Nephrol Dial Transplant (2010) 25: 2502–2510
doi: 10.1093/ndt/gfq111
Advance Access publication 17 March 2010
Original Articles
Protein-enriched diet increases water absorption via the aquaporin-2
water channels in healthy humans
Thomas Guldager Lauridsen1, Henrik Vase1, Jørn Starklint1, Jesper N. Bech1 and Erling B. Pedersen1,2
1
Department of Medicine, Department of Medical Research, Regional Hospital Holstebro, Lægaardvej 12, 7500 Holstebro, Denmark
and 2University of Aarhus, 8000 Aarhus C, Denmark
Correspondence and offprint requests to: Thomas Guldager Lauridsen; E-mail: [email protected]
Abstract
Background. According to animal experiments, a proteinenriched diet increased renal absorption of sodium and water. We wanted to test the hypothesis that a protein-enriched
diet would increase the expression of the aquaporin-2 water
channels and the epithelial sodium channels in the distal
part of the nephron using biomarkers for the activity of
the two channels.
Methods. We performed a randomized, placebo controlled
crossover study in 13 healthy humans to examine the effect of a protein-enriched diet on renal handling of water and sodium during baseline condition and during
hypertonic saline infusion. We measured the effect of the
protein-enriched diet on urinary excretions of aquaporin-2
(u-AQP2), the β-fraction of the epithelial sodium channels (u-ENaCβ), free water clearance (CH2O), fractional
excretion of sodium and vasoactive hormones.
Results. During baseline conditions, u-AQP2 increased,
and C H2O decreased during the protein-enriched diet,
whereas u-ENaCβ was unchanged, although the urinary
sodium excretion increased. During hypertonic saline infusion, the response in the effect variables did not deviate
between protein-enriched and normal diet. Plasma concentrations of angiotensin II and aldosterone increased as well
as pulse rate. Vasopressin in plasma was unchanged, and
prostaglandin E2 fell during the protein-enriched diet.
Conclusions. The protein-enriched diet increased water
absorption via an increased transport via the aquaporin-2
water channels. The increased u-AQP2 might be due to a
reduced prostaglandin level. The increase in renal sodium
excretion seems to be mediated in another part of the nephron than the epithelial sodium channels.
Keywords: aldosterone; angiotensin II; epithelial sodium channels
free water clearance; vasopressin
Introduction
In studies of healthy subjects, children, elderly, hypertensive patients and during pregnancy, a protein-enriched diet
and amino acid infusion increased glomerular filtration
rate (GFR) and renal plasma flow, which corresponds to
the renal reserve capacity [1–7]. According to studies of
isolated nephrons from rats, a protein-enriched diet suppressed the tubuloglomerular feedback mechanism (TGF)
[8,9] and the increased tubular absorption of sodium and
water. Thus, the amount of sodium reaching the macula
densa was reduced, and the increase in GFR was attributed
to a decrease in TGF.
In rats, the mechanism for increased sodium absorption
was attributed to the increased Na+–K+–ATP-ase activity in
the thick ascending limb of Henle's loop, whereas the
mechanism of increased water absorption has not been
clarified [10]. Studies in healthy humans and patients with
chronic glomerulonephritis showed that amino acid infusion caused a reduction in the proximal tubular absorption
of sodium and water and an increase in distal tubular
absorption resulting in an unchanged sodium and water
excretion [7,11,12].
A protein-enriched diet affects hormones that are involved in regulation of the expression of aquaporin-2
(AQP2) water channels and the epithelial sodium channels
(ENaC), such as vasopressin, the prostaglandin system, the
renin–angiotensin–aldosterone system, the natriuretic peptide system and the sympathetic nervous system [13–20].
It seems likely that changes in the function of AQP2 and
ENaC channels could be involved in the increased water
and sodium absorption during the protein-enriched diet.
AQP2 and ENaC are expressed in the distal tubules, and
u-AQP2 and u-ENaC reflect the activity of these channels
for water and sodium transport via the principal cells. The
role of these channels has not previously been studied during a diet with high-protein content.
In the present study, we measured the effect of proteinenriched diet on u-AQP2 and u-ENaCβ. We wanted to test
the hypothesis that a protein-enriched diet firstly would increase u-AQP2 and u-ENaCβ during baseline condition,
and secondly change the renal response to hypertonic saline
infusion.
We performed a randomized, placebo controlled crossover study in healthy humans to examine the effect of
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Protein-enriched diet and aquaporin-2 and epithelial sodium channels
protein-enriched diet on renal handling of water and sodium during baseline condition and during hypertonic saline infusion. We measured the effect of protein-enriched
diet on u-AQP2, u-ENaCβ, fractional urinary excretion of
sodium (FENa), urinary excretion of prostaglandin E2
(u-PGE2), urinary excretion of cyclic AMP (u-c-AMP),
free water clearance (CH2O), and plasma concentrations
of renin (PRC), angiotensin II (p-Ang II), aldosterone
(p-Aldo), vasopressin (p-AVP), atrial natriuretic peptide
(p-ANP) and brain natriuretic peptide (p-BNP).
Materials and methods
Participants
2503
Procedure
Subjects were studied on five consecutive days.
Day 1–3: The participants ate the specified diet, drank fluid (35 mL/kg
body weight) and maintained normal physical activity.
Day 4: As Day 1–3. In addition, the subjects collected urine during
24 h.
Day 5: The participants arrived at 7:30 a.m. in the laboratory. An intravenous catheter was placed in fossa cubiti on each side; one for collection of blood samples, the other for infusion of the 51Cr–EDTA and
hypertonic saline. Urine was collected from 7:00–9:30 a.m. to measure
the effect variables. Afterwards, urine was collected at the following
seven periods: 9:30–10:00 a.m. (P-1), 10:00–10:30 a.m. (P-2),
10:30–11:00 a.m. (P-3), 11:00–11:30 a.m. (P-4), 11:30–12:00 p.m.
