , increases sodium reabsorption in the collecting duct synergistically with aldosterone -LSB- 24 , 25 -RSB- . Angiotensin II -LRB- AII -RRB- increases sodium reabsorption directly through activation of AT1 receptors coupled to phospholipase C , independently of the induction of aldosterone release -LSB- 26 -RSB- . Insulin-like growth factor I -LRB- IGF-1 -RRB- used in the treatment of insulin resistance in type 2 diabetes can induce sodium retention and edema -LSB- 27 -RSB- , likely through its stimulatory action on sodium transport along the collecting duct -LSB- 28 -RSB- . In mouse collecting duct cells , tumor necrosis factor alpha -LRB- TNFα -RRB- increases membrane expression of Na,K-ATPase -LSB- 29 -RSB- , and administration of etanercept , a TNFα receptor antagonist , to diabetic rats induces natriuresis -LSB- 30 -RSB- . More recently , the thiazolidinedione agonists of peroxisome proliferator-activated receptor γ -LRB- PPARγ -RRB- were reported to induce sodium retention -LSB- 31 -RSB- through a stimulatory effect on the collecting duct -LSB- 32 -RSB- . Quite unexpectedly , inhibition of nitric oxide synthase promotes sodium excretion in cirrhotic rats with ascites -LSB- 33 -RSB- . Except for AII , all maneuvers aimed at blocking these different pathways had no effect on sodium retention in nephrotic syndrome -LRB- Fig. 2 -RRB- . Only the AT1 blocker irbesartan was able to improve sodium excretion , although not up to normal level . However , this effect was limited to the initial sodium retention observed 2 -- 4 days after PAN administration and did not alter the main effect observed at days 5 and 6 . This suggests that AII , independently of aldosterone , may be involved in the early retention of sodium observed after the peak of sodium excretion at day 1 . Fig. 2Profile of sodium excretion in PAN nephrotic rats . Daily urinary sodium excretion , expressed as a function of urinary creatinine excretion , following administration of puromycin aminonucleoside -LRB- 150 mg/kg body wt , intravenously -RRB- in normal rats -LRB- dotted lines -RRB- or genetically modified or pharmacologically treated rats -LRB- solid lines -RRB- . Arrows or grey boxes show the time of treatment . Brattleboro rats genetically lack vasopressin secretion . JB1 , an inhibitor of IGF-1 receptors , was continuously administered via subcutaneous mini-pumps at a dose of 12 μg/100 g body wt per day , starting on day 3 . The antagonist of AT1 receptor , irbesartan , was administered per os at a dose of 2 mg/100g body wt per day . Etanercept , a chimeric antibody directed against TNF receptor , was administered twice -LRB- days − 1 and 2 -RRB- at a dose of 0.2 mg/100g body wt . The inhibitor of nitric oxide synthase , L-NAME , was administered twice daily by gavage -LRB- 0.5 mg/100 g body wt per 12 h -RRB- throughout the study . The antagonist of PPARγ , SR202 , was given per os at a dose of 20 mg/100 g body wt . All controls were treated in parallel with the vehicle . Values are means ± SE from 4 -- 5 ratsAs a matter of fact , sodium retention in PAN nephrosis unlikely results from the presence of an abnormal blood concentration of any factor , since only the treated kidney displays increased sodium reabsorption in unilateral PAN-nephrotic rats . Rather , alterations in collecting duct function may be accounted for either by a direct effect of PAN on CCD or by the abnormal presence of an unidentified stimulatory factor in the luminal fluid that reaches the distal nephron . The presence of this factor may result from its abnormal filtration secondarily to the alteration of the glomerular filtration barrier -LRB- e.g. , a high molecular weight protein -RRB- , or from its generation in the proximal tubule secondarily to alteration of protein/peptide handling . Na,K-ATPase is the primary target of sodium retention in PAN nephrosis Analysis of corticosteroid-clamped rats not only demonstrated that hyperaldosteronemia is not involved in the etiology of edemas in NS , but it also provided insights into the primary molecular target of sodium retention in the CCD . Indeed , in CCDs from corticosteroid-clamped PAN rats , Na,K-ATPase is induced as in adrenal-intact nephrotic rats -LSB- 13 -RSB- . In contrast , targeting of ENaC to the apical membrane is fully blunted -LSB- 11 , 13 -RSB- , and the residual channels expressed at the apical membrane are drastically inhibited , as their open probability is reduced > 10-fold -LSB- 13 -RSB- . Thus , induction of Na,K-ATPase is primarily responsible for sodium retention in PAN nephrotic syndrome . This conclusion likely applies to all experimental models of nephrotic syndrome and , possibly , to the disease in humans , since there is a significant reverse correlation between Na,K-ATPase activity in collecting duct and urinary sodium excretion during the phase of sodium retention in rats with nephrotic syndrome induced by PAN , Adriamycin or mercury chloride -LSB- 1 -RSB- . Edema formation in nephrotic syndrome Most sodium-retaining states are associated with high blood pressure but not with development of edema or ascites . Edema formation in NS results from the asymmetry of extracellular volume expansion brought about by sodium retention : the vascular volume is not , or only slightly , modified , whereas water and solutes accumulate in the interstitium . Distribution of fluid between vascular and interstitium compartments is governed by fluid exchanges across the capillary wall and by lymphatic draining . Capillary filtration capacity is increased almost twofold in nephrotic patients -LSB- 34 -RSB- . This fluid leakage is governed by several