Am J Physiol Renal Physiol 289: F2–F7, 2005;
doi:10.1152/ajprenal.00041.2005.
Invited Review
DNA damage and osmotic regulation in the kidney
Natalia I. Dmitrieva, Maurice B. Burg, and Joan D. Ferraris
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and
Blood Institute, Department of Health and Human Services, Bethesda, Maryland
hypertonicity; renal medulla; apoptosis; cell cycle delay
BACKGROUND: DNA DAMAGE AND REPAIR
MANY EXOGENOUS AND ENDOGENOUS stresses damage DNA.
Examples include ionizing radiation, UV radiation, chemical
carcinogens, reactive oxygen species (ROS), alkylating agents,
and more. All cells have DNA damage-response pathways that
can repair DNA accurately and prevent accumulation of mutations. This is essential for preventing diseases caused by both
genetically transmitted and acquired mutations. The general
organization of the DNA damage response is shown in Fig. 1.
DNA damage rapidly arrests the cell cycle, and DNA repair,
specific to the type of DNA damage, is activated. The cell cycle
arrest generally persists until the DNA is repaired. Failure to
repair the DNA damage activates apoptosis (programmed cell
death), which eliminates potentially malignant cells. Thus
DNA is under constant surveillance by the DNA damageresponse network, and coordinated activity of all its components is required to ensure genomic stability and to minimize
mutations. The exact nature of the response depends on the
type and extent of the DNA damage. Not all DNA breaks are
pathological. Transient breaks occur during normal DNA replication, transcription, and recombination. These breaks are
rapidly repaired by the same players that respond to DNA
damage of exogenous origin (66). Therefore, accumulation of
DNA damage and/or mutations can occur even in the absence
of obvious DNA damaging stress if any of the components of
the repair network are compromised. Many severe human
diseases are recognized that are linked to mutations in genes
Address for reprint requests and other correspondence: M. B. Burg, NHLBI,
Bethesda, MD 20892-1603 (e-mail: [email protected]).
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encoding proteins that participate in DNA repair and cell cycle
control (reviewed in Ref. 52). Furthermore, studies of transgenic mice have identified many genes involved in DNA repair
or cell cycle control whose malfunction is lethal (42, 71, 77) or
leads to premature aging, due to accelerated accumulation of
DNA damage and mutations (15, 30, 52).
With this as background, recent observations of cells in the
renal inner medulla are particularly striking. Renal inner medullary cells contain many DNA breaks in vivo and their DNA
repair is defective under normal conditions, yet the cells do not
suffer the expected consequences (21, 23). The high osmolality
in the renal inner medulla is responsible for the DNA breaks
and for inhibition of their repair.
HYPERTONICITY-INDUCED DNA DAMAGE IN CELL CULTURE
AND IN THE RENAL MEDULLA IN VIVO
Realization that high NaCl is genotoxic has evolved over
many years (reviewed in Refs. 21, 22, 38, 39). Most recently,
acute elevation of NaCl was observed to cause cell cycle arrest
(41, 46) and to activate such important responders to DNA
damage as GADD45 (41) and p53 (17, 18) (Fig. 2). Acute
elevation of osmolality from 300 to 500 – 600 mosmol/kgH2O
by adding NaCl causes DNA breaks in mIMCD3 cells (40).
This discovery led to further studies of how cells respond to
DNA damage induced by high NaCl. Initially, there is transient
arrest at all stages of the cell cycle. Activation of G2/M arrest
depends on p38 kinase (20), similar to the response to UV
radiation (6). In the presence of high NaCl, overexpression of
Gadd45 proteins inhibits mitosis and promotes G2/M arrest
(43). G1 arrest depends on p53 (18), as is the case with many
other DNA-damaging agents (reviewed in Ref. 24). After
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Dmitrieva, Natalia I., Maurice B. Burg, and Joan D. Ferraris. DNA damage
and osmotic regulation in the kidney. Am J Physiol Renal Physiol 289: F2–F7,
2005; doi:10.1152/ajprenal.00041.2005.—Renal medullary cells normally are exposed to extraordinarily high interstitial NaCl concentration as part of the urinary
concentrating mechanism, yet they survive and function. Acute elevation of NaCl
to a moderate level causes transient cell cycle arrest in culture. Higher levels of
NaCl, within the range found in the inner medulla, cause apoptosis. Recently, it was
surprising to discover that even moderately high levels of NaCl cause DNA
double-strand breaks. The DNA breaks persist in cultured cells that are proliferating
rapidly after adaptation to high NaCl, and DNA breaks normally are present in the
renal inner medulla in vivo. High NaCl inhibits repair of broken DNA both in
culture and in vivo, but the DNA is rapidly repaired if the level of NaCl is reduced.
