REVIEWS
PHYSIOLOGY 24: 186–191, 2009; doi:10.1152/physiol.00005.2009
Hypertonic Stress in the Kidney:
A Necessary Evil
Min Seong Kwon, Sun Woo Lim,
and H. Moo Kwon
The interstitium of the renal medulla is hypertonic, imposing deleterious effects on
Department of Medicine,
University of Maryland, Baltimore, Maryland
[email protected]
local cells. At the same time, the hypertonicity provides osmotic gradient for water
reabsorption and is a local signal for tissue-specific gene expression and differentiation of the renal medulla, which is a critical organ for water homeostasis.
Definition of Hypertonicity
Operation of the urinary concentrating mechanism
produces hyperosmolality in the interstitial fluid of the
renal medulla. Osmolality of the renal medulla routinely reaches over 1,000 mosmol/kg in humans and
3,000 mosmol/kg in rodents. The hyperosmolality is
due to accumulation of salt and urea. Salt and urea differ in their osmotic action. At the cellular level, salt is
an effective solute, whereas urea is not. This means
that, when the ambient osmolality is raised by addition of salt, cells shrink due to osmosis (movement of
water across the plasma membrane). Urea has high
membrane permeability comparable to water and
thus does not induce significant osmosis. In the renal
186
medulla, where complex arrangements of tubules,
some of which have regulated urea permeability, in
combination with dynamics of counter current flow of
tubular fluids confer osmotic effectiveness to urea for
water reabsorption. In this discussion, however, we
will ignore this and define hypertonicity as hyperosmotic salt because we will focus on cellular effects.
Why is Hypertonicity Bad?
When cells are exposed to a hypertonic solution, cells
loose water rapidly due to osmosis. Two immediate
consequences of exposure to hypertonicity are 1)
reduced cell volume and 2) increased ionic strength. It
has been proposed that these changes perturb protein
function due to macromolecular crowding (43)
induced by cell shrinkage and elevated ionic strength
(61). This concept was recently proved in an elegant
body of work using the model organism C. elegans (5).
In this study, a combination of the genome-wide RNAi
screening and sophisticated cell biological and biophysical assays was used to show that hypertonicity
induced protein damage (misfolding and aggregation)
and that accumulation of damaged proteins caused
death. Although the C. elegans study provide a proofof-concept that hypertonicity damages proteins,
whether the same mechanism causes cell death in the
mammalian renal medullary cells is not known. What
is clear is that hypertonicity also kills mammalian cells
in a dose-dependent manner (42). Both apoptosis and
necrosis are involved in the cell death.
TonEBP is a Master Regulator in
Protection from Hyperosmolality in
the Renal Medulla: Hypertonicity
and Urea
All organisms except the extreme halophilic archebacteria use organic osmolytes for survival when challenged with hypertonic conditions (61). Organic
osmolytes are polyhydric alcohols, amino acids and
their derivatives, and methylamines. Unlike electrolytes, organic osmolytes are compatible with protein, i.e., osmolytes do not disrupt protein structure
and function. Cellular accumulation of organic
osmolytes removes the two sources of stress by hypertonicity—cell shrinkage and elevated ionic strength;
1548-9213/09 8.00 ©2009 Int. Union Physiol. Sci./Am. Physiol. Soc.
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Water is the most abundant molecule in the body—
60% by weight. Since the majority of biochemical reactions take place in water, maintenance of optimal volume and composition—water homeostasis—becomes
critical. It is no wonder mammals, like other terrestrial
animals, possess powerful and elaborate mechanisms
for water homeostasis. Mammalian kidneys have the
ability to adjust the urine output in response to a wide
range of water intake by producing from very dilute
urine, below 100 mosmol/kg, to highly concentrated
urine, over 4,000 mosmol/kg in case of laboratory
mice and rats, without changing the amount of total
solute excretion. Remarkably, the interstitium of the
renal medulla stays hypertonic throughout the dramatic fluctuations in the urinary osmolality. It is safe
to state that the renal medullary cells are constantly
bathed in variable but hypertonic interstitial solution.
