Urea protects from the proapoptotic effect of
NaCl in renal medullary cells
Zheng
Zhang,
Wei
Tian, and
David M.
Cohen
Divisions of Nephrology and Molecular Medicine, Oregon Health
Sciences University and the Portland Veterans Affairs Medical
Center, Portland, Oregon 97201
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ABSTRACT |
Hypertonic NaCl upregulated two sensitive and specific
biochemical indices of apoptosis, caspase-3 activation and annexin V
binding, in a time- and dose-dependent fashion in renal medullary mIMCD3 cells. Pretreatment with urea (200 mM for 30 min) protected from
the proapoptotic effect of hypertonic stress (200 mosmol/kgH2O) in this model. The protective effect of urea
was dose dependent and was effective even when applied a short time
(
1 h) following NaCl exposure; this protective effect was not
observed in the nonrenal 3T3 cell line. In both mIMCD3 and 3T3 cells,
urea failed to protect from the proapoptotic stressor, ultraviolet
(UV)-B irradiation. The ability of urea to protect from hypertonic
stress was approximately comparable to the protective effect of peptide mitogens epidermal growth factor and insulin-like growth factor (IGF), but it potentiated the IGF effect. Interestingly, the
tyrosine kinase inhibitor, genistein, potentiated the proapoptotic
effect of urea yet abrogated the proapoptotic effect of hypertonic
stress. In aggregate, these data indicate that urea protects from the proapoptotic effect of hypertonic stress in a potentially cell type-specific and stimulus-specific fashion.
apoptosis; hypertonicity; caspase; annexin; stress; osmotic
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INTRODUCTION |
ON AN OSMOLAR BASIS,
urea and NaCl are the principal constituent solutes of the renal inner
medullary interstitium. Each activates a unique subset of signaling
intermediates and thereby regulates downstream expression of effector
gene products in a relatively solute-specific fashion (1,
3). The interrelationship of these two solutes has come
under increasing scrutiny in the context of medullary cell survival in
this inhospitable milieu. Hypertonic NaCl appears to limit the
proapoptotic effect of elevated concentrations of urea
(19, 22). In the course of investigating
signaling pathways conferring osmotic resistance, we uncovered the
converse relationship: urea protects from the proapoptotic effect of
hypertonic NaCl in the renal medullary mIMCD3 cell line, but not in
nonrenal 3T3 cells.
Aberrant regulation of apoptosis has been implicated in developmental
anomalies, neoplasia, and response to toxic or ischemic injury. The
osmotic strength of the renal inner medulla is sufficient to cause
apoptosis in diverse cell types in vitro. Resident cells of the inner
medulla tolerate a wide range of ambient osmolarities, in part because
of regulated accumulation of organic osmolytes (reviewed in Ref. 12).
Hyperosmotic urea and NaCl induce distinct programs of osmolyte
accumulation (12). Urea signaling exhibits hallmarks of a
peptide mitogen-like signaling pathway in cells of the renal medulla,
including transcription (9) and expression (5) of immediate-early genes and activation of the
signaling intermediates and receptor tyrosine kinase effectors ERK and
Elk-1 (4); Shc, Grb2, SOS, and Ras (24,
27); phospholipase-C
(8); and PI3K, Akt,
and p70 S6 kinase (27). Because it has recently
been shown that the peptide mitogen insulin-like growth factor (IGF) is
protective of hypertonicity-inducible apoptosis in cells of neural
origin (17), the ability of urea to mediate an analogous
phenomenon in renal medullary cells was investigated.
Earlier investigations relied upon relatively nonspecific endpoints of
cell viability. The caspases, members of a family of aspartate-directed
cysteine proteases, are responsible for much of the execution of the
apoptotic program (reviewed in Ref. 23). The tetrapeptide recognition
sequence differs among members of the caspase family. Caspase-3
specifically recognizes and cleaves the ASP-GLU-VAL-ASP (DEVD)
motif (23); a modified peptide substrate incorporating
this motif serves as the basis for a sensitive fluorometric assay of
caspase-3 protease activity. An early consequence of initiation of the
apoptotic program is disorganization of membrane lipid polarity.
