Stress Response Inhibits the Nephrotoxicity of Cisplatin

Marie H. Hanigan1, Mei Deng2, Lei Zhang1, Peyton T. Taylor Jr. 2 and Maia G. Lapus1

1Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

73104 and 2Department of Obstetrics and Gynecology, Division of Gynecologic Oncology,

University of Virginia Health Sciences Center, Charlottesville, VA 22908

Running Head: Stress Response and Cisplatin

Corresponding Author: Marie H. Hanigan, Ph.D., Biomedical Research Center Room 264, 975

N.E. 10th Street, Oklahoma City, Oklahoma 73104, USA (E-mail: marie-hanigan@ouhsc.edu).

Telephone: 405-271-3832; FAX 405-271-3813

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Articles in PresS. Am J Physiol Renal Physiol (September 7, 2004). doi:10.1152/ajprenal.00041.2003

Copyright © 2004 by the American Physiological Society.

Abstract. 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 the 3 hr cisplatin exposure. Further experiments revealed that minor variations in the

sodium concentration among standard tissue culture media modulated cisplatin nephrotoxicity.

Sodium chloride 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.

Key Words: kidney, cis-diamminedichloroplatinum, toxicity, proximal tubule, LLC-PK1 cells

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INTRODUCTION

The discovery of the potent 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 three hours (6). Renal

toxicity, as determined by elevated levels of blood urea nitrogen, is not evident until 72 to 96 hours

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 re-constituted 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).

Despite the wide-spread 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 mouse data indicated that the sodium ion is the

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important molecule, while Earhart and coworkers reported data from rat studies suggesting that the

chloride is the protective ion (10; 21).

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 hours 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.

MATERIALS AND METHODS

Cell Culture

LLC-PK1 (ATCC CRL 1392), a pig kidney cell line, was purchased from the American Type

Culture Collection (Manassas,VA). LLC-PK1 cells were grown in DMEM supplemented with 4

mM L-glutamine (DMEM, GIBCO/BRL, Grand Island, NY), 5% fetal bovine serum (FBS, Hyclone

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Laboratories, Logan, UT), penicillin (5 U/ml), and streptomycin (5 µg/ml, GIBCO/BRL). Cells

were maintained in 5% CO2 and 95% air at 37oC. Subconfluent cultures were passaged every 3 to 4

days. For toxicity experiments, LLC-PK1 cells were seeded in 96 well plates at 5x103 cells/well.

The cells formed confluent monolayers on the 3rd day after plating. The medium was replaced with

fresh medium on the 4th day and the cells were used for experiments on the 7th day after plating.

Cisplatin Toxicity

Cisplatin (Sigma Chemical Co, 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

Hank's Balanced Salt Solution (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 37oC in 5% CO2 , 95% air, except cells in L-15 medium which

were incubated at 37oC without CO2. After 3 hrs, 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 hrs at 37oC 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

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bicarbonate and the pH adjusted to pH 7.4. Saline was prepared with 2mM sodium phosphate

buffer , pH 7.4. Cisplatin was diluted in each test solution. The cells were exposed to cisplatin for 3

hrs at 37oC in an air atmosphere. Cells treated with the test solution that did not contain cisplatin

served as controls. After 3 hrs, the cisplatin-containing solutions were removed from the cells and

replaced with DMEM containing 5% FBS and antibiotics. Cell viability was assayed 69 hrs after

the cisplatin-solution was removed.

Solutions of NaCl were prepared with 5 mM HEPES, pH 7.4. Cells were exposed to 100 µM

cisplatin in the NaCl solutions for 3 hrs at 37oC 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

mannitol or N-methyl-D-glucamine as the concentration of NaCl increased to maintain the

osmolality of the solution at 288 mosmol/kg H2O. 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 x104 cells/well. On the 5th day, 100 µM 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 hrs. The cells were then washed twice with phosphate-buffered

saline (PBS). The cells in each well were digested in 150 µL concentrated HNO3 at 70°C for 4 hr.

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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 hrs prior to cisplatin treatment, the media was changed to fresh DMEM

containing 5% FBS, antibiotics and various concentrations of NaCl. After 3 hrs the media was

removed and the cells treated with 100 µM cisplatin in DMEM. At the end of the 3 hr cisplatin

exposure the cisplatin was removed and cells returned to DMEM containing 5% FBS and

antibiotics for 69 hrs. The number of viable cells was determined with the MTT assay.

