A loss-of-function nonsynonymous polymorphism
in the osmoregulatory TRPV4 gene is associated
with human hyponatremia
Wei Tiana,f, Yi Fua,b, Anna Garcia-Eliasc, Jose ´ M. Ferna ´ndez-Ferna ´ndezc, Rube ´n Vicentec, Patricia L. Kramerd,
Robert F. Kleinb,e, Robert Hitzemannb,f, Eric S. Orwollb,e, Beth Wilmotg, Shannon McWeeneyg, Miguel A. Valverdec,
and David M. Cohena,b,1
Department of Medicine, Divisions ofaNephrology and Hypertension, andeEndocrinology and Metabolism,dDepartments of Neurology,fBehavioral
Neuroscience, andgPublic Health and Preventive Medicine, Oregon Health and Science University, Portland, OR 97239;bPortland VA Medical Center,
Portland, OR 97239; andcLaboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, Universitat
Pompeu Fabra, Barcelona, Spain
Edited by Maurice B. Burg, National Heart, Lung and Blood Institute, Bethesda, MD, and approved June 30, 2009 (received for review April 14, 2009)
Disorders of water balance are among the most common and
morbid of the electrolyte disturbances, and are reflected clinically
as abnormalities in the serum sodium concentration. The transient
receptor potential vanilloid 4 (TRPV4) channel is postulated to
comprise an element of the central tonicity-sensing mechanism in
in vitro. A nonsynonymous polymorphism in the TRPV4 gene gives
rise to a Pro-to-Ser substitution at residue 19. We show that this
polymorphism is significantly associated with serum sodium con-
centration and with hyponatremia (serum sodium concentration
<135 mEq/L) in 2 non-Hispanic Caucasian male populations; in
addition, mean serum sodium concentration is lower among sub-
jects with the TRPV4P19Sallele relative to the wild-type allele.
Subjects with the minor allele were 2.4?6.4 times as likely to
exhibit hyponatremia as subjects without the minor allele (after
inclusion of key covariates). Consistent with these observations, a
human TRPV4 channel mutated to incorporate the TRPV4P19Spoly-
morphism showed diminished response to hypotonic stress (rela-
tive to the wild-type channel) and to the osmotransducing lipid
and likely influences systemic water balance on a population-wide
association study ? osmoregulation ? sodium ?
transient receptor potential ? cell volume regulation
associated with neurological dysfunction and death. Even subtle
changes in systemic osmolality cause reversible defects in coordi-
nation and cognition (1, 2). Clinically, water balance is reflected in
the serum (or plasma) sodium concentration. Water excess relative
to total body sodium content results in hyponatremia, the most
in refs. 3 and 4).
In mammals, systemic water balance is regulated via the renal
water-conserving role of the hormone arginine vasopressin. Re-
lease of arginine vasopressin from the posterior pituitary into the
circulation is governed by the hypothalamic sensor(s) of systemic
osmolality. Ample evidence suggests that the transient receptor
potential channel, TRPV4, comprises an element of the central
sensor of low osmolality. TRPV4 is the mammalian ortholog of a
C. elegans osmosensing protein (5, 6). In rodents, the channel is
expressed in the osmosensing nuclei of the brain (5), among other
sites. In vitro, TRPV4 is activated by hypotonicity (5–7) and by a
channel also participates in cell regulatory volume decrease (9, 10).
Osmotic and mechanical sensitivity of TRPV4 is ultimately con-
ferred by the arachidonic acid metabolites and epoxyeicosatrienoic
ystemic osmolality is among the most tightly regulated of
physiological parameters. In humans, aberrant water balance is
acids (EET), following phospholipase A2activation (11–13). Other
signaling pathways involving inositol trisphosphate (14, 15), SRC-
family tyrosine kinases (16, 17), and sensitization by coapplication
of different stimuli (18, 19) also impact the TRPV4 response to
the regulation of arginine vasopressin release in mouse, where
targeted deletion of the TRPV4 gene gives rise to aberrant systemic
osmoregulation (20, 21).
