Lung Fluid Balance in Humans: Impact
of hypoxia & the β2 Adrenergic Receptor
Bioastronautics Seminar Series
USRA Division of Space Life Sciences
Bruce D. Johnson, PhD
Professor of Medicine
Human Integrative and Environmental Physiology
Mayo Clinic
Ongoing Research
Synergies between altitude adaptation & heart failure
Healthy altitude adaptation
Heart Failure–adaptation
-↑ VE drive
-↑ VE drive
-hypocapnia
-hypocapnia
-pulmonary htn
-pulmonary htn
-neurohumoral activation
-neurohumoral activation
-periodic breathing-sleep
-Cheyne-Stokes, CSA
-↓ diastolic heart function
-high filling pressures
-HAPE
-pulmonary edema
“lung fluid regulation”
↓PIO2
hypoxic mechanisms
-hypoperfusion
-VA/QC mismatch
-↑ O2 extraction
Background: Healthy lungs and
Lung Water
• Alveoli need to remain relatively fluid-free for
adequate gas exchange
• Estimated that there is 3.8 ml/kg of fluid in the
healthy human lungs*
• Fluid regulation in the lungs requires a balance
between factors influencing accumulation and
those affecting removal
• Constant small flux of fluid from the capillariesÆ
interstitiumÆ alveoliÆ lymph
*Wallin et al. J Appl Physiol. 76:1868-1875, 1994.
Factors Influencing Accumulation
• Fluid accumulation in the lung follows
the forces described by Starling*
• Microvascular pressure (driving fluid out of the
•
•
vasculature)
Hydrostatic pressure of interstitium (opposes
microvascular pressure)
Osmotic pressure of the capillary (microvascular
absorptive pressure)
*Starling. Journal of Physiology (London). 19:312-326, 1896
Factors Influencing Removal
• Lymph flow acts to remove fluid from
the interstitial space
• Epithelial Sodium Channels (ENaC)
on type II alveolar cells act to clear
fluid from the alveoli
• mediated by the beta-2 adrenergic
receptors
Na+
Lung Fluid
Regulation
ß2AR
ENaC
Na+ K+
Alveolus
Na+
Type-II Cell
Type-I
Cell
AQP3
Na+
σ
AQP1
AQP5
AQP4
Type-II Cell
Pi
Capillary
Pc
σ
Net fluid out = K[(Pc-Pi)-σ(πc-πi)]
Lymphatic
πi
πc
Symptoms: >2
-rest dyspnea
- cough
- weakness
- chest tightness or
congestion
Signs: >2
- crackles/wheezing
- central cyanosis
- tachypnea
- tachycardia
Hypoxia
Altitude Pulmonary Edema
Lungs
↑ Sympathetic Activity
 Pap
 PBV
Uneven HPV
focal or regional over perfusion
pulmonary venous constriction
altered vascular permeability
clearance Na+ & H20
 capillary pressure
capillary leak
HAPE
HACKETT PH, ROACH RC. N Engl J Med, Vol. 345, 2001
Hypoxia and lung fluid balance:
Initial Study Aims
1. Develop techniques to assess lung fluid changes
in humans (are they sensitive enough?)
2. Model for altering lung fluid balance
a.
Role of short term hypoxic exposure on lung fluid balance
in healthy adults; evidence for fluid accumulation?
3. Examine the variability in fluid accumulation
across subjects exposed to hypoxia.
4. Factors that contribute to variability across
subjects.
a.
Role of nighttime SaO2 and ∆PASP in the development of
pulmonary edema.
Hypoxia and lung fluid balance:
Hypotheses
1. Techniques would be sensitive to small changes in
lung water (acute fluid loading)
2. Short duration hypoxic exposure would result in mild
fluid accumulation in most people.
3. Low nighttime SaO2 values & ↑PASP would correlate
with indices of fluid accumulation.
4. Some subjects would demonstrate more marked ↑ in
fluid accumulation:
•
HAPE susceptible*
•
abnormal  in PASP
•
blunted hypoxic VE response-↓SaO2
•
smaller lung volumes
*Bartsch P. et. al., Lancet 2002; 360: 571.
Normobaric Hypoxia (↓FIO2)
Colorado Altitude Tent
Inspired O2 =
Rochester
734-47 x 0.2093
= 144 mmHg
Tent
734-47 x 0.12
= 82 mmHg
~14,000 ft.