DNA Platination

LLC-PK1 cells were seeded in P-60 plates at 5.7x105 cells/plate. On the 5th day, the confluent cells

were exposed to 100 µM cisplatin in 125 to150 mM NaCl with 5 mM HEPES, pH 7.4, at 37°C

without CO2 for 3 hrs. The cells were washed with PBS and trypsinized. DNA was purified by the

method of McKeage and coworkers (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,

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with addition of 100 µg/ml RNase and incubation at 37°C for 1 hr. 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 to the toxicity of cisplatin in L-15 by a 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 post test

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 Inc. 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 hrs, then returned to complete DMEM. Toxicity was assessed at 72

hrs. The media on the cells in the four experimental groups varied only during the 3 hr cisplatin

exposure. Prior to and following cisplatin treatment, all cells were in complete DMEM. The data in

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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 µM and 150 µM, 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 hr and toxicity was measured at 72 hrs. 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 I. There was a

direct correlation between the concentration of NaCl in the solution and the reduction in cisplatin

toxicity: saline>L-15>HBSS>F-12>DMEM>RPMI . The higher the concentration of NaCl the less

toxic cisplatin was to the cells. The 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 amount in F-12 or RPMI (14

mM and 24 mM respectively). NaHCO3 increased the Na+ ion concentration in DMEM media by

39 mM in comparison to the Na+ ion concentration 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 hrs in a

100 µM solution of cisplatin containing 130 mM to 155 mM NaCl and 5 mM HEPES, pH 7.4. As

shown in Fig.2, increasing the concentration of NaCl from 130 mM to 155 mM increased cell

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survival in 100 µM cisplatin from 22+3% to 98+3 % (p<0.001). In the absence of cisplatin, the 3 hr

incubation in the NaCl solutions had no effect on cell viability (p=0.6).

Effect of Sodium Chloride Concentration on Cisplatin Accumulation

Sodium chloride 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 mM 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 were undertaken to determine whether the effect of NaCl on cisplatin

toxicity was due to interaction of the NaCl with the cisplatin or due to an effect of the salt on the

cells. The cells were incubated in media with various concentrations of NaCl for 3 hrs 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 was

removed and all the cells were treated with 100 µM 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 hrs in DMEM with 5% FBS and antibiotics. The data in

Fig. 4 show that the concentration of 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.

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Controls showed that the pretreatment with the media alone (no cisplatin) had no significant effect

on cell viability (p=0.18).

Higher concentrations of NaCl during the pretreatment period resulted in increased sensitivity to

cisplatin toxicity (Fig. 4), while 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, prior to the cisplatin exposure. The medium

was then changed to solutions with 130 mM 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 hrs prior to the cisplatin exposure. All of the cells were then treated with 100 µM 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

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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 II). 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 hrs in a 100 µM solution of cisplatin containing 115 mM

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/kg H2O. 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 hr

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 3hr in solutions containing 100 µM solution of cisplatin, 115

mM NaCl, 5 mM HEPES, pH 7.4 and increasing concentrations of mannitol (a sugar), sucrose (a

sugar), N-methyl-D-glucamine (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 hr incubation in the solutions had no effect on cell viability (data not

shown). The osmolality range tested in these experiments (214 to 288 mosm/kg H2O) is equivalent

to the osmolality range of the NaCl solutions tested in Fig 5 (open diamonds, 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

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Platinum levels were measured in confluent monolayers of cisplatin-treated cells incubated in

115mM NaCl with mannitol. Increasing the mannitol concentration from 19 mM to 75 mM

increased the osmolality of the solution from 232 to 288 mosm/kg H2O. 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 mM 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 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 exerts its protective effect.

DISCUSSION

Small variations in the osmolality among tissue culture media account for their modulation of

cisplatin nephrotoxicity. Sodium chloride 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

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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 of saline intravenously

per day (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 3h 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

135-145mM. Sodium concentrations below 120 mM or above 160 mM are considered pathologic

(13). Physiologic saline (0.85% NaCl) is 145 mM NaCl. The sodium concentration in the tissue

culture media we tested ranged from 138 mM 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

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protection. This scenario has been demonstrated with aminoglycosides which are also toxic to the

proximal tubules. Ticarcillin has been shown to protect against gentamicin nephrotoxicity. Sabra

and Branch demonstrated that the salt load associated with ticarcillin administration was the

primary mechanism by which ticarcillin protected against aminoglycoside nephrotoxicity (31).

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 NaClconcentration was varied prior to 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 3h. 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).

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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 re-adjust their volume by regulatory volume 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 reported that mammalian cells

grown at pH 6.7 had elevated levels of HSP27 and were resistant to cisplatin (36). Changes in the

osmolality of the extracellular medium have also been shown to alter chromatin structure and free-

radical induced 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 occur

extracellularly, catalyzed by the cell surface enzymes GGT and aminodipeptidase. The cysteine-

16

conjugates are metabolized to reactive thiols intracellularly by cysteine-S-conjugate beta-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.