(22, 23) or water retention (24), although without major repercus-
sion at the population level. To date, no human mutation in an
osmosensing TRP channel has been shown to impact osmoregula-
tion, and no polymorphisms impacting systemic water balance have
been reported for any gene. We hypothesized that a nonsynony-
balance on a population-wide basis.
We tested for an association between serum sodium concentration
and a nonsynonymous single nucleotide polymorphism (SNP) in
the TRPV4 gene. This polymorphism, rs3742030, gives rise to a
19; it was the only TRPV4 nonsynonymous SNP for which minor
at the time these studies commenced (25). Banked genomic DNA
from a panel of healthy elderly subjects (the Healthy Aging cohort)
in the National Institutes on Aging-funded Layton Aging and
Alzheimer’s Disease Center database at Oregon Health and Sci-
ence University (26) was genotyped for the presence of this allele
(see Methods). Characteristics of this cohort are presented in Table
1. Among non-Hispanic Caucasian subjects in this cohort (n ? 219
successful genotypes), the TRPV4P19Sallele appeared to be over-
represented in subjects with the lowest serum sodium concentra-
tions (Fig. 1A). A similar phenomenon was observed in the African
American subjects from this cohort (Fig. 1B; n ? 72). Of note, the
prevalence of the heterozygous state for this allele in Caucasian
Author contributions: A.G.-E., J.M.F.-F., R.H., M.A.V., and D.M.C. designed research; W.T.,
Y.F., A.G.-E., J.M.F.-F., R.V., and D.M.C. performed research; P.L.K., R.F.K., and E.S.O.
contributed new reagents/analytic tools; W.T., A.G.-E., J.M.F.-F., B.W., S.M., M.A.V., and
D.M.C. analyzed data; and M.A.V. and D.M.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
and Hypertension, Oregon Health and Science University, 3314 SW U.S. Veterans Hospital
Road, Portland, OR 97239. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 18, 2009 ?
vol. 106 ?
subjects (i.e., Utah residents with ancestry from northern and
western Europe; CEU) and in the Yoruba in Ibadan, Nigeria
(YRI), in the International HapMap Project data set (25) is 0.017
and 0.100, respectively.
Prevalence of hyponatremia (serum sodium concentration ?135
mEq/L) by genotype and ethnicity is shown in Fig. 1E. Hypona-
respectively, via Pearson’s ?2analysis). For each cohort, the mean
serum sodium concentration was significantly lower in the
TRPV4P19S-positive subjects (by 1.6 and 2.4 mEq/L; P ? 0.05 and
0.014 for the non-Hispanic Caucasian and African American
populations, respectively, by t test; Fig. 1F). We sought to further
quantify the effect of this allele upon systemic water balance in the
larger non-Hispanic Caucasian population using the covariates of
age, sex, and serum glucose concentration; glucose exerts an
osmotic effect independent of serum sodium concentration (27)
and age may be associated with hyponatremia (28). The strength of
association between serum sodium concentration (as a continuous
variable) and rs3742030 genotype was determined by linear regres-
sion using the above covariates. Presence of the rs3742030 minor
allele (i.e., the TRPV4P19Sallele) was significantly associated with
serum sodium concentration for males (P ? 0.0024) but not for
females (P value ? 0.40). Prevalence ratio calculations indicated
that male subjects with the minor allele were 6.45 times as likely to
exhibit hyponatremia as male subjects without the minor allele
(95% CI: 1.22–34.25; P value ? 0.029) after inclusion of the
covariates; for female subjects, the prevalence ratio was 1.76 (95%
CI: 0.52–6.0; P value ? 0.37; Table 2).
A larger male population was sought for replication of these
findings. Banked genomic DNA was obtained from subjects en-
rolled in the Osteoporotic Fractures in Men Study (MrOS; see
Methods), a prospective U.S. cohort study of 5,995 community-
dwelling men aged 65 years and over (29). Subjects with abnormal
kidney function (i.e., serum creatinine ? 1.3 mg/dL) and serum
marked hyperglycemia independently impact serum sodium con-
centration (27, 30). In addition, the majority of participants in
MrOS were non-Hispanic Caucasian; only subjects of this self-
reported ethnicity were selected for the replication study. Charac-
teristics of these subjects (n ? 4305) are shown in Table 3. Serum
sodium concentration followed a roughly normal distribution (Fig.