Phosphatidylserine, normally restricted to the inner leaflet of the
cell membrane, translocates to the cell surface (16) where
it can be labeled and detected by a fluorescent conjugate of the
phosphatidylserine binding protein, annexin V (reviewed in Ref. 21).
Herein, we used these sensitive and specific endpoints to examine the
ability of urea to influence the proapoptotic effect of hypertonic and
ultraviolet stress.
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METHODS |
Cell culture and treatment.
Cells in culture (mIMCD3, Ref. 20; and 3T3) were maintained,
propagated, and treated with solute as previously described (5, 24). Briefly, cells were treated with
gentle dropwise addition of 1/20 volume stock of 2.1 M NaCl or 4.2 M
urea. For experiments requiring the use of other solutes (e.g.,
mannitol; see Fig. 4), a complete medium change to premade medium
supplemented with the relevant concentration of solute was made for all
samples. For consistency with mIMCD3 signaling data, cells were
serum-deprived overnight prior to solute exposure. For ultraviolet
(UV)-B exposure, cells in confluent monolayers were irradiated from
below with a UV transilluminator (IBI model TF-20M; peak emission 312 nm; 6 tubes at 15 W) for the indicated interval. Because the surface of
the transilluminator rapidly became warm to the touch (>42°C by
thermistor-based measurement), dishes to be treated were elevated above
the surface by ~0.5 cm and cooled by a constant room-temperature air
jet between the dishes and the transilluminator surface to eliminate the confounding variable of heat shock. In pilot experiments, this configuration permitted maintenance of dishes at 37°C (±0.4°C by thermistor-based measurement) for longer than the 10-min interval required for maximal UV administration (12,000 J/m2) in the
depicted experiments. UV flux was monitored at 300 and 250 nm using a
UVX digital radiometer and UVX-25 and UVX-31 sensors (UVP) in
accordance with the manufacturer's directions as described by Iordanov
et al. (13). The bottom of the tissue-culture dish attenuated the exposure at 300 nm by ~55%; appropriate dose
adjustments were made. Absorbance of the tissue-culture dish across the
UV spectrum was determined spectrophotometrically (13).
Caspase-3 assay.
Caspase-3 (cpp32) microfluorescence assay was performed
according to a modification of the method of Enari et al.
(11). Briefly, cells were washed twice with ice-cold PBS,
scraped into 50 µl of extraction buffer (50 mM PIPES-NaOH, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, 20 µM
cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, 1 µg/ml pepstatin A, 50 µg/ml antipain, and 10 µg/ml
chymopapain), and lysed with five freeze-thaw cycles. Following
centrifugation at 10,000 g for 12 min at 4°C, supernatants were assayed for protein concentration as above. Cell lysate (25 µg)
was incubated in a reaction volume of 50 µl with fluorogenic substrate [N-acetyl-DEVD-MCA; 10 µM (BioMol)], 100 mM
HEPES-KOH, pH 7.5, 10% sucrose, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM
dithiothreitol, and 0.1 mg/ml ovalbumin for 60 min at 30°C in a
96-well microtiter plate (Falcon). Enzyme activity was detected by
Cytofluor II (PerSeptive Biosystems, Framingham, MA) at an excitation
wavelength of 360 nm and an emission wavelength of 460 nm. Among the
~40+ independent caspase-3 assays performed, the basal level of
caspase activity varied between 7 and 45 U/20 µg lysate. This
variability appeared to correlate with the elapsed interval since prior
medium change as well as unknown variables (data not shown). Although
not uniformly observed, there was a tendency for activation in response
to solute stress (expressed as fold induction) to be greater in
experiments exhibiting low basal caspase-3 activity. In this and other
assays, statistical significance was assigned to P < 0.05 with respect to the indicated control (e.g., vehicle treatment) of
data from three or more independent experiments compared via
t-test (Excel, Microsoft). The qualitative effects reported
herein (and their ordinal relationships) were observed in virtually all
experiments; magnitude of error bars is largely reflective of varying
degrees of induction of caspase-3 and annexin V binding in response to
applied stressors.
Annexin V binding assay.