Traditionally, the term “hypertonicity” has been often
used in conjunction with “stress,” since much of the
interest has been focused on the detrimental action of
hypertonicity. In this review, salubrious action of
hypertonicity in the renal medulla will be discussed.
Hypertonicity is an important local signal for the differentiation and maintenance of the renal medulla in
addition to its role in the osmotic action in the urinary
concentration. In genetically modified mice and in
certain pathological conditions in which the
medullary hypertonicity is compromised, the renal
medulla loses tissue-specific gene expression profile,
highlighting the importance of local hypertonicity in
the maintenance of the renal medulla.
rising concent
ume by osmos
C. elegans tol
are first allow
organic osmol
tonicity (28).
tonicity allow
hypertonicity
ic osmolytes o
The process
expression is i
scription facto
enhancer bind
al regulator fo
osmolytes (45
conditions, its
ty rises (see be
lar accumulati
renal medulla
membrane
cotransperter
cotransporter
cotransporter
enzymes—ald
ropathy target
glycerophosph
58). The organ
sorbitol, and g
inside the cel
sized because
In C. elega
encodes glyce
key event in th
mulation (29)
induction of g
involved in the
form of TonEB
there is no To
Despite the la
sible that the m
naling pathwa
the stimulatio
Urea also ca
exceeds 300 m
a role in prote
by stimulating
(HSP70) (49).
HSP70 gene in
and stimulate
gle cell in the
TonEBP (4, 30
by gene delet
nant negative
renal medulla
hypertonicity
threatening de
water via urin
ing ability. To
REVIEWS
ysiol.00005.2009
Woo Lim,
Moo Kwon
ment of Medicine,
timore, Maryland
e.umaryland.edu
solution, cells
wo immediate
onicity are 1)
nic strength. It
erturb protein
rowding (43)
ionic strength
in an elegant
C. elegans (5).
me-wide RNAi
gical and biohypertonicity
d aggregation)
oteins caused
ovide a proofges proteins,
ll death in the
known. What
mmalian cells
apoptosis and
tor in
olality in
onicity
hilic archebacal when chal(61). Organic
ino acids and
Unlike elecible with protein structure
n of organic
ress by hyperonic strength;
. Physiol. Soc.
protection from the harmful effects of hyperosmolality
in the renal medulla (FIGURE 1).
TonEBP Contributes to the
Urinary Concentration
Independent of Vasopressin
Although the antidiuretic hormone vasopressin is the
major regulator of the urinary concentration by virtue of
stimulating expression of aquaporin-2 (AQP2) water
channels (50) and the UT-A urea transporters (22), there
clearly is a vasopressin-independent component. For
example, in Brattleboro rats, which lack endogenous
vasopressin, expression of AQP2 is stimulated by manipulations that raise the medullary tonicity (32). In
primary cultures (55) and established lines (15) of collecting duct principal cells, AQP2 expression increases
in response to an increase in medium tonicity in a manner independent of vasopressin. There is a binding site
for TonEBP in the promoter region of the AQP2 that
mediates transcriptional stimulation in response to
hypertonicity (16). Likewise, the promoter of the UT-A
urea transporter gene is also stimulated by TonEBP (48).
It should be pointed out that, throughout the collecting
duct where AQP2 and UT-A are expressed, TonEBP is
highly expressed, including the cortical segments (4, 30).
In TonEBP-deficient transgenic mouse models, expression of AQP2 and UT-A is severely reduced (27, 36). Thus
TonEBP is involved in the urinary concentrating process
independent of vasopressin (FIGURE 1).
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ts of tubules,
rmeability, in
current flow of
ess to urea for
however, we
ty as hyperosular effects.
rising concentration of organic osmolytes restores volume by osmosis and lowers the cellular ionic strength.