(P-5), 12:00–12:30 p.m. (P-6) and 12:30–01:00 p.m. (P-7). Urine
was analysed for u-AQP-2, u-ENaC, u-osmolality ratio (Osm), u-Na,
u-K, u-creatinine (crea), u-c-AMP, u-PGE2 and u-51Cr–EDTA. The
subjects voided in the standing or sitting position. Otherwise, they
were in the supine position during the examination. Blood samples
were taken every 30 min starting at 9:30 a.m. for analysis of p-AVP,
p-Osm, p-K, p-Na, p-crea, p-albumin and p-51Cr–EDTA. In addition,
the blood samples for measurements of p-ANP, p-BNP, p-PRC, p-Ang
II and p-Aldo were drawn at 8:00 a.m., 11:00 a.m., 12:00 p.m. and
1:00 p.m. A total amount of 350-mL blood was drawn during the each
study day. The blood drawn at blood sampling was immediately substituted with isotonic saline. From 11:00 a.m. to 11:30 a.m. (P-4), 3%
saline was infused (7 mL/kg body weight). Blood pressure and pulse
rate were measured every 30 min during the examination.
Inclusion criteria were as follows: both males and females, age 18–
65 years and body mass index <30. Exclusion criteria were as follows:
clinical signs or history of disease in the heart, lungs, kidneys or endocrine organs; abnormal laboratory tests [blood haemoglobin, white cell
count, platelet counts, plasma concentrations (sodium, potassium, creatinine, albumin, bilirubin, alanine aminotransferase and cholesterol); blood
glucose; and albumin and glucose in urine]; malignancies; arterial hypertension (i.e. casual blood pressure >140/90 mmHg); alcohol abuse (>21
drinks per week for males and >14 for females); medical treatment; pregnancy; breastfeeding; lack of oral contraceptive treatment to women in the
fertile age; intercurrent diseases; problems with blood sampling or urine
collection; medicine abuse; donation of blood <1 month before the study;
and unwillingness to participate. Withdrawal criterion is a development of
one or more of the exclusion criteria.
The participants were weighed before and after the examination on
Day 5.
Ethics
Effect variables
The local medical ethics committee approved the study. All participants
received written information and gave their consent by signature.
The main effect variables were u-AQP2 and u-ENaCβ. The other effect
variables were u-PGE2, u-c-AMP, p-AVP, CH2O, urine volume, FENa, pOsm, u-Osm, p-PRC, p-Ang II, p-Aldo, p-ANP, p-BNP, pulse rate and
blood pressure.
Design
The study was randomized, placebo-controlled, single-blind and overcrossed. There was a time interval of 2 weeks between the two examinations. Each examination took 5 days.
Recruitment
Participants were recruited by advertisements in public and private institutions.
Number of participants
The relevant difference between protein-enriched and normal diet was
considered to be an increase in u-AQP2 of 0.2 ng/min. The standard deviation was estimated to be 0.14 ng/min. With a level of significance of
5% and a power of 90%, it could be calculated that 12–14 healthy subjects
needed to be included in the trial.
Measurements
Diet and fluid intake
The normal energy requirement was calculated with the formula: weight
(kilogram) × 100 (kilojoules) × activity factor (AF). AF ranged from 1.3
to 2.4 with a possible extra of 0.3 for physical activity in the spare time,
e.g. 30 min of sport 5–6 times a week. AF of 1.3 indicates no physical
activity, of 1.4–1.5 indicates sedentary work without physical activity in
the spare time, of 1.6–1.7 indicates work with walking during work hour
and/or physical activity in the spare time, of 1.8–1.9 corresponds to shop
assistant (standing/walk all day), and of 2.0–2.4 indicates hard physical
activity with or without physical activity in the spare time.
The diet had an energy amount according to weight and physical activity. The diet contained 12 000 kJ/day, if the estimated energy requirement was 10 000 kJ/day or above. It contained 8000 kJ/day, if the
estimated energy requirement was lower. The normal diet specifications
were: 55% of the total energy content from carbohydrates, 15% from proteins and 30% from lipids. The protein-enriched diet specifications were:
40% of the total energy content from carbohydrates, 30% from protein
and 30% from lipids. Sodium content was 60–70 mmol/day in both diets.
The diet contained three main meals and three small meals. The participants were not allowed to add any spices or sodium to the meals or to divide
the meals into bigger or smaller portions. The participants drank fluid according to weight (35 mL/kg/24 h). The fluid was tap water and one-half
litre of low-fat milk (energy content of milk was included in the calculation
of the diet). The participants maintained normal physical activity during the
study. The hospital catering officer prepared all the meals.
Glomerular filtration rate was measured using the constant infusion clearance technique with51Cr–EDTA as a reference substance.
u-AQP-2 was measured by radioimmunoassay as previously described,
and antibodies were raised in rabbits to a synthetic peptide corresponding
to the 15 COOH terminal amino acids in human AQP2 to which was
added an NH2 terminal cysteine for conjugation and affinity purification
[21]. Minimal detection level was 32 pg/tube. The coefficients of variation were 11.7% (inter-assay) and 5.9% (intra-assay).
U-c-AMP was measured using a kit obtained from R & D Systems, Minneapolis, MN, USA. Minimal detection level was 12.5 pmol/tube. The coefficients of variation were 6.9% (inter-assay) and 5.3% (intra-assay).
ENaCβ was measured by a newly developed radioimmunoassay (RIA).