Annexin binding was determined using the ApoAlert annexin V apoptosis
kit (Clontech) in accordance with the manufacturer's directions for
adherent cells (full details available at
http://www.clontech.com/techinfo/manuals/PDF/PT3050-1.pdf). Cells
were stained with propidium iodide (PI) or 7-amino actinomycin D
(7AAD) and annexin V-FITC as directed and sorted (FL1 for
annexin V-FITC, FL2 for PI, or FL3 for 7AAD; see below) on a
Becton-Dickinson Calibur instrument, following gating upon a
representative population of cells established by forward- and
side-angle light scatter. Apoptosis (early) was ascribed to cells
exhibiting annexin staining >40 fluorescence units (FL1) and PI
staining <300 fluorescence units (FL2). For PI staining, cells labeled
with only PI were used to compensate the FL2 signal out of FITC (FL1),
and cells labeled with only FITC-annexin V were used to compensate the
FITC signal out of FL2. Because the PI concentration was kept low, there was no excess signal overlapping into FL1. For the 7AAD protocol,
FL3 was used as the DNA channel, and there was no need to compensate.
Similar to the caspase-3 assay described above, there was a tendency
for activation in response to solute stress to be greater in
experiments exhibiting low basal annexin V binding activity. Subsequent
to these studies, a preliminary observation was made that
trypsinization, which is recommended by the manufacturer of the
ApoAlert kit and was performed in these studies, may influence phosphatidylserine localization in the cell membrane
(25). ApoAlert product literature indicated that the
annexin binding assay may appear to overestimate apoptosis relative to
other indices (Clontech). Whether this is a consequence of
trypsinization or other manipulations is unexplored. We have been
unable to reliably demonstrate annexin V-FITC binding to confluent
(untrypsinized) monolayers by fluorescence microscopy, even when
markedly proapoptotic stimuli (e.g., NaCl 400 mosmol/kgH2O)
were applied for 1-24 h (data not shown). We have, however,
qualitatively observed an increase in the abundance of PI-stained
nuclei and of condensed PI-stained nuclei in confluent monolayers
subjected to NaCl 200 mosmol/kgH2O relative to control treatment that was consistent with PI (or 7AAD) positivity in the
trypsinized and sorted cells (see Fig. 2; two top quadrants).
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RESULTS |
NaCl induces apoptosis in mIMCD3 cells.
To discriminate potentially subtle differences in proapoptotic events,
a highly quantitative approach was adopted with respect to apoptosis
detection. The renal medullary mIMCD3 cell line exhibits a
dose-dependent increase in activity of the pivotal apoptotic protease,
caspase-3, (as determined by cleavage of a fluorogenic substrate) in
response to hypertonic NaCl at 100 mosmol/kgH2O (50 mM) and
above (Fig. 1), consistent with the
proapoptotic effect observed by others in this model (22).

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Fig. 1.
NaCl increases caspase-3 activity in a dose-dependent
fashion. mIMCD3 monolayers were treated with the indicated
concentrations of NaCl (expressed in mosmol/kgH2O) for
4 h. Caspase-3 (cpp32) activity (calculated as U/20 µg protein
and calibrated with recombinant standard) is expressed relative to
control. P < 0.05 with respect to control (absence
of supplemental NaCl).
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To corroborate data obtained through the use of this assay, a second
index of apoptosis was examined in parallel in critical experiments.
Annexin V binding to disordered membrane phosphatidylserine orientation
(a hallmark of apoptosis) was examined via fluorescence-activated cell
sorting (FACS, Becton-Dickinson) analysis of cells labeled with fluorescein-conjugated annexin V. Under control conditions, comparatively few cells (4%) sorted to the "apoptotic quadrant" (Fig. 2A, bottom
right; high annexin V/low 7AAD labeling). When cells were
subjected to severe hypertonic stress as a positive control (NaCl 400 mosmol/kgH2O for 4 h), there was a marked increase in
the percentage of cells sorted to the bottom right quadrant (39%; Fig. 2B). These data were consistent with
NaCl-inducible apoptosis. Lesser degrees of hypertonic stress
exerted a lesser effect upon annexin V binding (see below).

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Fig. 2.