C. elegans tolerate extreme hypertonicity when they
are first allowed to accumulate glycerol, the major
organic osmolyte for this animal, in moderate hypertonicity (28). Likewise, slow increase in ambient
tonicity allows mammalian cells to adapt to extreme
hypertonicity because cellular accumulation of organic osmolytes occurs over the course of many hours.
The process is slow because stimulation of gene
expression is involved. In mammalian cells, the transcription factor named TonEBP (tonicity-responsive
enhancer binding protein) is the master transcriptional regulator for the cellular accumulation of organic
osmolytes (45). While TonEBP is active under isotonic
conditions, its activity increases when ambient tonicity rises (see below for more). TonEBP promotes cellular accumulation of the major organic osmolytes in the
renal medulla by transcriptional stimulation of plasma
membrane
transporters—sodium/myo-inositol
cotransperter (SMIT), sodium/chloride/betaine
cotransporter (BGT1), and sodium/chloride/taurine
cotransporter (TauT)—and rate-limiting biosynthetic
enzymes—aldose reductase (AR) for sorbitol and neuropathy target esterase (NTE, a phospholipase B) for
glycerophosphorylcholine (FIGURE 1) (11, 17, 44, 47,
58). The organic osmolytes—inositol, betaine, taurine,
sorbitol, and glycerophosphorylcholine—are trapped
inside the cell once they are transported or synthesized because they have low membrane permeability.
In C. elegans, induction of the gpdh genes that
encodes glycerol 3-phosphate dehydrogenase is the
key event in the hypertonicity-induced glycerol accumulation (29). Responsible transcription factor for the
induction of gpdh genes is not known. TonEBP is not
involved in the regulation because tonicity-responsive
form of TonEBP is found only in vertebrates (26) and
there is no TonEBP homolog in the nematode (14).
Despite the lack of TonEBP in the nematode, it is possible that the mammals and the nematode share a signaling pathway conveying the hypertonicity signal to
the stimulation of transcription.
Urea also causes cell death when its concentration
exceeds 300 mM (42). Interestingly, TonEBP also plays
a role in protection from the deleterious effects of urea
by stimulating the expression of heat shock protein 70
(HSP70) (49). TonEBP binds to the promoter of the
HSP70 gene in sites distinct from heat shock factor 1
and stimulates transcription of HSP70 (60). Every single cell in the renal medulla expresses high levels of
TonEBP (4, 30). Genetic inhibition of TonEBP, either
by gene deletion (36) or by overexpression of dominant negative form of TonEBP (27), results in severe
renal medullary atrophy because of failure to adapt to
hypertonicity and urea. These animals suffer from lifethreatening dehydration and fail to thrive as they loose
water via urine due to the lack of urinary concentrating ability. TonEBP is a master regulator in cellular
FIGURE 1. Physiology of TonEBP in the renal medulla
NKCC2, ROMK, and Na,K-ATPase in the thick ascending limb (TAL) drives
the sodium accumulation in the medullary interstitium. The resulting
hypertonicity stimulates TonEBP. Enhanced expression of SMIT, BGT1,
TauT, AR, and NTE promotes cellular accumulation of organic osmolytes
and protection from the harmful effects of hypertonicity. AQP2 water
channels and UT-A urea transporters contribute to the urinary concentration by promoting the collecting duct water permeability and urea secretion into the medullary interstitium. High expression of HSP70 protects
cells from the deleterious effects of urea.
PHYSIOLOGY • Volume 24 • June 2009
• www.physiologyonline.org
187
REVIEWS
Hypertonicity is a Local Signal for
Differentiation and Maintenance of
the Renal Medulla
“The expression of TonEBP in isotonic organs and tissues indicates that tonicity, i.e., isotonicity, is a widely
used signal for transcription.”
