Urea sensitizes mIMCD3 cells to heat
shock-induced apoptosis: protection by NaCl
Chantal
Colmont,
Stéphanie
Michelet,
Dominique
Guivarc'h, and
Germain
Rousselet
Service de Biologie Cellulaire, Commissariat à
l'Énergie Atomique, Centre d'Études Nucléaires
de Saclay, 91191 Gif-sur-Yvette, France
 |
ABSTRACT |
Urea, with NaCl, constitutes the osmotic
gradient that allows water reabsorption in mammalian kidneys. Because
NaCl induces heat shock proteins, we tested the responses to heat shock
of mIMCD3 cells adapted to permissive urea and/or NaCl concentrations. We found that heat-induced cell death was stronger after adaptation to
250 mM urea. This effect was reversible, dose dependent, and, interestingly, blunted by 125 mM NaCl. Moreover, we have shown that
urea-adapted cells engaged in an apoptotic pathway upon heat shock,
as shown by DNA laddering. This sensitization is not linked to a defect
in the heat shock response, because the induction of HSP70 was similar
in isotonic and urea-adapted cells. Moreover, it is not linked to the
presence of urea inside cells, because washing urea away did not
restore heat resistance and because applying urea and heat shock at the
same time did not lead to heat sensitivity. Together, these results
suggest that urea modifies the heat shock response, leading to
facilitated apoptosis.
hyperosmolarity; sodium chloride; adaptation
 |
INTRODUCTION |
IN MAMMALS, the
urinary concentrating mechanism relies on water reabsorption driven by
the renal corticopapillary osmotic gradient. Cells from the renal
medulla are thus uniquely exposed to fluctuating concentrations of NaCl
and urea, in which they are able to survive and function. Long-term
effects of hyperosmotic NaCl have been widely studied and include a
general heat shock response (7, 12, 16) and a specific
osmotic response (reviewed in Ref. 2). Induction of heat
shock protein expression and accumulation of compatible organic
osmolytes allow cell survival. Adaptation to hypertonic NaCl has also
been shown to protect cells from urea toxicity (12), an
observation that led to the notion that hyperosmolarity is better
tolerated when both NaCl and urea are present than when it is mediated
by either NaCl or urea ("transprotection"). This protective effect
of NaCl is linked to its capacity to induce expression of the HSP70
heat shock protein (13). We have also shown that chronic
hyperosmolar NaCl and urea were able to synergistically induce the
expression of the UT-A2 urea transporter mRNA (9), confirming a molecular interaction between the long-term effects of
hyperosmolar NaCl and urea. However, after experimental analysis of
acute exposure of cells to death-inducing hyperosmolar media, similar
results were interpreted in different ways (11, 15). One
group compared results obtained with the same final osmolarity, which,
as the actual water reabsorption driving force, is physiologically relevant. This led to a confirmation of the transprotection under acute
conditions (15). The other group considered the
concentrations of the individual solutes, which, because NaCl is
hypertonic whereas urea is freely membrane permeant, is relevant from
the cell biology point of view. No transprotection could then be
evidenced (11). Surprisingly, using the same experimental
approach, Zhang et al. (18) recently demonstrated that
urea can inhibit the induction of caspase 3 activity by acute NaCl
treatment, although a link with an inhibition of cell death, or a
modification of the cell death pathway, was not provided.
Interestingly, several morphological and biochemical features of the
cell deaths triggered by progressive or acute osmolarity increases are
actually different (10).
Beyond its physicochemical effects on protein structure and function
(3, 17), hyperosmolar urea has been shown to acutely regulate multiple signaling events in renal medullary cells in vitro.
In particular, a pathway exhibiting hallmarks of a receptor tyrosine
kinase pathway is triggered by urea. This includes activation of
phospholipase C-
(6), activation of
phosphatidylinositol 3-kinase and its effectors Akt and p70 S6 kinase
(20), activation of Shc with recruitment of Grb2
(20), and induction of immediate-early genes
(5). Urea is also able to activate a signaling pathway leading to the activation of extracellular signal-regulated kinases (4). Urea also exerts a prooxidant effect necessary for
increased expression of the stress-responsive gene Gadd153
(19). However, little is known about the cellular effects
of hyperosmotic urea. Acute hyperosmotic urea shocks have been reported
to induce apoptosis (11). We have recently shown
that the cell death pathways triggered by acute or progressive urea
increases are different, with typical apoptosis resulting from
progressive urea increases (10). To our knowledge, other
cellular effects of permissive urea concentrations (i.e.,
concentrations that allow mIMCD3 cells to grow in vitro) have not been
addressed. As mentioned above, urea imposes a stress on target cells.