parameters according to Starling 's law : where Jv is the trans-capillary flux of fluid , Lp the hydraulic conductivity of capillaries , S the exchange surface , Pc and Pi the hydrostatic pressure of the capillary and interstitium , respectively , σ the reflection coefficient of proteins across the capillary wall , and Πc and Πi the oncotic pressure in the capillaries and interstitium , respectively . Unexpectedly , data from the literature indicate that the increase in Jv in NS is not accounted for by change in the oncotic pressure gradients , as intuitively thought , but by changes in the intrinsic properties of the capillary walls that govern their hydraulic conductance and reflexion coefficient for proteins . This means that the capillary wall is a direct target in NS , to the same right as the glomerular filtration barrier . The trans-capillary gradient of oncotic pressure -LRB- Πc -- Πi -RRB- is unchanged in NS The lack of edema and ascites in analbuminemic rats and patients -LSB- 16 , 35 -RSB- has questioned the importance of low plasma oncotic pressure in the genesis of edema in NS . As a matter of fact , the trans-capillary gradient of oncotic pressure is unchanged in analbuminemic rats , owing to a parallel decrease in plasma and interstitium oncotic pressure -LSB- 35 -RSB- . Experiments in dogs , in which plasma oncotic pressure was progressively decreased by 50 % through plasmapheresis , showed that the interstitium oncotic pressure decreased in parallel and that the trans-capillary gradient remained unaffected . Extracellular volume increased transiently during the phase of variation of plasma oncotic pressure , in response to hyperaldosteronemia and renal sodium retention , but returned to normal level during the period of stabilized low plasma oncotic pressure -LSB- 36 -RSB- . Essentially similar observations were made in humans . During NS , it was also observed that the oncotic pressure of the interstitium decreases in parallel with that of the vascular compartment , so that the trans-capillary gradient is only slightly reduced -LSB- 37 -RSB- . In addition , diuretic treatments -LSB- 37 -RSB- or extracorporeal ultrafiltration -LSB- 38 -RSB- allow the withdrawal of significant amounts of edema without significant change in the trans-capillary gradient of oncotic pressure.Thus , decrease in plasma oncotic pressure in animal models as well as in nephrotic patients does not alter significantly the trans-capillary gradient of oncotic pressure and is neither a determinant parameter in the genesis of edema nor a resistance factor to edema withdrawal . The trans-capillary gradient of hydrostatic pressure -LRB- Pc -- Pi -RRB- is unchanged in NS Capillary pressure is unchanged in nephrotic patients -LSB- 34 -RSB- . Because soft tissues display an almost infinite compliance , their interstitium pressure increases by only 2 mmHg with their fluid filling -LSB- 39 -RSB- . In nephrotic patients interstitial pressure in edematous and non-edematous sectors differs by < 4 mmHg -LSB- 40 -RSB- . Thus , it can be admitted that the trans-capillary gradient of hydrostatic pressure is not significantly altered in soft edematous tissues during NS . The capillary hydraulic conductivity Lp is increased in NS The threshold of venous pressure triggering fluid transfer across capillaries is significantly reduced in NS -LSB- 34 -RSB- , suggesting that endothelial Lp is decreased . The main determinants of the hydraulic conductivity of capillaries are the occlusive junctions -LRB- constituted by occludin , claudins and proteins ZO -RRB- and adhesive junctions -LRB- made of cadherins , catenins and actinin -RRB- between endothelial cells . These proteins are direct targets for intracellular signaling cascades , in particular protein kinase C -LRB- PKC -RRB- , which phosphorylates occludin -LSB- 41 -RSB- and alters the endothelial permeability -LSB- 42 -RSB- . In NS this pathway may be activated through two mechanisms . Firstly , hypoalbuminemia increases Lp via an increase in intracellular calcium -LSB- 43 -RSB- . Secondly , high plasma levels of TNFα observed in patients with minimal change disease -LSB- 44 -RSB- activate PKC and increase Lp -LSB- 45 -RSB- . The reflexion coefficient of proteins -LRB- σ -RRB- is increased in NS Increased coefficient of reflexion of macromolecules in NS is evidenced by the higher rate of leakage of technetium-labeled albumin towards the interstitium in nephrotic patients than in controls -LSB- 46 -RSB- . Because this change in reflexion coefficient is observed in patients with NS of different origin , it is likely not accounted for by the circulating permeability factor of lymphocyte origin responsible for glomerular hyperfiltration in minimal change disease . Increased σ was also reported in blood -- peritoneal barrier permeability in PAN-nephrotic rats -LSB- 47 -RSB- . Therapeutic implications Nephrotic edema results from the combination of renal sodium retention and increased capillary permeability . Although treatment of either of these two alterations would prevent edema , treatment of capillary permeability alone would lead to hypertension . Thus , treatments of edema must primarily target renal sodium retention . Different therapeutic strategies are needed to prevent edema formation in children with chronic proteinuria and to treat massive edema . In the first case it is sufficient to prevent sodium accumulation , either through limiting dietary sodium uptake or by natriuretic drugs that target the collecting duct , e.g. , amiloride . In clinical practice the daily uptake of sodium is usually limited to 0.5 mmol/kg . In