1D). All subjects with the lowest serum sodium concentration (i.e.,
?138 mEq/L, corresponding to the lowest decile, or ?1.5 SD units
below the population mean) and highest serum sodium concentra-
tion (i.e., ?145 mEq/L, approximating the highest decile, or ?1.5
SD units above the population mean) were genotyped for the
TRPV4P19Sallele, as was as a random selection of subjects with
sodium concentration approximating the sample mean (141–142
mEq/L; see Methods). This approach was adopted to ensure max-
imal representation of the population extremes, vis-a `-vis serum
sodium concentration; note that not all members of the ‘‘low’’
sodium MrOS group have hyponatremia (i.e., serum sodium con-
centration ?135 mEq/L), and our subsequent analysis takes this
3. Prevalence of the TRPV4P19Sallele expressed as a function of
serum sodium concentration is shown in Fig. 1C; prevalence of the
TRPV4P19Sallele in the ‘‘low,’’ ‘‘mean,’’ and ‘‘high’’ serum sodium
concentration groups in this cohort was 6.1% (n ? 444), 4.2% (n ?
448), and 3.7% (n ? 408), respectively (Fig. 1D Inset). Mean serum
sodium concentration was 0.9 mEq/L lower in subjects with the
TRPV4P19Sallele (Fig. 1F; P ? 0.04 via t test). Serum sodium
concentration was again significantly associated with the
TRPV4P19Sallele (P ? 0.019), as determined by linear regression
analysis using the covariates of age, serum glucose, serum
creatinine, and recruitment center (Table 2). Subjects with the
minor allele were 2.43 times as likely to exhibit hyponatremia as
subjects without the allele (95% CI: 1.17–5.06; P value ? 0.017)
after inclusion of the covariates in this exclusively male population
with the TRPV4P19Sallele was not attributable to another poly-
morphism in tight linkage disequlibrium. All TRPV4 exons and
exon-intron boundaries were resequenced from 10 hyponatremic
subjects with the TRPV4P19Sminor allele; no other synonymous or
otherwise functional polymorphisms were detected. In addition, no
polymorphisms in strong linkage disequilibrium with rs3742030
impacting coding or splicing were identified in haplotype analysis
(http://www.hapmap.org/; International HapMap Project, release
21) (25). Of note, no subjects with 2 copies of the rs3742030 minor
(TRPV4P19S) allele were identified in the Healthy Aging or MrOS
be causal for hyponatremia in the study cohorts. We hypothesized
that the variant channel would be less responsive to hypotonicity in
permissive with respect to water excess. Therefore, we set out to
functionally evaluate the impact of the TRPV4P19Spolymorphism
in a heterologous expression system. We first tested the subcellular
distribution of TRPV4WTand TRPV4P19Sby confocal immunoflu-
orescence microscopy in HEK293 cells transiently transfected with
a cDNA coding for full-length wild-type human TRPV4, or with a
cDNA mutated to incorporate the TRPV4P19Spolymorphism.
TRPV4WTand TRPV4P19Sshowed similar levels of expression and
localization to the plasma membrane, as determined via confocal
immunofluorescence microscopy (supporting information (SI) Fig.
S1) and via cell surface biotinylation experiments (Fig. S2). No
Table 1. Characteristics of the Healthy Aging cohort and of the non-Hispanic Caucasian and African American subjects who were
Ethnicity% Male AgeSerum Na?
Healthy Aging (total) 46580.9% Non-Hispanic Cauc.
18.1% African American
0.4% Native American
100% African American
37.680.7 ? 9.1a
138.2 ? 2.7105 ? 33
Genotyped non-Hispanic Caucasian
Genotyped African American
2193781.8 ? 9.2a
138.5 ? 3.2101 ? 23
722472.8 ? 6.5137.7 ? 2.3 118 ? 58
Characteristics of the Healthy Aging cohort and of the successfully genotyped non-Hispanic Caucasian and African American subjects, including number of
subjects per group (n), self-reported ethnicity, percent of subjects that were male, age (mean ? SD), and laboratory values (mean ? SD). Not all subjects were
genotyped, owing to availability of genomic DNA. Subgroups include (i) all successfully genotyped non-Hispanic Caucasian subjects and (ii) all successfully
genotyped African American subjects.
aTwo subjects were excluded from these calculations because a numerical value for age was not reported. Serum creatinine was not determined in this cohort.