The inhibition of DNA repair is associated with suppressed activity of some DNA
damage-response proteins like Mre11, Chk1, and H2AX but not that of others, like
GADD45, p53, ataxia telangiectasia-mutated kinase (ATM), and Ku86. In this
review, we consider possible mechanisms by which the renal cells escape the
known dangerous consequences of persistent DNA damage. Furthermore, we
consider that the persistent DNA damage may be a sensor of hypertonicity that
activates ATM kinase to provide a signal that contributes to protective osmotic
regulation.
Invited Review
DNA DAMAGE AND OSMOTIC REGULATION
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damage response, and how the cells manage to cope (see also
other recent reviews in Refs. 21 and 22).
HYPERTONICITY INHIBITS THE USUAL DNA
DAMAGE RESPONSE
Fig. 1. General structure of the classic DNA damage response. DNA damage
causes rapid cell cycle arrest and activates a DNA repair program specific to
the type of DNA damage. The cell cycle resumes when the DNA is repaired.
If the DNA damage is too extensive or cannot be repaired, apoptosis eliminates
the cell before dangerous mutations can occur.
Fig. 2. Effects of hypertonicity. Hypertonicity causes DNA
damage. Transient cell cycle delay occurs through the
concerted actions of GADD45, p53, and p38, affording
immediate protection. However, in contrast to the classic
DNA damage response (Fig. 1), DNA repair is inhibited,
and the cells proliferate with persistent DNA damage and
without activating apoptosis. Ameliorating factors include
accumulation of compatible organic osmolytes [sorbitol,
betaine, inositol, and glycerophosphocholine (GPC)], expression of heat shock proteins, and activation of Ku86.
HSPs, heat shock proteins. See the text for a detailed
description.
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several hours, the cells begin proliferating again, despite the
continued presence of high NaCl (9, 56). This sequence is very
similar to the classic DNA damage response, as depicted in
Fig. 1, which led us to assume initially that cell cycle arrest
provides time for repair of DNA damage, as is the case
following ionizing radiation. However, to our surprise, cells in
culture that have adapted to high NaCl, and are proliferating
rapidly, still contain numerous DNA breaks (23). Despite the
DNA breaks, the adapted cells appear normal and exhibit little,
if any, apoptosis (9, 23, 56). The DNA breaks persist as long
as the level of NaCl remains high, but, when NaCl is lowered,
DNA repair activates immediately, and the DNA breaks are
repaired (19, 23). This pattern is not restricted to tissue culture.
Amazingly, numerous DNA breaks are present in vivo in cells
of the normal mouse renal inner medulla (23), which can be
attributed to the high level of NaCl that is normally present in
renal medullary interstitial fluid (3). Despite the presence of
DNA breaks in the inner medulla, apoptosis is rare there (63,
74). As in cell culture, DNA breaks persist in the renal medulla
as long as the NaCl concentration remains high. However, the
breaks are rapidly repaired when inner medullary osmolality is
decreased by the diuretic furosemide (23).
