Oxygen

kinetics

and

debt

during

recovery

from

expiratory

flow-limited

exercise

in

healthy

humans

In

healthy

subjects

expiratory

flow

limitation

-LRB-

EFL

-RRB-

during

exercise

can

lower

delivery

to

the

working

muscles

We

hypothesized

that

if

this

affects

exercise

performance

it

should

influence

kinetics

at

the

end

of

exercise

when

the

debt

is

repaid

We

performed

an

incremental

exercise

test

on

six

healthy

males

with

a

Starling

resistor

in

the

expiratory

line

limiting

expiratory

flow

to

l

s

to

determine

maximal

EFL

exercise

workload

-LRB-

Wmax

-RRB-

In

two

more

square-wave

exercise

runs

subjects

exercised

with

and

without

EFL

at

Wmax

for

min

while

measuring

arterial

saturation

-LRB-

-RRB-

end-tidal

pressure

of

-LRB-

-RRB-

and

breath-by-breath

consumption

taking

into

account

changes

in

stored

in

the

lungs

Over

the

last

minute

of

EFL

exercise

mean

-LRB-

mmHg

-RRB-

was

significantly

higher

-LRB-

P

-RRB-

compared

to

control

-LRB-

mmHg

-RRB-

At

the

end

of

EFL

exercise

fell

significantly

by

When

exercise

stopped

EFL

was

removed

and

we

continued

to

measure

During

recovery

there

was

an

immediate

step

increase

in

so

that

repayment

of

EFL

debt

started

at

a

higher

than

control

Recovery

kinetics

after

EFL

exercise

was

best

characterized

by

a

double-exponential

function

with

fundamental

and

slow

time

constants

of

and

s

compared

to

control

values

of

and

s

respectively

EFL

debt

was

greater

than

control

-LRB-

vs.

l

-RRB-

We

conclude

that

EFL

exercise

increases

the

debt

and

leads

to

hypoxemia

in

part

due

to

hypercapnia

Introduction

In

spite

of

the

fact

that

dynamic

hyperinflation

-LRB-

Calverley

and

Koulouris

O'Donnell

et

al.

-RRB-

and

peripheral

muscle

deconditioning

-LRB-

Maltais

et

al.

Richardson

et

al.

-RRB-

are

important

factors

limiting

exercise

in

COPD

there

is

evidence

that

inadequate

oxygen

delivery

to

working

locomotor

and

respiratory

muscles

may

also

play

an

important

role

-LRB-

Aliverti

and

Macklem

Aliverti

et

al.

Iandelli

et

al.

-RRB-

This

situation

has

been

modeled

in

normal

humans

by

imposing

expiratory

flow-limitation

-LRB-

EFL

-RRB-

during

exercise

which

limits

exercise

performance

by

intense

dyspnea

-LRB-

Aliverti

et

al.

Iandelli

et

al.

-RRB-

while

decreasing

arterial

oxygen

saturation

and

cardiac

output

-LRB-

Aliverti

et

al.

-RRB-

This

in

turn

leads

to

premature

competition

between

respiratory

and

locomotor

muscles

for

available

energy

supplies

and

the

early

onset

of

mixed

respiratory

and

metabolic

acidosis

-LRB-

Aliverti

and

Macklem

Kayser

et

al.

-RRB-

As

a

result

of

these

previous

experiments

-LRB-

Aliverti

and

Macklem

Aliverti

et

al.

Kayser

et

al.

-RRB-

we

predicted

that

EFL

exercise

in

normal

subjects

should

increase

the

oxygen

debt

by

decreasing

systemic

oxygen

delivery

to

exercising

muscles

Accordingly

to

test

this

hypothesis

we

measured

kinetics

during

repayment

of

the

debt

after

control

and

EFL

exercise

in

young

normal

male

subjects

Our

results

revealed

that

EFL

increased

net

debt

by

an

average

and

led

to

hypoxemia

in

part

due

to

hypercapnia

Material

and

methods

Subjects

We

studied

six

healthy

male

individuals

whose

baseline

characteristics

are

shown

in

Table

All

subjects

gave

signed

informed

consent

and

the

protocol

was

approved

by

the

University

of

Athens

Ethics

Committee

where

the

experiments

were

conducted.Table

characteristicsSubjectAge

-LRB-

years

-RRB-

Height

-LRB-

m

-RRB-

Weight

-LRB-

kg

-RRB-

-LRB-

l

-RRB-

-LRB-

predicted

-RRB-

Dyspnea

-LRB-

Borg

scale

-RRB-

1381.76783.90971551.752221.73754.231001702.583191.69593.93971001.674221.94865.471121252.555221.88805.731091002.056341.66603.5296901.87

forced

expiratory

volume

in

s

in

absolute

and

as

percentage

predicted

maximal

workload

when

EFL

was

imposed

during

the

incremental

cycling

test

maximal

oxygen

uptake

when

EFL

was

imposed

during

the

incremental

test

Pulmonary

function

tests

Spirometry

for

the

determination

of

forced

expiratory

volume

in

s

-LRB-

-RRB-

was

performed

by

a

Vmax

system

-LRB-

Sensor

Medics

Anaheim

CA

USA

-RRB-

-LRB-

Table

-RRB-

Exercise

protocols

Three

exercise

tests

were

performed

for

each

subject

on

an

electromagnetically

braked

cycle

ergometer

-LRB-

Ergoline

Sensor

Medics

-RRB-

with

the

subjects

maintaining

a

pedaling

frequency

of

rpm

At

the

beginning

an

incremental

test

was

performed

to

establish

the

individual

subject

's

maximal

workload

when

EFL

was

imposed

by

the

application

of

a

Starling

resistor

in

the

expiratory

line

limiting

flow

to

approximately

l

s

After

min

of

baseline

measurements

followed

by

min

of

unloaded

pedaling

the

work

rate

was

increased

every

min

by

W

to

the

limit

of

tolerance

-LRB-

i.e.

the

point

where

the

work

rate

could

not

be

tolerated

due

to

severe

sensation

of

dyspnea

see

Table

-RRB-

On

a

separate

day

two

square-wave

protocols

were

performed

the

first

without

EFL

-LRB-

control

-RRB-

and

the

second

with

EFL

After

min

of

baseline

measurements

followed

by

min

of

unloaded

pedaling

the

work

rate

was

set

and

maintained

at

for

min

in

both

protocols

At

the

end

of

exercise

EFL

was

removed

and

recovery

was

monitored

for

an

additional

min

-LRB-

Fig.

-RRB-

Fig.

example

of

volume

changes

of

the

chest

wall

during

min

of

baseline

measurements

-LRB-

QB

quiet

breathing

-RRB-

min

of

unloaded

cycling

-LRB-

W/u

warming

up

-RRB-

min

of

exercise

at

the

pre-determined

and

min

of

recovery

Arrows

indicate

end

of

expiration

and

inspiration

Data

acquisition

was

interrupted

for

min

every

min

in

order

to

allow

data

storage

Gas

exchange

and

flow

measurements

Instantaneous

inspired

and

expired

and

concentrations

end-tidal

values

for

and

-LRB-

respectively

-RRB-

and

flow

at

the

mouth

were

recorded

breath-by-breath

-LRB-

Vmax

Sensor

Medics

-RRB-

throughout

both

control

and

EFL

exercise

and

recovery

runs

Flow

displaced

at

the

mouth

was

also

measured

by

a

screen

type

pneumotachograph

-LRB-

Hans

Rudolph

Kansas

City

MO

USA

-RRB-

and

from

these

measurements

the

following

parameters

were

obtained

minute

ventilation

tidal

volume

-LRB-

VT

-RRB-

breathing

frequency

-LRB-

fb

-RRB-

inspiratory

-LRB-

Ti

-RRB-

expiratory

-LRB-

Te

-RRB-

total

respiratory

-LRB-

Ttot

-RRB-

cycle

time

and

duty

cycle

-LRB-

Ti/Tot

-RRB-

Cardiac

frequency

-LRB-

fc

-RRB-

and

percentage

arterial

oxygen

saturation

-LRB-

-RRB-

were

determined

using

the

R

R

interval

from

a

on-line

electrocardiogram

-LRB-

Marquette

Max

Marquette

Hellige

GmbH

Germany

-RRB-

and

a

pulse

oximeter

-LRB-

Nonin

Nonin

Medical

USA

-RRB-

respectively

The

modified

Borg

Scale

-LRB-

Borg

-RRB-

was

used

to

rate

the

magnitude

of

dyspnea

and

leg

discomfort

every

min

throughout

exercise

Operational

chest-wall

volume

measurements

Simultaneously

throughout

the

square

wave

exercise

and

recovery

protocols

the

volume

of

the

chest

wall

was

measured

by

optoelectronic

plethysmography

-LRB-

OEP

system

BTS

Milan

Italy

-RRB-

as

previously

described

-LRB-

Aliverti

et

al.