Urine samples were kept frozen at −20°C until assayed. ENaCβ was synthesized and purchased by Lofstrand Labs Limited—Gaithersburg, MD,
USA. The β-ENaC antibody was raised against a synthetic peptide in
rabbits and affinity purified as previously described [22]. Iodination of
ENaCβ was performed by the chloramine T method using 40 μg of
ENaCβ and 37 MBq 125I. The reaction was stopped by the addition
of 20% human serum albumin. 125I-labelled ENaCβ was separated from
the iodination mixture by the use of a Sephadex G-25 Fine column. The
assay buffer was 40 mM sodium phosphate (pH = 7.4), 0.2% human
albumin, 0.1% Triton X-100 and 0.4% EDTA. A 1.5% solution of gamma globulins from pig (Sigma) and 25% polyethylene glycol 6000
(Merck) also containing 0.625% Tween 20 (Merck) was prepared using
the 0.4 M phosphate buffer. Urine samples were kept frozen at −20°C.
2504
After thawing out, the urine samples were centrifuged for 5 min at
1.6 × 100 g (3000 rpm). The supernatant was extracted using Sep-Pak
C18. The elution fluid was 4 mL of a mixture comprising 90% methanol,
0.5% acetic acid and 9.5% demineralized water. The eluates were freeze
dried and kept at −20°C until assayed. The mixture of 300 μL of standard or freeze-dried urine eluates redissolved in 300 μL assay buffer, and
50 μL of antibody was incubated for 24 h at 4°C. Thereafter, 50 μL of the
tracer were added, and the mixture was incubated for a further 24 h at 4°C.
Gamma globulin from pigs (100 μL) and 2 mL polyethylene glycol 6000
were added. The mixture was centrifuged at 3500 rpm for 20 min at 4°C.
The supernatant (free fraction) was poured off, and the precipitate (bound
fraction) was counted in a gamma counter. The unknown content in urine
extracts was read from a standard curve. For 13 consecutive standard
curves, the zero standard was 70 ± 1.6%, and for increasing amounts of
ENaCβ standard, the binding inhibition was: 69 ± 1.4% (15.6 pg/tube), 66 ±
1.5% (31.25 pg/tube), 62 ± 1.6% (62.5 pg/tube), 54 ± 1.5% (125 pg/tube),
40 ± 1.4% (250 pg/tube), 26 ± 1.2% (500 pg/tube), 14 ± 0.6% (1000 pg/
tube), 8.2 ± 0.4% (2000 pg/tube) and 5.1 ± 0.3% (4000 pg/tube). The ID
50, i.e. the concentration of standard needed for 50% binding inhibition,
was 322 ± 12 pg/tube (n = 13). The non-specific binding determined by
performing the RIA without antibody was 1.3 ± 0.3% (n = 13). The interassay variation was determined by quality controls from the same urine
pool spiked with ENaCβ standard. In consecutive assays, the coefficient
of variation was: at a mean level of 78 pg/tube 12% (12 assays), at a mean
level of 155 pg/tube 10% (12 assays) and at a mean level of 394 pg/tube
17% (10 assays). The intra-assay variation was determined on samples
from the same urine pool in several assays at different concentration levels.
At a mean level of 180 pg/tube (n = 10) and 406 pg/tube (n= 10), the coefficients of variation were 6.4% and 9.0%, respectively. In addition, the
coefficients of variation were calculated on the basis of duplicate determinations in different assays to 9.1% (n = 22) in the range 58–101 pg/tube,
8.6% (n = 26) in the range 143–203 pg/tube, 8.7% (n = 20) in the range
205–421 pg/tube and 10.0% (n = 68) in the whole range 58–421 pg/tube.
The sensitivity calculated as the smallest detectable difference at the 95%
confidence limit was 10 pg/tube in the range 58–101 pg/tube (n = 22),
20 pg/tube in the range 143–203 pg/tube (n = 26), 48 pg/tube in the range
205–421 pg/tube (n = 20) and 28 pg/tube in the whole range 58–421 pg/
tube (n = 68). The lower detectable limit of the assay was 34 pg/tube. It was
calculated using the average zero binding for 13 consecutive assays minus
2 SD. The volume of urine used for extraction from the same pool was
varied (18 different volumes in the range 250–6000 μL), and the mean
concentration measured was 89 ± 6 pg/mL. There was a highly significant
correlation between the extracted volume of urine and the amount of picogram per tube (r = 0.99, n = 18). Recovery of the labelled tracer during
the extraction–freezing drying procedure was 94 ± 3% (n = 13), 95 ± 3%
(n = 13), 95 ± 2% (n = 10) and 95 ± 2% (n = 7) in four different pools
used in several extraction procedures. When ENaCβ in the range of 62.5–
250 pg was added to urine, a highly significant correlation was found
between the measured and the expected values (r = 0.981, n = 12, P <
0.001). We measured u-ENaCβ in nine patients with arterial hypertension
treated with amiloride.
During the study day, the patients collected a urine sample at 08:00 a.m.
and 11:00 a.m. They took no medication in the morning before the first
urine sample. Immediately after the first urine sampling, the patients took
their usual dose of amiloride 5 or 10 mg. In the whole group, a significant correlation was found between the changes in u-Na/crea and changes
in u-ENaCβ/crea (ρ = −0.720, n = 9, P = 0.029).
u-PGE2 was measured by a kit from Assay Designs, Inc., Ann Arbor,
MI, USA. The coefficients of variations were 10.9% (inter-assay) and
6.3% (intra-assay).
The blood samples were centrifuged for 15 min at 3000 rpm at 4°C.
Plasma was separated from blood cells and kept frozen at −20°C until
assayed. AVP, ANP, BNP and Ang II were extracted from plasma with
C18 Sep-Pak (Water associates, Milford, MA, USA) and subsequently determined by radioimmunoassays [23,24]. The antibody against AVP was a
gift from Professor Jacques Dürr, Miami, FL, USA. Minimal detection
level was 0.5 pmol/L. The coefficients of variation were 13% (inter-assay)
and 9% (intra-assay). Rabbit anti-ANP antibody was obtained from the
Department of Clinical Chemistry, Bispebjerg Hospital, Denmark. Minimal detection level was 0.5 pmol/L, and the coefficients of variation were
12% (inter-assay) and 10% (intra-assay). Rabbit anti-BNP antibody without cross-reactivity with urodilatin and α-ANP was used. Minimal detection level was 0.5 pmol/L plasma. The coefficients of variation were 11%
(inter-assay) and 6% (intra-assay). The antibody against Ang II was ob-
T.G. Lauridsen et al.
tained from the Department of Clinical Physiology, Glostrup Hospital,
Denmark. Minimal detection level was 2 pmol/L. The coefficients of variation were 12% (inter-assay) and 8% (intra-assay).