Tian et al.PNAS ?
August 18, 2009 ?
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no. 33 ?
TRPV4 was immunodetectable in HEK293 cells transfected only
with GFP (Fig. S3).
Whole-cell cationic currents recorded from HEK293 cells ex-
in osmolality) were markedly diminished in the TRPV4P19Strans-
2 A and B), whereas the response to the synthetic TRPV4 agonist
4?-phorbol 12,13-didecanoate (4?PDD) was unaffected by the
polymorphism (Fig. 2 A and B). Plots of current-voltage relation-
ship obtained at the indicated time points in Fig. 2 A and B are
shown in Fig. 2 C and D, respectively. Whole-cell cationic currents
activated by 4?PDD (in TRPV4WT- and TRPV4P19S- expressing
rectification similar to that previously described for TRPV4 (6, 8,
31). In aggregated data, mean current density in response to the
allele than in wild-type TRPV4 (Fig. 2E). Interestingly, the re-
significantly between the 2 alleles. Exposure to 30% hypotonic
solution did not activate significant whole-cell cationic currents in
GFP-transfected HEK293 cells (Fig. S4). Importantly, the
TRPV4P19Sallele exerted a dominant negative effect with respect
to the wild-type allele. When cotransfected in a 1:1 ratio (i.e.,
mimicking a human subject heterozygous for the TRPV4P19Sallele
of this autosomal gene), the variant allele suppressed the response
of the wild-type allele to 15% hypotonicity (Fig. 2E).
TRPV4P19Schannels also showed a decreased response to the
osmotransducing messenger, epoxyeicosatrienoic acid (156 nM;
for a variant channel that exhibits reduced responsiveness to mild
hypotonic stress (i.e., such as that likely to be encountered in vivo)
and to the intracellular lipid messenger, epoxyeicosatrienoic acid,
but not to more-pronounced hypotonic stress or to the synthetic
We find that a nonsynonymous polymorphism in the central sensor
of hypotonicity, TRPV4, is associated with hyponatremia (i.e.,
relative water excess) and with serum sodium concentration in 2
male non-Hispanic Caucasian cohorts. Subjects with the minor
allele were 6.45 or 2.43 times as likely to exhibit hyponatremia as
subjects without the minor allele (after inclusion of key covariates).
Mean serum sodium concentration among subjects with one copy
of the minor allele was significantly lower (by 0.9–2.4 mEq/L). No
other functional polymorphisms in linkage disequilibrium with
rs3742030 were identified. Consistent with these data, the variant
allele was associated with loss of channel function in response to
modest reductions in osmolality and to the osmotransducing lipid
messenger epoxyeicosatrienoic acid, but not to marked hypotonic-
ity or the synthetic agonist 4?PDD. In addition, insofar as oli-
further suggest a dominant-negative effect of the variant
We infer that this nonsynonymous polymorphism is likely to be
causal for hyponatremia, potentially via reducing the hypothalamic
osmolality set point. It is unclear why such an association would be
stronger in male subjects. Little is known about the relationship
between sex and water balance. In one study, male sex was
associated with mild or moderate hyponatremia at presentation to
hospital or during hospitalization (32); however, women may be
more susceptible to permanent brain damage in response to acute
hyponatremia (33). It is conceivable that sex hormones influence
TRPV4 function in vivo, as has been observed for TRPM6 in
Although mammalian osmoregulation is incompletely under-
stood, abundant data point to a role for TRPV4 as a component of
the central osmosensing mechanism. TRPV4 was cloned on the
basis of its homology with the C. elegans neural tonicity-sensing
channel OSM-9 (5, 6). TRPV4 is activated by hypotonicity in vitro,
and perturbations of even a few mOsmol/kg H2O were sufficient to
achieve this effect (5–7); such exquisite sensitivity closely parallels
the in vivo mechanism whereby a change of only a few mOsmol/kg
is expressed in the blood–brain barrier-deficient central osmosens-
ing nuclei (5, 35), and targeted deletion of the TRPV4 gene gives
rise to aberrant osmoregulation in murine models (20, 21). In
addition, an as-of-yet unidentified splice variant of the closely
related TRPV1 channel likely represents the central sensor of
hypertonicity (36). In aggregate, these data strongly support a role
for TRPV4 in the regulation of systemic water balance.