We (10) and others (12) have shown that the long-term effects of this stress are not sufficient to protect cells
from subsequent hyperosmotic injuries. However, it could not be
excluded that urea-induced stress was able to modify cell responses to
another stress, a point that would give important indications on the
long-term cellular effects of urea at the molecular level. Phenotypic
interactions between cell responses to different kinds of stress have
already been reported, essentially because of the potential beneficial
protective consequences of nonlethal stresses, a phenomenon called
hormesis (1). In addition, exploring the interactions
between hyperosmotic NaCl and/or urea and another kind of stress could
help in individualizing the respective effects of global
hyperosmolarity and of the different solutes. Therefore, we have
analyzed the responses of urea-adapted cells to heat shock.
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METHODS |
Cell culture.
Mouse inner medullary collecting duct (mIMCD3) cells (14)
were obtained from the American Type Culture Collection. The cells were
maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and
F-12 medium (Life Technologies) supplemented with 10% fetal bovine
serum (Sigma) and 2 mM L-glutamine (Sigma; referred to as
iso medium) at 37°C with 5% CO2. Cells were adapted to
hyperosmotic media with different solutes (NaCl, urea, glycerol, or a
combination of NaCl and urea). Cells grown in iso medium were
subcultured at a ratio of 1 to 10 and grown overnight, and the medium
was exchanged for a freshly prepared hyperosmotic medium. Osmolarity increases were never >200 mosmol/lH2O, whatever the solute
used. Cells were then grown to confluence in this hyperosmotic
conditions, subcultured at a ratio of 1 to 5, and grown overnight, and
another osmolarity increase could be performed. When the desired
osmolarity was reached, cells were grown and subcultured in these
hyperosmotic conditions for 10-15 passages (1.5-2 mo).
Experiments were performed with cells adapted to the desired osmolarity
for at least 1 wk. Hyperosmotic media were prepared extemporaneously by
adding the desired volumes of solute stock solutions (2.5 M NaCl, 5 M
glycerol, or 5 M urea; all stock solutions were prepared in iso medium
and sterile filtered). It should be noted that urea stock solutions were kept no longer than 3 days to avoid spontaneous degradation. Viable cells were assayed by manual counting of at least 100 trypsinized adherent cells in a trypan blue solution. Heat shocks were
performed by placing culture flasks in a prewarmed 42°C incubator in
the presence of 5% CO2.
Analysis of genomic DNA by agarose electrophoresis and flow
cytometry.
Genomic DNA was extracted from detached cells by lysis in 20 mM Tris,
10 mM EDTA, and 0.5% Triton X-100, pH 7.5. After repeated pipetting,
cell lysates were centrifuged for 5 min at 15,000 g, and the
supernatants were treated with 0.2 mg/ml proteinase K and 0.2 mg/ml
RNase A for 1 h at 42°C. Samples were analyzed on an agarose
electrophoresis gel in the presence of ethidium bromide and visualized
by ultraviolet fluorescence. For the analysis of cell DNA content,
floating and adherent cells were pooled, washed with phosphate-buffered
saline (PBS; Sigma), fixed in 1× PBS with 70% ethanol at
20°C for
10 min, washed once in PBS, and treated with 100 µg/ml RNase A for 30 min at 37°C. Samples were then incubated for 10 min in the dark with
20 µg/ml propidium iodide and analyzed with a FACScalibur flow
cytometer (Becton Dickinson). Cells were first selected on a forward
scatter/side scatter dot plot, and propidium iodide fluorescence was
analyzed on the FL2 channel. Single cells were selected on a
FL2-A/FL2-W dot plot, and the DNA content was determined as FL2-H.
Western blotting.