Aldosterone in plasma was determined by radioimmunoassay using a
kit from Diagnostic Systems Laboratories Inc., Webster, TX, USA. Minimal detection level was 22 pmol/L. The coefficients of variations were
8.2% (inter-assay) and 3.9% (intra-assay).
PRC was determined by radioimmunoassay using a kit from CIS Bio
International, Gif-Sur-Yvette Cedex, France. Minimal detection level was
1 pg/mL. The coefficients of variation were 14.5% (inter-assay) and 4.5%
(intra-assay).
Plasma and urinary osmolality were measured by freezing-point depression (Advanced Model 3900 multisampling osmometer).
Blood pressure was measured with UA-743 digital blood pressure metre (A&D Company, Tokyo, Japan).
Plasma and urinary concentrations of sodium and potassium were
measured by routine methods at the Department of Clinical Biochemistry,
Holstebro Hospital, Denmark.
All clearances were standardized to a body surface area of 1.73 m2 [10].
Statistics
SPSS statistical software (version 17.0) was used for all the analyses. The
level of significance was P < 0.05 in all analyses. We used a general linear
model with repeated measures for comparison between protein diet treatment and recommended diet when several measurements were done during the examination. A paired t-test was used for comparison between the
two groups. Bonferroni correction was used when appropriate. Values are
given as mean ± 1 SD.
Results
Demographics
Sixteen subjects were allocated to the study. Three participants were excluded, because they could not accept
the protein-enriched diet. Thirteen participants were included in the study, seven women and six men with a
mean age of 26 ± 6 years. Blood pressure was 120/70 ±
9/5 mmHg. Blood samples showed: β-haemoglobin 8.2 ±
0.89 mmol/L, p-sodium 139 ± 1.5 mmol/L, p-potassium
4.0 ± 0.1 mmol/L, p-albumin 44 ± 2.9 g/L, p-creatinine
77 ± 11 μmol/L, p-bilirubin 10 ± 4 μmol/L, p-alanine
aminotransaminase 21 ± 8 U/l, p-glucose 5.0 ±
0.8 mmol/L, p-cholesterol 4.4 ± 0.7 mmol/L.
Table 1. The effect of protein-enriched diet on urine volume (u-vol),
urinary excretions of sodium (u-Na), prostaglandin E2 (u-PGE2), cyclic
AMP (u-c-AMP), aquaporin-2 (u-AQP2), epithelial sodium channel
fraction β (u-ENaC β ), urine osmolality (u-Osm) and free water
clearance (CH2O) in the 24-h urine collection in a randomized, placebocontrolled, crossover study in 13 healthy subjects
u-vol (mL/24 h)
u-Na (mmol/24 h)
u-PGE2 (pg/min)
u-c-AMP (pmol/min)
u-AQP2 (ng/μmol creatinine)
u-ENaC (pg/μmol creatinine)
u-Osm (mosm/kg H2O)
CH2O (mL/min)
Normal diet
Protein-enriched
diet
P
2423 (432)
52 (19)
1016 (1071)
3590 (767)
92.5 (14)
13.5 (3)
305 (83)
−0.002 (0.37)
2376 (840)
75 (27)
6174 (10 527)
3916 (738)
164.0 (41)
13.4 (3)
569 (162)
−1.38 (0.79)
NS
<0.001
NS
NS
<0.001
NS
<0.001
<0.001
Values are means and (1 SD). A paired t-test was used for comparison of
means. NS, not significant.
Protein-enriched diet and aquaporin-2 and epithelial sodium channels
3,0
P<0.001
U-AQP2 ng/min
2,5
2,0
2505
Table 2. The effect of protein-enriched diet on urine volume (u-vol),
urinary excretions of aquaporin-2 (u-AQP2), epithelial sodium channel
fraction β (u-ENaCβ), prostaglandin E2 (u-PGE2), cyclic AMP (u-cAMP), and free water clearance (CH2O), serum osmolality (s-Osm),
urine osmolality (u-Osm) and plasma arginine vasopressin (p-AVP)
during a 2.5-h urine collection between the 24-h urine collection and
the hypertonic saline infusion study in a randomized, placebocontrolled, crossover study in 13 healthy subjects
1,5
Normal diet
Protein-enriched
diet
P
414
91.3
1.20
11.8
114.4
1201
5470
2.74
286
159
0.82
504
156.6
2.1
12.8
121.5
878
6478
0.13
289
341
0.98
NS
<0.001
<0.02
NS
NS
<0.002
NS
<0.02
<0.04
<0.006
<0.02
1,0
0,5
0,0
normal
protein
Paired t-test
Fig. 1. Effect of protein-enriched diet on urinary excretion of aquaporin-2
(u-AQP2) in a 24-h urine collection in a randomized, placebo-controlled,
crossover study in 13 healthy subjects.
Effect of protein-enriched diet on u-AQP2, u-ENaCβ,
u-PGE2, u-c-AMP, CH2O, FENa and p-AVP
Table 1 shows the effect of protein-enriched diet during a
24-h period of urine collection. u-AQP2/u-crea was increased (77%, P < 0.001), and u-AQP2 (95%, P < 0.001)
(Figure 1). CH2O were significantly reduced (P < 0.001).
u-ENaC β /u-crea and u-ENaC β were not statistically
changed by the protein-enriched diet. Urinary sodium excretion was increased significantly (43%, P < 0.001) during the protein-enriched diet. Urine volume and u-c-AMP
were unchanged. u-PGE2 was unchanged, but the variation
in u-PGE2 was very high.
