Am J Physiol Renal Physiol 288: F125–F132, 2005.
First published September 7, 2004; doi:10.1152/ajprenal.00041.2003.
Stress response inhibits the nephrotoxicity of cisplatin
Marie H. Hanigan,1 Mei Deng,2 Lei Zhang,1 Peyton T. Taylor, Jr.,2 and Maia G. Lapus1
1
Department of Cell Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma; and 2Department of Obstetrics and Gynecology, Division
of Gynecologic Oncology, University of Virginia Health Sciences Center, Charlottesville, Virginia
Submitted 31 January 2003; accepted in final form 1 September 2004
kidney; cis-diamminedichloroplatinum; toxicity; proximal tubule;
LLC-PK1 cells
Despite the widespread use of salt, the mechanism by which
salt protects the kidney has never been identified. Several
theories have been proposed to explain the protective effect of
hydration against cisplatin nephrotoxicity. Some investigators
have suggested that hypertonic saline and saline hydration
protect the kidney by increasing the rate of cisplatin excretion
(9). Others have proposed that the salt provides a high concentration of chloride ions, which prevents the dissociation of
the chloride ions from the platinum molecule, thereby reducing
the formation of the reactive, aquated species of cisplatin (5,
10). There has been debate as to whether the sodium or the
chloride ion is responsible for the protective effect. Litterst’s
(21) mouse data indicated that the sodium ion is the important molecule, whereas Earhart and co-workers (10) reported data from rat studies suggesting that the chloride is
the protective ion.
As part of ongoing studies on the mechanism of cisplatin
nephrotoxicity, we developed an in vitro model system of
cisplatin-induced toxicity toward proximal tubule cells (33).
Confluent monolayers of cells are treated for 3 h with cisplatin.
The drug is removed and cell viability is assessed at 3 days.
This treatment mimics the in vivo exposure. In this system,
cisplatin toxicity is dose and time dependent. During the course
of our studies, we found that treating the cells with cisplatin in
RPMI-1640 medium was more toxic than treating the cells
with cisplatin in Dulbecco’s Modified Eagle Medium
(DMEM). Both RPMI-1640 medium and DMEM are commonly used to maintain mammalian cells in culture. Their
formulations are very similar. In this study, we analyzed the
effect of four commonly used tissue culture media on the
toxicity of cisplatin. We tested individual components and
found that the NaCl concentration in the media modulated
cisplatin toxicity. The robust effect of NaCl on cisplatin toxicity in this in vitro system is similar to that observed in vivo.
This model system enabled us to test directly several of the
hypotheses regarding the mechanism by which NaCl protects
the kidney from cisplatin toxicity.
antitumor activity of cisplatin
provided an effective drug for the treatment of germ cell
tumors and ovarian cancer (11). It is also used as a radiosensitizer for cervical cancer and as a component of consolidation
therapy for many types of solid tumors. The efficacy of
cisplatin is limited, however, by the cumulative nature of its
dose-limiting nephrotoxicity. Cisplatin is toxic to the renal
proximal tubules (8). The drug is excreted in the urine in a
diphasic manner; most of the drug is excreted within the first
3 h (6). Renal toxicity, as determined by elevated levels of
blood urea nitrogen, is not evident until 72 to 96 h after the
initial administration (8). Studies in rodents revealed that
administering cisplatin in hypertonic salt reduced its nephrotoxicity (21). Hydration with saline was shown to be protective
in dogs (24). These data have been applied in the clinic. The
drug is reconstituted in 0.9% sodium chloride. Vigorous hydration with saline and simultaneous administration of mannitol before, during, and after cisplatin administration significantly reduced the cisplatin-induced nephrotoxicity and is now
the accepted standard of care (4).
Cell culture. LLC-PK1 [American Type Culture Collection
(ATCC) CRL 1392], a pig kidney cell line, was purchased from
ATCC (Manassas, VA). LLC-PK1 cells were grown in DMEM
supplemented with 4 mM L-glutamine (DMEM, GIBCO/BRL, Grand
Island, NY), 5% FBS (Hyclone Laboratories, Logan, UT), penicillin
(50 U/ml), and streptomycin (50 ␮g/ml, GIBCO/BRL). Cells were
maintained in 5% CO2-95% air at 37°C. Subconfluent cultures were
passaged every 3 to 4 days. For toxicity experiments, LLC-PK1 cells
Address for reprint requests and other correspondence: M. H. Hanigan,
Biomedical Research Center Rm. 264, 975 N.E. 10th St., Oklahoma City, OK
73104.
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.
THE DISCOVERY OF THE POTENT
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MATERIALS AND METHODS
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Hanigan, Marie H., Mei Deng, Lei Zhang, Peyton T. Taylor Jr.,
and Maia G. Lapus. Stress response inhibits the nephrotoxicity of
cisplatin. Am J Physiol Renal Physiol 288: F125–F132, 2005. First
published September 7, 2004; doi:10.1152/ajprenal.00041.2003.—
Salt loading and saline hydration are used to protect patients from
cisplatin-induced nephrotoxicity. The mechanism by which salt exerts
its protective effect is unknown. As part of an ongoing study of
cisplatin nephrotoxicity, an in vitro assay system was developed that
models the in vivo exposure and response of proximal tubule cells to
cisplatin. In this study, it was discovered that the toxicity of cisplatin
toward LLC-PK1 cells varied dramatically according to the tissue
culture media used for 3-h cisplatin exposure. Further experiments
revealed that minor variations in the sodium concentration among
standard tissue culture media modulated cisplatin nephrotoxicity.