These discoveries raise numerous questions. They make us
wonder how cells exposed to high NaCl can cope with the
continuous presence of DNA breaks. How can the cells transcribe broken DNA efficiently, and how can they replicate it
safely? Why do the cells apparently ignore the breaks? Why
don’t they remain in cell cycle arrest, repair the DNA, or else
undergo apoptosis, as with other stresses? Is continued presence of the breaks necessary for adaptation to high NaCl? Is
there an alternative DNA damage response, modified by NaCl
at the molecular level, so that the cells can survive and function
despite the DNA breaks? We do not yet have the answers to
those questions, but we are beginning to have some understanding. In what follows, we present our current view of how
high NaCl induces DNA breaks, how it inhibits the DNA
High NaCl modifies the DNA damage response at the
molecular level in several ways. Mre11 exonuclease is a
nuclear protein that normally binds rapidly to DNA breaks,
initiating the process of repair (14). However, high NaCl
causes Mre11 to move from the nucleus to the cytoplasm,
where it is unavailable to initiate DNA repair (19), and
Mre11 stays in the cytoplasm even after the cells adapt to
high NaCl (23). Chk1 becomes phosphorylated immediately
after induction of DNA breaks by other genotoxic agents,
initiating cell cycle arrest (5, 32). However, Chk1 is not
phosphorylated in response to the DNA breaks that are
induced by high NaCl (19, 23). Histone H2AX becomes
phosphorylated at DNA double-strand breaks induced by ionizing radiation (55), which is a prerequisite for successful DNA
repair (10, 54). The DNA breaks caused in cell culture by high
NaCl, however, do not induce phosphorylation of histone
H2AX (23). Furthermore, despite the existence of DNA
breaks, H2AX is not phosphorylated in mouse renal inner
medullas in vivo. We propose that inhibition by high NaCl of
repair of normally occurring DNA breaks may cause them to
accumulate, explaining how DNA becomes damaged in the
presence of high NaCl.
Remarkably, all the aspects of the DNA damage response
that are lacking in cells as long as they are exposed to high
NaCl become activated immediately when NaCl is reduced.
Mre11 moves into the nucleus, histone H2AX and Chk1
become phosphorylated, and the DNA breaks disappear within
a few hours (19, 23). Thus at least some components of the
classic DNA damage response are inhibited by high NaCl.
However, others are not. DNA damage-response proteins that
are upregulated by high NaCl include GADD45 (11, 41, 43),
p53 (17), and ataxia telangiectasia (AT)-mutated kinase
(ATM) (33).
We speculate that high NaCl activates a nonclassic DNA
damage response in which the DNA breaks are not religated
but are modified to maintain DNA function without inducing
chronic cell cycle arrest or apoptosis. That would involve an
alternative protein complex that stabilizes the DNA breaks and
maintains chromatin structure in cells exposed to high NaCl.
Our preliminary results suggest that the Ku86/Ku70 heterodimer might be an essential part of such a complex (16).
Cells deficient in Ku86 are hypersensitive to high NaCl, as
manifested by profound inhibition of proliferation, aberrant
Invited Review
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DNA DAMAGE AND OSMOTIC REGULATION
mitosis, and increased chromatin fragmentation (16). Ku is a
heterodimeric DNA end-binding complex composed of two
proteins, Ku70 and Ku86. It is a major component of the
complex involved in nonhomologous end joining of DNA
double-strand breaks. It binds with high affinity to DNA ends,
independent of their sequence or structure (45, 53, 62). Biochemical studies and the structure of the Ku heterodimer
suggest that it acts as an alignment factor (67). We suppose that
it helps stabilize the DNA breaks in cells adapted to high NaCl
in a way that maintains DNA function, but that additional,
unidentified proteins are involved. Further studies are needed
to detail the molecular mechanisms involved in response to
DNA breaks induced by high NaCl and to understand how cells
cope despite their continued presence.
Not only are DNA breaks continuously present in renal
medullary cells as long as they are exposed to high NaCl, but
it now appears that the breaks may be sensors of hypertonicity
that contribute to the protective osmoregulatory response.
Other known effects of hypertonicity were previously proposed
to be the sensors, including alterations in cell volume, in
intracellular ionic strength, and in molecular crowding (28).