Serum [Na+], mEq/l
Number of subjects
Low Mean High
Percent with hyponatremia
Serum [Na+] less than or equal to
134135136 137 138All
Healthy Aging - Cauc
Serum [Na+] less than or equal to
Healthy Aging - AA
132 133 134 135 136 137 138
Serum [Na+] less than or equal to
MrOS - Cauc
Prevalence of the TRPV4P19Sallele among genotyped non-Hispanic Caucasian
(Cauc) (A) and African American (AA) (B) subjects in the Healthy Aging cohort
from the Layton Center for Aging and Alzheimer’s Disease, and from the
genotyped non-Hispanic Caucasian subjects in the Osteoporotic Fractures in
Depicted is the prevalence of the heterozygous genotype as percent of total
number of subjects with serum sodium concentration at or below the indi-
cated level. ‘‘All’’ denotes prevalence for the entire genotyped population.
Note the change in y-axis scale in A–C. (D) Distribution of serum sodium
concentration (binned as integers) for all Caucasian subjects in the MrOS
cohort with creatinine ?1.3 mg/dL and glucose ?150 mg/dL (n ? 4,409).
Prevalence of the TRPV4P19Spolymorphism is shown in the inset bar graph,
where Low corresponds to [Na?] ? 138 mEq/L (?lowest decile), Mean is [Na?]
141 or 142 mEq/L, and High represents [Na?] ? 145 mEq/L (?highest decile);
bars are keyed to the frequency distribution via shading (see Methods). (E)
Prevalence of hyponatremia, defined as serum sodium concentration ?135
mEq/L, among subjects with one (P19S-positive) or no (P19S-negative)
TRPV4P19Salleles in non-Hispanic Caucasian (Cauc; n ? 219) and African
American (AA; n ? 72) subjects in the Healthy Aging cohort, and in non-
Hispanic Caucasian MrOS subjects (n ? 1,300). (F) Mean serum sodium con-
centration (?SD) in the 3 cohorts, expressed as a function of the presence or
(by 0.9–2.4 mEq/L) in all 3 groups of TRPV4P19S-positive subjects relative to
TRPV4P19S-negative subjects; P ? 0.05, 0.014, and 0.04 via t test for the
non-Hispanic Caucasian and African American Healthy Aging subjects and for
the non-Hispanic Caucasian MrOS cohort, respectively.
Presence of the TRPV4P19Sallele is associated with hyponatremia.
www.pnas.org?cgi?doi?10.1073?pnas.0904084106Tian et al.
be the expected phenotype, based upon the in vitro hypotonicity
responsiveness of both heterologously expressed (5–7) and natively
expressed (37, 38) TRPV4 channels. Reduced sensitivity to sys-
temic hypotonicity would fail to trigger corrective measures (i.e.,
release of arginine vasopressin). However, TRPV4-null mice ex-
hibit a variable phenotype. Mizuno et al. (20) noted no difference
in plasma sodium concentration or in circulating levels of arginine
vasopressin in TRPV4?/?mice, relative to their wild-type litter-
mates. Provocative testing with water loading (via gavage) also
failed to uncover a defect. Hyperosmotic challenge in this model—
via simultaneous water restriction and i.p. propylene glycol—
and Friedman (21) similarly noted no gross difference in plasma
when mice were single housed and fluid deprived, the TRPV4?/?