NaCl has been shown to protect against cisplatin-induced nephrotoxicity in vivo but has never before been demonstrated in vitro. NaCl did
not alter the cellular accumulation of cisplatin. NaCl altered the
osmolarity of the external media, and its effect was replicated by
substituting equiosmolar concentrations of impermeant anions or
cations. The change in osmolarity triggered a stress response within
the cell that modulated sensitivity to cisplatin. These data resolve
several long-standing controversies regarding the mechanism by
which salt loading protects the kidney from cisplatin-induced nephrotoxicity.
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treatment, the medium was changed to fresh DMEM containing 5%
FBS, antibiotics, and various concentrations of NaCl. After 3 h, the
media were removed and the cells treated with 100 ␮M cisplatin in
DMEM. At the end of the 3-h cisplatin exposure, the cisplatin was
removed and cells returned to DMEM containing 5% FBS and
antibiotics for 69 h. The number of viable cells was determined with
the MTT assay.
DNA platination. LLC-PK1 cells were seeded in P-60 plates at
5.7 ⫻ 105 cells/plate. On day 5, the confluent cells were exposed to
100 ␮M cisplatin in 125 to150 mM NaCl with 5 mM HEPES, pH 7.4,
at 37°C without CO2 for 3 h. The cells were washed with PBS and
trypsinized. DNA was purified by the method of McKeage and
co-workers (22). Briefly, cells were lysed (10 mM Tris, 10 mM
EDTA, 0.15 M NaCl, and 0.5% SDS, pH 8.0) in the presence of 1
mg/ml proteinase K. The lysis mixture was incubated at 60°C for 10
min and then 37°C overnight. DNA was purified by phenol extraction
and ethanol precipitation. The DNA pellet was resuspended in 10 mM
Tris, 1 mM EDTA, pH 8.0, with addition of 100 ␮g/ml RNase and
incubation at 37°C for 1 h. DNA was then reprecipitated and hydrolyzed in 0.2% HNO3. DNA concentration was estimated by measuring
the absorbance at 260 nm, and the platinum content was measured by
FAAS as described above.
Data analysis. Each experiment was done a minimum of three
times. Each data point within the experiment represents the mean
value of three to six replicate wells. To detect statistically significant
effects of medium or inorganic salt solutions on cisplatin nephrotoxicity, the toxicity of each concentration of cisplatin in each of the
solutions was compared with the toxicity of cisplatin in L-15 by a
Student’s t-test. Significant correlations between the salt concentration
in the solution and cisplatin toxicity, platinum accumulation or platinum-DNA binding were detected by one-way ANOVA and posttest
for a linear trend. Statistically significant difference between curves
were detected by an F-test. All statistical analyses were done using
GraphPad Prism version 3 (GraphPad Software, San Diego, CA).
RESULTS
Effect of medium on cisplatin toxicity. Tissue culture media
modulated the toxicity of cisplatin toward proximal tubule
cells. LLC-PK1 cells were grown to confluence and maintained
in DMEM with 5% FBS and antibiotics. The cells were treated
with cisplatin for 3 h and then returned to complete DMEM.
Toxicity was assessed at 72 h. The media on the cells in the
four experimental groups varied only during the 3-h cisplatin
exposure. Prior to and following cisplatin treatment, all cells
were in complete DMEM. The data in Fig. 1A show that
cisplatin in RPMI or F-12 media was significantly more toxic
to LLC-PK1 cells than equimolar cisplatin in DMEM or L-15
media. A dose-dependent toxicity of cisplatin was observed
with RPMI and F-12 media containing cisplatin. At 100 and
150 ␮M, cisplatin was significantly less toxic in DMEM or
L-15 media (P ⬍ 0.001).
To determine which components of the media modulated
cisplatin toxicity, components of the media were tested with
the same protocol used for the media. The cells were exposed
to cisplatin in the test solutions for 3 h, and toxicity was
measured at 72 h. Data from experiments in which the inorganic salt components of the media were tested showed that the
salt solutions modulated cisplatin toxicity (Fig. 1B). The composition of the solutions tested is shown in Table 1. There was
a direct correlation between the concentration of NaCl in the
solution and the reduction in cisplatin toxicity: saline⬎L15⬎HBSS⬎F-12⬎DMEM⬎RPMI. The higher the concentration of NaCl, the less toxic cisplatin was to the cells. The
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were seeded in 96-well plates at 5 ⫻ 103 cells/well. The cells formed
confluent monolayers on day 3 after plating. The medium was replaced with fresh medium on day 4, and the cells were used for
experiments on day 7 after plating.
Cisplatin toxicity. Cisplatin (Sigma, St. Louis, MO) was prepared
as a 3.3 mM stock solution in 0.9% saline daily. RPMI-1640 Medium
(RPMI, Cat. No. 31800), DMEM (Cat. no. 12800), Leibovitz’s L-15
Medium (L-15, Cat. no. 41300), Nutrient Mixture Ham’s F-12 (F-12,
Cat. no. 21700), and HBSS (Cat. no. 11201) were prepared from
powder (GIBCO/BRL). L-15 was supplemented with 18 mM HEPES
and 0.2% BSA. The pH of all solutions and media was adjusted to 7.4.
For toxicity experiments, the media on the cells was changed to the
test solution and cisplatin was added to the wells. Cells treated with
only the test solution served as controls. The cells were incubated at
37°C in 5% CO2-95% air, except cells in L-15 medium that were
incubated at 37°C without CO2. After 3 h, the cisplatin-containing
solution was removed from the cells and replaced with DMEM
containing 5% FBS and antibiotics. The cells were incubated for an
additional 69 h at 37°C in 5% CO2. The number of viable cells was
then determined with the MTT assay (23). A standard curve showed
a direct correlation between the number of viable LLC-PK1 cells and
the OD570 in the MTT assay.