Table 2 shows the effect variables in the 2.5-h urine collection made between the 24-h collection and the start of
the saline infusion study. We found significantly increased
levels of u-AQP2/u-crea (70%, P < 0.001) and u-AQP2
(75%, P < 0.02) during the protein-enriched diet. CH2O
was significantly decreased (P < 0.02), and p-AVP was
significantly increased 19% (P < 0.02) during the proteinenriched diet compared with the normal diet.
u-ENaCβ/u-crea and u-ENaCβ were not significantly
changed by the protein-enriched diet. Serum osmolality
(s-Osm) was significantly increased (1%, P < 0.04) during the protein-enriched diet, whereas u-PGE2 was significantly reduced by 27% (P < 0.002). We found no
significant changes in u-c-AMP and urine volume.
Effect of protein-enriched diet on the response to
hypertonic saline infusion
Table 3 shows the effect variables before, during and after
hypertonic saline infusion between the protein-enriched
diet and the normal diet. u-AQP2, u-Osm and p-Osm levels
were significantly increased during the protein-enriched
diet. During both diets, u-AQP2, u-Osm and p-Osm increased significantly after hypertonic saline infusion,
but there were no statistical difference between the two
diets regarding the response to hypertonic saline infusion
(Figure 2). The protein-enhanced diet significantly reduced
u-vol (mL/150 min)
u-AQP2 (ng/μmol creatinine)
u-AQP2 (ng/min)
u-ENaC (pg/μmol creatinine)
u-ENaC (pg/min)
u-PGE2 (pg/min)
u-c-AMP (pmol/min)
CH2O (mL/min)
s-Osm (mosm/kg H2O)
u-Osm (mosm/kg H2O)
p-AVP (pmol/L)
(262)
(16)
(0.5)
(2)
(42)
(664)
(360)
(2.48)
(2.9)
(163)
(0.2)
(323)
(43)
(1.0)
(4)
(62)
(633)
(4122)
(2.72)
(3.9)
(182)
(0.3)
Values are means and (1 SD). A paired t-test was used for comparison of
means. NS, not significant.
u-PGE2 and CH2O. Both u-PGE2 and CH2O were reduced
after hypertonic saline infusion, but there was no statistical
difference in the response to hypertonic saline infusion. In
addition, we found no statistical differences in p-AVP,
u-ENaCβ, urine volume, u-c-AMP and GFR. The levels
of p-AVP, u-ENaCβ and u-c-AMP all increased significantly after hypertonic saline infusion, but there were no statistical significant differences between the protein-enriched
diet compared with the normal diet in the response to hypertonic saline period. Urine volume fell significantly after
hypertonic saline infusion and to the same extent in the two
dietary groups. GFR was unchanged.
Figure 3 shows that FENa increased significantly after hypertonic saline infusion, but we found no statistical difference in the response to hypertonic saline between the two
dietary groups. Figure 4 shows that u-ENaCβ increased significantly in both groups during hypertonic saline infusion.
FENa rose faster than u-ENaCβ. Thus, FENa level was significantly higher already during the hypertonic saline infusion,
whereas u-ENaCβ first increased significantly at 60 min after during both diets. There was a tendency to a higher FENa
after hypertonic saline infusion during the protein-enriched
diet, but it did not deviate significantly from the normal diet.
Effect of protein-enriched diet on vasoactive hormones at
baseline, before and after hypertonic saline infusion
p-Aldo was significantly increased during the proteinenriched diet (Table 4). p-Aldo was significantly increased
before, during and after hypertonic saline infusion in subjects on protein-enriched diet compared with recommended
diet. During both protein-enriched diet and normal diet, pAldo decreased significantly after 3% saline infusion, and
there was no difference between the two groups in response
to hypertonic saline infusion.
p-Ang II was significantly increased during the proteinenriched diet compared with the normal diet. The difference disappeared during the hypertonic saline infusion.
2506
T.G. Lauridsen et al.
Table 3. The effect of protein-enriched diet on urinary excretions of aquaporin-2 (u-AQP2) and epithelial sodium channel fraction β (u-ENaCβ), plasma
arginine vasopressin (p-AVP), urine osmolality (u-Osm), serum osmolality (s-Osm), urinary excretion of prostaglandin E2 (u-PGE2), free water clearance
(CH2O), urine volume (u-vol), urinary excretion of c-AMP (u-c-AMP) and glomerular filtration rate (GFR); and the baseline values before hypertonic
saline infusion and 30, 60, 90 and 120 min after hypertonic saline infusion in a randomized, placebo-controlled, crossover study in 13 healthy subjects
Baseline
30 min
60 min
90 min
120 min
P
u-AQP2 (ng/µmol creatinine)
Protein-enriched diet
135.3 (33)
Normal diet
96.1 (17)
P
0.01
143.4 (32)*
104.1 (25)*
0.01
168.9 (40)*
109.8 (31)*
0.01
171.8 (47)*
114.1 (28)*
0.01
177.0 (40)*
118.0 (23)*
0.01
0.003
u-ENaCβ (pg/µmol creatinine)
Protein-enriched diet
10.6 (4.0)
Normal diet
9.5 (3.2)
11.1 (2.6)**
9.5 (1.9)
12.7 (2.5)**