Hypertonicity shrinks cells by osmosis, elevating the concentration of all intracellular components. Intracellular ionic
strength rises, and intracellular macromolecules become more
crowded, altering the activity of proteins, DNA, and other
cellular macromolecules. Thus reduction of cell volume by
hypertonicity alters the activity of numerous cellular constituents. Cells generally respond to hypertonicity by two successive processes, regulatory volume increase (RVI) (31) and
accumulation of compatible organic osmolytes (73). The rapid
initiation of RVI normalizes cell volume by accumulation of
inorganic ions, followed by an osmotic influx of water. However, that still leaves high intracellular ionic strength. Accumulation of compatible organic osmolytes lowers intracellular
ionic strength while maintaining cell volume. The benefit to the
cells is that compatible organic osmolytes do not perturb
macromolecular function like inorganic ions do. The kidneys
maintain osmolality of the extracellular fluid constant at ⬃290
mosmol/kgH2O by regulating the concentration and volume of
the urine. Therefore, most cells do not normally experience
hypertonicity. However, operation of the urinary concentrating
mechanism entails a high concentration of NaCl in renal
medullary interstitial fluid. The lowest normal concentration of
inorganic salts (mainly NaCl) in renal medullary interstitial
fluid is ⬃600 mosmol/kgH2O, which occurs during prolonged
Fig. 3. Hypertonicity activates tonicity-responsive enhancer (TonE)/osmotic response element (ORE) binding
protein (TonEBP/OREBP), resulting in the protective osmoregulatory responses. Several protein kinases contribute
to activation of TonEBP/OREBP. All are required for the
full response, but no one of them, alone, fully activates.
Cytoskeletal alterations, DNA damage, and other, as yet
unknown, stimuli activate the kinases. SMIT, sodium-myoinositol cotransporter; UTA, urea transporter; BGT1, betaine-␥-aminobutyric acid transporter. See the text for a
detailed description.
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OSMOTIC REGULATION
water diuresis (2). The concentration of NaCl increases during
prolonged antidiuresis, accompanied by accumulation of molar
concentrations of urea, making the renal medulla a site of
extreme osmotic stress. A vital part of the protective response
is accumulation of compatible organic osmolytes. The predominant renal medullary organic osmolytes are sorbitol, glycine
betaine (betaine), myo-inositol (inositol), and glycerophosphocholine (GPC) (7). Renal cells exposed to high salt in tissue
culture accumulate the same compatible organic osmolytes
(48). If accumulation of the compatible organic osmolytes is
prevented, growth and survival of renal medullary cells exposed to high salt are impaired in tissue culture (72) and
survival is impaired in vivo (35). Although accumulation of
compatible organic osmolytes evidently protects cells from
important harmful effects of hypertonicity, the protection is not
complete. The DNA damage response is still inhibited, and
DNA damage still remains.
In addition to organic osmolytes, renal inner medullary
epithelial cells accumulate heat shock proteins when exposed
to high salt both in vivo and in cell culture (8, 12, 50, 58). The
heat shock protein response to hypertonicity is rapid, which
provides protection while organic osmolytes are being accumulated (12). Furthermore, hsp70 is directly antiapoptotic. It
inhibits apoptosis through several mechanisms, including prevention of formation of the apoptosome (4, 70). Targeted
disruption of hsp70.1 sensitizes cells to osmotic stress in vitro
and in vivo. Also, hsp70.1-deficient mouse embryonic fibroblasts tolerate less osmotic stress than do wild-type fibroblasts,
and hsp70.1-deficient mice have increased apoptosis in their
renal medullas (61).
Hypertonicity increases transcription of HSP70 and also
of the genes responsible for accumulation of organic osmolytes, namely, aldose reductase (AR), which catalyzes the
conversion of glucose to sorbitol, the betaine-␥-aminobutyric acid transporter (BGT1), and the sodium-myo-inositol
cotransporter (SMIT), which transport betaine and inositol,
respectively, into the cells (7) (Fig. 3). The increased
transcription is mediated by an enhancer, osmotic response
element/tonicity-responsive enhancer (ORE/TonE), present
in their 5⬘-flanking regions (7, 69). The urea transporter
gene, UTA, is similarly regulated by hypertonicity (49) (Fig.
3) and is essential for optimal operation of the urinary
concentrating mechanism (25). Hypertonicity-induced increases in transcription of these genes results from activation of the transcription factor TonE/ORE binding protein
(TonEBP/OREBP) (37, 47, 69).