Effect of NaCl concentration and osmolality on cisplatin toxicity.
Solutions containing the inorganic salt components of L-15, F-12,
RPMI, HBSS, and DMEM were prepared according to their formulations in the GIBCO/BRL catalogue, but without sodium bicarbonate
and the pH adjusted to pH 7.4. Saline was prepared with 2 mM
sodium phosphate buffer, pH 7.4. Cisplatin was diluted in each test
solution. The cells were exposed to cisplatin for 3 h at 37°C in an air
atmosphere. Cells treated with the test solution that did not contain
cisplatin served as controls. After 3 h, the cisplatin-containing solutions were removed from the cells and replaced with DMEM containing 5% FBS and antibiotics. Cell viability was assayed 69 h after the
cisplatin-solution was removed.
Solutions of NaCl were prepared with 5 mM HEPES, pH 7.4. Cells
were exposed to 100 ␮M cisplatin in the NaCl solutions for 3 h at
37°C without CO2. Cells incubated in the NaCl solutions without
cisplatin served as controls for each concentration of NaCl. Cisplatin
was prepared in 0.9% NaCl. The solution for each set of triplicate
wells was made separately and constituted such that the NaCl concentration in the cisplatin-containing solution was the same as its
corresponding control. Solutions containing NaCl and mannitol or
NaCl and N-methyl-D-glucamine were prepared with 5 mM HEPES,
pH 7.4. Equiosmolar solutions were prepared by decreasing the
concentrations of mannitol or N-methyl-D-glucamine as the concentration of NaCl increased to maintain the osmolality of the solution at
288 mosmol/kgH2O. The osmolality of the tissue culture media and
salt solutions was determined with a Wescor 5520 VAPRO Vapor
Pressure Osmometer (Logan, UT).
Cisplatin uptake. LLC-PK1 cells were seeded in 24-well plates at
5 ⫻ 104 cells/well. On day 5, 100 ␮M cisplatin in 125 to 150 mM
NaCl/5 mM HEPES, pH 7.4, was added to the confluent monolayers
and incubated at 37°C without CO2 for 3 h. The cells were then
washed twice with PBS. The cells in each well were digested in 150
␮l concentrated HNO3 at 70°C for 4 h. The concentration of HNO3
was adjusted to 3.25 M with distilled water, and Triton X-100 was
added to a final concentration of 1%. Platinum levels were quantified
by flameless atomic absorption spectrometry (FAAS). A Varian
SPECTRAA-220Z Graphite Furnace Double Beam Atomic Absorption Spectrophotometer (Houston, TX) with Zeeman background
correction was used. The platinum standard included equivalent
amounts of HNO3 and Triton X-100. The number of cells in triplicate
wells was determined as previously described (16).
Pretreatment with salt. DMEM was prepared according to the
formulation in the GIBCO/BRL catalogue with the exception of NaCl,
which was omitted from the stock solution so that the concentration
could be varied within the experiment. Three hours before cisplatin
STRESS RESPONSE AND CISPLATIN
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concentrations of the other components did not correlate with
cisplatin toxicity. It should be noted that DMEM reduced the
toxicity of cisplatin (Fig. 1A). However, when the inorganic
salt solution of DMEM was tested, it potentiated cisplatin
toxicity (Fig. 1B). DMEM contains 44 mM NaHCO3, which is
higher than the concentration in F-12 or RPMI (14 and 24 mM,
respectively). NaHCO3 increased the Na⫹ ion concentration in
DMEM media by 39 mM compared with the Na⫹ ion concentration in the DMEM inorganic salt solution tested. The inorganic salt fraction of L-15 is also somewhat less protective than
L-15 medium. There may be additional components in L-15
medium that modulate cisplatin toxicity.
To directly analyze the effect of NaCl on the toxicity of
cisplatin, cells were incubated for 3 h in a 100 ␮M solution of
cisplatin containing 130 to 155 mM NaCl and 5 mM HEPES,
pH 7.4. As shown in Fig. 2, increasing the concentration of
NaCl from 130 to 155 mM increased cell survival in 100 ␮M
cisplatin from 22 ⫾ 3 to 98 ⫾ 3% (P ⬍ 0.001). In the absence
of cisplatin, the 3-h incubation in the NaCl solutions had no
effect on cell viability (P ⫽ 0.6).
Effect of NaCl concentration on cisplatin accumulation.
NaCl did not alter the accumulation of cisplatin in LLC-PK1
cells. Platinum levels were measured in confluent monolayers
treated with cisplatin solutions containing NaCl. Increasing the
NaCl concentration from 125 to 155 mM had no significant
effect on platinum accumulation (Fig. 3), despite the dramatic
effect on toxicity (Fig. 2).
Preconditioning cells with various concentrations of NaCl.
A series of experiments was undertaken to determine whether
the effect of NaCl on cisplatin toxicity was due to interaction
of the NaCl with the cisplatin or to an effect of the salt on the
cells. The cells were incubated in media with various concentrations of NaCl for 3 h before the cisplatin treatment. The
preincubation media consisted of DMEM formulated with
various concentrations of NaCl plus 5% FBS. At the end of the
preincubation period, the media were removed and all the cells
were treated with 100 ␮M cisplatin in DMEM containing the
standard amount of NaCl. After the cisplatin treatment, the
cisplatin-containing solution was removed and all cells were
incubated for an additional 69 h in DMEM with 5% FBS and
antibiotics. The data in Fig. 4 show that the concentration of
Table 1. Composition of salt solutions tested in Fig. 1 B
Medium
Component
Saline
L-15
HBSS
F-12
DMEM
RPM1
CaCl2
KCl
KH2PO4
MgCl2
MgSO4
NaCl
Na2HPO4
NaH2PO4
HCl to adjust pH
NaOH to adjust pH
Na⫹
Cl⫺
—
—
—
—
—
145.4
1.5
0.5
—
0.3
149.2
145.4
1.3mM*
5.4
—
1.0
0.8
136.9
1.3
—
0.2
—
139.5
147.1
1.3
5.4
0.4
—
0.8
136.9
0.3
—
—
0.3
137.8
144.9
0.3
3.0
—
0.6
—
130.0
1.0
—
0.1
—
132.0
134.9
1.8
5.4
—
—
0.8
109.5
3.9
1.2
—
0.6
119.1
118.5
—
5.4
—
—
0.4
102.7
5.6
—
1.0
—
113.9
109.1
*All concentrations are mM.