Genetic deletion of the UT-A1 and UT-A3 urea transporters also has the same effect (9) since these proteins are the major route of urea reabsorption in the
inner medulla. Lowering the urea concentration in the
renal medulla does not affect the morphology or function of the renal medulla other than reducing urinary
concentrating ability.
In contrast, the hypertonic salt concentration in the
medullary interstitium is difficult to change. It is
impossible to lower the interstitial hypertonicity on a
long-term basis without affecting the integrity of the
Table 1. Function of TonEBP and target genes involved
Function
Target Genes
References
Urinary concentration
AQP2, UT-A, ROMK
12, 16, 48
Adaptation to hypertonicity
SMIT, BGT1, AR, TauT,
NTE, SNAT2
11, 17, 44,
47, 58
Adaptation to high urea
HSP70, Osp94 (?)
24, 60
Adaptive immunity
TNF-␣, lymphotoxin ␤
35
Skeletal muscle differentiation
Cyr61
51
Metabolism of xenobiotics
Cytochrome P450 2 and
3 subfamilies
18, 25
Nucleus
Lamin A and C,
nucleoporin 88
2, 8
Tumor metastasis
Cyr61
19, 34
Viral replication
HIV-1 LTR
52
Diabetic nephropathy
AR
21, 62
188
PHYSIOLOGY • Volume 24 • June 2009
• www.physiologyonline.org
Bidirectional Regulation of TonEBP
by Ambient Tonicity
TonEBP is also known as NFAT5—nuclear factor of
activated T cells. In T cells, TonEBP is markedly
induced on T-cell receptor activation (57). TonEBP
regulates cytokines such as TNF-␣ and lymphotoxin-␤
in T-cells (35). Not surprisingly, the cytokine production is stimulated by hypertonicity. Transgenic mice
expressing inhibitory TonEBP in T cells (58) and
TonEBP knockout mice (13) reveal that TonEBP is
required for T-cell proliferation and adaptive immunity.
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As discussed above, the two major solutes in the renal
medulla—salt and urea—differ in their osmotic
action. Their effects on the functional and morphological differentiation of the renal medulla are also distinct. Urea concentration in the renal medulla can be
easily manipulated without obvious or detrimental
effects on the renal medulla. For example, low protein
diet (via reducing the production of urea) (23) or
increased water intake (via reducing circulating levels
of vasopressin) (53) is quite effective in dramatically
reducing the urea concentration in the renal medulla.
renal medulla. For example, a dramatic water diuresis
with a 10-fold increase in the urine flow for several days
does not affect the interstitial sodium concentration in
the renal medulla (53, 54). Loop diuretics work acutely,
but its effectiveness decreases over time because the
kidney is vigorously adapting to the salt loss (7). For
chronic reduction of the interstitial sodium concentration, life-threatening maneuvers such as continuous
administration of high doses of loop diuretics are
required (54). In mouse models of Bartter’s syndrome
made by deletion of the gene encoding the Na-K-2Cl
cotransporter type 2 (NKCC2) (56) or the ROMK potassium channel (37), renal medulla cannot be examined
due to rampant hydronephrosis. There are, however,
mouse models made by deletion of the AQP1 (39) or
ClC-K1 gene (1) that exhibit significant decreases in the
interstitial sodium concentration while maintaining
intact renal medulla. In these animals, gene expression
profile in the renal medulla is clearly altered (46), providing strong evidence that the local hypertonicity is an
important signal for the medullary-specific gene expression. For example, expression of UT-A1 is dramatically
decreased in these animals, most likely due to reduced
activity of TonEBP. Another example is that those proteins normally enriched in the renal cortex such as the
␤- and ␥-subunits of the ENaC channels are enriched in
the inner medulla of the AQP1- or ClC-K1-deficient
mice. Likewise, in cyclosporin-induced nephropathy
(33) and hypokalemic animals (20), reduced TonEBP
expression and activity in the renal medulla are
observed in correlation with downregulation of NKCC2.