Severe NaCl-inducible hypertonic stress markedly
increases annexin V binding. After receiving control treatment
(A) or hypertonic NaCl [400 mosmol/kgH2O (N400)
for 4 h; B], mIMCD3 monolayers were trypsinized,
incubated with 7AAD and FITC-labeled annexin V, and subjected to
fluorescence-activated cell sorting (FACS) analysis. 7AAD
(y-axis in A and B) stains nuclei of
dead (permeabilized) cells, and FITC-annexin V (x-axis in
A and B) binds phosphatidylserine translocated to
the outer leaflet of the cell membrane. Apoptotic cells (early phase)
appear in the bottom right quadrant (4% of total for
control vs. 39% of total for N400 for 4 h), corresponding to
relatively high FITC-annexin V binding and relatively low 7AAD labeling
(see METHODS). Representative data are depicted in
C, where the y-axis indicates percent of cells
staining with FITC-annexin V.
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NaCl (100 mM/200 mosmol/kgH2O) activated
caspase- 3 in mIMCD3 cells in a time-dependent fashion (Fig.
3), peaking at 4 h of treatment. Greater degrees of hypertonic stress resulted in a similar
time course of caspase-3 activation (data not shown). Hypertonic NaCl
(100 mM) also increased annexin V binding (Fig. 3); the kinetics of
this upregulation closely paralleled that of NaCl-inducible caspase-3
activation.

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Fig. 3.
Hypertonic NaCl increases caspase-3 activity and annexin
V binding in a time-dependent fashion. mIMCD3 cells were treated with
NaCl (200 mosmol/kgH2O) for the indicated interval (in
hours), prior to determination of caspase-3 activity (expressed as
units of cpp32 activity/20 µg protein) or annexin V binding
[expressed as percentage of cells exhibiting both propidium iodide
(PI) impermeance and FITC fluorescence by FACS analysis].
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Unlike impermeant solutes, urea fails to exert a proapoptotic
effect in mIMCD3 cells.
The effect of each of a panel of membrane-permeant and -impermeant
solutes, applied in an equiosmolar fashion, was examined with respect
to caspase-3 activity. The effect of the functionally impermeant solute
mannitol was indistinguishable from that of NaCl (Fig.
4). Urea (200 mM) and the
nonphysiological permeant solute glycerol (200 mM) failed to increase
caspase-3 activity. The effect of glucose (200 mM) was similar to that
of mannitol (n = 2 experiments; data not shown).

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Fig. 4.
Urea fails to activate caspase-3. mIMCD3 cells were
treated with the indicated permeant (urea, glycerol) or functionally
impermeant (NaCl, mannitol) solutes (200 mosmol/kgH2O for
4 h) prior to determination of caspase-3 activity (expressed
relative to control treatment). Depicted data are means ± SE for
4 separate experiments. P < 0.05 with respect to
control.
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Urea pretreatment protects from NaCl-associated apoptosis in mIMCD3
cells.
Others have suggested that NaCl may protect from the adverse effects of
elevated urea concentration. In light of the ability of peptide
mitogens to confer protection from hypertonic stress in cells of neural
origin (17), the ability of urea to protect from
hypertonicity was investigated in this cell line. Treatment of mIMCD3
monolayers with NaCl (200 mosmol/kgH2O for 4 h)
resulted in a 2.5-fold increase in caspase-3 activity (Fig.
5A). Pretreatment of control
cells treated with urea (200 mM for 30 min prior to sham treatment;
4.5 h total urea exposure) decreased caspase-3 activity by 23%, a
degree that, although reproducible, did not achieve statistical
significance. Pretreatment of mIMCD3 monolayers with urea (200 mM for
30 min) prior to NaCl treatment, however, inhibited caspase-3
activation by 37% (Fig. 5A). This represented a 61%
inhibition of the increment in caspase-3 activity inducible by NaCl
treatment. Urea also protected from the proapoptotic effect of nonionic
solutes such as mannitol (data not shown). The protective effect of
urea was evident out to 48 h of treatment (the last time point
examined), suggesting that urea was not operating through a delay of
apoptosis (data not shown).

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Fig. 5.
Urea protects from NaCl-inducible apoptosis in mIMCD3
cells. mIMCD3 cells received either no pretreatment ( Urea) or
urea pretreatment (200 mM urea for 30 min and thereafter; +Urea) prior
to exposure to hypertonic stress (200 mosmol/kgH2O for
4 h). Cells were harvested for determination of caspase-3 activity
(units of cpp32 activity/20 µg protein expressed relative to control
activity; A) or annexin V binding (percentage of cells
exhibiting PI impermeance and FITC fluorescence by FACS analysis,
expressed relative to control; B). Depicted data are
means ± SE for 4-12 separate experiments.