AJP-Renal Physiol • VOL
Fig. 2. Influence of sodium chloride concentration on cisplatin nephrotoxicity.
Confluent monolayers of LLC-PK1 cells were incubated for 3 h in solutions
containing 100 ␮M cisplatin, with 5 mM HEPES, pH 7.4 and varying amounts
of NaCl (E) or in the HEPES/NaCl solutions without cisplatin (F). The test
solutions were removed at the end of the 3-h exposure and fresh DMEM
medium with 5% FBS and antibiotics was added to the cells. Cell survival was
assayed at 72 h. Each point is the average of 3 values ⫾ SD.
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Fig. 1. Nephrotoxicity of cisplatin in tissue culture media (A)
and inorganic salt solutions (B). A: confluent monolayers of
LLC-PK1 cells were incubated for 3 h in cisplatin in RPMI (䊐),
F-12 (Œ), DMEM ({), or L-15 medium with 18 mM HEPES and
0.2% BSA (F). B: confluent monolayers of LLC-PK1 cells were
incubated for 3 h in cisplatin in the solutions containing the
inorganic salt component of RPMI (䊐), DMEM with 5 mM
NaPO4 ({), F-12 (Œ), HBSS (horizontal bars), L-15 (F), and
saline buffered with 2 mM NaPO4 (Xs). All solutions were
adjusted to pH 7.4. For experiments in both A and B, the
cisplatin solutions were removed at the end of the 3-h exposure
and fresh DMEM medium with 5% FBS and antibiotics was
added to the cells. Cell survival was assayed at 72 h. Each point
is the average of 3 to 6 values ⫾ SD. Values that differed
significantly from the survival in L-15 medium (A) or L-15
inorganic salt solution (B) are indicated by * (P ⬍ 0.01) or **
(P ⬍ 0.001).
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NaCl in the preincubation medium had a dramatic effect on the
toxicity of cisplatin. Pretreatment of the cells with higher
concentrations of Na⫹ ions in DMEM was correlated with
greater cisplatin toxicity. Cell survival decreased from 80.4 ⫾
7.6 to 2.4 ⫾ 2.9%. The linear trend was highly significant (P ⬍
0.0001). All of the cells were treated with the same cisplatin
solution. These data show that the NaCl concentration is
modulating cisplatin toxicity through its effect on the cell
rather than through a direct interaction with the cisplatin.
Controls showed that the pretreatment with the media alone (no
cisplatin) had no significant effect on cell viability (P ⫽ 0.18).
Fig. 4. Effect of preconditioning cells with various concentrations of NaCl on
cisplatin nephrotoxicity. Confluent monolayers of LLC-PK1 cells were incubated for 3 h in fresh DMEM containing 5% FBS, antibiotics, and various
concentrations of NaCl. After 3 h, the media were removed and all the cells
were treated with 100 ␮M cisplatin in DMEM (E) or DMEM alone (F). The
cisplatin solutions were removed at the end of the 3-h exposure and fresh
DMEM with 5% FBS and antibiotics was added to the cells. Cell survival was
assayed at 72 h. Each point is the average of 3 values ⫾ SD.
AJP-Renal Physiol • VOL
Table 2. Osmolality of tissue culture media
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Medium
Osmolality, mosmol/kgH2O
RPMI
F-12
L-15
DMEM
286
298
330
350
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Fig. 3. Effect of NaCl on the accumulation of platinum. Confluent monolayers
of LLC-PK1 cells were incubated for 3 h in solutions containing 100 ␮M
cisplatin, with 5 mM HEPES, pH 7.4 and varying amounts of NaCl. At the end
of 3 h, the cisplatin was rinsed off the cells and the amount of platinum that had
accumulated in the cells was determined by flameless atomic absorption
spectrometry (FAAS). Each point is the average of the mean value from 3
experiments ⫾ SE.
Higher concentrations of NaCl during the pretreatment period resulted in increased sensitivity to cisplatin toxicity (Fig.
4), whereas higher NaCl concentrations during the cisplatin
exposure were protective (Fig. 2). These data suggest a correlation between osmotic induction of changes in cell volume
and sensitivity to cisplatin. In the experiment shown in Fig. 2,
all of the cells were incubated in DMEM, which contains 156
mM sodium, before the cisplatin exposure. The medium was
then changed to solutions with 130 to 155 mM NaCl during the
cisplatin exposure. The decrease in extracellular salt concentration may have caused cell swelling. The data show that there
was a dose-response relationship between the increase in cisplatin susceptibility and the decrease in extracellular salt.