Cala

et

al.

Vogiatzis

et

al.

-RRB-

In

brief

the

motion

of

retro-reflective

markers

placed

front

and

back

over

the

chest

wall

from

clavicles

to

pubis

was

recorded

Each

marker

was

tracked

by

six

video

cameras

three

in

front

of

the

subject

and

three

behind

Subjects

grasped

handles

positioned

at

mid-sternum

level

which

lifted

the

arms

away

from

the

rib

cage

so

that

lateral

markers

could

be

visualized

Dedicated

software

which

recognizes

the

markers

on

each

camera

in

real

time

reconstructed

their

co-ordinates

by

stereophotogrammetry

and

using

Gauss

's

theorem

calculated

inspiratory

and

expiratory

tidal

volume

variations

Thus

breathing

pattern

was

measured

both

plethysmographically

and

by

integration

of

flow

Data

acquisition

from

the

OEP

system

was

interrupted

for

min

every

min

-LRB-

Fig.

-RRB-

in

order

to

allow

data

storage

Calculation

of

breath-by-breath

oxygen

consumption

The

volume

of

exchanged

at

the

mouth

differs

from

the

volume

of

taken

up

by

pulmonary

capillaries

if

the

amount

of

stored

within

the

lung

changes

This

occurs

if

the

volume

inspired

is

different

from

the

volume

expired

in

a

given

respiratory

cycle

and/or

if

alveolar

concentrations

of

change

-LRB-

Capelli

et

al.

-RRB-

This

is

particularly

important

during

on

and

off-exercise

transients

-LRB-

Cautero

et

al.

-RRB-

The

changes

in

stored

within

the

lung

must

be

subtracted

from

the

volume

of

exchanged

at

the

mouth

in

order

to

obtain

the

amount

of

exchanged

at

the

alveolar

level

This

can

be

done

by

combining

OEP

with

independent

measures

of

the

subdivisions

of

lung

volume

and

continuously

measuring

changes

in

the

absolute

gas

volume

by

OEP

throughout

all

exercise

tests

This

method

was

recently

described

in

detail

-LRB-

Aliverti

et

al.

-RRB-

Accordingly

in

the

present

study

breath-by-breath

oxygen

consumption

was

computed

throughout

using

the

data

acquired

by

OEP

flow

at

the

mouth

and

the

continuous

inspired

and

expired

and

concentration

recordings

Using

this

method

corrections

were

made

for

-LRB-

-RRB-

pneumotachograph

integrator

drift

-LRB-

-RRB-

the

sampling

rates

of

the

and

meters

-LRB-

-RRB-

the

time

delay

of

the

analyzer

and

the

flow

through

the

and

sampling

lines

-LRB-

-RRB-

precise

synchronization

of

concentration

and

volume

signals

and

-LRB-

-RRB-

any

changes

in

stored

within

the

lung

Thus

in

contrast

to

commercial

devices

we

measured

at

the

level

of

the

pulmonary

capillaries

not

at

the

mouth

Curve

fitting

To

characterize

the

kinetics

of

the

response

during

square-wave

exercise

and

recovery

single

and

double-exponential

functions

were

applied

respectively

to

the

data

using

a

non-linear

least-squares

fitting

procedure

-LRB-

Figs.

-RRB-

without

omitting

the

first

s

of

the

response

for

either

the

on

or

off-transient

The

equations

applied

-LRB-

Linnarsson

Ozyener

et

al.

-RRB-

were

where

in

Eq

the

response

is

the

sum

of

two

components

each

described

by

an

exponential

decay

with

two

time

constants

τ1

-LRB-

the

fundamental

component

-RRB-

and

τ2

-LRB-

the

slow

component

-RRB-

In

the

same

equation

a

and

c

are

the

initial

values

of

these

two

components

and

their

sum

-LRB-

a

c

-RRB-

represents

the

initial

value

of

the

total

response

i.e.

the

value

of

at

time

zero

The

adequacy

of

the

fitting

model

was

tested

by

calculating

values

and

residuals

for

single

and

double

exponentials.Fig

example

of

breath-by-breath

during

control

-LRB-

closed

circles

-RRB-

and

EFL

exercise

-LRB-

open

circles

-RRB-

and

recovery

Upon

completion

of

exercise

the

Starling

resistor

was

removed

Note

that

time

is

aligned

on

the

precise

instant

in

which

the

recovery

started

-LRB-

time

zero

-RRB-

Fig.

example

of

breath-by-breath

during

recovery

from

control

-LRB-

closed

circles

-RRB-

and

EFL

exercise

-LRB-

open

circles

-RRB-

Data

are

fitted

with

double-exponential

curves

Measurement

of

oxygen

debt

Oxygen

debt

was

calculated

by

integrating

for

each

subject

the

fitting

curves

between

time

zero

-LRB-

start

of

recovery

-RRB-

and

an

arbitrary

time

of

s

-LRB-

Fig.

-RRB-

Then

the

value

of

mean

at

baseline

multiplied

by

the

same

period

of

time

was

subtracted

-LRB-

Aliverti

et

al.

-RRB-

Estimation

of

cardiac

output

and

arterio-venous

oxygen

difference

To

estimate

the

cardiac

output

-LRB-

CO

-RRB-

and

the

arterio-venous

oxygen

-LRB-

a-v

-RRB-

difference

during

EFL

and

control

exercise

the

following

equations

-LRB-

Stringer

et

al.

-RRB-

were

used

Statistical

analysis

Data

are

presented

as

mean

SD

Significant

differences

in

recorded

variables

over

the

last

minute

of

the

square-wave

exercise

protocols

between

control

and

EFL

tests

were

assessed

by

the

Wilcoxon

signed-rank

test

as

were

recovery

data

from

control

and

EFL

exercise

tests

Linear

regression

analysis

was

performed

using

the

least-squares

method

A

statistical

significance

of

was

used

for

all

analyses

Results

Square-wave

protocols

Figure

displays

a

typical

example

of

breath-by-breath

during

control

and

EFL

exercise

protocols

Individual

and

mean

values

of

sustained

during

the

two

square-wave

protocols

are

shown

in

Table

whereas

the

gas

exchange

and

ventilatory

pattern

parameters

are

shown

in

Table

tended

to

be

lower

-LRB-

by

-RRB-

at

the

end

of

EFL

compared

to

control

exercise

albeit

not

significantly

-LRB-

Table

-RRB-

However

EFL

compared

to

control

exercise

was

associated

with

significantly

lower

values

for

fb

and

Ti/Tot

Te

at

the

end

of

EFL

exercise

was

significantly

higher

compared

to

control

-LRB-

Table

-RRB-

Over

the

last

minute

of

EFL

exercise

mean

values

for

fc

-LRB-

mmHg

-RRB-

and

dyspnea

scores

were

significantly

larger

compared

to

control

exercise

-LRB-

Fig.

-RRB-

whereas

mean

-LRB-

mmHg

-RRB-

was

significantly

lower

-LRB-

Fig.

-RRB-

The

mean

fall

-LRB-

P

-RRB-

in

at

the

end

of

EFL

exercise

was

-LRB-

Fig.

-RRB-

Leg

discomfort

at

the

end

of

EFL

exercise

was

not

different

to

that

at

the

end

of

control

exercise

-LRB-

Fig.