Hypoxia and lung fluid balance:
General Protocol
Visit 1 – Screening Tests (healthy young adults)
Maximal Exercise Test
Pulmonary Function Testing
Complete Blood Count
Visit 2 – Fluid Loading - saline (sensitivity of methods)
0700 hrs – Pre loading
saline loading (15-20 mn)
0900 hrs – post measures
Physiology (Dm, Vc, Q, Spirometry)
CT Scanning (EILV)
CT Scanning (TLC and EELV)
Blood Draw (catecholamines)
Blood Draw (catecholamines)
Physiology (Dm, Vc, Q spirometry)
Visit 3 – Hypoxia Exposure
1300 hr–Pre Hypoxia measures
12% FIO2 tent (17-18 hrs)
Physiology (Dm, Vc, Q, Spirometry)
Echocardiography (PASP)
CT imaging
Blood Draw (catecholamines)
0700 post measures
Physiology (Dm, Vc, Q, Spirometry)
Echocardiography (PASP)
CT imaging
Blood Draw (catecholamines)
Exercise
repeat
Key Methods:
Determining changes in lung fluid
Vc = pulmonary capillary blood volume
DM = alveolar-capillary membrane conductance
Lung density
tissue volume
*Determined from
diffusing capacities of
the lungs for carbon
monoxide & nitric oxide
(DLCO & DLNO)
Determined from CT
imaging
Subject monitoring
Q = cardiac output – acetylene rebreathe
BP = blood pressure
HR = heart rate
Symptoms = Lake Louise altitude questions
*Guenard et al, Respir Physiol. 1987 Oct;70(1):113-20.
Piiper et al, Adv Exp Med Biol. 1988;222:491-5.
Borland and Higenbottam, Eur Respir J. 1989 Jan;2(1):56-63.
Tamhane et. al., Chest.2001;120:1850-1856.
Lung Diffusing Capacity-CO
DLco
conductance of CO from alveolar air Æ capillary hgb
Reciprocal
resistance to gas transfer across the barrier
1/DLCO
sum of resistances imposed by the (50%) alveolarcapillary membrane (1/DM) & (50%) blood [(1/)θVc]
1
DL
=
1
DM
+
1
θ x Vc
θ = rate of gas uptake/mmHg pressure gradient/ml whole blood [reaction rate of gas with hgb =
(0.73 + 0.0058 PAO2) x 14.6/Hb].
Gas Diffusion Across the AlveolarCapillary Membrane
Alveolar Basement Membrane
Alveolus
Interstitial
Space
Alveolar epithelium
Capillary basement membrane
Alveolar fluid
Capillary endothelium
104 mmhg
40 mmhg
Diffusing of CO2
Diffusing of O2
40 mmhg
1/DM
45 mmhg
Red blood cell
1/θVc
Capillary
Lung Diffusing Capacity-NO
• Ferrous binding site of hgb
• scavenge NO with an affinity ~8,000x CO
• Reaction velocity NO 250x that for CO
• Diffusion coefficient in H2O for NO & CO are similar
• Solubility of NO in H2O only 2x that for CO
• DLNO is predominantly limited by resistance
at the alveolar-capillary membrane (DM).
DLCO/DLNO technique for determination
of DM and Vc
Rebreathe Technique for Lung Diffusion
VT
He &
C2H2
CO &
NO
Computed Tomography-Lung
Density and Tissue Volume
Computed Tomography cont’d
Lung Segmentation
Fluid Loading using a rapid saline
infusion (30 ml/kg over 15-20 min)
Use of Vivometrics LifeShirtTM
Physiological Monitoring in the Altitude Tent
-Ventilation
-Breathing pattern
-Oxygen saturation
-Heart rate
Results – hypoxia and lung fluid balance
N = 25
95% Pred
Snyder et al, J. Appl. Physiol. 2006
Results: Physiological Responses to Saline Infusion,
Hypoxia and Hypoxic exercise (n=25, mean ± SD)
Baseline
HR, beats/min
60±8
Cardiac output,
l/min
4.3±1.1
SBP, mmHg
115±11
DBP, mmHg
79±8
O2 saturation, % 98±1.6
Pulm. Art. Pres.
mmHg
21±8
Intervention
Fluid Challenge
Hypoxia
End Exercise
72±15*
76±11*
166±18*
5.4±1.4*
125±14*
79±11
98±1
4.1±0.8
119±13
71±3
83±3*
150±65*
51±23*
81±4*
2,287 ± 522 ml
20 ± 2 min
37±8*
17±1 h
15±0.4 min
__________________________________________________________________________
Values are means ± SD; n=25. *P<0.05 compared with baseline.