Conversely, in the experiment shown in Fig. 4, the extracellular NaCl was varied for 3 h before the cisplatin exposure. All
of the cells were then treated with 100 ␮M cisplatin in a
solution containing 156 mM sodium. Cells that were pretreated
with media containing less than 156 mM sodium experienced
an increase in extracellular salt when switched to the cisplatin
solution. These cells may have responded by cell shrinkage
which protected them from cisplatin toxicity. The cells that had
been pretreated with media containing more than 156 mM
sodium experienced a decrease in extracellular salt when
switched to the cisplatin solutions. These cells may have
responded by swelling which made them more susceptible to
cisplatin toxicity. The data in Fig. 3 demonstrate that the
changes in extracellular salt concentration alters the sensitivity
of the cell to cisplatin without altering the accumulation of
platinum in the cell. The changes in cell volume may have
modulated stress responses within the cell that altered sensitivity to cisplatin.
Effect of osmolality on cisplatin toxicity. An analysis of the
osmolality of the four tissue culture media tested in Fig. 1A
revealed a correlation between the osmolality of the media and
the toxicity of cisplatin (Table 2). These data suggested that the
NaCl concentration in the extracellular solution may be modulating cisplatin toxicity through an osmotic effect. To compare the effect of NaCl vs. osmolality on cisplatin toxicity, one
set of cells was treated for 3 h in a 100 ␮M solution of cisplatin
containing 115 to 155 mM NaCl and 5 mM HEPES, pH 7.4.
Two additional sets of cells were incubated in the same
solutions balanced with mannitol or N-methyl-D-glucamine
such that the osmolality of each solution was 288 mosmol/
kgH2O. As shown in Fig. 5, maintaining the osmolality of the
solution with either mannitol or N-methyl-D-glucamine protected the cells from cisplatin toxicity, indicating that the
osmotic effect of NaCl modulated cisplatin toxicity. In the
absence of cisplatin, the 3-h incubation in the salt solutions had
no effect on cell viability (data not shown).
Four compounds were tested for their ability to modulate
cisplatin toxicity by modulating the osmolality. Cells were
incubated for 3 h in solutions containing 100 ␮M cisplatin, 115
STRESS RESPONSE AND CISPLATIN
Fig. 6. Effect of osmolality on cisplatin toxicity. Various concentrations of
mannitol (F), sucrose (Œ), N-methyl-D-glucamine (䊐), and sodium gluconate
(filled bar) were added to 115 mM NaCl with 5 mM HEPES. Confluent
monolayers of LLC-PK1 cells were incubated for 3 h in each of the solutions
containing 100 ␮M cisplatin. The cisplatin solutions were removed at the end
of the 3-h exposure and fresh DMEM medium with 5% FBS and antibiotics
was added to the cells. Cell survival was assayed at 72 h. Each point is the
average of 3 values ⫾ SD.
the 65% decrease in cisplatin toxicity over this same range of
extracellular NaCl. However, altered DNA platination may be
part of the mechanism by which changes in extracellular NaCl
exert its protective effect.
DISCUSSION
Fig. 5. Effect of NaCl vs. osmolality on cisplatin toxicity. Confluent monolayers of LLC-PK1 cells were incubated for 3 h in 100 ␮M cisplatin in 5 mM
HEPES containing NaCl alone ({), NaCl and mannitol (F), or NaCl and
N-methyl-D-glucamine (䊐). The varying amounts of NaCl were balanced with
mannitol or N-methyl-D-glucamine such that the final osmolality of each
mannitol and each N-methyl-D-glucamine solution was 288 mosmol/kgH2O.
The cisplatin solutions were removed at the end of the 3-h exposure, and fresh
DMEM with 5% FBS and antibiotics was added to the cells. Cell survival was
assayed at 72 h. Each point is the average of 3 values ⫾ SD.
AJP-Renal Physiol • VOL
Small variations in the osmolality among tissue culture
media account for their modulation of cisplatin nephrotoxicity.
NaCl and mannitol have been shown to protect against cisplatin-induced nephrotoxicity in vivo, but not in vitro (5). Our
data have shown that the NaCl does not alter the accumulation
of cisplatin. The salt did not exert its effect by interacting
directly with cisplatin, but rather by altering the osmolality of
the extracellular solution, thereby modulating a stress response
within the cell.
It has been proposed that volume repletion of patients with
isotonic saline protects against cisplatin-induced nephrotoxicity by flushing the cisplatin out of the body, limiting uptake
into the proximal tubule cells (9). Patients treated with cisplatin
are generally given up to 6 l saline/day iv (28, 34). The diuretic
mannitol is also commonly used to decrease the nephrotoxicity
of cisplatin. In our assay system, NaCl and mannitol protected
the cells from cisplatin toxicity. The amount of cisplatin in the
medium was constant during the 3-h treatment; therefore, the
protective effect of these compounds was not due to decreased
exposure of the cells to the drug.
The in vitro studies presented here demonstrated a large
effect of NaCl on cisplatin toxicity over a narrow concentration
range (130 to 155 mM). The normal reference range for
sodium in serum is 135–145 mM. Sodium concentrations
below 120 or above 160 mM are considered pathological (13).
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mM NaCl, 5 mM HEPES, pH 7.4 and increasing concentrations of mannitol (a sugar), sucrose (a sugar), N-methyl-Dglucamine (an impermeant cation), or gluconate (an impermeant anion). At equivalent osmolality, all four compounds
reduced cisplatin toxicity to a similar level (Fig. 6). In the
absence of cisplatin, the 3-h incubation in the solutions had no
effect on cell viability (data not shown). The osmolality range
tested in these experiments (214 to 288 mosm/kgH2O) is
equivalent to the osmolality range of the NaCl solutions tested
in Fig. 5 ({, 115 to 155 mM). These data show that an osmotic
stress response modulates the sensitivity of renal cells to
cisplatin toxicity.