-RRB-

The

on-transient

data

were

best

fitted

with

a

mono-exponential

function

where

the

time

constant

of

the

fundamental

component

was

not

significantly

different

during

EFL

exercise

as

compared

to

control

exercise

-LRB-

Table

-RRB-

Table

data

at

the

end

of

the

min

protocols

with

and

without

EFL

Control

exerciseEFL

exerciseWork

rate

-LRB-

W

-RRB-

fc

-LRB-

beats

min

-RRB-

VT

-LRB-

l

-RRB-

fb

-LRB-

breaths

min

-RRB-

Ti

-LRB-

s

-RRB-

Te

-LRB-

s

-RRB-

Ti/Tot

-LRB-

-RRB-

Values

are

means

SD

oxygen

uptake

carbon

dioxide

output

fc

cardiac

frequency

minute

ventilation

VT

tidal

volume

fb

breathing

frequency

Ti

inspiratory

time

Te

expiratory

time

Ti/Tot

duty

cycle

Significant

differences

-LRB-

P

-RRB-

between

EFL

and

control

exerciseTable

and

mean

data

on

the

time

constant

of

the

fundamental

and

slow

component

of

the

response

for

on

and

off-transientsSubjectOn-transient

τ1

-LRB-

s

-RRB-

Off-transient

τ1

-LRB-

s

-RRB-

Off-transient

τ2

-LRB-

s

-RRB-

.866.234.729.21,4291027.5250.562.932.123.11,428930.7349.054.633.932.8

P

Fundamental

-LRB-

τ1

-RRB-

time

constant

of

the

on-transient

response

during

control

and

EFL

exercise

-LRB-

first

and

second

columns

-RRB-

Fundamental

-LRB-

τ1

-RRB-

and

slow

-LRB-

τ2

-RRB-

time

constants

of

the

off-transient

response

after

control

and

EFL

exercise

-LRB-

third

to

sixth

column

-RRB-

Values

values

for

heart

rate

-LRB-

fc

a

-RRB-

-LRB-

b

-RRB-

-LRB-

c

-RRB-

-LRB-

d

-RRB-

dyspnea

-LRB-

e

-RRB-

and

leg

discomfort

-LRB-

f

-RRB-

at

the

end

of

control

and

EFL

exercise

protocols

Recovery

oxygen

kinetics

and

oxygen

debt

Figure

displays

a

typical

example

of

breath-by-breath

data

fitted

with

a

double-exponential

function

during

recovery

from

control

and

EFL

exercise

square-wave

tests

The

values

of

the

fitted

curves

applied

to

the

recovery

breath-by-breath

data

were

not

significantly

different

between

EFL

-LRB-

-RRB-

and

control

exercise

-LRB-

-RRB-

Immediately

after

removing

the

Starling

resistor

at

the

end

of

EFL

exercise

there

was

a

step

increase

in

so

that

repayment

of

debt

started

at

a

higher

after

EFL

than

control

Figure

demonstrates

individual

values

of

the

a

c

and

a

c

parameters

obtained

from

Eq

All

three

parameters

were

significantly

higher

after

EFL

compared

to

control

exercise

-LRB-

a

P

c

P

a

c

P

-RRB-

Also

shown

in

Fig.

is

the

total

debt

which

was

l

after

EFL

exercise

compared

to

the

control

value

of

l

an

increase

of

-LRB-

P

-RRB-

caused

by

EFL

In

order

to

verify

possible

problems

originated

by

the

curve

fitting

oxygen

debt

and

deficit

were

also

calculated

directly

from

the

original

breath-by-breath

data

and

did

not

differ

significantly

compared

to

the

data

obtained

by

the

fitted

curves

-LRB-

average

difference

-RRB-

Fig.

values

of

the

parameters

given

in

Eq

a

-LRB-

a

-RRB-

c

-LRB-

b

-RRB-

a

c

-LRB-

c

-RRB-

and

calculated

gross

values

for

the

oxygen

debt

-LRB-

d

-RRB-

after

the

EFL

and

control

exercise

protocols

Individual

and

mean

values

for

the

off-transient

time

constants

of

the

fundamental

and

slow

components

are

shown

in

Table

The

fundamental

time

constant

was

significantly

-LRB-

P

-RRB-

shorter

during

recovery

from

EFL

compared

to

control

exercise

-LRB-

Table

-RRB-

The

off-transient

slow

component

during

recovery

from

EFL

tended

-LRB-

P

-RRB-

to

be

shorter

compared

to

that

after

control

exercise

however

in

recovery

from

control

exercise

the

slow

component

was

not

evident

in

subjects

and

-LRB-

Table

-RRB-

Table

reports

the

individual

and

mean

data

of

the

on

and

off

transients

for

EFL

and

control

exercise.Table

and

mean

data

of

the

on

and

off

transientsSubjectOn-transientOff-transientAControl

-LRB-

l

min

-RRB-

aEFL

-LRB-

l

min

-RRB-

aControl

-LRB-

l

min

-RRB-

aEFL

-LRB-

l

min

-RRB-

cControl

-LRB-

l

min

-RRB-

cEFL

-LRB-

l

min

-RRB-

11.9441.4721.912.420.670.7821.7512.0592.334.930.720.9130.9931.0401.852.090.490.6041.8061.7421.872.620.410.6550.9811.7431.403.840.680.9461.5711.4541.291.890.500.67

P

For

the

on-transient

after

control

and

EFL

exercise

the

value

for

a

was

obtained

from

Eq

For

the

off-transient

after

control

and

EFL

exercise

the

values

for

a

and

c

were

obtained

from

Eq

Estimation

of

cardiac

output

and

arterio-venous

oxygen

difference

From

Eq

-RRB-

COEFL

-LSB-

l

min

-LRB-

-RRB-

-RSB-

l

min

From

Eq

a-v

-LRB-

-RRB-

ml

per

ml

of

blood

In

addition

a-v

-LRB-

-RRB-

ml

per

ml

of

blood

Based

on

the

above

calculations

the

mixed

venous

blood

content

-LRB-

-RRB-

for

EFL

would

be

equivalent

to

ml

per

ml

blood

or

ml

l

For

control

would

be

equivalent

to

ml

per

ml

blood

or

ml

l

Taking

into

account

that

of

blood

volume

is

located

in

the

venous

compartment

at

rest

and

during

moderate

exercise

that

figure

falls

to

approximately

-LRB-

Astrand

and

Rodahl

-RRB-

then

during

EFL

exercise

with

a

cardiac

output

of

l

min

the

will

be

-LSB-

-LRB-

l

min

-RRB-

l

-RSB-

l

or

ml

For

control

exercise

the

will

be

-LSB-

-LRB-

l

min

-RRB-

-RSB-

l

or

ml

Thus

the

difference

in

between

EFL

and

control

will

be

ml

Hence

from

the

l

difference

in

oxygen

debt

between

EFL

and

control

calculated

in

Table

-LRB-

l

-RRB-

l

-LRB-

i.e.

approximately

one-third

-RRB-

would

account

for

the

replenishment

of

the

blood

oxygen

stores.Table

deficit

and

debt

for

control

and

EFL

exercise

Control

exerciseEFL

deficit

-LRB-

ml

-RRB-

debtfast

component

-LRB-

ml

-RRB-

debtslow

component

-LRB-

ml

-RRB-

debttotal

-LRB-

ml

-RRB-

-LRB-

ml

-RRB-

debtnet

-LRB-

ml

-RRB-

Values

are

means

SD

oxygen

uptake

Significant

differences

-LRB-

P

-RRB-

between

EFL

and

control

exercise

Discussion

There

is

growing

evidence

that

an

inadequate

oxygen

supply

to

meet

demand

may

play

a

more

dominant

role

in

limiting

exercise

capacity

in

some

patients

with

advanced

COPD

than

impaired

lung

function

-LRB-

Aliverti

et

al.

Maltais

et

al.

Oelberg

et

al.

Potter

et

al.

Richardson

et

al.

Stark-Leyva

et

al.

-RRB-

In

healthy

subjects

limiting

expiratory

flow

by

a

Starling

resistor

or

increasing

expiratory

load

by

imposing

a

constant

pressure

at

the

mouth

during

expiration

have

been

shown

to

significantly

reduce

cardiac

output

during

exercise

-LRB-

Aliverti

et

al.

Stark-Leyva

et

al.

-RRB-

This

results

from

the

enforced

decrease

in

the

velocity

of

shortening

of

expiratory

muscles

and

hypercapnia

both

of

which

contribute

to

increased

expiratory

muscle

force

and

decreased

duty

cycle

so

that

expiration

acts

like

a

Valsalva

maneuver

with

inadequate

time

to

recover

during

inspiration

-LRB-

Aliverti

et

al.

Iandelli

et

al.

-RRB-

Furthermore

the

decreased

duty

cycle

magnifies

the

effect

of

high

expiratory

pressures

when

averaged

over

the

whole

respiratory

cycle

Our

present

results

show

that

EFL

increased

debt

by

an

average

and

led

to

hypoxemia

in

part

due

to

hypercapnia

This

supports

our

hypothesis

that

the

reduction

in

cardiac

output

during

EFL

exercise

decreases

supply

to

working

locomotor

and

respiratory

muscles

resulting

in

an

increased

debt

We

conclude

that

the

decreased

cardiac

output

is

important

in

limiting

EFL

exercise

performance

in

healthy

subjects

EFL

as

a

model

for

COPD

The

experimental

model

used

in

this

study

was

designed

to

simulate

in

part

the

flow-limitation

commonly

experienced

by

COPD

patients

during

exercise

The

pros

and

cons

of

this

model

have

been

previously

extensively

discussed

-LRB-

Aliverti

et

al.

Iandelli

et

al.

-RRB-

The

model

has

demonstrated

that

exercise

with

EFL

induces

intolerable

dyspnea

retention

impaired

exercise

performance

expiratory

muscle

recruitment

-LRB-

Kayser

et

al.

-RRB-

blood

shifts

from

trunk

extremities

a

reduced

duty

cycle

arterial

desaturation

and

a

decrease

in

cardiac

output

-LRB-

Aliverti

et

al.