PAN-nephrotic rats administration of amiloride starting before the onset of sodium retention fully prevents sodium retention and formation of ascites -LSB- 5 -RSB- . More classically , clinicians are confronted with patients who already display massive edema . Treatment aims both at limiting further sodium retention and at promoting the excretion of the mass of sodium and water sequestered in edema . While amiloride is well suited to block further sodium retention , it is inefficient in promoting massive sodium excretion , because , under normal conditions , sodium reabsorption along the collecting duct is quantitatively low . Recourse to more potent diuretics , such as loop diuretics , is restricted by the functional resistance of nephrotic patients to the natriuretic effect of furosemide . Several explanations have been proposed to account for this resistance , but none has been confirmed : The pharmacokinetics of furosemide urinary elimination is not significantly altered in nephrotic children -LSB- 48 -RSB- Binding of furosemide to albumin in the tubular fluid does not account for furosemide resistance , since , despite controversial data -LSB- 49 , 50 -RSB- , inhibitors of furosemide -- albumin binding do not improve sodium excretion significantly.The intrinsic sensitivity of the Na/K/2Cl transporter of the loop of Henle , the molecular target of furosemide , is not altered in nephrotic rats -LSB- 5 -RSB- . Based on our present knowledge of the site and cellular mechanism of sodium retention in NS , another explanation of furosemide resistance , and a therapeutic strategy to circumvent it , can be proposed . In non-nephrotic patients , furosemide decreases sodium reabsorption along the thick ascending limb of the loop of Henle , which increases sodium delivery to the distal nephron . Because the sodium reabsorption capacity of the distal nephron is rather limited , only a small fraction of the overload of sodium is reabsorbed , and the major fraction is excreted in the urine , accounting for the natriuretic effect of loop diuretics . Note that following long-term treatment with furosemide , there are adaptations of the distal nephron which increase its sodium reabsorption capacity and , thereby , reduce the natriuretic effect of furosemide . Because nephrotic patients display a huge sodium reabsorption capacity along their connecting and cortical collecting tubules , most of the overload of sodium brought about by furosemide-induced inhibition of transport in the thick ascending limb is reabsorbed , thereby blunting the natriuretic effect . Thus , the apparent resistance of nephrotic patients to loop diuretics can be circumvented by the inhibition of distal sodium reabsorption with amiloride . As a matter of fact , co-administration of furosemide and amiloride to nephrotic children increases urinary sodium excretion , induces a negative sodium balance and promotes complete edema withdrawal within 1 week -LSB- 21 -RSB- . Conclusion Whatever their etiology , nephrotic syndromes are always associated with renal retention of sodium . Renal sodium retention results from enhanced sodium reabsorption along the connecting and cortical collecting ducts and from blunted responsiveness of medullary collecting ducts to the natriuretic response to atrial natriuretic peptide . Induction of de novo synthesis of Na,K-ATPase is the primary effector of increased sodium reabsorption . It is not accounted for by any circulating factor , in particular aldosterone , known to stimulate sodium reabsorption along the distal nephron . New research strategies will be required to identify the unknown regulatory pathway that is dysregulated in NS . Sodium retention in NS does not lead to high blood pressure but leads to an asymmetric expansion of the interstitium , while the vascular volume remains unchanged in most patients . This asymmetry of extracellular volume expansion is accounted for by changes in the intrinsic properties of the endothelial capillary barriers , i.e. , an increase in its hydraulic conductivity and permeability to proteins , rather than to an imbalance of Starling 's forces . Thus , the pathophysiology of nephrotic syndrome relies on at least three disorders : a major alteration of the glomerular filtration barrier responsible for proteinuria and hypoalbuminemia , an induction of distal nephron Na,K-ATPase responsible for sodium retention , and alterations in the capillary permeability accounting for the asymmetry of volume expansion . Although causal relationships between these three events have not been formally established , it is assumed that the glomerular defect engenders both the tubular and the capillary alterations . This conclusion is based on the facts that : -LRB- a -RRB- whatever its origin , alteration of the glomerular filtration barrier always leads to sodium retention and edema formation , i.e. , to the tubular and capillary defects , and -LRB- b -RRB- loss of function mutations of nephrin , which is expressed in the glomerular slit diaphragm but in neither the collecting duct nor capillary endothelial cells , is sufficient to promote proteinuria , sodium retention and edema -LSB- 51 -RSB- . Curiously , in PAN-induced nephrosis , sodium retention precedes proteinuria , suggesting that it is not secondary to the glomerular dysfunction . It should be stressed , however , that microproteinuria may appear sooner and concomitantly with sodium retention .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O

sodium retention
collecting duct
aldosterone
capillary hydraulic conductivity
diuretics

na,k-atpase
epithelial sodium channels (enacs)