Proteins were extracted by lysis for 60 min at 4°C in cell lysis
buffer [1× PBS, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] in the presence of protease inhibitors (1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1 µg/ml aprotinin), followed by centrifugation at 15,000 g for 20 min at 4°C. Protein content was assayed
with the Micro BCA bicinchoninic acid kit (Pierce). Samples (1 µg) were loaded onto a 10% acrylamide SDS-PAGE gel after denaturation in
Laemmli sample buffer (15 min at 65°C). After transfer onto a
polyvinylidene difluoride membrane (Polyscreen; NEN), blots were
saturated for 2 h at room temperature in PBS supplemented with
0.3% (vol/vol) Tween 20 and 5% (wt/vol) nonfat dry milk (PBS-TM). The
anti-HSP70 monoclonal antibody (clone C92F3A-5; Stressgen) was diluted
in the same buffer and incubated on the membrane for 1.5 h. After
five washes, the secondary antibody (peroxidase-coupled goat
anti-mouse; Promega) was added after dilution in PBS-TM and incubated
for 45 min at room temperature. The membrane was washed three times in
PBS-TM, twice in PBS-T, and then once in PBS before bound antibodies
were revealed with the ECL+ enhanced chemiluminescence kit (Amersham)
according to the manufacturer's instructions.
Statistical analysis.
Simple comparisons were performed by unpaired Student's
t-test. Multiple comparisons were performed by ANOVA,
followed by Fisher' protected least significant difference test.
Statistical significance was set at 5% (P < 0.05).
 |
RESULTS |
Urea sensitizes mIMCD3 cells to heat shock-induced
apoptosis.
mIMCD3 cells were first adapted (as described in METHODS)
to media made hypertonic with 250 mosmol/lH2O NaCl [125 mM
NaCl added, final osmolality 540 ± 7 mosmol/kgH2O
(mean ± SD, n = 17); referred to as Na medium],
or 250 mosmol/lH2O urea (250 mM urea added, final
osmolality 558 ± 12 mosmol/kgH2O, n = 14; U medium), or both (final osmolality 804 ± 20 mosmol/kgH2O, n = 63; NaU medium). The
osmolality of iso medium was found to be 317 ± 7 mosmol/kgH2O (n = 27). The proliferation
rates of these cells were similar (doubling time ~15 h), except for
cells adapted in NaU medium, which exhibited a doubling time of ~21 h
(see Fig. 2). Adapted cells were then seeded at a density of 6 × 105 in 25-cm2 flasks, grown for 16-20 h at
37°C, and submitted to a 42°C heat shock for various periods of
time. Results are presented as the percentage of viable adherent cells
relative to the initial number of cells (Fig.
1). In the absence of heat shock, we
observed a similar proliferation rate for cells grown in iso
medium (178 ± 37% of seeded cells at the end of the experiment;
mean ± SE, n = 7) and U medium (137 ± 22%,
n = 6). The number of cells in Na medium is
statistically different from the number of cells in iso medium
(P < 0.04), but not from the number of cells in U
medium (P = 0.58). This is probably due to the
anisosmotic passage conditions (PBS wash and trypsinization). After a
2-h heat shock, however, there was no difference between cells grown in
iso and Na media (P = 0.08), whereas the difference
between cells grown in iso and U media is statistically significant
(P < 0.05). After a 5-h heat shock, cells in U medium
were more sensitive (41 ± 8%, n = 5) than cells
grown in iso medium (101 ± 14%, n = 6;
P = 0.0005) or in Na medium (74 ± 9%,
n = 7; P < 0.04). Only 9 ± 7%
(n = 6) of cells grown in U medium resisted an 8-h heat
shock. Again, this value is statistically different from that of both cells grown in iso medium (81 ± 9%, n = 7;
P < 0.0001) and cells grown in Na medium (78 ± 11%, n = 8; P < 0.0001).

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Fig. 1.
Urea adaptation sensitizes mouse inner medullary
collecting duct (mIMCD3) cells to heat shock. mIMCD3 cells were
maintained in isotonic medium (iso), or adapted to Na medium (Na), U
medium (U), or NaU medium (NaU), as detailed in METHODS.
They were seeded at 6 × 105 cells per
25-cm2 flask and incubated for 16-20 h at 37°C.
Cells were then heat shocked at 42°C for the indicated periods of
time and allowed to recover at 37°C. The duration of heat shock plus
recovery period was kept at 8 h. Cells were then trypsinized and
counted in a trypan blue solution. Values presented are percentages of
viable adherent cells relative to the number of seeded cells. Results
are presented as means ± SE of at least 5 independent
experiments.