Hypoxia exposure - Most common symptoms included headache, nausea, trouble
sleeping with average Lake Louise Score of 1.6
Results: Changes in Pulmonary Function in
Volume (L) or Flow (L/s)
Response to Saline Infusion and 17 hr hypoxia
FVC
FEV1
FEF50
6
5.5
5
*
*
*
*
*
4.5
4
3.5
3
2.5
PreSaline
PostSaline
PreHypoxia Post Hypoxia
Condition
* = p < 0.05
Results: Changes in alveolar-capillary conductance
(DM) relative to capillary blood volume (Vc) in response
to fluid loading, hypoxia, & hypoxic exercise
Results: Mean lung density in response to saline
infusion, 17-h rest hypoxia, and after hypoxic exercise
water
air
For CT, air = -1000, water = 0
Results: Lung tissue volume at baseline, post
saline, post hypoxia, and post hypoxic exercise from CT
↑ fluid
↓ fluid
Estimation of Lung Water
• Lung Water =
(Pst TV-Vc(ml’s))-(Pre IP TV-Vc(ml’s))
• Pst TV is the given tissue volume post•
•
intervention (in ml’s)
IP TV is the interpolated tissue volume for that
lung volume (in ml’s)
Vc is the pulmonary capillary blood volume
(in ml’s) of that condition
Results: Estimated lung water changes in response to
fluid loading, 17hr hypoxia, & after hypoxic exercise
Results: Change in tissue volume from lung apex Æ base in
response to fluid loading, 17hr hypoxia, & after hypoxic exercise
Summary of findings
• Rapid fluid loading – (expected)
•
•
•
↓ maximal lung volumes & flow rates
↓ in DM and DM/Vc (evidence for reduced gas conductance)
↑ in lung tissue volume
techniques are sensitive to changes in lung water
• Short duration 17-hr hypoxic exposure - (unexpected)
•
•
•
↑ maximal lung volumes & flow rates
↑ in DM and DM/Vc (evidence for improved gas conductance)
↓ in lung tissue volume
“Short duration normobaric hypoxia causes
a loss of lung water in healthy adults”
Snyder et al., J Appl Physiol 101: 1623-1632, 2006.
Loss of lung water (normobaric hypoxia)
Causes vs Study Limitations?
• Hypoxia or recubency related diuresis
• no evidence for significant diuresis (minimal ∆ plasma volume, ↓1.3%)
• Hypoxia exposure not long enough to induce ↑ in lung water
• HAPE, 1st 24-48 hrs at altitude vs 17 hr exposure in present study
• Hypoxia not severe enough
• abrupt exposure to 4300m (no acclimatization period)
• Slept at 4300m
• Normal variation observed across subjects
• Difference between normobaric and hypobaric hypoxia?
Time course of alveolar fluid clearance (A) &
lung water volume (B) in rats exposed to 10%
hypoxia
Normobaric
Hypoxia
Sakuma et. al., Effects of
hypoxia on alveolar fluid
transport capacity in rat lungs.
J Appl Physiol 91: 1766–1774,
2001.
Role of barometric pressure in pulmonary
fluid balance & O2 transport in sheep
hypobaric hypoxia
Normoxic hypobaria
Normobaric hypoxia
Levine B, et al., JAP.1988
Incidence of High Altitude Pulmonary
Edema (HAPE)
• ~15% at 4500m (by stethoscope or X-ray)
• (reports of 4%-70%-definition?)
•
•
•
•
•
Related to rate of ascent
Final altitude achieved
Altitude where one sleeps
Physical activity at altitude
Interindividual susceptibility – genetic variation?
Lancet Jan. 24, 2002,
Hultgren, Herb, High Altitude Medicine Copyright© 1997
Hypoxia Exposure
Interindividual Variation in Lung Fluid Balance
12% subjects
simulated 4300m
with ↑ lung water
Relationship of lung water changes
to TR velocity – hypoxia
100.0
Change in Lung Water (ml)
50.0
0.0
-50.0
-100.0
-150.0
r = 0.24
-200.0
0.0
20.0
40.0
60.0
80.0
Change in TR Velocity (%)
100.0
120.0
Index of PASP
Relationship of Nighttime Oxygen
Saturation to the change in lung water
100.0
Change in Lung Water (ml)
50.0
0.0
-50.0
-100.0
-150.0
-200.0
-250.0
-300.0
76
78
80
82
84
86
Average nighttime O2 saturation (%)
88
90
Individual susceptibility & Genetic Variation
Importance of β2AR ÆStimulation of ENaC
alveolar space
Na+
Agonist
Na+
AC
B2AR
α
Po?
cAMP
Channel
Insertion?