Invited Review
DNA DAMAGE AND OSMOTIC REGULATION
REGULATION OF TonEBP/OREBP BY PROTEIN KINASES
multimer in which each partner blocks and inactivates the
kinase domain of the other (1). Within moments after DNA
double-strand breaks (DSBs), ATM undergoes intermolecular
autophosphorylation at S1981, releasing fully active monomers
(1). Nuclear ATM is recruited to sites of DSBs soon after DNA
damage occurs. However, the initial signal for ATM activation
may be the accompanying alteration of chromatin rather than
the DSBs themselves (1, 60). Even a very few DSBs result in
autophosphorylation of ATM throughout the nucleus, not just
at DSBs, and perturbation of chromatin structure activates
ATM even in the absence of DNA damage (reviewed in Ref.
60). When ATM is activated by DSBs, it phosphorylates and
activates numerous targets, such as p53, MDM2, Chk2,
BRCA1, and NBS1, that mediate cell cycle arrest, DNA repair,
and apoptosis (60). As discussed above, high NaCl not only
causes DSBs but inhibits their repair. Some DNA damageresponse proteins, like Mre11, are inhibited by high NaCl,
whereas others, like Ku86, are not. Recently, we found that
elevating NaCl activates ATM and that ATM contributes to the
tonicity-dependent activation of TonEBP/OREBP (33). Activation of ATM by high NaCl could be due to the DNA damage
itself or to perturbation of chromatin structure, both of which
could result from high NaCl (38, 40). TonEBP/OREBP contains amino acid sequences around S1197, S1247, and S1367
that match the consensus for ATM phosphorylation sites.
These serines are contained in a region of TonEBP/OREBP
(amino acids 548 –1531), whose phosphorylation is increased
by high NaCl (26). Wortmannin, which inhibits phosphatidylinositol kinase-related kinases including ATM, decreases tonicity-dependent activation of TonEBP/OREBP. Involvement
of DNA-activated protein kinases in the response to hypertonicity had previously been suspected based on effects of
LY-294002, an inhibitor similar to wortmannin (40). Wortmannin reduces activity of an ORE/TonE luciferase reporter
and abundance of BGT1 mRNA (33). Studies of AT cells,
which lack functional ATM, demonstrated that ATM is specifically involved. High NaCl-dependent TonEBP/OREBP
transcriptional activity and transactivation are reduced in these
cells, and these activities are restored when the AT cells are
reconstituted with wild-type ATM, but not with ATM rendered
inactive by an S1981A mutation (33). As mentioned above,
TonEBP/OREBP has several serines that are putative ATM
phosphorylation sites. Overexpression of wild-type TonEBP/
OREBP in HEK293 cells stimulates ORE-dependent transcription much more than does overexpression of TonEBP/OREBP
mutated at the putative ATM phosphorylation sites, indicating
that these sites are important for TonEBP/OREBP activity
(33). Finally, ATM physically associates with TonEBP/
OREBP. ATM coimmunoprecipitates with TonEBP/OREBP
and vice versa. Also, an antibody against ATM supershifts
TonEBP/OREBP in electrophoretic mobility shift assays, indicating that the physical association is retained in the transcription complex.
REGULATION OF TonEBP/OREBP BY ATM, A DNA
DAMAGE-RESPONSE PROTEIN
PERSPECTIVE
There is no reason to believe that the protein kinases already
mentioned are linked to DNA damage, but such a link is
provided by ATM kinase, which is a DNA damage-response
protein, whose gene is mutated in the human genetic disorder
AT. In unstressed cells, ATM is a homodimer or higher-order
We propose that the DNA DSBs (or the associated perturbation of chromatin) that are caused by hypertonicity activate
ATM and that this contributes to hypertonicity-induced activation of TonEBP/OREBP (Fig. 3). It has been questionable
whether there is a single sensor of hypertonicity that signals
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Like many other transcription factors, TonEBP/OREBP is
controlled at multiple levels. Hypertonicity affects its distribution between nucleus and cytoplasm, its phosphorylation, its
protein abundance, and its transactivation. Thus, at osmolalities near 300 mosmol/kgH2O, TonEBP/OREBP is present in
both the nucleus and the cytoplasm. Hypertonicity shifts it to
the nucleus, where it is available to regulate transcription, and
hypotonicity shifts it to the cytoplasm (68). High NaCl transactivates TonEBP/OREBP, resulting in increased transcriptional activity (27). Hypertonicity also rapidly increases phosphorylation of TonEBP/OREBP on tyrosines and serines (13).