P < 0.05 with respect to Urea.
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In similar fashion, NaCl treatment (100 mM) increased annexin V binding
by 2.1-fold relative to control (Fig. 5B). Urea treatment alone decreased annexin V binding by 18%; however, this did not achieve statistical significance. Urea pretreatment blocked
NaCl-inducible annexin V binding by 34% (Fig. 5B). This
represented a 63% inhibition of the increment in annexin V
binding inducible by NaCl treatment. Therefore, urea pretreatment was
protective of NaCl-inducible apoptosis with respect to two unrelated
biochemical indices of apoptosis. Importantly, pretreatment with an
impermeant solute (such as mannitol) in place of urea dramatically
potentiated the proapoptotic effect of NaCl (data not shown).
The dose dependence of the protective effect of urea with respect to
NaCl-inducible apoptosis is shown in Fig.
6. Statistical significance (protective
effect) could not be demonstrated with urea concentrations less than
100 mM. Urea concentrations of 400 mM or greater exerted a modest
proapoptotic effect (data not shown). Urea pretreatment (200 mM) also
protected from NaCl 400 mosmol/kgH2O; higher doses of urea
(e.g., 400 mM) were ineffective in protecting from this greater degree
of hypertonic stress (data not shown). The ability of urea (200 mM) to
protect from NaCl (100 mM/200 mosmol/kgH2O) was not steeply
time dependent (Fig. 7), suggesting the
presence of a fairly broad window of protective effect. Urea was
effective whether it was added 30 min before or up to 60 min after the
imposition of hypertonic (NaCl) stress; however, it was not
consistently effective when applied at later time points. These data
were inconsistent with a "conditioning" effect of urea, as may
occur when a mild heat shock protein-inducing stress precedes a more
severe one and thereby limits the severity of the subsequent insult.
Pretreatment for 30 min with an osmolality control (e.g., NaCl or
mannitol; 200 mosmol/kgH2O) in place of urea failed to prevent caspase-3 activation by hypertonic stress, further arguing against a conditioning effect of urea. Only at 5 min of pretreatment did urea protection from caspase-3 activation fail to achieve statistical significance.

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Fig. 6.
Urea protection from NaCl-inducible apoptosis is dose
dependent. Effect of pretreatment of mIMCD3 cells with the indicated
concentration of urea for 30 min, prior to treatment with NaCl (200 mosmol/kgH2O for 4 h) upon caspase-3 activity,
expressed as percent protection from hypertonicity. Depicted data are
means ± SE of 4-6 separate experiments. P < 0.05 with respect to control (0 urea).
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Fig. 7.
Urea protection from NaCl-inducible apoptosis is
operative over a broad temporal window. First three bars depict effect
upon relative caspase-3 activity of control treatment (C) and treatment
with 200 mosmol/kgH2O urea (U) or NaCl (N). Remaining bars
( 30 to +60 min) indicate the effect of pretreatment with urea (200 mosmol/kgH2O) for an interval commencing at the indicated
time with respect to the application of hypertonic NaCl
(mosmol/kgH2O); 30, 30-min pretreatment with urea; 0, simultaneous addition of urea and NaCl; +60, urea treatment starting 60 min after application of NaCl stress. Data are expressed relative to
treatment with NaCl 200 mosmol/kgH2O; depicted data are
means ± SE of 4-6 separate experiments. P < 0.05 with respect to NaCl treatment alone (N).
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Of note, the protective effect of urea was not a universal phenomenon.
In the fibroblastic 3T3 cell line, hypertonic NaCl (200 mosmol/kgH2O) markedly increased caspase-3 activity (Fig. 8). Urea (100 mM) modestly increased
caspase-3 activity, whereas 200 mM urea markedly increased caspase-3
activity. In contrast to the mIMCD3 data, pretreatment with either 100 or 200 mM urea failed to protect from the proapoptotic effect of NaCl
in this fibroblastic model.

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Fig. 8.