The local hypertonicity in the renal medulla
appears to form early in the renal development. In
mouse, expression of NKCC2 starts around E14 and is
followed by TonEBP expression (30). Expression of
TonEBP target genes such as UT-A and AR starts after
the initiation of TonEBP expression. When the neonatal pups are treated with loop diuretic to inhibit the
activity of NKCC2, developmental processes of the
inner medulla are blocked (30). These observations
demonstrate that the differentiation of the renal
medulla requires local hypertonicity. Thus the interstitial hypertonicity of the renal medulla is an important local signal not only for specific gene expression
profile but also for development of the renal medulla.
Interestingly,
spleen are mod
In addition, To
under the mil
inducing sodi
(SNAT2), which
acids as organ
for cellular ad
and extreme (r
cellular accum
TonEBP is a
57). In brain, T
(38). Interesti
TonEBP increa
ia (systemic h
tion of ~190 g
that TonEBP is
hypertonicity.
in isotonic con
is involved in
its target gene
cated in tumo
and possibly
TonEBP stim
immunodefici
long-terminal
In cultured
ditions based
Furthermore,
both an increa
ity: TonEBP ac
is lowered, wh
ty is elevated
controls the
expression of
indicates that
used signal fo
Molecular
Regulatio
At least three
bidirectional r
tion, transacti
complex for i
dance. Althou
es in respon
phosphorylati
defined, and s
Nuclear loc
ambient tonic
the range of
25–55% of cell
function of ton
twofold (25–55
ulation is com
localization s
(FIGURE 3). I
REVIEWS
TonEBP
clear factor of
is markedly
(57). TonEBP
ymphotoxin-␤
okine producansgenic mice
ells (58) and
hat TonEBP is
tive immunity.
Interestingly, lymphoid tissues such as thymus and
spleen are moderately hypertonic by ~40 mosmol/kg (13).
In addition, TonEBP is required for proliferation of T cells
under the mild hypertonicity (13). This is achieved by
inducing sodium-coupled amino acid transporter-2
(SNAT2), which mediates cellular accumulation of amino
acids as organic osmolytes (58). Thus TonEBP is critical
for cellular adaptation to both mild (lymphoid tissues)
and extreme (renal medulla) hypertonicity by promoting
cellular accumulation of distinct organic osmolytes.
TonEBP is abundant in organs that are isotonic (41,
57). In brain, TonEBP expression is limited to neurons
(38). Interestingly, nuclear localization of neuronal
TonEBP increases rapidly in response to hypernatremia (systemic hypertonicity) in association with induction of ~190 genes in the brain (38, 40). This suggests
that TonEBP is involved in the neuronal adaptation to
hypertonicity. In other organs, TonEBP is clearly active
in isotonic conditions (Table 1). For example, TonEBP
is involved in the differentiation of skeletal muscle via
its target genes such as Cyr61 (51). Cyr61 is also implicated in tumor metastasis of the gastric cancer (34)
and possibly breast cancer (19). In monocytes,
TonEBP stimulates the replication of the human
immunodeficiency viruses (HIV-1) by binding to their
long-terminal repeat (LTR) (52).
In cultured cells, TonEBP is active in isotonic conditions based on expression of its target genes.
Furthermore, TonEBP responds bidirectionally to
both an increase and a decrease in the ambient tonicity: TonEBP activity decreases when ambient tonicity
is lowered, whereas it increases when ambient tonicity is elevated (59). Ambient tonicity is a rheostat that
controls the activity of TonEBP (FIGURE 2). The
expression of TonEBP in isotonic organs and tissues
indicates that tonicity, i.e., isotonicity, is a widely
used signal for transcription.
Molecular Mechanism of TonEBP
Regulation by Ambient Tonicity
At least three discrete pathways are involved in the
bidirectional regulation of TonEBP: nuclear localization, transactivation (stimulation of RNA polymerase
complex for inititation of transcription), and abundance. Although phosphorylation of TonEBP increases in response to hypertonicity (31), changes in
phosphorylation in response to hypotonicity are not
defined, and sites of phosphorylation are not known.