ATP
PKA
K+
Na+
Na+K+ ATPase
Alveolar type II Cell
Interstitial space
β γ
ENaC on type II alveolar cells clear
fluid from alveoli Æ mediated by β2AR
• β2AR stimulation of ENaC:
•
•
•
•
•
↑ the number of ENaC’s on the apical membrane^
↑ the probability of an open ENaC+
↑ fluid clearance from the alveoli in animal models*
β2AR over-expression in transgenic models ↑ lung fluid
clearance with or without catecholamines
potentially ↓ incidence of high-altitude pulmonary edema in
humans#
^Xi-Juan et al. American J. Phys. - Lung Cell. & Mol. Phys. 282:L609-L620, 2002
+Matalon et al. J Appl Physiol. 93:1852-1859, 2002.
*Dumasius et al. Circ Res. 89:907-914, 2001
#
Sartori et al. NEJM vol. 346, 2002
Inhaled β-agonist clears basal lung water at sea level
Alveolar-capillary conductance (DM) relative to capillary volume (Vc)
over time after nebulized Albuterol 2.5mg/3ml saline
1
0.9
Healthy adults, n=28, age=28±5
*
*
DM/Vc
0.8
0.7
0.6
0.5
0.4
0.3
Baseline
15-minutes
Post
Snyder and Johnson. FASEB Journal. 21(5), April 2007.
30-minutes
Post
Time Point
45-minutes
Post
60-minutes
Post
Beta-2 Adrenergic Receptor-Common
Polymorphisms
From: Ligget. Am. J. Respir. Crit. Care Med., Volume 161, Number 3, March 2000, S197-S201
Frequencies of β2AR polymorphisms
in normal subjects, RFHS*
Position
Genotype
Frequency (%)
16
Arg homozygous
Arg/Gly
Gly homozygous
Gln homozygous
Gln/Glu
Glu homozygous
Thr homozygous
Thr/Ile
Ile homozygous
14.6
46.6
38.8
31.6
52.9
15.5
97.1
2.9
0
27
164
*RFHS = Rochester Family Heart Study, Bray et al, Circ. 101:2877-2882. 2000
Array of two-locus genotypes in a subset of
1495 adults (797 women and 698 men, RFHS)
Codon
Arg16Arg
Arg16Gly
Gly16Gly
Totals
Gln27Gln
216
211
57
484 (32%)
Gln27Glu
2
499
243
744 (50%)
Bray et. al., Circulation 101:2877-2882. 2000
Glu27Glu
0
16
251
267 (18%)
Totals
218 (15%)
726 (48%)
551 (37%)
1495
β2AR Polymorphisms and
Physiological Function
From Dishy et al. N Engl J Med. 2001 Oct 4;345(14):1030-5
Influence of the Arg16Gly Polymorphism of the
β2AR on Airway Function During Exercise
A rg
G ly
15
FEF50 (% change)
Gly16
10
n=26
5
*
*
n=16
Arg16
0
0
3
6
9
12
15
18
-5
Tim e (m inutes)
Snyder et. al., Chest, March 1, 2006; 129(3): 762 - 770.
21
24
27
30
Influence of β2AR
Genotype on
Cardiac Function
in Healthy Adults
(n = 64)
Snyder et al, J. Physiol (London)
571.1, 2006, 121-130
Influence of β2AR Genotype on Lymphocyte
Receptor Density (Arg16 n=15, Gly16 n=15)
*
Snyder et. al,. Med Sci Sports Exerc 38(5), 882–886. 2006
Relationship of β2AR density (on lymphocytes)
to cardiac function in healthy adults (n=30)
10
9
Arg16
Gly16
Cardiac Output (L/min)
8
7
6
5
r=0.428
p=0.009
4
3
2
1
0
500
1000
1500
2000
Receptor Density (Receptors/Lymphocyte)
Snyder et. al,. Med Sci Sports Exerc 38(5), 882–886. 2006
2500
Hypothesis
• The Arg16Gly polymorphism of the β2AR
will influence lung fluid balance
• Subjects homozygous for Arg at position
16 will have a reduced ability to clear fluid
from the lung
• Baseline due to differences in receptor
density
• With catecholamine stimulation due to
enhanced desensitization
Methods
• Healthy young adults
• Genotyping
• Vc and Dm
• DLCO, DLNO
• Lung density and tissue volume
• CT imaging
• Pulmonary pressure
• Echocardiography
• Subject monitoring
• HR, BP, Q, symptoms
Hypoxia and lung fluid balance:
General Protocol