Urea fails to prevent NaCl-inducible caspase-3 activation
in 3T3 cells. 3T3 cells were treated with 100 or 200 mM urea (open
bars; U100 and U200, respectively) or 200 mosmol/kgH2O NaCl
(light gray bar; N200). Pretreatment with urea 100 or 200 mM (U100/N or
U200/N) prior to treatment with 200 mosmol/kgH2O NaCl is
indicated by black bars.
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Others have shown that peptide growth factors may protect from the
proapoptotic influence of other stressors (e.g., hypertonicity or UV
stress) through an unknown mechanism (14,
17). Because of the similarity between urea signaling and
mitogen signaling, the relationship among these phenomena was explored
in preliminary fashion. Two peptide mitogens, IGF and epidermal growth
factor (EGF), protected mIMCD3 cells from hypertonic NaCl (200 mosmol/kgH2O) and to a degree approximately equivalent to
urea (Fig. 9). When data were subjected
to paired analyses, the effect of IGF exceeded that of urea in all
experiments (P < 0.05), and the effect of urea
exceeded that of EGF in all experiments (P < 0.05).
Concentrations of urea and IGF affording maximal protection were used
in these studies; interestingly, urea pretreatment substantially
enhanced the protective effect of IGF (Fig. 9). It is difficult to
establish whether the combined effects are less than additive,
additive, or synergistic, because "percent protection" is maximal
at 100. The ordinal relationship among these protectors (IGF > urea > EGF) was preserved in the setting of more extreme degrees
of hypertonic stress (400 mosmol/kgH2O NaCl). Under these
conditions, IGF protected by 39%, urea protected by 13%, and EGF
protected by a statistically insignificant 4% (data not shown).

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Fig. 9.
Peptide mitogens protect from NaCl-inducible caspase-3
activation. mIMCD3 cells were pretreated for 30 min with urea (200 mM),
IGF (30 nM), EGF (100 nM), or a combination of urea + IGF
(+IGF/Urea). Data are expressed as percent protection from NaCl, where
100% indicates complete protection (i.e., restoration of caspase-3
activity to control value) and 0% indicates the absence of protection,
relative to NaCl treatment (200 mosmol/kgH2O for 4 h)
alone. P < 0.05 with respect to urea pretreatment.
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UV-B stress is associated with apoptosis in diverse cell culture
models. To determine the universality of the
protective/antiapoptotic effect of urea in the mIMCD3 cell line with
respect to adverse stimuli, the effect of urea upon UV-B-inducible
apoptosis was examined. A UV transilluminator (peak emission ~312 nm)
was calibrated and found to deliver ~5 mW/cm2 UV-B at 300 nm (see METHODS). When monitored through a plastic tissue-culture dish, this value was attenuated to ~2.3
mW/cm2. On this basis, it was calculated that a 5-min
exposure would provide ~6,000 J/m2 UV-B. Possibility of
UV-C contamination was excluded by measuring absorbance of the tissue
culture plastic across the UV spectrum (Fig.
10A). UV-C transmission was
blocked quantitatively by the tissue-culture plastic (Fig.
10A); therefore, virtually all of the cell exposure
represented UV-B. As anticipated, increasing doses of UV-B produced
increasing degrees of caspase-3 activation in the mIMCD3 cell model
(Fig. 10B). Urea pretreatment, however, failed to protect
mIMCD3 cells from the adverse effect of UV-B irradiation. Similar
results were obtained in the nonrenal 3T3 cell line (data not shown).

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Fig. 10.
Urea
pretreatment fails to prevent UV-B-inducible caspase-3 activation.
A: absorbance of the bottom of the tissue culture dish
(Falcon 3003) across the UV spectrum, determined
spectrophotometrically. Arrowhead denotes wavelength of maximal UV
emission by transilluminator (see METHODS). B:
effect of no pretreatment or urea pretreatment (200 mM for 30 min) upon
UV-B-inducible caspase-3 activity expressed relative to control. UV-B
dose, expressed as J/m2 at 300 nm, is indicated in
brackets.