13.0 (4.1)**
12.5 (3.0)**
12.7 (3.0)**
12.4 (3.1)**
12.9 (4.1)**
NS
0.89 (0.2)
0.90 (0.3)
2.16 (0.7)*
1.84 (0.9)*
1.38 (0.4)*
1.48 (0.5)*
1.27 (0.4)*
1.14 (0.4)**
1.1 (0.3)**
1.1 (0.4)**
NS
177 (53)
128 (35)
0.01
344 (86)*
274 (68)*
688 (187)*
616 (95)*
0.05
806 (72)*
659 (80)*
0.01
762 (122)*
663 (90)*
0.003
289 (3.1)
285 (2.7)
0.01
293 (3.5)*
289 (2.4)*
0.05
296 (3.6)*
293 (2.3)*
0.05
294 (3.3)*
291 (2.5)*
291 (3.1)*
290 (3.0)*
0.03
690 (380)
1019 (497)
0.05
588 (250)
1004 (528)
0.01
631 (463)
1370 (1759)
463 (173)**
920 (504)
0.05
543 (380)**
902 (478)
0.01
0.02
2.6 (1.8)
3.7 (1.3)
−0.3 (0.8)*
0.3 (0.6)*
0.05
−1.9 (0.7)*
−1.6 (0.5)*
−2.6 (0.5)*
−1.8 (0.6)*
0.01
−2.5 (0.7)*
−2.0 (0.6)*
0.01
<0.0003
u-vol (mL/min)
Protein-enriched diet
Normal diet
P
6.2 (1.8)
6.6 (1.3)
3.2 (1.1)*
3.2 (1.5)*
1.6 (0.5)*
1.6 (0.7)*
1.5 (0.3)*
1.5 (0.6)*
1.6 (0.3)*
1.7 (0.8)*
NS
u-c-AMP (pmol/min)
Protein-enriched diet
Normal diet
P
4960 (672)
5147 (741)
5824 (1255)**
5551 (2004)**
5362 (823)
6115 (1272)
4908 (633)
4943 (859)
4704 (581)
5129 (1083)
NS
GFR (mL/min)
Protein-enriched diet
Normal diet
P
100 (9)
100 (7)
99 (18)
91 (28)
101 (8)
105 (22)
104 (8)
95 (11)
102 (11)
101 (12)
NS
p-AVP (pg/mL)
Protein-enriched diet
Normal diet
u-Osm (mosm/kg water)
Protein-enriched diet
Normal diet
P
s-Osm (mosm/kg water)
Protein-enriched diet
Normal diet
P
u-PGE2 (pg/min)
Protein-enriched diet
Normal diet
P
CH2O (mL/min)
Protein-enriched diet
Normal diet
P
Values are means and (1 SD). P indicates significant difference between the protein-enriched diet and the normal diet using a general linear model with
repeated measures. A paired t-test was used for comparison of means when differences were found between the two treatments. NS, not significant.*P <
0.01, **P < 0.05 from baseline.
p-ANP, p-BNP and p-Renin were the same during between protein-enriched diet and normal diet. p-ANP and
p-BNP increased significantly after hypertonic saline infusion, whereas p-Renin decreased significantly after hypertonic saline infusion. However, we found no significant
difference in the response to hypertonic saline infusion between the two groups.
Effect of protein-enriched diet on blood pressure and pulse
rate
At baseline, blood pressure was 113/66 ± 12/8 mmHg, and
pulse rate was 64 ± 11 beats/min in the protein-enriched
dietary group; the corresponding values in the normal dietary group were the same (blood pressure: 116/63 ± 10/
7 mmHg, pulse rate: 62 ± 10 beats/min).
The systolic blood pressure increased significantly with
an average of 5 mmHg for both groups (P < 0.03 proteinenriched diet, and P < 0.01 normal diet). Diastolic blood
pressure was unchanged in both groups.
Figure 5 shows that pulse rate increased significantly
during the protein-enriched diet compared with normal
diet. No statistically significant differences were seen
in the response to 3% saline infusion between the two
groups. The increase was 6 beats/min for both groups
(P < 0.003).
Protein-enriched diet and aquaporin-2 and epithelial sodium channels
2,0
2507
180
P<0.003
160
u-ENaCβ pg/min
u-AQP2 ng/min
1,8
1,6
1,4
1,2
140
NS
120
100
1,0
Normal
Protein
0,8
Before
30
60
Time
90
80
normal
protein
60
120
Before
Fig. 2. Effect of protein-enriched diet on urinary excretion aquaporin-2
(u-AQP2) before hypertonic saline infusion, and 30, 60, 90 and
120 min after hypertonic saline infusion compared with normal diet in
a randomized, placebo-controlled, crossover study in 13 healthy subjects.
30
60
Time
90
120
Fig. 4. Effect of protein-enriched diet on urinary excretion of epithelial
sodium channel fraction β (u-ENaCβ) before hypertonic saline infusion,
and 30, 60, 90 and 120 min after hypertonic saline infusion compared
with normal diet in a randomized, placebo-controlled, crossover study
in 13 healthy subjects.
2,5
2,0
FENa %
NS
1,5
1,0
0,5
normal
protein
0,0
Before
30
60
90
120
Fig. 3. Effect of protein-enriched diet on fractional excretion of sodium
before hypertonic saline infusion, and 30, 60, 90 and 120 min after
hypertonic saline infusion compared with normal diet in a randomized,
placebo-controlled, crossover study in 13 healthy subjects.
Effect of protein-enriched diet on body weight
Body weight was unchanged after both protein-enriched
diet [before: 73.2 ± 12.6 kg, after: 73.8 ± 12.8 kg, not significant (NS)] and normal diet (before: 72.5 ± 12.3 kg, after: 72.9 ± 12.5 kg, NS).
Discussion
The present study of healthy subjects showed that the
protein-enriched diet clearly increased u-AQP2 and reduced free water clearance. Thus, our hypothesis that
a protein-enriched diet would increase u-AQP2 could
not be rejected, but the protein-enriched diet did not
change the response in u-AQP2 to hypertonic saline. We
did not find a significant change in u-ENaCβ during the
protein-enriched diet compared with the normal diet.