Effect of osmolality on cisplatin accumulation. Platinum
levels were measured in confluent monolayers of cisplatintreated cells incubated in 115 mM NaCl with mannitol. Increasing the mannitol concentration from 19 to 75 mM increased the osmolality of the solution from 232 to 288 mosmol/
kgH2O. Over this concentration range, there appears to be a
decrease in platinum accumulation per cell (Fig. 7A). However,
when the data are graphed with a volume correction to account
for the osmolarity-induced change in cell volume, the data
show that the platinum accumulation does not change over this
osmolality range (Fig. 7B).
Effect of NaCl on platinum bound to DNA. Increasing the
NaCl concentration resulted in a decrease in the amount of
platinum bound to DNA. Increasing the NaCl concentration
from 125 to 150 mM decreased the level of DNA platination
from 153 ⫾ 11 to 98 ⫾ 15 ng/mg DNA, a 36% decrease (Fig.
8, P ⬍ 0.0001). The decrease in DNA platination is less than
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Fig. 7. Effect of osmolality on the accumulation of platinum.
Confluent monolayers of LLC-PK1 cells were incubated for 3 h
in solutions containing 100 ␮M cisplatin, with 115 mM NaCl,
5 mM HEPES, pH 7.4. Mannitol was used to vary the osmolality of the solutions. At the end of 3 h, the cisplatin was rinsed
off the cells and the amount of platinum that had accumulated
in the cells was determined by FAAS. Each point is the average
of 3 to 6 values ⫾ SD. Data are expressed as the platinum
accumulation per cell (A) and platinum accumulation per cell
with a volume correction (B).
Fig. 8. Effect of NaCl on platinum bound to DNA. Confluent monolayers of
LLC-PK1 cells were incubated for 3 h in solutions containing 100 ␮M
cisplatin, with 5 mM HEPES, pH 7.4 and varying amounts of NaCl. At the end
of 3 h, the cisplatin was rinsed off the cells, DNA was isolated, and the amount
of platinum bound to the DNA was determined by FAAS. Each point is the
average of 3 values ⫾ SD.
AJP-Renal Physiol • VOL
are also toxic to the proximal tubules. Ticarcillin has been
shown to protect against gentamicin nephrotoxicity. Sabra and
Branch (31) demonstrated that the salt load associated with
ticarcillin administration was the primary mechanism by which
ticarcillin protected against aminoglycoside nephrotoxicity.
Clinical protocols have administered cisplatin in solutions
containing as much as 3% NaCl (2, 28). Some investigators
have proposed that the salt provides a high concentration of
chloride ions, which prevents the dissociation of the chloride
ions from the platinum molecule, thereby reducing the formation of the reactive, aquated species of cisplatin (5). In vivo
NaCl must be injected shortly before or in the same solution as
the cisplatin to exert its nephroprotective effect (5). Our studies
in which the NaCl concentration was varied before cisplatin
treatment showed that the extracellular NaCl is affecting the
cell and not interacting directly with the cisplatin.
Both NaCl and mannitol protected against cisplatin-induced
toxicity. At equiosmolar concentrations, they were equally
protective. Studies of platinum accumulation showed that increasing the extracellular concentration of NaCl did not change
the total amount of platinum in cells at 3 h. In contrast,
increasing the extracellular concentration of mannitol decreased cellular platinum accumulation. These data indicate
that total platinum accumulation is not the mechanism by
which osmolality alters cisplatin toxicity, as these two compounds differed in their effect on accumulation while having
the same effect on toxicity. There have been conflicting data
regarding the mechanism by which cisplatin is taken up into
cells (1). Recently, two groups have shown that cisplatin can be
transported into the cell by the copper transporter Ctr1, an
energy and sodium-independent transporter (17, 19, 20). Permeant ions such as sodium and chloride and impermeant ions
such as mannitol have different effects on membrane permeability and transport systems (12).
We propose that osmotic stress responses are induced in
proximal tubule cells by increased or decreased osmolality of
the extracellular solution. These responses may be triggered by
cell swelling or cell shrinkage as the osmolality of the extracellular solution changes. The volume changes are transient.
Epithelial cells can readjust their volume by regulatory volume
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Physiological saline (0.85% NaCl) is 145 mM NaCl. The
sodium concentration in the tissue culture media we tested
ranged from 138 to 156 mM. In calculating the sodium and the
chloride concentration in each medium, the total amount of
each ion included the ions contributed by the sodium salts of
amino acids and the sodium hydroxide or hydrochloric acid
solutions used to adjust the pH. Studies analyzing the protective effect of a variety of compounds on cisplatin toxicity often
do not take into account the amount of sodium added when the
sodium salt of the compound is used. Sodium selenite protected rats against cisplatin-induced nephrotoxicity without
changing the pharmacokinetics of the cisplatin (35). The investigators demonstrated that selenite does not react with the
cisplatin and speculated that the selenite requires bioactivation
to exert its protective effect. It is possible that the sodium
rather than the selenite is responsible for the protection. This
scenario has been demonstrated with aminoglycosides, which
STRESS RESPONSE AND CISPLATIN
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Siribhinya Benyajati in the Department of
Physiology at The University of Oklahoma Health Sciences Center for helpful
discussions during the preparation of this manuscript.
GRANTS
This work was supported by Grant RO1-CA57530 (to M. H. Hanigan) from
The National Cancer Institute, National Institutes of Health, and a grant from
the Presbyterian Health Foundation (Oklahoma City, OK).
REFERENCES
1. Andrews PA. Cisplatin accumulation. In: Platinum-Based Drugs in Cancer Therapy, edited by Kelland LR and Farrell NP. Totowa, NJ: Humana,
2000, p. 89 –113.
2. Bajorin D, Bosl GJ, and Fein R. Phase-I trial of escalating doses of
cisplatin in hypertonic saline. J Clin Oncol 5: 1589 –1593, 1987.