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With respect to the role of specific urea-inducible signaling events in
the protective effect of urea, studies were performed with the tyrosine
kinase inhibitor, genistein, which inhibits some urea-inducible
signaling events (e.g., Ref. 8). Interestingly, genistein pretreatment
(100 µM for 30 min) converted the modest ability of urea to diminish
apoptosis under control conditions to a markedly proapoptotic effect
(129 ± 45% increase in caspase-3 activity; n = 3; data not shown). The opposite phenomenon was observed with respect
to NaCl-inducible apoptosis; specifically, genistein pretreatment
substantially inhibited the proapoptotic effect of NaCl (28 ± 13%; n = 4; data not shown). Genistein appeared to
have no effect upon the ability of urea to protect from NaCl-inducible apoptosis; however, this may have represented a combined effect of
potentiation of urea-inducible apoptosis and inhibition of NaCl-inducible apoptosis. Therefore, urea and NaCl signaling exhibit markedly dissimilar responses to genistein pretreatment.
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DISCUSSION |
Using two independent biochemical indices of apoptosis, we show
that pretreatment with urea protects renal medullary cells from the
proapoptotic effect of hypertonic stress. The caspase-3 and annexin V
assays were used rather than less quantitative approaches to maximize
the likelihood of detecting a potentially subtle protective effect of
urea. This protective effect is not complete; however, it does block
greater than 60% of the increment in apoptosis inducible by modest
degrees of hypertonic stress (e.g., 200-400
mosmol/kgH2O) and is a function of the applied urea
concentration. Whether the ability of 200 mM urea to protect most
effectively from 200 mosmol/kgH2O NaCl is a consequence of
the equiosmolar solute ratio rather than the absolute concentration of
urea remains unclear. A ratio-dependent protective effect has been
described in the context of the counteracting osmolytes and urea stress
(12). Detailed investigation of this possibility was
precluded by the relative inability of very low doses of NaCl to exert
a demonstrable apoptotic effect (Fig. 1) and by the proapoptotic effect
associated with concentrations of urea markedly in excess of 200 mM.
Urea, NaCl, and cell death.
Others have described a relationship among urea and NaCl concentration
and cell viability that is converse to the present one
(19, 22). Our studies, in contrast, utilized
as outcome measures biochemical indices unique to apoptosis. Santos et
al. (22) recently observed in the mIMCD3 model that high
concentrations of either urea or NaCl decreased cell viability and that
combining the two solutes enhanced cell survival of an osmotic shock.
Neuhofer et al. (19) showed that pretreatment of renal
epithelial MDCK cells with hypertonic stress (a phenomenon known to
induce heat shock protein expression; Ref. 10) was associated with
subsequent enhanced survival of urea stress. This group later
implicated NaCl-inducible hsp72 expression in this
preconditioning effect (18). In our studies, medullary
cells were much less sensitive to urea than they were to NaCl (on a
mosmol/kgH2O basis) and subapoptotic doses of urea
protected from proapoptotic doses of NaCl. This protective effect of
the "activating" solute urea paralleled in many respects the
ability of peptide growth factors to protect medullary cells from
hypertonic stress (Fig. 9).
Growth factor protection from hypertonic stress.
Matthews et al. (17) showed that IGF-I confers protection
from hypertonic stress in a human neuroblastoma cell line. In Rat-1
fibroblasts, IGF-I protects from the proapoptotic effect of UV
irradiation, and this phenomenon is Akt/PI3K dependent
(14). We have previously implicated a PI3K-dependent
signaling pathway in acquisition of resistance to both urea and
hypertonic stress (27). Interestingly, in the Rat-1 model,
overexpression of the relevant cell surface IGF receptor resulted in
PI3K independence, implicating involvement of a second signaling
pathway (14). We were unable to examine the role of these
pathways in the present model, because pharmacological inhibition of
PI3K-dependent signaling sharply sensitized medullary cells to osmotic
stressors (27). Specifically, whereas PI3K inhibition
exerted only a modest proapoptotic effect upon control-treated mIMCD3
cells in culture, it markedly potentiated the proapoptotic effect of
high-dose urea (>400 mM) and both moderate (200 mosmol/kgH2O) and severe (400 mosmol/kgH2O) hypertonic stress (27). It was previously concluded on
this basis that a PI3K-dependent pathway was potentially instrumental in protecting medullary cells from the adverse effects of hypertonic and urea stress. We sought to establish parallels between peptide mitogen-associated protection and that of urea in the renal medullary model. The tyrosine kinase inhibitor genistein inhibits elements of
urea-mediated signaling in renal medullary cells (8);
therefore, the effects of this compound were examined with respect to
the protective effect of urea. Although the general tyrosine kinase inhibitor genistein failed to influence the protective effect of urea
upon hypertonic stress, these data were confounded by the striking
ability of genistein to prevent hypertonicity-inducible caspase-3
activation and by the ability of this inhibitor to potentiate the
adverse effect of urea exposure upon this index of apoptosis. These
data serve to further underscore the dissimilar physiological responses
engendered by equiosmolar concentrations of urea and NaCl.