noted a blunted arginine vasopressin response to osmotic challenge
(albeit with a different stimulus, i.p. hypertonic NaCl) (21). Im-
portantly, during chronic treatment with exogenous vasopressin
response and much more dramatic fall in blood osmolality; the net
own baseline and that of their wild-type littermates (21). In sum,
they drink too little in the absence of unregulated vasopressin and
too much in the presence of unregulated vasopressin. Therefore,
hypofunctioning of the TRPV4 allele(s) predisposes to hyponatre-
mia in the presence of constitutive vasopressin action. These data,
coupled with our own, suggest that presence of the TRPV4P19S
allele may synergize with human conditions marked by chronically
upregulated vasopressin level or vasopressin effect in promoting
The molecular mechanism through which the Pro-to-Ser substi-
tution at residue 19 reduces osmoresponsiveness of the human
rise to a high-probability context for protein phosphorylation
(NetPhos prediction server; http://www.cbs.dtu.dk/services/
NetPhos/) (39), it is tempting to speculate that this residue under-
goes posttranslational modification only in the variant allele.
In the present cohorts, the presence of hyponatremia was as-
mic at the time their laboratory studies were performed. Although
the rate of change in serum sodium concentration may impact the
development of symptoms (40), even modest ‘‘stable’’ hyponatre-
mia leads to impaired functioning of the central nervous system
(e.g., refs. 1 and 2). Screening for the presence of the TRPV4P19S
allele may be justified in human subjects as an index of propensity
to aberrant water balance, irrespective of their present serum
sodium concentration. Although we have no direct evidence that
in the development of overt hyponatremia, screening may be
valuable in subjects predisposed to hyponatremia by virtue of their
postoperative state, medication usage, or recreational activities (4).
Of note, the greater frequency of this allele among the Yoruba of
Nigeria (25) and among African American subjects (present data)
may reflect the selective advantage of a modest water excess (i.e.,
a lower set point for systemic osmolality) in conferring protection
from symptomatic water deficit in hot environments in which water
access may be unpredictable.
Healthy Aging cohort of the National Institutes on Aging-funded Layton Aging
and Alzheimer’s Disease Center database at Oregon Health and Science Univer-
sity (26). Individuals in this Healthy Aging cohort represented nondemented
control subjects for longitudinal studies of the determinants of Alzheimer’s
disease and other dementing conditions in the elderly (26). Genomic DNA was
subjected to phi29-based whole-genome amplification (REPLI-g kit; QIAGEN).
The TRPV4 exon of interest was PCR amplified using primers bracketing the
TRPV4P19Spolymorphism (rs3742030); the amplicon was then subjected to se-
quencing with one of the original amplification primers in an automated se-
quencing platform (Applied Biosystems; Vollum Institute for Advanced Biomed-
Table 2. Association of serum sodium concentration with presence of the TRPV4P19Sallele
Healthy Aging Age, glucoseM
6.45 (1.22–34.25); P ? 0.029
1.76 (0.52–6.0); P ? 0.37
2.43 (1.17–5.06); P ? 0.017MrOS Age, glucose, creatinine, recruitment center1,300
in the Healthy Aging and MrOS cohorts, as tested via linear regression analysis on available covariates (shown) and stratified by sex (see
Methods). For the Healthy Aging cohort, subjects with missing age data (n ? 2) were excluded from this analysis; in addition, subjects with
ratios (and 95% confidence intervals) were calculated for the presence of hyponatremia (serum sodium concentration ?135 mEq/L) as a
Aging and MrOS cohorts were 6.45 and 2.43 times as likely, respectively, to exhibit hyponatremia as were subjects lacking the allele.
Table 3. Characteristics of the MrOS cohort and of the serum sodium concentration subgroups successfully genotyped
% Male AgeEthnicity
MrOS Low Sodium
MrOS Mean Sodium
MrOS High Sodium
74.3 ? 6.0
73.5 ? 5.7
73.5 ? 5.9
73.5 ? 5.8
136.6 ? 2.0
141.5 ? 0.5
145.6 ? 1.0
141.4 ? 2.7
0.95 ? 0.17
0.97 ? 0.15
0.97 ? 0.15
0.97 ? 0.15
102 ? 12
100 ? 12
100 ? 12
101 ? 12
Characteristics of the MrOS cohort and of the MrOS Low, Mean, and High serum sodium concentration subgroups, including number of subjects per group
(n), percent of subjects that were male, age (mean ? SD), self-reported ethnicity, and laboratory values (mean ? SD). The last 6 columns indicate percent of
subjects from each MrOS recruitment site, where BI, MN, PA, PI, PO, and SD represent the MrOS Birmingham, Minneapolis, Palo Alto, Pittsburgh, Portland, and
San Diego recruitment sites, respectively. Subgroups include all successfully genotyped non-Hispanic Caucasian subjects; they were drawn from the ?All MrOS?