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Cells grown in NaU medium clearly suffered from passaging because in
the absence of heat shock, only 88 ± 5% (n = 8)
of seeded cells were viable. This number is different from the number
obtained in iso medium (P < 0.007). After a 5-h heat
shock, the number of remaining viable cells is not statistically
different between Na and NaU media (P = 0.19), but it
also is not different between U and NaU media (P = 0.32). However, cells grown in NaU medium tolerated an 8-h heat shock
better than cells grown in U medium (48 ± 10% vs. 9 ± 7%;
P < .01). This finding shows that Na is able to
inhibit the greater heat sensitivity of cells adapted to hyperosmotic
urea. The inhibition was not complete because the difference between
the number of cells in Na and NaU medium was found to be significant
(P = 0.03). It should be noted that, in this
experimental setting, Na on its own had no effect on the heat
sensitivity of mIMCD3 cells (P values of 0.08, 0.05, and 0.82 after a 2-, 5-, or 8-h heat shock, respectively).
To determine whether these results could be detected after short heat
shocks, we treated cells for 2 h at 42°C and then incubated them
for different periods of time at 37°C. The number of viable cells as a function of time is presented in Fig.
2,
A-D. Again, urea-adapted cells showed a greater
sensitivity to this reduced heat shock (Fig. 2C). At 48 h, only 5 ± 3% (n = 5) of the urea-adapted cells
were still viable, compared with 123 ± 20% for cells grown in
iso medium (n = 6; P < 0.05). This
effect of urea adaptation was blocked by NaCl, because cells grown in
NaU medium (Fig. 2D) behaved essentially like cells grown in
isotonic conditions (Fig. 2A) except for their basal
proliferation rate. The calculated doubling time for cells grown in NaU
medium in the absence of heat shock was 21 h, compared with
14 h for cells grown in iso medium. It should be noted that, in
this experimental setting, there was no obvious difference between
cells adapted to hypertonic NaCl (Fig. 2B) and cells
maintained in iso medium (Fig. 2A). Genomic DNA was
extracted from nonadherent cells at 24 and 48 h and analyzed on an
agarose gel (Fig. 2E). The observation of an apoptotic
DNA laddering pattern indicated that urea-adapted cells were engaged in
an apoptotic pathway after the 2-h heat shock. A quantitative analysis of the heat-shock induced apoptosis could thus be
performed by quantifying via flow cytometry the number of events in the sub-G1 peak of propidium iodide labeled cells after heat
shock (Fig. 2F). The number of heat shock-dependent
apoptotic events could then be calculated by subtracting the number
of apoptotic events in the absence of heat shock. Urea adaptation
raised this value from 9.7% to 50.7%.

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Fig. 2.
Urea-adapted cells engage in an apoptotic pathway
upon heat shock. A-D: cells adapted to isotonic medium
(A) or to Na (B), U (C), or NaU medium
(D) were seeded at 105 cells per
25-cm2 flask and incubated 16-20 hrs at 37°C. They
were then heat shocked (+ HS) for 0 ( ) or 2 h
( ) and incubated at 37°C for the indicated periods of
time. Values presented are percentages of viable adherent cells
relative to the number of seeded cells. Results are presented as
means ± SE of at least 5 independent experiments. E:
at 24 and 48 h after a 2-h heat shock, genomic DNA was extracted
and analyzed on an agarose gel electrophoresis. F: the
number of heat shock-induced apoptotic events was estimated by
analyzing the DNA content of cells grown in iso medium or adapted to
urea 48 h after a 5-h heat shock (iso + HS or U + HS,
respectively). As a control, the same experiment was performed on
heat-shocked cells (iso and U). Identification of the different peaks
(sub-G1, G1, and G2/M) is
indicated. PI, propidium iodide.
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Cells were then adapted for at least 1 wk to various urea
concentrations, and the response of adapted cells to an 8-h heat shock
was analyzed (Fig. 3A). No
effect of urea could be detected at urea concentrations under 50 mM.
Cells revealed a greater sensitivity to heat shock at urea
concentrations of 100 mM (P < 0.003 between heat
shocked and non-heat shocked cells) and higher. It should be noted that
this concentration of urea can be reached in the plasma of uremic
patients.

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Fig. 3.
Dose response and reversibility of the urea effect on the
heat shock response. A: cells were adapted for at least 1 wk
to media made hyperosmotic with the indicated concentrations of urea.
The response to heat shock was analyzed as described in Fig. 1, with a
heat shock length of 8 h. Results are presented as means ± SE of at least 5 independent experiments. B: cells adapted
to U medium were shifted (U/iso) or not shifted back (U) to iso medium
for 24 h, and the heat shock response was analyzed as in
A. Cells grown in iso medium were used as a control. Results
are presented as means ± SE of 3 independent experiments.