ACKNOWLEDGEMENT

We gratefully acknowledge Dr. Siribhinya Benyajati in the Department of Physiology at The

University of Oklahoma Health Sciences Center for her helpful discussions during the preparation

of this manuscript. This work was supported by RO1 CA57530 (M.H.H.) from The National

Cancer Institute, NIH and a grant from the Presbyterian Health Foundation, Oklahoma City, OK.

17

REFERENCES

Reference List

1. Andrews PA. Cisplatin accumulation. In: Platinum-Based Drugs in Cancer Therapy, edited

by Kelland LR and Farrell NP. Totowa, New Jersey: Humana Press, 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.

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 beta-lyase pathway. In: Advances in

18

Pharmacology, Vol.27, edited by Anders MW and Dekant W. New York: Academic Press

Inc., 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 U S A 99: 4592-4595,

2002.

12. Filipovic D and Sackin H. Stretch- and volume-activated channels in isolated proximal

tubule cells. Am J 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.

19

14. Hanigan MH, Gallagher BC, Taylor PTJr and Large MK. Inhibition of gamma-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.

Gamma-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 gamma-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 U S A

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.

20

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.

21

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 extended-spectrum 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.

22

34. Townsend DM and Hanigan MH. Inhibition of gamma-glutamyl transpeptidase or

cysteine S-conjugate beta-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 WB, 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.

23

__________________________________________________________________________________________ Table I. Composition of Salt Solutions Tested in Fig 1B __________________________________________________________________________________________ Medium Component Saline L-15 HBSS F12 DMEM RPMI __________________________________________________________________________________________ CaCl2 - 1.3mM* 1.3 0.3 1.8 - KCl - 5.4 5.4 3.0 5.4 5.4 KH2PO4 - - 0.4 - - - MgCl2 - 1.0 - 0.6 - - MgSO4 - 0.8 0.8 - 0.8 0.4 NaCl 145.4 136.9 136.9 130.0 109.5 102.7 Na2HPO4 1.5 1.3 0.3 1.0 3.9 5.6 NaH2PO4 0.5 - - - 1.2 - HCl to adjust pH - 0.2 - 0.1 - 1.0 NaOH to adjust pH 0.3 - 0.3 - 0.6 - Na+ 149.2 139.5 137.8 132.0 119.1 113.9 Cl- 145.4 147.1 144.9 134.9 118.5 109.1 ___________________________________________________________________________________________ * all concentrations are mM

______________________________________ Table II. Osmolality of Tissue Culture Media ______________________________________ Osmolality Medium (mosmol/kg H2O) ______________________________________ RPMI 286 F-12 298 L-15 330 DMEM 350 ______________________________________

Fig. 1 Nephrotoxicity of Cisplatin in Tissue Culture Media (A) and Inorganic salt

Solutions (B). Panel A:Confluent monolayers of LLC-PK1 cells were incubated for 3 hrs

in cisplatin in RPMI (open squares), F-12 (closed triangles), DMEM (open diamonds ) or

L-15 medium with 18 mM HEPES and 0.2% BSA (closed circles). Panel B: Confluent

monolayers of LLC-PK1 cells were incubated for 3 hrs in cisplatin in the solutions

containing the inorganic salt component of RPMI (open squares), DMEM with 5mM

NaPO4 (open diamonds), F-12 (closed triangles), HBSS ((horizontal bars), L-15 (closed

circles) and saline buffered with 2mM NaPO4 (Xs). All solutions were adjusted to pH

7.4. For experiments in both panel A and B, the cisplatin solutions were removed at the

end of the 3 hr exposure and fresh DMEM medium with 5% FBS and antibiotics was

added to the cells. Cell survival was assayed at 72 hrs. Each point is the average of 3 to

six values + S.D. Values that differed significantly from the survival in L-15 medium

(panel A) or L-15 inorganic salt solution (Panel B) are indicated by * (p<0.01) or ** (p<

0.001).

0

20

40

60

80

100

0 50 100 150

Cisplatin (mM)

Su

rviv

al(

%)

0

20

40

60

80

100

120

140

0 50 100 150

Cisplatin (mM)S

urv

iva

l(%

)

A B

**

** **

**

***

0

20

40

60

80

100

125 135 145 155

NaCl (mM)

Su

rviv

al(%

)

Fig. 2 Influence of Sodium Chloride Concentration on Cisplatin Nephrotoxicity.