-RRB-

In

the

present

study

we

confirmed

many

of

these

results

and

showed

that

the

increased

debt

was

accompanied

by

a

reduction

in

by

and

a

fall

in

by

at

the

end

of

EFL

exercise

thus

leading

to

a

reduction

in

systemic

delivery

of

in

close

agreement

with

the

reduction

reported

by

Aliverti

et

al.

-LRB-

-RRB-

It

is

reasonable

to

assume

that

the

reduction

in

systemic

delivery

during

EFL

exercise

would

be

associated

with

the

progressive

recruitment

of

fast-twitch

fibers

and

hence

the

premature

onset

of

the

lactate

threshold

The

latter

has

been

shown

to

be

the

case

during

exercise

with

EFL

-LRB-

Aliverti

et

al.

-RRB-

When

exercise

workloads

exceed

the

lactate

threshold

energy

supplies

are

inadequate

to

meet

demands

The

resulting

competition

between

working

locomotor

and

respiratory

muscles

for

the

available

energy

supplies

regulated

by

autonomic

reflex

mechanisms

-LRB-

Harms

et

al.

-RRB-

would

be

substantially

worsened

by

the

decrease

in

the

available

and

should

further

increase

the

lactate

production

It

is

therefore

likely

that

the

muscle

and

blood

lactate

levels

-LRB-

not

measured

in

the

present

study

-RRB-

would

be

appreciably

higher

during

EFL

exercise

and

consequently

the

lactate

related

metabolic

cost

could

significantly

contribute

to

the

repayment

of

the

debt

in

recovery

from

EFL

exercise

In

addition

to

the

reduction

in

systemic

delivery

the

present

study

shows

that

application

of

EFL

during

exercise

significantly

decreased

minute

ventilation

compared

to

control

exercise

causing

hypercapnia

This

confirms

earlier

results

that

show

that

a

vicious

circle

is

induced

whereby

increasing

central

ventilatory

drive

increases

expiratory

pressure

which

further

reduces

alveolar

ventilation

and

cardiac

output

-LRB-

Aliverti

et

al.

Iandelli

et

al.

-RRB-

Furthermore

the

observed

elevated

heart

rate

during

EFL

exercise

possibly

reflects

a

reduction

in

stroke

volume

secondary

to

the

decrease

in

venous

return

In

the

study

by

Stark-Leyva

et

al.

-LRB-

-RRB-

expiratory

loading

during

exercise

increased

heart

rate

in

an

attempt

to

minimize

the

effects

of

the

reduced

stroke

volume

on

cardiac

output

Thus

the

reduced

cardiac

output

secondary

to

the

high

expiratory

pressures

would

also

be

expected

to

contribute

importantly

to

the

greater

oxygen

debt

that

was

measured

after

EFL

exercise

In

summary

EFL

exercise

in

healthy

subjects

reproduces

many

features

of

COPD

including

acute

respiratory

failure

a

condition

resembling

cor

pulmonale

dyspnea

and

impairment

of

exercise

performance

Furthermore

it

has

led

to

testable

predictions

one

of

which

is

the

rationale

for

this

study

and

which

to

date

have

been

proven

to

be

accurate

-LRB-

Aliverti

et

al.

b

-RRB-

Off-transient

kinetics

The

increase

in

debt

by

that

we

found

during

EFL

exercise

impacted

as

predicted

on

kinetics

during

recovery

Accordingly

the

repayment

of

debt

after

EFL

exercise

started

at

a

higher

compared

to

control

and

the

off-transient

time

constant

of

the

fundamental

and

slow

components

were

shorter

after

EFL

exercise

Collectively

the

findings

describing

the

EFL

off-transient

kinetics

reflect

a

more

rapid

replenishment

of

blood

stores

-LRB-

fundamental

component

-RRB-

and

a

faster

repayment

of

tissue

debt

-LRB-

slow

component

-RRB-

both

of

which

could

result

from

the

greater

deficit

during

EFL

exercise

as

opposed

to

control

exercise

Indeed

if

we

considered

that

cardiac

output

during

EFL

exercise

was

lower

than

control

and

was

not

significantly

different

between

EFL

and

control

then

it

would

be

reasonable

to

expect

that

during

exercise

with

EFL

that

is

known

to

decrease

pulmonary

blood

flow

-LRB-

Aliverti

et

al.

-RRB-

the

arterial

to

mixed

venous

blood

difference

would

be

larger

and

the

mixed

venous

blood

content

would

be

lower

This

notion

was

confirmed

by

our

calculations

of

arterial

to

mixed

venous

blood

When

at

the

end

of

exercise

EFL

was

removed

the

sudden

increase

in

cardiac

output

and

in

blood

perfusing

the

lungs

brought

about

a

refilling

of

the

stores

of

mixed

venous

blood

hence

the

sudden

increase

of

-LRB-

marking

the

replenishment

of

the

body

's

deprived

oxygen

stores

-RRB-

a

faster

kinetics

and

a

larger

debt

in

EFL

-LRB-

Table

-RRB-

The

larger

depletion

of

the

inner

oxygen

stores

is

also

suggested

by

the

longer

time

constants

of

the

on

and

off-transient

response

during

EFL

exercise

-LRB-

Table

-RRB-

Our

calculations

showed

that

approximately

of

the

debt

difference

between

EFL

and

control

exercise

was

due

to

the

blood

replenishment

-LRB-

Table

-RRB-

caused

by

the

reduced

cardiac

output

during

EFL

whereas

the

rest

was

due

to

other

possible

mechanisms

described

below

In

line

with

the

results

of

Cunningham

et

al.

-LRB-

-RRB-

and

Ozyener

et

al.

-LRB-

-RRB-

describing

kinetics

after

heavy

exercise

the

off-transient

data

after

EFL

exercise

were

best

fitted

by

a

two

component

exponential

function

The

fundamental

off-transient

component

after

control

exercise

-LRB-

s

-RRB-

was

similar

to

that

-LRB-

s

-RRB-

described

by

Cunningham

et

al.

-LRB-

-RRB-

and

Ozyener

et

al.

-LRB-

-RRB-

following

very

heavy

exercise

lasting

as

in

the

present

study

for

min

On

the

other

hand

the

time

constant

of

the

fundamental

component

after

EFL

exercise

-LRB-

s

-RRB-

was

significantly

shorter

than

that

of

control

exercise

thus

confirming

replenishment

of

the

body

's

deprived

oxygen

stores

upon

removal

of

EFL

In

addition

the

shorter

time

constant

calculated

after

EFL

is

the

result

of

fitting

the

data

from

higher

starting

points

as

evidenced

by

the

significantly

higher

a

c

values

shown

in

Table

and

Fig.

Furthermore

the

time

constant

of

the

slow

component

after

EFL

exercise

-LRB-

s

-RRB-

was

twofold

longer

than

the

one

-LRB-

s

-RRB-

described

previously

for

very

heavy

exercise

-LRB-

Ozyener

et

al.

-RRB-

possibly

reflecting

the

additive

effects

of

the

EFL-induced

reduction

in

systemic

delivery

on

the

repayment

of

tissue

debt

Although

at

present

the

mechanism

-LRB-

s

-RRB-

of

the

slow

component

is

not

fully

understood

there

are

important

factors

that

could

influence

the

slow

component

after

EFL

exercise

These

include

the

blood

lactate

concentration

-LRB-

Poole

et

al.

-RRB-

the

influence

of

the

metabolic

acidosis

on

the

dissociation

curve

-LRB-

Wasserman

et

al.

-RRB-

the

increased

respiratory

and

cardiac

muscle

energy

requirement

associated

with

EFL

exercise

-LRB-

Aaron

et

al.

Harms

et

al.

-RRB-

the

progressive

recruitment

of

type-II

fibers

-LRB-

Coyle

et

al.

-RRB-

and

to

a

lesser

extent

the

increased

levels

of

circulating

catecholamines

-LRB-

Gaesser

et

al.

-RRB-

associated

with

the

greater

cardiovascular

response

during

EFL

exercise

Importantly

excessive

expiratory

muscle

recruitment

has

been

shown

in

patients

with

airflow

limitation

to

increase

the

oxygen

cost

of

breathing

threefold

-LRB-

Aliverti

et

al.

-RRB-

Accordingly

it

is

reasonable

to

assume

that

the

higher

cost

of

breathing

during

EFL

exercise

would

significantly

enhance

the

dept

as

compared

to

exercise

without

EFL

In

the

present

study

we

utilized

a

double-exponential

function

to

describe

the

off-transient

kinetics

not

only

for

EFL

exercise

but

also

for

control

exercise

-LSB-

typically

fitted

by

a

mono-exponential

function

-LRB-

Cunningham

et

al.