3. Choi JY, Shah M, Lee MG, Schultheis PJ, Shull GE, Muallem S, and
Baum M. Novel amiloride-sensitive sodium-dependent proton secretion
in the mouse proximal convoluted tubule. J Clin Invest 105: 1141–1146,
2000.
4. Cornelison TL and Reed E. Nephrotoxicity and hydration management
for cisplatin, carboplatin, and ormaplatin. Gynecol Oncol 50: 147–158,
1993.
AJP-Renal Physiol • VOL
5. Daley-Yates PT and McBrien DCH. A study of the protective effect of
chloride salts on cisplatin nephrotoxicity. Biochem Pharmacol 34: 2363–
2369, 1985.
6. DeConti RC, Toftness BR, Lange RC, and Creasey WA. Clinical and
pharmacological studies with cis-diamminedichloroplatinum (II). Cancer
Res 33: 1310 –1315, 1973.
7. Dekant W, Vamvakas S, and Anders MW. Formation and fate of
nephrotoxic and cytotoxic glutathione S-conjugates: cysteine conjugate
␤-lyase pathway. In: Advances in Pharmacology, Vol. 27, edited by
Anders MW and Dekant W. New York: Academic, 1995, p. 115–162.
8. Dobyan DC, Levi J, Jacobs C, Kosek J, and Weiner MW. Mechanism
of cis-platinum nephrotoxicity. II. morphological observations. J Pharmacol Exp Ther 213: 551–556, 1980.
9. Dumas M, de Gislain C, d’Athis P, Chadoint-Noudeau V, Escousse A,
Guerrin J, and Autissier N. Influence of hydration on ultrafilterable
platinum kinetic and kidney function on patients treated with cis-diamminedichloroplatinum (II). Cancer Chemother Pharmacol 26: 278 –282,
1990.
10. Earhart RH, Martin PA, Tutsch KD, Erturk E, Wheeler RH, and Bull
FE. Improvement in the therapeutic index of cisplatin (NSC 119875) by
pharmacologically induced chloruresis in the rat. Cancer Res 43: 1187–
1194, 1983.
11. Einhorn LH. Curing metastatic testicular cancer. Proc Natl Acad Sci USA
99: 4592– 4595, 2002.
12. Filipovic D and Sackin H. Stretch- and volume-activated channels in
isolated proximal tubule cells. Am J Physiol Renal Fluid Electrolyte
Physiol 262: F857–F870, 1992.
13. Finley PR, Grady HJ, Olsowka ES, and Tilzer LL. Chemistry. In:
Laboratory Test Handbook, edited by Jacobs DS, DeMott WR, Finley PR,
Horvat RT, Kasten BL, and Tilzer LL. Hudson, OH: Lexi-Comp, 1993, p.
94 –350.
14. Hanigan MH, Gallagher BC, Taylor PT Jr, and Large MK. Inhibition
of ␥-glutamyl transpeptidase activity by acivicin in vivo protects the
kidney from cisplatin-induced toxicity. Cancer Res 54: 5925–5929, 1994.
15. Hanigan MH, Lykissa ED, Townsend DM, Ou CN, Barrios R, and
Lieberman MW. ␥-Glutamyl transpeptidase-deficient mice are resistant
to the nephrotoxic effects of cisplatin. Am J Pathol 159: 1889 –1894, 2001.
16. Hanigan MH and Pitot HC. Isolation of ␥-glutamyl transpeptidase
positive hepatocytes during the early stages of hepatocarcinogenesis in the
rat. Carcinogenesis 3: 1349 –1354, 1982.
17. Ishida S, Lee J, Thiele DJ, and Herskowitz I. Uptake of the anticancer
drug cisplatin mediated by the copper transporter Ctr1 in yeast and
mammals. Proc Natl Acad Sci USA 99: 14298 –14302, 2002.
18. Kruidering M, van de WB, Zhan Y, Baelde JJ, Heer E, Mulder GJ,
Stevens JL, and Nagelkerke JF. Cisplatin effects on F-actin and matrix
proteins precede renal tubular cell detachment and apoptosis in vitro. Cell
Death Differ 5: 601– 614, 1998.
19. Lee J, Pena MM, Nose Y, and Thiele DJ. Biochemical characterization
of the human copper transporter Ctr1. J Biol Chem 277: 4380 – 4387, 2002.
20. Lin X, Okuda T, Holzer A, and Howell SB. The copper transporter
CTR1 regulates cisplatin uptake in Saccharomyces cerevisiae. Mol Pharmacol 62: 1154 –1159, 2002.
21. Litterst CL. Alterations in the toxicity of cis-dichlorodiammineplatinum-II and in tissue localization of platinum as a function of NaCl
concentration in the vehicle of administration. Toxicol Appl Pharmacol
61: 99 –108, 1981.
22. McKeage MJ, Abel G, Kelland LR, and Harrap KR. Mechanism of
action of an orally administered platinum complex [ammine bis butyrato
cyclohexylamine dichloroplatinum (IV) (JM221)] in intrinsically cisplatin-resistant human ovarian carcinoma in vitro. Br J Cancer 69: 1–7, 1994.
23. Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Methods
65: 55– 63, 1983.
24. Ogilvie GK, Krawiec DR, Gelberg HB, Twardock AR, Reschke RW,
and Richardson BC. Evaluation of a short-term saline diuresis protocol
for the administration of cisplatin. Am J Vet Res 49: 1076 –1078, 1988.
25. Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, and Morishima S.
Receptor-mediated control of regulatory volume decrease (RVD) and
apoptotic volume decrease (AVD). J Physiol 532: 3–16, 2001.