The increase in phosphorylation within amino acids 548 –1531
coincides with enhanced transactivation (27). Finally, after
several hours, hypertonicity increases the abundance of
TonEBP/OREBP mRNA and protein (37, 47).
Several different protein kinases are known to be involved in
tonicity-induced activation of TonEBP/OREBP (Fig. 3) (59).
Their combined activity is necessary for full tonicity-induced
activation, but no one of them, alone, is sufficient. Furthermore, while we know that each of these kinases is activated by
high NaCl, we understand less about how the tonicity is sensed
and about possible interrelations between the kinases. The
specific protein kinases known to be activated by hypertonicity
include p38, Fyn, PKA, and ATM.
Hypertonicity increases phosphorylation of p38, accompanied by an increase in its kinase activity (29, 34, 44). Inhibition
of p38 reduces tonicity-dependent activation of TonEBP/
OREBP (36, 57). Cytoskeletal effects of hypertonicity, mediated by Rac, are one sensor for hypertonic activation of p38
(Fig. 3) (65). It seems likely that this OSM pathway is involved
in p38-mediated activation of TonEBP/OREBP, although there
is not yet direct evidence that interrupting the pathway reduces
hypertonic activation of TonEBP/OREBP.
Fyn is a nonreceptor tyrosine kinase that is activated by
hypertonicity. Inhibition of Fyn by the pyrazolopyrimidine
derivative PP2 or transfection of dominant-negative Fyn partially blocks hypertonic activation of an ORE/TonE reporter
(36). Furthermore, hypertonic activation of an ORE/TonE
reporter is attenuated in Fyn-deficient cells. Thus Fyn contributes to hypertonicity-induced activation of TonEBP/OREBP
(Fig. 3).
High NaCl also activates PKA, which contributes to activation of TonEBP/OREBP (26). H-89, an inhibitor of PKA,
reduces tonicity-dependent increases in ORE/TonE reporter
activity, induction of AR and BGT1 mRNAs, and TonEBP/
OREBP transactivation. Overexpression of the catalytic subunit of PKA (PKAc) increases the activity of an ORE/TonE
reporter and transactivates TonEBP/OREBP, while dominantnegative PKAc reduces activity of an ORE/TonE reporter.
Finally, TonEBP/OREBP and PKAc coimmunoprecipitate,
demonstrating physical association (26).
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through a network of interrelated kinases or whether hypertonicity perturbs numerous cellular functions, leading to a unique
combination of signals that together fully activate TonEBP/
OREBP. We favor the latter. ATM is one of several kinases
that contribute to activation of TonEBP/OREBP. Each is necessary for full activation, but none, alone, is sufficient. Strikingly, simply activating ATM by damaging DNA, does not of
itself activate TonEBP/OREBP. Ionizing and ultraviolet radiation both damage DNA and activate ATM, but they do not
activate TonEBP/OREBP (33). Additional signals are necessary. One signal is transmitted through the p38 pathway, likely
triggered by cytoskeletal reorganization. Another trigger may
be the oxidative stress that accompanies hypertonicity (75, 76),
but the downstream components of this pathway are not yet
known.
Sensing hypertonicity in mammalian cells has been variously attributed to changes in intracellular ionic strength (51,
64), cell volume (51), and molecular crowding (28). Now,
additional perturbations have to be considered, including DNA
damage, cytoskeletal alterations, and oxidative stress. Considering that most other functions of mammalian cells are much
more complicated than those of yeast, the additional complexity of multiple sensors of hypertonicity and multiple pathways
signaling osmotic regulation is not surprising.
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