Ozyener

et

al.

-RRB-

-RSB-

in

order

to

allow

adequate

comparisons

of

relevant

parameters

of

recovery

for

both

exercise

tests

Accordingly

the

slow

component

observed

after

control

exercise

was

not

discernible

in

two

subjects

using

the

double-exponential

function

This

is

in

accordance

with

previously

reported

data

by

Cunnnigham

et

al.

-LRB-

-RRB-

who

exercised

healthy

subjects

at

a

similar

work

rate

-LRB-

W

-RRB-

yielding

similar

exercise

-LRB-

l

min

-RRB-

as

the

one

reached

in

the

present

study

during

control

exercise

In

conditions

where

exercise

is

sustained

at

a

moderate

intensity

as

in

the

control

test

the

off-transient

slow

component

is

often

not

discernible

such

that

kinetics

can

retain

first-order

characteristics

-LRB-

Cunnnigham

et

al.

Gerbino

et

al.

Ozyener

et

al.

-RRB-

In

conclusion

the

results

of

the

present

study

provide

further

evidence

that

expiratory

flow

limitation

during

exercise

reduces

systemic

delivery

enhances

the

debt

and

leads

to

hypoxemia

in

part

due

to

hypercapnia

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


What is the mechanism of microalbuminuria in diabetes : a role for the glomerular endothelium ? Microalbuminuria is an important risk factor for cardiovascular disease and progressive renal impairment . This holds true in the general population and particularly in those with diabetes , in whom it is common and marks out those likely to develop macrovascular disease and progressive renal impairment . Understanding the pathophysiological mechanisms through which microalbuminuria occurs holds the key to designing therapies to arrest its development and prevent these later manifestations . Introduction The associations between microalbuminuria , cardiovascular disease and progressive renal impairment are well described , but how these are linked mechanistically is something of a conundrum -LSB- 1 -RSB- . Here we focus on the pathogenesis of microalbuminuria in patients with diabetes , in whom it occurs commonly and has particular significance . In type 1 diabetes the prevalence gradually increases from onset of disease -LRB- 6 % after 1 -- 3 years -RRB- , reaching over 50 % after 20 years -LSB- 2 -RSB- . In type 2 diabetes the prevalence is 20 -- 25 % in both newly diagnosed and established diabetes -LSB- 3 -RSB- . However , it is also instructive to review the general epidemiology of microalbuminuria , including those conditions with which it is associated and those for which it is a risk factor . Such an analysis reveals generalised endothelial dysfunction as a common denominator in microalbuminuria in both the general and diabetic populations . In 1989 , this observation led to the hypothesis that a common process underlies both microalbuminuria and generalised endothelial dysfunction in diabetes . This process was suggested to be the dysregulation of enzymes involved in metabolism of extracellular matrix , the ` Steno hypothesis ' -LSB- 4 -RSB- . Nearly 20 years on from the Steno hypothesis , the determinants of selective glomerular permeability to proteins at the cellular and molecular level are much better understood . In particular , the importance of podocyte-specific proteins in the regulation of selective permeability has been recognised . Similarly , much is now known about the biochemical derangements important in the pathogenesis of diabetic complications . We draw together these elements to consider the pathophysiological mechanisms through which diabetes exerts its effects on glomerular permeability in the initiating stages of diabetic nephropathy , i.e. at or before the appearance of microalbuminuria . These early changes establish the milieu in which the more advanced changes of overt diabetic nephropathy develop . Defining the mechanistic links from biochemical derangements to the appearance of increased urinary albumin highlights key elements in the pathophysiological pathway of the development of both diabetic nephropathy and micro - and macrovascular disease elsewhere . We hold with the established view that increased transglomerular passage of albumin is the major source of microalbuminuria -LSB- 5 -RSB- . While other hypotheses have been advanced , for example , failure of tubular reuptake of albumin , none are sufficiently robust to seriously challenge this position . In both general and diabetic populations , conditions associated with endothelial damage predispose to microalbuminuria In the general -LRB- non-diabetic -RRB- population , hypertension is the major risk factor for microalbuminuria , and the prevalence of microalbuminuria in essential hypertension is around 25 % . Individuals with essential hypertension who develop microalbuminuria have a higher incidence of biochemical disturbances , implying that hypertension per se may not be the cause of microalbuminuria , but , rather , these additional derangements -LSB- 6 -RSB- . Microalbuminuria is strongly associated with vascular disease in hypertensive patients , suggesting that it is a marker of vascular and/or endothelial damage in this condition -LSB- 7 -RSB- . The insulin resistance syndrome describes a clustering of disorders the underlying pathology of which is thought to be related to insulin resistance and/or endothelial dysfunction -LSB- 8 -RSB- . Microalbuminuria is associated with several of the disturbances found in the insulin resistance syndrome , including endothelial dysfunction and obesity , in addition to type 2 diabetes . Proinflammatory cytokines produced by visceral adipocytes -LRB- adipokines -RRB- have recently emerged as important mediators of the increased cardiovascular risk associated with the insulin resistance syndrome . These adipokines represent a possible link from insulin resistance and obesity to microalbuminuria in the non-diabetic population . Microalbuminuria can be detected in patients undergoing major surgery , particularly when complicated by sepsis -LSB- 9 -RSB- , and is associated with other inflammatory states , including rheumatoid arthritis and inflammatory bowel disease -LSB- 10 -RSB- . Microalbuminuria can also be detected in a significant proportion of the normal non-diabetic , normotensive population -LRB- 6.6 % in one study -LSB- 11 -RSB- -RRB- , where it also associates with cardiovascular disease . Male sex -LSB- 11 -RSB- and hormone replacement therapy in women -LSB- 12 -RSB- seem to increase susceptibility to microalbuminuria , and although the basis for this is not clear , the fact that men have a higher incidence of vascular disease in general implies a common aetiology . Hypertension is approximately twice as frequent in individuals with diabetes as in those without , and hypertensive individuals are predisposed to the development of diabetes -LSB- 13 -RSB- . Hypertension is certainly a major determinant of microangiopathy in diabetes , but the relationship between hypertension and microalbuminuria in diabetes is complex . Hypertension and microalbuminuria often coexist in diabetic patients , and reducing blood pressure reduces microalbuminuria in type 1 diabetes -LSB- 14 -RSB- . However , it is unclear whether hypertension contributes to the development of microalbuminuria in diabetes . At least in type 1 diabetes , hypertension and microalbuminuria appear to develop together : in longitudinal studies there is no evidence that hypertension develops before microalbuminuria -LSB- 15 -RSB- . This is more difficult to demonstrate in type 2 diabetes , perhaps because of the heterogeneity of the disease . One third of patients with type 1 diabetes develop advanced nephropathy , and the renal status of probands of diabetic patients makes a difference of nearly 50 % in risk -LSB- 16 -RSB- . A family history of hypertension also predisposes to microalbuminuria in diabetes -LSB- 17 -RSB- . These observations suggest a genetic predisposition in individuals with type 1 diabetes who develop diabetic nephropathy , but as yet no gene has been identified . Perhaps most significant is that this and other evidence suggests that the susceptibility of type 1 diabetic patients to diabetic nephropathy is increased in response to increased genetic risk of insulin resistance -LSB- 18 -RSB- . Consequently , it is the combination of this genetic risk with hyperglycaemia and its sequelae that leads to microalbuminuria and , eventually , diabetic nephropathy . Thus , in the diabetic as well as in the general population the risk factors for the development of microalbuminuria can be grouped into those associated with vascular disease , including endothelial dysfunction , inflammation and insulin resistance . This implies that microalbuminuria may also , at least in these situations , result from endothelial dysfunction . This does not preclude significant damage to other components of the vessel wall , including basement membrane , pericytes , matrix components -LRB- collagen , elastin -RRB- and vascular smooth muscle cells . However , as the principal regulators of vascular permeability , endothelial cells are likely to be involved in systemic increases in permeability . Microalbuminuria is a risk factor for macro - and microangiopathy , including advanced diabetic nephropathy Microalbuminuria is a significant risk factor for cardiovascular mortality in type 1 -LSB- 19 -RSB- and 2 diabetes -LSB- 20 -RSB- , as well as in the non-diabetic population -LSB- 21 -RSB- . In type 1 diabetes , microalbuminuria is closely associated with microangiopathy elsewhere -LRB- e.g. retinopathy -RRB- . Although this association is also present in type 2 diabetes , in a proportion of these patients microalbuminuria does not appear to reflect generalised microvascular damage -LSB- 22 -RSB- . Microalbuminuria predicts the development of overt diabetic nephropathy in type 1 and 2 diabetes ; however , the relationship in type 2 diabetes is less clear because of the greater heterogeneity of this condition and the presence of other risk factors for microalbuminuria in these , usually elderly , patients -LSB- 23 -RSB- . Microalbuminuria invariably precedes overt diabetic nephropathy , and although microalbuminuria may regress spontaneously in a proportion of cases , it remains the best documented predictor for high risk of development of diabetic nephropathy in both type 1 and type 2 diabetes -LSB- 24 -RSB- . The relationship between microalbuminuria and vascular disease suggests a common causality , as established risk factors explain at most a small part of these associations -LSB- 25 -RSB- . Unifying mechanisms , such as generalised endothelial dysfunction or inflammation , are therefore implicated -LSB- 8 -RSB- . Indeed , AER is correlated with endothelial dysfunction in type 1 and type 2 diabetes , and in the non-diabetic population -LSB- 26 -RSB- . Markers of chronic low-grade inflammation , including C-reactive protein -LRB- CRP -RRB- , are also correlated with microalbuminuria in type 1 and type 2 diabetes -LSB- 27 -RSB- , and increasing evidence from the non-diabetic population indicates the importance of inflammation in the pathogenesis of cardiovascular disease -LSB- 28 -RSB- . Generalised and glomerular endothelial dysfunction Markers of endothelial dysfunction , including elevated serum von Willebrand factor -LRB- vWF -RRB- and increased transcapillary albumin escape rate , are present before the onset of microalbuminuria in type 1 diabetes and worsen in association with it -LSB- 27 , 29 -RSB- . Type 2 diabetes is often complicated by the presence of other risk factors for vascular disease , and discerning the contribution of hyperglycaemia and its sequelae to endothelial dysfunction in this condition is more difficult . While in some type 2 diabetic patients microalbuminuria may occur in the absence of evidence of endothelial dysfunction , in others , vWF levels predict its development -LSB- 30 -RSB- . The close association between endothelial dysfunction and microalbuminuria in type 1 diabetes may underlie the predictability of development of diabetic nephropathy and the greater susceptibility to micro - and macrovascular disease in other organs . As endothelial dysfunction is an important antecedent of microalbuminuria in both types of diabetes -LRB- albeit with a less predictable relationship in type 2 -RRB- , it provides an attractive explanation for the association between microalbuminuria and vascular disease in diabetes -LRB- Fig. 1 -RRB- . But to what extent can endothelial dysfunction be said to cause microalbuminuria ? As the glomerular endothelium is exposed to the same diabetic milieu as other endothelia , it is highly likely that it is also dysfunctional . This begs the question of how glomerular endothelial dysfunction could lead to microalbuminuria . To address this we now turn to consider the structure and function of the glomerular filtration barrier -LRB- GFB -RRB- . Fig. 1The relationship between hyperglycaemia , insulin resistance , endothelial dysfunction , macrovascular disease and microalbuminuria in type 1 and type 2 diabetes . Proposed major pathways are represented by red arrows ; those of less certain significance by black arrows . The diagram illustrates , for the example , a possible mechanism for the increased risk of microalbuminuria in patients with type 1 diabetes and a susceptibility to insulin resistance . Particularly in type 2 diabetes , other pathways , not directly involving endothelial dysfunction , are likely in the pathogenesis of macrovascular disease and may also contribute to microalbuminuria -LRB- broken arrows -RRB- The glomerular filtration barrier is a complex biological sieve Unlike other capillaries , glomerular capillaries have a high permeability to water -LRB- hydraulic conductivity -RRB- yet , like other capillaries , are relatively impermeable to macromolecules . These fundamental permeability properties depend on the unique , three-layer structure of the GFB : the endothelium with its glycocalyx , the glomerular basement membrane -LRB- GBM -RRB- and podocytes -LRB- glomerular epithelial cells ; Fig. 2 -RRB- -LSB- 31 -RSB- . Fig. 2Representation of a cross-section through the GFB showing the three-layer structure consisting of glomerular endothelium and glycocalyx , glomerular basement membrane -LRB- GBM -RRB- and podocyte foot processes . Albumin , represented by orange ellipses , does not pass through the normal GFB in significant amounts Glomerular endothelial cells Glomerular endothelial cells are highly specialised cells with regions of attenuated cytoplasm punctuated by numerous fenestrae , circular transcellular pores 60 -- 80 nm in diameter -LSB- 31 , 32 -RSB- . Initially these fenestrations were thought of as empty and to therefore provide little barrier to the passage of proteins . Standard fixation protocols for electron microscopy do not preserve the glycocalyx , but newer fixation techniques have allowed the demonstration of a glomerular endothelial glycocalyx of 200 -- 400 nm in thickness -LSB- 33 , 34 -RSB- . This glycocalyx covers both fenestral and inter-fenestral domains of the glomerular endothelial cell luminal surface.Studies of the systemic endothelial glycocalyx are instructive here . The glycocalyx is a dynamic , hydrated layer largely composed of glycoproteins and proteoglycans with adsorbed plasma proteins . Heparan sulphate proteoglycans -LRB- HSPGs -RRB- are largely responsible for the negative charge characteristics of the glycocalyx . Removal of the glycocalyx increases vascular protein permeability , providing evidence that it hinders the passage of macromolecules -LSB- 35 , 36 -RSB- . Compared with those without , capillaries with fenestrations are much more permeable to water and small solutes , but not to proteins . These characteristics can only be explained by the glycocalyx -LSB- 37 -RSB- . Therefore , the presence of a significant glomerular endothelial glycocalyx implies that the glomerular endothelium significantly contributes to the barrier to macromolecules -LSB- 31 , 32 , 38 -RSB- . Experimental data support this view : in mice treated with glycocalyx-degrading enzymes the distance between luminal lipid droplets and the glomerular endothelium was decreased , and this was accompanied by an increase in AER -LSB- 39 -RSB- . In rats , under normal perfusion conditions albumin is confined to the glomerular capillary lumen and endothelial fenestrae -LSB- 40 -RSB- . Endothelial glycocalyx has the correct anatomical distribution -LRB- on the surface of endothelial cells , including in fenestral openings -RRB- to explain this distribution . Reactive oxygen species -LRB- ROS -RRB- , which are known to disrupt the glycocalyx -LSB- 41 -RSB- , cause torrential proteinuria without any identifiable structural changes in the GFB using standard electron microscopy techniques -LSB- 42 -RSB- . Furthermore , the ability of human glomerular endothelial cell glycocalyx to form a permeability barrier to macromolecules can be directly demonstrated in vitro -LSB- 43 -RSB- . The GBM The GBM is a basal lamina specialised for the structural requirements of the GBM and its filtration function . It is a hydrated meshwork of collagens and laminins to which negatively charged HSPGs are attached . Traditional concepts have therefore characterised the GBM as a charge-selective barrier . However , more recent analyses indicate that it only makes a small direct contribution to the barrier to protein passage -LSB- 31 -RSB- . Podocytes Podocytes , or , more specifically , their interdigitating foot processes , form the outer layer of the GFB -LRB- Fig. 2 -RRB- . The gaps between adjacent foot processes , the ` filtration slits ' -LRB- 25 -- 60 nm -RRB- , are spanned by the slit diaphragm . This is a molecular structure thought to form the most restrictive barrier to the passage of water and macromolecules . The effect of mutations in podocyte-specific proteins -LRB- e.g. nephrin mutations result in congenital nephrotic syndrome -RRB- indicate the importance of podocytes in resisting the passage of protein -LSB- 44 -RSB- . However , exactly how podocytes and their foot processes contribute to selective permeability is not yet clear . Electron microscopy studies have suggested that the slit diaphragm has a porous structure with a pore half-width of 2 nm . However , this figure is too small to account for the experimentally observed passage of solutes of various radii , emphasising that our understanding of slit diaphragm function is incomplete -LSB- 45 -RSB- . Podocyte biology has been reviewed in detail elsewhere -LSB- 44 -RSB- . Passage of albumin across the normal GFB Filtration Water filters across the GFB via gaps through or between cells rather than through the cell cytoplasm . Therefore , the high hydraulic conductivity of the GFB depends on the presence of fenestrae and filtration slits . Fifty per cent of the hydraulic resistance of the GFB is afforded by the two cell layers -LRB- endothelium and podocyte foot processes -RRB- , and 50 % by the GBM -LSB- 46 -RSB- . Solute flux Solute flux -LRB- including albumin -RRB- from the glomerular capillary occurs by a combination of convection -LRB- i.e. being swept along by the filtration of water -RRB- and diffusion -LSB- 31 -RSB- . The rate of convection of a solute depends on the filtration rate and on its reflection coefficient . Reflection coefficients are related to solute size , larger solutes having higher reflection coefficients . A value of 1 indicates that a molecule is totally excluded . The rate