pool based upon serum sodium concentration, as explained in Methods. ?All MrOS? includes all subjects from the original MrOS cohort (n ? 5,995) who fulfilled
the following criteria: (i) serum sodium, creatinine, and glucose concentrations were determined; (ii) ethnicity was self-reported as non-Hispanic Caucasian; (iii)
serum creatinine was ?1.3 mg/dL; and (iv) serum glucose was ?150 mg/dL.
Tian et al.PNAS ?
August 18, 2009 ?
vol. 106 ?
no. 33 ?
ical Research). Presence of the TRPV4P19Sallele was detected by inspection of
electropherograms using FinchTV software (Geospiza). Genomic DNA from a
total of 299 subjects was genotyped for the presence of the TRPV4P19Sallele. For
8 subjects, genotyping was not successful (i.e., insufficient sample), leaving 291
successful genotypes (219 Caucasian and 72 African American subjects).
Genotyping: Osteoporotic Fractures in Men (MrOS) Study. The Osteoporotic
Fractures in Men (MrOS) Study was designed to assess the determinants of
age (29). Subjects were recruited from 6 centers (see Table 3); details were
parent study from 5,532 subjects; all were male. Serum sodium, creatinine, and
glucose were measured in all subjects on a single instrument using thawed,
previously frozen serum (Clinical Laboratory, Portland VA Medical Center). Sub-
jects with serum creatinine ?1.3 were excluded from further analysis because
abnormal renal function may lead to impaired water excretion (e.g., ref. 30).
Subjects with serum glucose ?150 mg/dL were excluded because the indepen-
dent osmotic effect of hyperglycemia depresses serum sodium concentration,
These represented subjects in 1 of 3 groups, based upon serum sodium concen-
tration (see Fig. 1D). The ‘‘low’’ sodium concentration group was designed to
groups approximated the lowest and highest deciles (or ?1.5 SD units) of the
for serum sodium concentration in nonexcluded non-Hispanic Caucasian MrOS
subjects (Table 3) was 141.4 mEq/L; for the ‘‘mean’’ group, we genotyped every
third subject when subjects with serum sodium concentration of 141 and 142
mEq/L were ordered by serum sodium concentration, and then by coded alpha-
of reporting by the clinical laboratory). Banked genomic DNA was subjected to
TRPV4P19Sallele in a blinded fashion using a custom-designed real-time PCR-
based assay directed against SNP rs3742030 (Applied Biosystems). Of 1,449 sam-
ples obtained from the parent study, 26 samples could not be genotyped (i.e.,
insufficient quantity of DNA). The successfully genotyped non-Hispanic Cauca-
sian subjects (n ? 1,304) were used for replication.
For both Healthy Aging and MrOS, All genotyping studies using human DNA
were approved by the Institutional Review Board of the Portland VA Medical
Title 45—Public Welfare, Department of Health and Human Services; Part 46—
Protection of Human Subjects; Paragraph 46.101(b)(4)—i.e., Exemption 4.
Statistical Analysis. Unadjusted association between one copy of the variant
vitro. Time courses for whole-cell currents at ?100 mV
and ?100 mV in HEK293 cells transfected with human
TRPV4WT(A) and TRPV4P19S(B) exposed to 15% hypo-
tonicity and then 4?-phorbol 12,13-didecanoate
(4?PDD; 10 ?M). (C and D) Corresponding whole-cell
current/voltage relationships recorded at the times
indicated by the color-coded boxes in A and B, respec-
tively. (E) Mean current density (expressed as pA/pF at
?100 mV) of TRPV4WT(WT) and TRPV4P19S(P19S) and
of an equimolar ratio of the 2 alleles (WT:P19S) upon
exposure to mild (i.e., 15%; Left) and more pro-
nounced (i.e., 30%; Right) hypotonicity. (F) Represen-
currents from TRPV4WTand TRPV4P19Stransfectants
loaded with 156 nM epoxyeicosatrienoic acid (EET) in
are expressed as the mean ? SEM of the number of
experiments shown in brackets. *P ? 0.05, relative to
other transfectants in presence of 15% hypotonicity,
via one-way ANOVA and Tukey post hoc.