26. Okuda M, Masaki K, Fukatsu S, Hashimoto Y, and Inui K. Role of
apoptosis in cisplatin-induced toxicity in the renal epithelial cell line
LLC-PK1. Implication of the functions of apical membranes. Biochem
Pharmacol 59: 195–201, 2000.
288 • JANUARY 2005 •
www.ajprenal.org
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.1 on June 14, 2017
decrease or regulatory volume increase, active processes involving ion channels and other cotransporter-mediated mechanisms (3, 25, 37). These transient changes in cell volume may
affect cisplatin nephrotoxicity by any of several mechanisms.
Changes in cell volume have been shown to modulate cell
survival and apoptotic pathways (32). Cisplatin kills LLC-PK1
cells by a caspase-3-dependent apoptosis (18, 26, 38). Resistance to cisplatin has been associated with induction of proteins
associated with other stress responses. Wachsberger and coworkers (36) reported that mammalian cells grown at pH 6.7
had elevated levels of HSP27 and were resistant to cisplatin.
Changes in the osmolality of the extracellular medium have
also been shown to alter chromatin structure and free radicalinduced DNA-protein crosslinks (27, 30). Alteration of chromatin structure may alter the accessibility of platinum to DNA.
The data in this study showed increased platinum-DNA binding at low NaCl concentrations, the conditions under which the
cells were the most sensitive to cisplatin toxicity. Alternatively,
an osmotic response may be modulating components of the
pathway through which cisplatin is metabolized to a nephrotoxin. We have shown that the nephrotoxicity of cisplatin is
due to the conjugation of cisplatin to glutathione and the
subsequent metabolism of that conjugate to a cysteinyl-glycine
conjugate, then to a cysteine conjugate, and finally to a reactive
thiol, which is toxic to the cell (14, 15, 33). This pathway is
analogous to the metabolic activation of nephrotoxic alkenes
(7). In this pathway, the glutathione conjugates are formed
intracellularly then excreted. The metabolism of the glutathione conjugate and the cysteinyl-glycine conjugate occurs extracellularly, catalyzed by the cell-surface enzymes GGT and
aminodipeptidase. The cysteine conjugates are metabolized to
reactive thiols intracellularly by the cysteine-S conjugate
␤-lyase. The osmotic stress response could be affecting any of
several steps in this pathway.
In the clinic, hydration by intravenous infusion of saline is
the method most commonly used to prevent cisplatin-induced
nephrotoxicity (29). Our ongoing studies into the molecular
mechanism of this stress response will elucidate this pathway
and provide novel strategies to block the nephrotoxic effects of
cisplatin.
F131
F132
STRESS RESPONSE AND CISPLATIN
AJP-Renal Physiol • VOL
34. Townsend DM and Hanigan MH. Inhibition of ␥-glutamyl transpeptidase or cysteine S-conjugate ␤-lyase activity blocks the nephrotoxicity of
cisplatin in mice. J Pharmacol Exp Ther 300: 142–148, 2002.
35. Vermeulen NP, Baldew GS, Los G, McVie JG, and De Goeij JJ.
Reduction of cisplatin nephrotoxicity by sodium selenite. Lack of interaction at the pharmacokinetic level of both compounds. Drug Metab
Dispos 21: 30 –36, 1993.
36. Wachsberger PR, Landry J, Storck C, Davis K, O’Hara MD, Owen
CS, Leeper DB, and Coss RA. Mammalian cells adapted to growth at pH
6.7 have elevated HSP27 levels and are resistant to cisplatin. Int J Hyperthermia 13: 251–255, 1997.
37. Wu KL, Khan S, Lakhe-Reddy S, Wang L, Jarad G, Miller RT,
Konieczkowski M, Brown AM, Sedor JR, and Schelling JR. Renal
tubular epithelial cell apoptosis is associated with caspase cleavage of the
NHE1 Na⫹/H⫹ exchanger. Am J Physiol Renal Physiol 284: F829 –F839,
2003.
38. Zhan Y, van de Water B, Wang Y, and Stevens JL. The roles of
caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of
renal epithelial cells. Oncogene 18: 6505– 6512, 1999.
288 • JANUARY 2005 •
www.ajprenal.org
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.1 on June 14, 2017
27. Oleinick NL, Chiu SM, Ramakrishnan N, and Xue LY. The formation,
identification, and significance of DNA-protein cross-links in mammalian
cells. Br J Cancer Suppl 8: 135–140, 1987.
28. Ozols RF, Corden BJ, Jacob J, Wesley MN, Ostchega Y, and Young RC.
High-dose cisplatin in hypertonic saline. Ann Intern Med 100: 19 –24, 1984.
29. Pinzani V, Bressolle F, Haug IJ, Galtier M, and Blayac JP. Cisplatin
induced renal toxicity and toxicity-modulating strategies: a review. Cancer Chemother Pharmacol 35: 1–9, 1994.
30. Proft M and Struhl K. MAP kinase-mediated stress relief that precedes
and regulates the timing of transcriptional induction. Cell 118: 351–361,
2004.
31. Sabra R and Branch RA. Role of sodium in protection by extendedspectrum penicillins against tobramycin-induced nephrotoxicity. Antimicrob Agents Chemother 34: 1020 –1025, 1990.
32. Terada Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa
W, Kasuga M, Sasaki S, and Marumo F. Hyperosmolality activates Akt
and regulates apoptosis in renal tubular cells. Kidney Int 60: 553–567, 2001.
33. Townsend DM, Deng M, Zhang L, Lapus MG, and Hanigan MH.
Metabolism of cisplatin to a nephrotoxin in proximal tubule cells. J Am
Soc Nephrol 14: 1–10, 2003.