Thus, we reject our hypothesis that a protein-enhanced diet would increase u-ENaCβ. The amount of ENaCβ in
urine is supposed to reflect the activity of sodium transport via the epithelial sodium channels just as u-AQP2 re-
flects the functional status of the AQP2 water channels
[21]. Our analyses showed that the assay has a satisfactory
reliability. In addition, we demonstrated a significant correlation between the changes in urinary sodium excretion
and the changes in u-ENaCβ. Thus, our results are in accordance with u-ENaCβ being a biomarker of the transport
of sodium via ENaC during acute studies, presumably reflecting the up- and downregulation of β-ENaC expression and sodium transport via ENaC. However, further
studies are necessary to elucidate more precisely to what
degree u-ENaCβ reflects the activity of ENaC.
We found that the protein-enriched diet did not increase
GFR. This might be due to the fact that our subjects were
under a water-loading condition, and with this condition,
another human study has also shown the lack of increase
in GFR [25].
At start of the study, the protein-enriched diet increased
p-AVP significantly, but after orally water loading and during hypertonic saline infusion, this increase disappeared.
Presumably, it is due to the fact that the combination of oral
water load and hypertonic saline infusion had produced a
stronger stimulus for AVP suppression than the potential
stimulatory effect of protein-enriched diet. Using a general
linear model with repeated measures, our analyses showed
that the levels of u-Osm and p-Osm were significantly
higher during the protein-enriched diet during hypertonic
saline infusion. However, there was no significant difference in the response in p-AVP to hypertonic saline infusion
between the groups during the protein-enriched and normal
diet. Consequently, the increase in u-AQP2 could not be
caused by an increase in p-AVP but must be secondary
to an AVP-independent mechanism. We found that the
protein-enriched diet reduced u-PGE2 both during 2.5 h
before the infusion experiment and during the infusion
study. However, no significant difference was measured
in the 24-h excretion of PGE2, but the variations in these
measurements were very high. Most likely, this could be attributed to addition of secrets from the prostate during this
long period of urine collection. The most important measurements of u-PGE2 were done during the observation in
2508
T.G. Lauridsen et al.
Table 4. The effect of protein-enriched diet on plasma atrial natriuretic peptide (p-ANP), plasma brain natriuretic peptide (p-BNP), plasma renin
(p-renin), plasma angiotensin II (p-Ang II) and plasma aldosterone (p-Aldo) at the end of fasting and before hypertonic saline infusion (baseline),
60 min after hypertonic saline infusion (60 min post) and 120 min after hypertonic saline infusion (120 min post) compared with normal diet in a
randomized, placebo-controlled, crossover study in 13 healthy subjects
At the end of fasting
Baseline
60 min post
120 min post
P
p-ANP (pmol/L)
Protein-enriched diet
Normal diet
7.2 (4.5)
6.6 (3.4)
8.5 (5.9)
6.1 (3.2)
13.5 (10.3)
8.9 (5.0)
10.9 (6.9)
8.0 (3.6)
NS
p-BNP (pmol/L)
Protein-enriched diet
Normal diet
0.98 (0.6)
0.96 (0.7)
1.17 (0.8)
1.10 (0.8)
1.47 (1.1)
1.24 (0.9)
1.40 (1.0)
1.28 (0.8)
NS
p-Renin (mIU/L)
Protein-enriched diet
Normal diet
26.9 (19.0)
23.2 (16.8)
22.1 (16.1)
16.8 (13.9)
11.8 (8.2)
12.0 (11.7)
10.9 (7.7)
8.3 (4.5)
NS
p-Ang II (pmol/L)
Protein-enriched diet
Normal diet
P
25.6 (12.0)
17.3 (10.9)
0.05
22.2 (10.7)
15.4 (6.6)
0.05
11.8 (5.5)
10.8 (6.6)
10.6 (5.3)
9.6 (6.6)
0.02
1512 (1082)
1125 (812)
792 (631)
441 (301)
0.05
351 (233)
275 (148)
335 (219)
239 (118)
0.05
0.044
p-Aldo (pmol/L)
Protein-enriched diet
Normal diet
P
Values are means and (1 SD). P indicates significant difference between the protein-enriched diet and normal diet using a general linear model with
repeated measures. A paired t-test was used for comparison of means when differences were found between the two treatments. NS, not significant.
Pulse rate (beats/min)
80
p<0.01
70
60
50
Normal
Protein
Morning before
30
60
90
120
Values are means (SD). P is calculated using a general linear model with repeated measures
Fig. 5. Effect of protein-enriched diet on pulse rate before hypertonic saline infusion, and 30, 60, 90 and 120 min after hypertonic saline infusion
compared with normal diet in a randomized, placebo-controlled, crossover study in 13 healthy subjects.
the laboratory, and the results from all measurements in
urine collected in the laboratory showed a reduction in
u-PGE 2 . This is in contrast to some rat studies which
showed that prostaglandin synthesis was increased in rats
kept on a high-protein diet [26,27]. This difference between
the present study and the rat studies might be due to species
differences, or to the fact that our study was conducted after
4 days of increased protein intake, whereas the rat studies
were done after an 8-week treatment with protein-enriched
diet. The reduced level of u-PGE2 and the increased u-AQP2
in the present study must have resulted in a changed balance
between the AVP stimulatory effect on the one hand and the
antagonizing effect of prostaglandins on the other regarding
AQP2 expression.
Other regulatory factors than vasopressin and prostaglandins may influence the expression of AQP2, i.e. the
renin–angiotensin–aldosterone system, the natriuretic peptide system and the sympathetic nervous system [28–35].
In our study, the protein-enriched diet increased p-Ang II,
p-Aldo and pulse rate significantly compared with the
normal diet. The increase in p-Ang II might have contributed to the increased levels of u-AQP2, since angiotensin
II stimulates/enhances AQP2 expression [32,35], and angiotensin II receptor blockade reduces AQP2 expression
[30]. It is more speculative whether aldosterone is involved in the increased level of u-AQP2. In a rat study,
a high aldosterone level increased basolateral AQP2 expression, and the authors suggested that the increased ba-
Protein-enriched diet and aquaporin-2 and epithelial sodium channels
solateral expression of AQP2 induced by aldosterone may
play a significant role in water absorption during conditions with increased sodium absorption in the collecting
ducts [29]. We also measured an increased level of p-Aldo
and a decreased FENa. However, u-ENaCβ was not significantly changed in the present study. Thus, an increased
sodium absorption via the epithelial sodium channels
seems unlikely. The increased tubular absorption of sodium must take place in another part of the nephron during
the protein-enriched diet.