of diffusion of a solute depends on the concentration gradient and its diffusivity -LRB- solute permeability -RRB- . The diffusivity of a solute decreases with increasing size . A measure of overall flux of a solute is given by the sieving coefficient : the filtrate to plasma concentration ratio . The sieving coefficient for albumin across the normal GFB is < 0.001 -LSB- 47 -RSB- . The ratio of the contribution of convective to diffusive flux -LRB- the Peclet number -RRB- increases with increasing molecular size . That is , for small molecules , diffusion dominates ; for larger molecules , convection dominates . The Peclet number for albumin across the normal GFB is near to unity , so both diffusion and convection contribute -LSB- 48 -RSB- . Hence , an increase in GFR will result in an increase in the convective flux of albumin . However , despite this , the best estimates indicate that even a 50 % increase in filtration rate would only increase urinary albumin in the sub-microalbuminuric range , as the majority of the excess albumin is reabsorbed by the tubules -LSB- 47 -RSB- . Therefore , for albumin flux to increase sufficiently to produce microalbuminuria -LRB- assuming normal tubular reuptake -RRB- , the GFB must be physically altered in such a way as to increase the sieving coefficient of albumin across it.Models have been developed to correlate the macromolecular sieving properties of the GFB with the structure and properties of its components -LSB- 31 -RSB- . These indicate that the GFB functions as a whole , with each layer having an important contribution to selective permeability . Whereas hydraulic resistances are essentially additive , sieving coefficients are multiplicative . This relationship means that a change in one element significantly affects the overall protein permeability to the same degree , i.e. a 10 % change in permeability of any one layer will produce a 10 % change in overall GFB permeability . Importantly , this means that , regardless of which layer is most restrictive , a change in the permeability of any layer of the GFB could potentially account for microalbuminuria . Structural alterations in the GFB associated with microalbuminuria in diabetes Glomerular structural changes typical of diabetic nephropathy are commonly established by the time microalbuminuria becomes apparent -LSB- 49 , 50 -RSB- . However , the changes seen are heterogeneous , and all may be found in normoalbuminuric diabetic patients -LSB- 51 -RSB- . Early changes described include an increase in glomerular size , GBM thickening , mesangial expansion and broadening of podocyte foot processes -LSB- 49 -- 51 -RSB- . The increase in glomerular size is due both to mesangial expansion and to enlargement in glomerular capillaries . The latter occurs at least in part through angiogenesis , an endothelium-dependent process -LSB- 52 , 53 -RSB- . Glomerular structural changes are less marked in type 2 diabetes , with only a third conforming to the classical pattern observed in type 1 diabetes -LSB- 22 -RSB- . Specific assessment of structural changes in glomerular endothelial cells and associated glycocalyx in diabetes has not been performed . Evidence is emerging , however , that total systemic glycocalyx volume is reduced by acute hyperglycaemia in humans -LSB- 54 -RSB- . Furthermore , type 1 diabetic patients have decreased systemic glycocalyx volume , and this correlates with the presence of microalbuminuria -LSB- 55 -RSB- . GBM thickening alone , without change in composition , does not significantly affect its protein permeability characteristics . Broadening of podocyte foot processes -LRB- effacement -RRB- is an indicator of podocyte injury but generally correlates poorly with degree of proteinuria -LSB- 56 -RSB- . Indeed , proteinuria may occur in the complete absence of structural changes to podocytes -LSB- 57 -RSB- . This is the case with diabetic microalbuminuria , which can occur in the absence of such changes , at least in type 2 diabetes -LSB- 58 -RSB- . Some have suggested that podocyte loss occurs in early diabetes and that this would contribute to the filtration barrier defect -LSB- 51 -RSB- . Others have argued that significant early podocyte loss does not occur -LSB- 59 -RSB- . A likely reconciliation is that the lower proportion of podocytes seen in early disease is due to a relative increase in mesangial and endothelial cells , while podocyte loss occurs at a later stage . Functional alterations in GFB selective permeability associated with microalbuminuria in diabetes Increased flux of albumin across the GFB in diabetic microalbuminuria can be confirmed in experimental models using isolated glomeruli -LSB- 60 -RSB- and by inhibition of tubular reabsorption of albumin -LSB- 61 -RSB- . Analysis of the permeability of the GFB to molecules of varying size and charge can be used to estimate whether the increased flux of albumin is due to loss of size or charge selectivity of the GFB . In animal models of diabetes the defect is primarily in charge selectivity -LSB- 61 -RSB- . In clinically healthy non-diabetic individuals with microalbuminuria , loss of both size and charge selectivity of the GFB can be demonstrated -LSB- 62 -RSB- . However , in type 1 diabetes , defects in charge selectivity occur earlier than loss of size selectivity -LSB- 63 -RSB- . Similarly , in Pima Indians with type 2 diabetes , microalbuminuria is associated with loss of charge selectivity . Loss of size selectivity is seen only in those developing macroalbuminuria -LSB- 58 -RSB- . We have already noted that physical alteration of the GFB is necessary to increase its permeability to albumin . Therefore , this confirmation of changes in GFB selective permeability in diabetic microalbuminuria in the absence of clearly identified structural correlates suggests that the key changes are yet to be elucidated . These considerations and the predominance of defects in charge selectivity point to alterations in the negatively charged glomerular endothelial glycocalyx as the missing link . We now move to consider what aspects of the diabetic milieu are responsible for these GFB changes and how they might cause endothelial , including glycocalyx , dysfunction . Metabolic pathways and effectors from hyperglycaemia to microalbuminuria There is now overwhelming evidence that hyperglycaemia is the major initiating factor in the pathogenesis of diabetic complications , including microalbuminuria . However , most adverse effects of glucose are mediated indirectly through diverse metabolic pathways . Four major hypotheses have highlighted the roles of AGEs , increased activity of the polyol pathway , activation of protein kinase C and increased flux through the hexosamine pathway -LRB- Fig. 3 -RRB- . Activation of these pathways in turn causes dysregulation of a number of effector molecules which cause cellular damage and dysfunction . The roles of these pathways and effectors have been studied in detail in overt diabetic nephropathy , but the importance of these elements individually in its initiation and the appearance of microalbuminuria are less clear . Fig. 3Pathways to microalbuminuria in diabetes . Hyperglycaemia , through increased mitochondrial superoxide production , dysregulates key intracellular metabolic pathways . These in turn lead to the production of effectors that directly cause glomerular endothelial cell -LRB- GEnC -RRB- dysfunction -LRB- particularly of the glycocalyx -RRB- and disturb podocyte -- endothelial cell communication . This results in microalbuminuria . Progression of these lesions and development of other glomerular changes , including podocyte damage , lead to overt diabetic nephropathy For example , TGFβ is an effector molecule with a clear role in the progression of established diabetic nephropathy , but there is no evidence that it is important in the mechanism of proteinuria per se . It is a key pro-fibrotic mediator , and in type 1 and type 2 diabetes , TGFβ is elevated in serum , urine and glomeruli from an early stage of disease . Its levels correlate with the degree of mesangial expansion , interstitial fibrosis and renal insufficiency , but not with microalbuminuria . Similarly , in animal models , blockade of TGFβ signalling inhibits the development of these pathological features , apart from the microalbuminuria -LSB- 64 , 65 -RSB- . Here we focus on selected intermediaries and effectors that have an identifiable role in microalbuminuria . In large part , dissection of these pathways and elucidation of their role in glomerular disease has necessarily relied on tissue culture or animal models . Such are described where human studies are lacking or where they provide additional significant insights . ROS Brownlee has proposed oxidative stress as a unifying mechanism whereby the above-mentioned four pathways are inter-linked in the pathogenesis of diabetic complications -LRB- Fig. 3 -RRB- -LSB- 66 -RSB- . Hyperglycaemia increases oxidative stress through overproduction of superoxide and other ROS by the mitochondrial electron transport chain . ROS have direct cellular effects -LRB- e.g. they increase activity of nuclear factor κB -LSB- NFκB -RSB- , a key inflammatory regulator -RRB- , increase oxidative stress and interact with the above four pathways . Normalisation of mitochondrial superoxide production blocks all four of these pathways implicated in hyperglycaemic damage and corrects a variety of hyperglycaemia-induced phenotypes in target cells of diabetic complications . Endogenous superoxide dismutase normally neutralises excess superoxide but is overwhelmed in the diabetic state.As well as forming a nexus of metabolic pathways dysregulated by hyperglycaemia , ROS can also be considered as effectors through direct cellular actions . ROS