The TRPV4P19Sallele is hypofunctioning in
www.pnas.org?cgi?doi?10.1073?pnas.0904084106Tian et al.
analysis with the Fisher exact probability test (see figure legends). In the case of
of the large size of the population. Comparison between mean serum sodium
via two-tailed t test for the Healthy Aging populations (non-Hispanic Caucasian
and African American), and via one-tailed t test for the confirmatory MrOS
population. Hyponatremia was defined as serum sodium concentration ?135
mEq/L. Prevalence ratios were calculated in SAS using all covariates (see below)
there was a lack of converge with the Poisson distribution).
For linear regression analysis to test the association between serum sodium
concentration as a continuous variable and rs3742030 genotype in the non-
Hispanic Caucasian Healthy Aging cohort, the data set was filtered to eliminate
genotyped subjects with glucose ?150 mg/dL (n ? 11) to preserve consistency
with the tested MrOS samples (where subjects with serum glucose ?150 mg/dL
were excluded). In addition, 2 subjects were missing data on age; therefore, the
final numbers for analysis were 76 males and 130 females. Covariates for linear
regression included sex, age, and serum glucose concentration; the latter 2 may
impact serum sodium concentration (27, 28). Note that there was no serum
creatinine determination for this data set. For female subjects, age and glucose
concentration were significantly associated with serum sodium concentration
(P ? 0.0001 and P ? 0.006, respectively); for male subjects, age was associated
with serum sodium concentration (P ? 0.0045).
In the non-Hispanic Caucasian subset of the MrOS cohort, the unadjusted
high sodium groups did not reach statistical significance (P ? 0.22, Pearson’s ?2
test). For linear regression analysis in this cohort, no subjects with serum glucose
level were significantly associated with hyponatremia (P value ? 0.026 and ?
0.0001, respectively). One recruitment center (BI, Birmingham) was associated
with hyponatremia (P ? 0.022), whereas glucose concentration and other re-
cruitment centers were not significantly associated with hyponatremia.
only polymorphism genotyped in these populations. Of note, no subjects ho-
mozygous for the TRPV4P19Sallele were identified in any study population,
although only approximately 3 would be expected by Hardy-Weinberg equilib-
rium using the allelic frequency in the largest population (MrOS).
Cell Transfection, Immunodetection, and Electrophysiological Recordings. Hu-
man TRPV4 cDNA was amplified from human kidney mRNA, cloned (with its
native stop codon intact) into the mammalian expression vector pcDNA3.1/V5-
entire cDNA was confirmed by sequencing. Cationic currents were registered
using the patch-clamp technique in whole-cell configuration. Full details are
provided in SI Methods.
ACKNOWLEDGMENTS. These studies were supported by grants from the Na-
tional Institutes of Health (R21AG029968 to D.M.C. and P30AG08017 to P.L.K.),
the American Heart Association (D.M.C.), the Department of Veterans Affairs
(D.M.C.), and the Spanish Ministry of Science and Innovation (Grants SAF2006–
13893-C02–02, SAF2006–04973, red HERACLES FIS RD06/0009 to M.A.V. and
J.M.F.), Marato ´ de TV3 (Grants 061331 and 080430), and Generalitat de Catalu-
nya. M.A.V. is an ICREA Academia researcher. Data obtained from the Layton
Institutes of Health (P30 AG008017 and P50 AT00066), the Department of Vet-
(OCTR), grant number UL1 RR024140 from the National Center for Research
Resources (NCRR), a component of the National Institutes of Health (NIH), and
NIH Roadmap for Medical Research. The Osteoporotic Fractures in Men (MrOS)
Medical Research Grants U01 AR45580, U01 AR45614, U01 AR45632, U01
AR45647, U01 AR45654, U01 AR45583, U01 AG18197, U01-AG027810, and UL1
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no. 33 ?