Mechanism of urea protection.
We originally hypothesized that the effect of urea upon NaCl-inducible
apoptosis would represent a signaling-dependent and not a
physicochemical effect. Data presented here both support and refute
this notion. The parallel abilities of urea, which exhibits selected
biochemical hallmarks of a renal epithelial cell growth factor, and of
the peptide mitogens, IGF and EGF, to protect from
hypertonicity-inducible apoptosis support a signaling-dependent mechanism of protection. Efforts to ascertain the contribution of
specific urea signaling effector pathways to the protective effect were
confounded by the proapoptotic effect of the inhibitors themselves as
described above (Ref. 27 and data not shown). Inhibition of the
urea-activatable (and peptide mitogen-activatable) Ras effector arm
through inducible high-level expression of a dominant negative-acting
N17Ras mutant failed to influence mIMCD3 cell resistance to either urea
or hypertonic stress (24). It could be argued, in
contrast, that the ability of urea to add with a
receptor-saturating dose of peptide mitogen does not support a common
mechanism. By extension, however, the additivity of IGF- and
EGF-inducible protection (n = 2; data not shown) from
hypertonicity-inducible apoptosis would similarly implicate two
distinct mechanisms, a less likely possibility. It would therefore
appear plausible that a submaximal protective response is activated by
either urea or peptide mitogen and that additivity need not equate to
independence of the responsible signaling events.
Limitations of the antiapoptotic effect of urea.
The ability of urea to protect from apoptosis is subject to at least
three constraints. First, based upon the limited sampling presented
here, it may exhibit cell-type specificity. Such specificity could
represent a consequence of the unique ability of renal epithelial cells
to respond to exogenous urea through the activation of receptor tyrosine kinase effector pathways. Activation of such effector pathways, in a fashion similar to the cell response to a peptide mitogen, could permit urea treatment to mimic the known protective effect of these mitogens in other contexts. We have been unable to
confirm a promitotic effect of urea in the mIMCD3 cell line, although
enhanced DNA synthesis and total DNA content have been observed in
other renal epithelial cell lines in the absence of an increase in cell
number (6, 7, 15). With respect
to cell-type specificity, it is of interest that concentrations of NaCl
and urea in the renal medulla are generally (but not universally) regulated in parallel. It is conceivable that urea protects from the
proapoptotic influence of hypertonic NaCl in this milieu in vivo. The
inability of urea to protect from NaCl stress in the nonrenal 3T3 cell
culture model supports this teleological explanation. Second, the
protective effect of urea is steeply dose dependent; urea
concentrations in excess of 50 mM are required for demonstrable protection from hypertonicity. Interestingly, this protection is not
confined to pretreatment; urea inhibited hypertonicity-inducible caspase-3 activation even when applied up to 60 min after initial exposure to hypertonic stress. Based upon this latter observation, it
appears likely that a substantial interval must be traversed before
biochemical commitment to irreversible caspase-3 activation (and
ultimately apoptosis) occurs. Third, the protective effect of urea
applies to only a subset of proapoptotic stimuli. Specifically, urea
protected from hypertonicity (both NaCl and mannitol induced) yet
failed to protect from the proapoptotic effect of UV-B irradiation. As
both urea (26) and UV irradiation (2) are
potential oxidative stressors, this failure of protection may represent
a consequence of additive pro-oxidant stress.
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-52494 and by the National Kidney
Foundation and American Heart Association.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. M. Cohen, Mail Code PP262, Oregon Health Sciences Univ., 3314 S.W. US
Veterans Hospital Rd., Portland, OR 97201 (E-mail:
cohend{at}ohsu.edu).
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. §1734 solely to indicate this fact.
Received 4 October 1999; accepted in final form 5 April 2000.
 |
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