Visit 1 – Screening Tests (healthy young adults)
Maximal Exercise Test
Pulmonary Function Testing
Complete Blood Count
Visit 2 – Fluid Loading - saline (sensitivity of methods)
0700 hrs – Pre loading
saline loading (15-20 mn)
0900 hrs – post measures
Physiology (Dm, Vc, Q, Spirometry)
CT Scanning (EILV)
CT Scanning (TLC and EELV)
Blood Draw (catecholamines)
Blood Draw (catecholamines)
Physiology (Dm, Vc, Q spirometry)
Visit 3 – Hypoxia Exposure
1300 hr–Pre Hypoxia measures
12% FIO2 tent (17-18 hrs)
Physiology (Dm, Vc, Q, Spirometry)
Echocardiography (PASP)
CT imaging
Blood Draw (catecholamines)
0700 post measures
Physiology (Dm, Vc, Q, Spirometry)
Echocardiography (PASP)
CT imaging
Blood Draw (catecholamines)
Exercise
repeat
Results: Subject Characteristics
Arg16
Gly16
_____________________________________________
N
14
15
Females (n)
4
3
Age (yrs)
30±7
30±8
Height (cm)
174±7
181±8
Weight (Kg)
76±15
82±11
Body Mass Index (kg/m2)
25±4
25±4
VO2peak (mL/kg/min)
37±7
40±7
% Pred
103±15
105±11
______________________________________________
* p<0.05 between genotype groups
Results: Physiological Responses to
Fluid Loading and Hypoxia
BASELINE
Post SALINE
Arg16 Gly16
Arg16
HR,
bpm
62±3
73±4*
SBP, mmHg
102±8 120±4
122±4* 127±4
114±4 124±3^
DBP, mmHg
78±2
77±5
79±3
79±4
68±1*
73±2*
MAP, mmHg
87±4
93±3
93±3
95±3
84±2*
90±2*^
SAO2, %
98±1
98±1
98±1
98±1
83±1*
83±1*
RVSP,
mmHg
28±3
30±2
39±3*
37±2*
58±2
Gly16
End HYPOXIA
68±4*
Arg16
Gly16
78±3*
72±3*
Epinephrine (%change
from baseline)
Results: Catecholamine Levels
2400
2000
1600
Arg16
Gly16
1200
800
400
0
Post Saline
Post Hypoxia
Condition
Post Hypoxic
Exercise
100.0
Results: DM/Vc
Arg16
Gly16
*
*
80.0
%(Dm/Vc)
60.0
40.0
20.0
0.0
-20.0
-40.0
*
Post Saline
Post Hypoxia
Condition
Post Hypoxic Exercise
Results: Lung Tissue Volume (CT)
*
Tissue Volume (% change from
baseline)
15.0
Arg16
Gly16
10.0
5.0
0.0
*
baseline
*
-5.0
-10.0
-15.0
-20.0
Post Saline
Post Hypoxia
Condition
Post Hypoxic Exercise
Results: Estimated Changes in
Lung Water
25
20
Arg16
Gly16
*
15
% Change
10
5
0
-5
-10
-15
*
-20
-25
Saline
Hypoxia
Condition
*
Hypoxic Exercise
Relationship of %∆ in β2AR density with
17hr hypoxia to ∆’s in lung water
Change in Lung Water Post Hypoxic
Exposure (ml)
50.0
0.0
-50.0
-100.0
-150.0
r = 0.64
-200.0
-40.0
- Arg16
- Gly16
-20.0
0.0
20.0
40.0
60.0
80.0
% change Beta 2 Adrenergic Receptor Density
100.0
Summary: Genetic Variation of the
β2ARs & Lung Fluid Balance
• Gene that encodes the β2AR has common functional
polymorphisms in humans
• Arg16Gly polymorphism of the β2AR directly or
indirectly influences receptor density & susceptibility to
agonist mediated desensitization.
• In healthy adults, subjects homozygous for Arg at aa 16
(and homozygous for Gln at aa 27) have a reduced
ability to clear fluid with rapid fluid loading or with
hypoxic exposure.
• mediated via ENaC or lymphatics
Unresolved Issues
• Are homozygous Arg16 subjects more
susceptible to HAPE?
• Is receptor function better explained by more
extensive haplotypes rather than sNPs?
• Other polymorphisms in lung fluid removal
pathways that may play a role (e.g., α subunit of
the ENaC, α1 & α2 isoforms of Na+K+ ATPase, PNMT)
• Why the decrease in lung water with hypoxia,
sustained into exercise?
• Exposure time, severity, normal variation,
barometric pressure ∆?