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Long-term phenotypic changes of cell lines maintained in vitro can
reflect either adaptation of the whole cell population or selection of
a subpopulation of cells. Because the second hypothesis would require
another interpretation of our data, in particular that of the
protective effect of NaCl, we analyzed the reversibility of urea
adaptation with the idea that selection of a subpopulation cannot be
reversed by shifting the cells back to the initial culture conditions.
Therefore, cells were adapted to U medium for at least 1 wk and then
either shifted or not shifted back to iso medium for 24 h before
being tested for their resistance to an 8-h heat shock (Fig.
3B). Cells grown in iso medium were analyzed in parallel. The heat-sensitive phenotype was essentially reversible. After an 8-h
heat shock, remaining viable cells were 37 ± 4% for urea-adapted cells and 278 ± 15% after reversion (P < 0.0001). It should be noted that the small difference between cells
grown in iso medium and cells reversed from U medium is actually
statistically significant (322 ± 6% vs. 278 ± 15%;
P < 0.02).
Urea adaptation does not block heat shock protein expression.
Because NaCl has been claimed to protect cells from subsequent stresses
by inducing a heat shock response, we reasoned that urea might
sensitize cells by inhibiting this heat shock response. Thus we
analyzed the expression of the HSP70 protein, as a marker of this
response, in cells adapted to different media, before and after heat
shock (Fig. 4). We first confirmed that
HSP70 was overexpressed in cells grown in media containing NaCl (Na
medium and NaU medium) but not in cells adapted to urea (Fig. 4, U,
lane 0). The expression of the HSP70 protein was induced by
heat shock in cells maintained in iso medium, as expected, and was
overinduced in cells grown in Na and NaU medium. More interestingly,
cells adapted to hyperosmotic urea were found to be able to overexpress HSP70 in response to heat shock at levels similar to those observed in
cells maintained in isotonic conditions (cf. Fig. 4, U, lanes 5 and 8 vs. iso, lanes 5 and 8).

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Fig. 4.
Urea adaptation does not block the heat shock-induced
expression of the heat shock protein HSP70. Cells adapted to different
anisotonic media were heat shocked for 0, 5, or 8 h. Proteins were
extracted as described in METHODS and analyzed for HSP70
expression by Western blot analysis. Data are from 1 representative
experiment of 4.
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Effect of acute urea changes.
One possible way to explain our data would be that urea and heat shock
could have qualitative and/or quantitative additive effects on protein
denaturation. Because urea is a freely permeant solute, such additive
effects should be detectable when urea and heat shock are applied at
the same time. We thus treated mIMCD3 cells with NaCl, urea, or
glycerol (as a control of the effect of an hyperosmotic shock with a
permeant solute) and incubated the cells immediately at 42°C for
8 h. As shown in Fig. 5A,
this treatment had no statistically significant effect on cell
survival. Reciprocally, washing urea away (with two 15-min washes in
isotonic medium) should have blocked the hypersensitivity of
urea-adapted cells. However, this was not the case (Fig. 5B,
Urea, closed vs. hatched bars). Again, glycerol-adapted cells were not
hypersensitive to heat shock either before or after the two isotonic
washes. Altogether, these experiments show that the cellular effects of urea adaptation are not immediately mediated by urea.

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Fig. 5.
Acute changes in urea concentration do not modify the
heat shock response. A: cells grown in isotonic condition
were seeded at 6 × 105 cells per 25-cm2
flask and incubated at 37°C for 16-20 h. Medium was then
replaced by isotonic medium alone or isotonic medium supplemented with
125 mM NaCl, 250 mM glycerol (Gly), or 250 mM urea, and cells were
immediately placed at 42°C for 8 h. After cells were counted in
trypan blue solution, values were standardized relative to the number
of seeded cells. Results are presented as means ± SE of at least
5 independent experiments. B: cells were first adapted for
at least 1 wk in isotonic medium or in medium supplemented with 250 mM
glycerol or 250 mM urea. They were then seeded at 6 × 105 cells per 25-cm2 flask and incubated for
16-20 h at 37°C. Cells were then washed twice for 15-min with
the same medium and incubated for 8 h at 37°C (open bars) or
42°C (closed bars) or washed twice in isotonic medium and incubated
for 8 h at 42°C (hatched bars). Cells were then counted in
trypan blue. Values were standardized relative to the number of seeded
cells. Results are presented as means ± SE of at least 5 independent experiments.