Confluent monolayers of LLC-PK1 cells were incubated for 3 hrs in solutions containing

100 mM cisplatin, with 5 mM HEPES, pH 7.4 and varying amounts of NaCl (open

circles) or in the HEPES/NaCl solutions without cisplatin (closed circles). The test

solutions were removed at the end of the 3 hr exposure and fresh DMEM medium with

5% FBS and antibiotics was added to the cells. Cell survival was assayed at 72 hrs. Each

point is the average of 3 values + S.D.

0

5

10

15

20

25

120 130 140 150 160

NaCl (mM)

Pt (n

g/10

5 cel

ls)

Fig. 3 The Effect of NaCl on the Accumulation of Platinum. Confluent monolayers of

LLC-PK1 cells were incubated for 3 hrs in solutions containing 100 µM cisplatin, with 5

mM HEPES, pH 7.4 and varying amounts of NaCl. At the end of 3 hrs 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 the mean value from 3 experiments ±

S.E.

0

20

40

60

80

100

100 120 140 160 180 200[Na+] (mM) in DMEM

during preincubation

Surv

ival

(%)

Fig. 4 Effect of Preconditioning Cells with Various Concentrations of NaCl on Cisplatin

Nephrotoxicity . Confluent monolayers of LLC-PK1 cells were incubated for 3 hr in fresh

DMEM containing 5% FBS, antibiotics and various concentrations of NaCl. After 3 hrs

the media was removed and all the cells were treated with 100 µM cisplatin in DMEM

(open circles) or DMEM alone (closed circles). The cisplatin solutions were removed at

the end of the 3 hr exposure and fresh DMEM with 5% FBS and antibiotics was added to

the cells. Cell survival was assayed at 72 hr. Each point is the average of 3 values ± S.D.

0

20

40

60

80

100

110 120 130 140 150 160

NaCl (mM)

Surv

ival

(%)

Fig. 5 Effect of NaCl vs. Osmolality on Cisplatin Toxicity. Confluent monolayers of

LLC-PK1 cells were incubated for 3 hr in100 µM cisplatin in 5 mM HEPES containing

NaCl alone (open diamonds), NaCl and mannitol (closed circles), or NaCl and N-methyl-

D-glucamine (open squares). The varying amounts of NaCl were balanced with mannitol

or N-methyl-D-glucamine such that the final osmolality of each solution was 288

mosmol/kg H2O. The cisplatin solutions were removed at the end of the 3 hr exposure

and fresh DMEM with 5% FBS and antibiotics was added to the cells. Cell survival was

assayed at 72 hr. Each point is the average of 3 values ± S.D.

0

20

40

60

80

100

200 220 240 260 280 300

Osmolality (mosmol/kg H2O)

surv

ival

(%)

Fig. 6. Effect of Osmolality on Cisplatin Toxicity. Various concentrations of mannitol

(closed circles), sucrose (closed triangles), N-methyl-D-glucamine (open squares) and

sodium gluconate (closed bar) were added to 115 mM NaCl with 5 mM HEPES.

Confluent monolayers of LLC-PK1 cells were incubated for 3 hr in each of the solutions

containing 100 µM cisplatin. The cisplatin solutions were removed at the end of the 3 hr

exposure and fresh DMEM medium with 5% FBS and antibiotics was added to the cells.

Cell survival was assayed at 72 hr. Each point is the average of 3 values ± S.D.

0

5

10

15

20

220 240 260 280 300

Osmolality (mOsm)

Pt (n

g/10

5 cel

ls)

0

500

1000

1500

2000

2500

3000

3500

4000

220 240 260 280 300

Osmolality (mOsm)

accu

mul

atio

n/(1

/osm

olal

ity)

A B

Fig. 7. The Effect of Osmolality on the Accumulation of Platinum. Confluent

monolayers of LLC-PK1 cells were incubated for 3 hrs in solutions containing 100 µM

cisplatin, with 115mM NaCl, 5 mM HEPES, pH 7.4. Mannitol was used to vary the

osmolarity of the solutions. At the end of 3 hrs 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 the mean value from three to six values + S.D. The data are

expressed as the platinum accumulation per cell (A) and platinum accumulation per cell

with a volume correction (B).

0

50

100

150

120 140 160

NaCl (mM)

Pt (

ng/m

g D

NA

)

`

Fig. 8. The Effect of NaCl on Platinum Bound to DNA. Confluent monolayers of LLC-

PK1 cells were incubated for 3 hrs in solutions containing 100 µM cisplatin, with 5 mM

HEPES, pH 7.4 and varying amounts of NaCl. At the end of 3 hrs 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 + S.D.