decrease glomerular HSPG production -LSB- 67 -RSB- , directly disrupt the endothelial glycocalyx -LSB- 68 -RSB- , interfere with nitric oxide bioavailability and activate NFκB.Glomerular ROS production is increased in experimental diabetes -LSB- 69 -RSB- and transgenic overexpression of superoxide dismutase attenuates renal injury , including increases in AER -LSB- 70 -RSB- . A superoxide dismutase mimetic also ameliorates increases in glomerular permeability both in vivo and ex vivo in non-diabetic models -LSB- 71 -RSB- . Little work has yet been done on the use of antioxidants in human diabetic nephropathy , but in vitro work confirms the importance of ROS in cells of the GFB . Podocytes produce ROS in response to high glucose -LSB- 72 -RSB- , and genetic overexpression of superoxide dismutase prevents inhibition of endothelial nitric oxide synthase -LRB- eNOS -RRB- activity by hyperglycaemia in endothelial cells -LSB- 73 -RSB- . Vascular endothelial growth factor Vascular endothelial growth factor -LRB- VEGF -RRB- is a key regulator of vascular permeability and angiogenesis and is implicated in the pathogenesis of diabetic retinal neovascularisation . In the glomerulus it is produced in large amounts by podocytes and is thought to be important in maintaining glomerular endothelial cell fenestrations -LSB- 74 -RSB- . In rat models of type 1 diabetes , VEGF is upregulated in podocytes throughout the course of disease -LSB- 75 -RSB- . VEGF inhibition attenuates glomerular hypertrophy and albumin excretion and prevents upregulation of eNOS production in glomerular endothelial cells -LSB- 76 -RSB- . Similar effects are observed in some , but not all , models of type 2 diabetes -LSB- 77 -RSB- . In human type 1 diabetes , serum VEGF concentrations vary according to glycaemic control , and higher levels are associated with microvascular complications , including microalbuminuria -LSB- 78 -RSB- . In type 2 diabetes , VEGF is upregulated early in the course of disease and urinary VEGF levels are correlated with microalbuminuria -LSB- 79 -RSB- . Further studies have confirmed initial upregulation of VEGF signalling in type 2 diabetes followed by a downregulation as podocyte loss and sclerosis develops -LSB- 80 -RSB- . VEGF has the potential to induce the new vessel growth seen in early diabetic nephropathy -LSB- 53 -RSB- and to alter the permeability characteristics of the endothelium . Indeed , there is increasing evidence of the importance of precise control of glomerular VEGF for normal GFB function : both transgenic podocyte-specific under - or overexpression result in glomerular abnormalities , including glomerular endothelial changes and proteinuria -LSB- 81 -RSB- . Retardation of albuminuria in experimental diabetes by angiogenesis inhibitors further implies the importance of angiogenesis and/or endothelial-related processes in the development of diabetic microalbuminuria -LSB- 52 , 82 -RSB- . The growth hormone/IGF system Dysregulation of the growth hormone/IGF system can be detected early in experimental diabetes and is associated with both glomerular hypertrophy and microalbuminuria -LSB- 83 -RSB- . All components of the growth hormone/IGF system can be detected at the mRNA level in the normal kidney . However , as detailed histochemical studies have not been performed , the exact location of system components with the glomerulus and the likely role of particular cell types can not be defined.The rapid renal growth in experimental diabetes is preceded by a rise in the renal concentration of IGF-1 -LSB- 84 -RSB- . Somatostatin analogues ameliorate the increase in IGF-1 and renal hypertrophy and reduce AER -LSB- 85 -RSB- . In human type 1 diabetes , serum growth hormone levels and urinary IGF-1 levels are elevated and correlate with microalbuminuria , while an IGF-1 gene polymorphism modifies the risk of development of microalbuminuria -LSB- 86 -RSB- . The somatostatin analogue octreotide reduces macroalbuminuria and endothelial dysfunction in type 2 diabetes -LSB- 87 -RSB- . Taken together , this evidence points to a role for the growth hormone/IGF system early in human diabetic nephropathy . However , there are few clues to its contribution to the pathogenesis of microalbuminuria at the structural level . IGF-1 activates intracellular intermediates , including NFκB , and an IGF-1 receptor inhibitor suppresses VEGF production , suggesting that VEGF is a downstream mediator of the effects of IGF-1 -LSB- 88 -RSB- . Proinflammatory cytokines and adipokines TNFα is a proinflammatory cytokine with diverse actions , including increased production of endothelial cell adhesion molecules and IL-6 , which in turn regulates CRP . TNFα also directly increases endothelial permeability and disrupts the glycocalyx -LSB- 41 , 89 -RSB- . In experimental diabetes , TNFα levels rise in urine and the renal interstitium prior to the onset of albuminuria and correlate with it -LSB- 90 -RSB- , as they do in human type 2 diabetes -LSB- 91 -RSB- . IL-6 levels also correlate with albuminuria in type 1 and 2 diabetes -LSB- 91 , 92 -RSB- . CRP , often thought of as a downstream marker of inflammation , is elevated in both type 1 and 2 diabetes -LSB- 27 , 93 -RSB- and correlates strongly with cardiovascular disease.Adipokines , which include leptin and adiponectin -LRB- and , arguably , TNFα and IL-6 -RRB- , also have the potential to contribute to the development of microalbuminuria in both non-diabetic and diabetic populations . Enlargement of fat cells in obesity is associated with a generalised proinflammatory state , including increased levels of TNFα and IL-6 , and hypersecretion of adipokines , with the exception of adiponectin , which is downregulated -LSB- 94 -RSB- . Leptin induces vascular permeability and synergistically stimulates angiogenesis with VEGF -LSB- 95 -RSB- . Its serum levels correlate strongly with nephropathy in type 2 diabetes -LSB- 96 -RSB- . Adiponectin , unlike other adipokines , appears to have protective effects in the vasculature in general by reducing endothelial cell activation and inflammation.What do these changes in regulation and expression of these mediators tell us about the mechanism of microalbuminuria ? It is clear that ROS and oxidative stress have a central role , given their importance in various metabolic pathways and direct cellular effects , including in the disruption of endothelial glycocalyx , which may be relevant in pathogenesis of microalbuminuria . There is strong evidence for a role of VEGF early in the course of diabetic nephropathy , probably through upregulation of production by podocytes and actions on glomerular endothelial cells disrupting their contribution to the GFB . The importance of dysregulation of angiogenic factors is emphasised by the protective effects of angiogenesis inhibitors . The growth hormone/IGF system is also dysregulated early in diabetes , and it appears that IGF-1 is an important contributor to microalbuminuria through VEGF . Involvement of inflammatory mediators again points to a role for glomerular endothelial cells through endothelial activation , and hence , an increase in permeability through disruption of the glycocalyx . Conclusions In summary , the various avenues of study of diabetic microalbuminuria reviewed converge on the glomerular endothelium . Our analysis therefore leads to the conclusion that this is the site of the initial damage that leads to the development of microalbuminuria in diabetes . The most important aspect of this damage is disruption of the endothelial glycocalyx through actions of mediators dysregulated by the diabetic milieu . Key players include ROS , VEGF and proinflammatory cytokines -LRB- Fig. 4 -RRB- . Disturbance of endothelial cell -- podocyte communication contributes to and amplifies the endothelial lesion . Progression of microalbuminuria to overt nephropathy is accompanied by predictable structural changes in the glomerulus , including podocyte damage and loss . This is the result of the ongoing diabetic milieu and disturbed cell -- cell communication , but is also secondary to increased penetration of the GFB by serum proteins -LSB- 97 -RSB- . Fig. 4Proposed mechanism of glomerular filtration barrier damage leading to diabetic microalbuminuria . High glucose causes dysregulation of mediators including TNFα and enhanced production of ROS , which directly damage the glomerular endothelial glycocalyx leading to microalbuminuria . Increased levels of pro-angiogenic molecules , including VEGF and inflammatory mediators , induce an activated and more permeable glomerular endothelial cell phenotype Thus recent evidence remains broadly supportive of the Steno hypothesis -LSB- 4 -RSB- but it points specifically to disturbance of the endothelial glycocalyx as the common process underlying both microalbuminuria and generalised endothelial dysfunction . While it would be overly simplistic , particularly in type 2 diabetes , to suggest that this is the only factor involved -LRB- Fig. 4 -RRB- , it follows nevertheless that microalbuminuria is an indicator of generalised endothelial dysfunction . Most interestingly , these conclusions imply that glycocalyx dysfunction is involved in the pathogenesis of other vascular disease , both microvascular and macrovascular . Therefore , therapies aimed at protecting or repairing endothelial cells and their glycocalyx would be expected to retard these diseases . Reduction or reversal of microalbuminuria implies resolution of generalised endothelial dysfunction and has potential to be a useful indicator of successful reduction of overall cardiovascular risk -LSB- 98 -RSB- .

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

diabetes
glomerular endothelial cell
glomerular filtration barrier
glycocalyx
microalbuminuria
podocyte