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 |
DISCUSSION |
Cells from the kidney medulla are generally exposed for long
periods of time to high osmolarities resulting from the reabsorption of
NaCl and urea. The long-term effects of NaCl on cell physiology have
been studied by different laboratories (2). They consist essentially of the triggering of both a general stress response (as
revealed by the induction of heat shock proteins) and a specific osmotic response leading to the cytoplasmic accumulation of compatible organic osmolytes. Together, these responses facilitate cell survival in this stressful environment. In contrast, little is known of the
long-term effects of hyperosmotic urea on cell physiology.
Our data demonstrate that cells adapted to hyperosmotic urea show a
greater sensitivity to heat shock than cells grown in isotonic
conditions. This sensitivity is linked to the engagement of a larger
proportion of urea-adapted cells into an apoptotic pathway upon
heat shock treatment. Of note is the fact that this effect was found to
be dose dependent and could be detected at urea concentrations of 100 mM. Such a concentration can be reached in uremic patients (G
Deschênes, personal communication), suggesting that nonrenal
cells are submitted to these hyperosmotic urea conditions under
pathophysiological situations. The clinical relevance of our findings
are not clear at this point, but our results suggest that they should
be explored not only in renal systems but also in nonrenal cells. In
any case, the findings stress the differences between acute and chronic
hyperosmolarity treatments. Only cells adapted to hyperosmolar urea
were hypersensitive to heat shock, whereas acute urea treatment did not
lead to similar results (Fig. 5A). We have previously shown
that progressive increases in NaCl or urea concentrations lead to cell
survival or typical apoptosis, respectively (10).
Again, this is in contrast with the results obtained by us and others
on acute osmolarity increases (10, 11, 15). Because in
vivo osmolarity changes are not acute, our data suggest that results
obtained from in vitro models of acute hyperosmolarity changes might
miss some aspects of the cellular responses to osmotic stress.
The functional bases of this long-term effect of urea require further
investigation to be fully understood at the molecular level. However,
three points can be stressed from our data. 1) Although urea
is known to denature proteins at a high concentration, the greater
sensitivity of urea-adapted cells to heat shock is not linked to an
additive effect of heat and urea on protein denaturation. Such an
additive effect should be detectable upon simultaneous treatment with
urea and heat, which is not the case. Furthermore, it should be blocked
by washing out urea, which also is not the case. Thus it is tempting to
suppose that the specific signaling pathways triggered by urea are able
to induce cellular phenotypic changes that might include this greater
heat sensitivity. 2) The long-term effect of urea is
specific in the sense that glycerol, another freely permeant solute,
does not sensitize cells to heat shock. In other words, hyperosmolarity
per se is not sufficient to induce the long-term effects induced by
hyperosmotic urea. This point again suggests that the specific
signaling events triggered by urea could play a major role in the
induction of a heat-sensitive phenotype. 3) The effect of
urea is not due to an inhibition of the heat shock response, because
the HSP70 induction is conserved in heat-sensitized cells. Thus the
mechanism of heat-sensitization by urea is different from the mechanism
of stress protection induced by NaCl, which relies most probably on the
induction of a heat shock response.
Interestingly, the urea-induced sensitization is blunted by NaCl. From
a physiological point of view, urea is both a waste product and a major
functional molecule involved in resistance to water limitation in
several metazoans. For instance, amphibians are able to increase their
urea plasma levels to retain water in their "milieu
intérieur." In mammals, this increase in
osmolarity is limited to the kidney medulla, where urea concentrations
can reach very high levels. Our results are in line with the emerging notion of a transprotection mechanism between NaCl and urea because they demonstrate that NaCl protects cells not only from the acute toxic
effects of urea but also from the long-term consequences of the
accumulation of this solute. Thus, beyond its role as a driving force
for water reabsorption, it appears that NaCl plays a role as a cellular
protectant against the deleterious effects of urea, most probably
because of its capacity to induce both an osmotic and a general stress
response. In particular, because the induction of HSP70 has been shown
to be a critical event in NaCl-mediated protection against urea
toxicity (13) and because this protein is known to induce
thermotolerance in several cellular models (8), it might
be speculated that the same protein is involved in NaCl-mediated
protection against urea sensitization to heat shock. Whether other
NaCl-induced mechanisms also play a role is an open question.
 |
ACKNOWLEDGEMENTS |
Present address of C. Colmont: University of Connecticut Health
Center, 263 Farmington Avenue, Farmington CT 06030.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: G. Rousselet, Bâtiment 532, Service de Biologie Cellulaire,
CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France (E-mail:
rousselet{at}dsvidf.cea.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 March 2000; accepted in final form 6 October 2000.
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