Nuclear localization of TonEBP is determined by
ambient tonicity in a highly predictable manner. Over
the range of tonicity from 135 to 500 mosmol/kg,
25–55% of cellular TonEBP is in the nucleus as a tight
function of tonicity (26). Thus the range of regulation is
twofold (25–55%) for the nuclear localization. This regulation is completely dependent on the single nuclear
localization signal (NLS) near the NH2-terminus
(FIGURE 3). Inactivation of the NLS by site-directed
FIGURE 2. Bidirectional regulation of TonEBP
TonEBP is active in isotonicity. Its activity is enhanced by
hypertonicity and suppressed by hypotonicity. This
means that tonicity (isotonicity) is a signal for TonEBP
action in many tissues.
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water diuresis
or several days
ncentration in
s work acutely,
e because the
t loss (7). For
um concentraas continuous
diuretics are
er’s syndrome
the Na-K-2Cl
ROMK potassibe examined
are, however,
AQP1 (39) or
ecreases in the
e maintaining
ene expression
ered (46), proertonicity is an
ic gene expresis dramatically
due to reduced
hat those proex such as the
are enriched in
C-K1-deficient
nephropathy
duced TonEBP
medulla are
ion of NKCC2.
enal medulla
velopment. In
und E14 and is
Expression of
AR starts after
en the neonato inhibit the
cesses of the
observations
of the renal
hus the intera is an imporne expression
enal medulla.
mutagenesis obliterates the tonicity-dependent changes in
the nuclear localization: 25% of the mutant TonEBP molecules with inactive NLS is in the nucleus regardless of the
ambient tonicity. When short fragments of TonEBP containing the NLS are fused to constitutively cytoplasmic proteins such as a triplet of green fluorescent protein, the
fusion proteins displays tonicity-dependent changes in
nuclear localization (26). These observations demonstrate
that the tonicity-dependent changes in the nuclear localization of TonEBP are autonomously controlled by the NLS
without involving other domains of TonEBP.
Phosphorylation does not play a role in the regulation of the
NLS because site-directed mutations that eliminate potential phosphorylation sites in the NLS display normal tonicity regulation.
The transactivating activity of TonEBP is also bidirectionally regulated by changes in ambient tonicity (10). There are
three activation domains—AD1, AD2, and AD3—that stimulate transcription (31) (FIGURE 3). In addition, there are two
modulation domains—MD1 and MD2—that do not stimulate transcription by themselves but enhance the activity of
activation domains. All the activation and modulations
domains act cooperatively to confer a tremendous transactivational potential to TonEBP. AD2 and MD1 are stimulated
FIGURE 3. Functional domains of TonEBP
RHD (Rel-homology domain) binds DNA. Ambient tonicity stimulates
NLS, AD2, and MD1. RHA (RNA helicase A) binding is inhibited by
ambient tonicity, leading to enhanced transactivation. NLS, nuclear
localization signal; AD1, AD2, and AD3, activation domains; MD1 and
MD2, modulation domains. See text for details.
PHYSIOLOGY • Volume 24 • June 2009
• www.physiologyonline.org
189
REVIEWS
Conclusion
Hypertonicity is essential for the development and function of the renal medulla. TonEBP is the key transcription factor that controls gene expression in response to
cues from ambient tonicity. As such, TonEBP is a master
regulator in the renal medulla not only in cellular protection from the harmful hyperosmolality but also in the
urinary concentration and development of the renal
medulla. Outside the kidney, TonEBP is active in isotonic tissues, contributing to a variety of physiological and
pathophysiological processes. 
This work is supported by National Institute of Diabetes
and Digestive and Kidney Diseases Grants DK-42479 and
DK-61677. S. W. Lim is supported by a fellowship from the
National Kidney Foundation.
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