We found that the protein-enriched diet increased pulse
rate during the entire study day, which is most likely due
to the increased sympathetic nerve activity. In denervated
rat kidneys, a reduced sympathetic nervous stimulation reduced AQP2 expression [31], and the authors suggested that
renal sympathetic nerve activity may play an excitatory role
in AQP2 regulation. Thus, it cannot be excluded that an increased sympathetic nervous activity contributed to the increase in u-AQP2 in the present study. However, doubts
exist regarding this point, because an animal study showed
that an activation of the sympathetic nervous system occurs
stepwise [36]. Thus, an increase in renin secretion appears
to take place at frequencies lower than those needed to increase sodium and water absorption, while a decrease in renal haemodynamics only occurs at high frequencies [37].
These stepwise changes in renal effects induced by increased renal sympathetic nerve activity have also been confirmed in healthy humans by cardiopulmonary baroreceptor
unloading with increasing levels of lower negative body
pressure [38]. The protein-enriched diet did not change pRenin in the present study. This may be seen as an argument
against the increased urinary AQP2 excretion in our study
should the origin is from the increased sympathetic nerve
activity. Further studies with direct measurements of plasma
noradrenaline might be necessary to clarify the problem.
During increased levels of u-AQP2, one would expect
a decrease in CH2O which is in good agreement with our
results during the protein-enriched diet. Other studies
have shown that a high-protein diet results in a need
for massive urea transport in the kidney, and as a consequence, there is a need for increased solute-free water to
be absorbed [10]. However, urinary output was not significantly changed. This means that there is a reduced absorption of water in other parts of the nephron than via
the principal cells during a protein-enriched diet.
In conclusion, a protein-enriched diet increases u-AQP2
in a healthy man. The mechanism for an increased u-AQP2
might be due to a reduced prostaglandin level, but we cannot exclude that the angiotensin–aldosterone system or the
sympathetic nervous system plays a role in the upregulation of AQP2 expression. The increase in u-AQP2 results
in reduced CH2O, but since, in urine, volume was unchanged, a reduced water absorption must occur in another
place along the nephron. The increase in renal sodium excretion seems to be mediated in another part of the nephron than the epithelial sodium channels.
Acknowledgements. We thank laboratory technicians Lisbeth Mikkelsen, Henriette Vorup Simonsen, Kirsten Nyborg and Anette Andersen
for skilful technical assistance and commitment. The catering officer
and the staff at the kitchen are thanked for making the diet. The trial
2509
was supported by grants from Ringkjobing County, Lundbeck Foundation
and Hoerslev Foundation.
Conflict of interest statement. None declared.
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Received for publication: 10.11.09; Accepted in revised form: 10.2.10
Nephrol Dial Transplant (2010) 25: 2510–2515
doi: 10.1093/ndt/gfq067
Advance Access publication 17 February 2010
Association between hypernatraemia acquired in the ICU and
mortality: a cohort study
Michael Darmon1,2, Jean-François Timsit3,4, Adrien Francais3, Molière Nguile-Makao3,
Christophe Adrie5, Yves Cohen6, Maïté Garrouste-Orgeas3,7, Dany Goldgran-Toledano8,
Anne-Sylvie Dumenil9, Samir Jamali10, Christine Cheval11, Bernard Allaouchiche12,
Bertrand Souweine13 and Elie Azoulay1,3
1
Medical Intensive Care Unit, Saint-Louis University Hospital, APHP, 1 Avenue Claude Vellefaux, 75010 Paris, France; Paris-7 ParisDiderot University, UFR de Médecine, 75010 Paris, France, 2Medical ICU, Saint-Etienne University Hospital, Avenue Albert Raymond,
42270, Saint-Priest-en-Jarez, France, 3INSERM Unit 823; Grenoble 1 University, BP 217, 38043 Grenoble Cedex 9, France, 4Medical
Polyvalent Intensive Care Unit, Grenoble University Hospital, BP 217, 38043 Grenoble Cedex 9, France, 5Department of Physiology,
Cochin University Hospital, APHP, 27 r Fbg St Jacques 75014 Paris France, 6Medical–Surgical ICU, Avicenne University Hospital, 125 r
Stalingrad 93000 Bobigny, France; Paris-13 University, 93000 Bobigny, France, 7Medical–Surgical ICU, Saint-Joseph Hospital, 185 rue
Losserand, 75014, Paris, France, 8Medical–Surgical ICU, Gonesse Hospital, 25 rue Pierre de Theilley BP 30071, 95503 Gonesse, France,
9
Surgical ICU, Antoine Béclère University Hospital, 157 rue Porte de Trivaux, 92140 Clamart, France, 10Medical–Surgical ICU,
Dourdan Hospital, 2 rue Potelet 91415 Dourdan, France, 11Medical–Surgical ICU, Hyères Hospital, Rue du Maréchal Juin 83407 Hyeres,
France, 12Surgical ICU, Edouard Herriot University Hospital, Hospices Civils de Lyon, 5 place Arsonval 69437 Lyon, France and
13
Medical ICU, Clermont-Ferrand University Hospital, 58 r Montalembert 63003 Clemont-Ferrand, France
Correspondence and offprint requests to: Michael Darmon; E-mail: [email protected]
Abstract
Background. The aim of this study is to describe the prevalence and outcomes of intensive care unit (ICU)-acquired
hypernatraemia (IAH).
Methods. A retrospective analysis was performed on a
prospectively collected database fed by 12 ICUs. Subjects
are unselected patients with ICU stay >48 h. Mild and
moderate to severe hypernatraemia were defined as serum
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