Toxicology, 58 (1989) 71--80 Elsevier Scientific Publishers Ireland Limited.

A possible cellular mechanism of cisplatin-induced nephrotoxicity*

Gurmit Singh

Ontario Cancer Foundation, Hamilton Regional Cancer Centre, and McMaster University, Department o f Pathology, Hamilton, Ontario LSN 3Z5 (Canada)

(Received December 27th, 1988; accepted March 8th, 1989)

Summary

Cisplatin, a relatively new antitumor agent, is associated with renal function impairment. The mechanism of cisplatin-induced nephrotoxicity is unknown. A mouse model was used to examine nephrotoxicity induced by cisplatin. This study demonstrates both morphologically and biochemically that mitochondrial damage may be associated with cisplatin-induced cellular toxicity. The morphological changes are evident after 72 h following a single i0 mg/kg i.p. dose of cisplatin. Biochemical changes also follow the morphological abbreviations. In vitro incubation of cisplatin with cells also shows a decline in Rhodamine 123 fluorescence with time, which is indicative of mitochondrial damage. The present findings suggest the possibility that the nephrotoxic effects of cisplatin may be related to a mitochondrial damage.

Key words: Cisplatin; Nephrotoxicity; Mitochondria

Introduction

C/s-diamminedichloroplatinum (cisplatin), a heavy metal chemotherapeutic agent, has been found to be important for treatment of several human malignancies [1,2]. However, its usefulness is limited due to significant impairment in renal function during prolonged use [3--5]. The cellular mechanism of this adverse reaction is unclear at present. Earlier studies indicate that cisplatin exerts its effect on the S-3 segment of the proximal tubule located in the outer stripe of the medulla [6,7]. This region has a large number of mito- chondria compared to other parts of the kidney [8].

Cisplatin has been shown to cause a depletion of sulfhydryl (SH) groups which has been implicated to cause nephrotoxicity [9]. However, trans-diaminedichlo- roplatinum also decreases SH groups in the kidney, but does not cause renal damage. Failure of SH-protective agents such as cystamine, penicillamine and N- acetylcysteine to prevent nephrotoxicity does not support the sulfhydryl depletion hypothesis. Since the histopathological profile of cisplatin nephrotoxicity appears similar to that of other heavy metals, it is often thought that cisplatin nephrotoxicity is related to the platinum moiety [9]. However, cisplatin

Address all correspondence to: Gurmit Singh, Ph.D., McMaster University, Department of Pathology HSC - - 3N26D, 1200 Main St. W., Hamilton, Ontario L8N 3Z5, Canada. *Supported by: Medical Research Council of Canada, Grant MA-8509.

0300-483X/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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nephrotoxicity is stereospecific to the cis and not the trans isomer, indicating that the platinum a tom is not the proximate nephrotoxicant.

The aim of this study was to correlate morphological and biochemical changes in mitochondria following cisplatin treatment because evidence suggests that changes in mitochondrial function may be central to the pathogenesis of cellular damage [10--12]. A multifaceted approach was used to show correlations between cisplatin treatment and mitochondrial damage. Further experiments are in progress to demonstrate the molecular mechanism of cisplatin-induced mitochondrial damage which could ultimately lead to nephrotoxicity.

Materials and methods

Animals and chemicals Male C57BL/6J mice (20--25 g) were obtained from Jackson Laboratories,

Maine. All mice were allowed to acclimatize for a period of at least 4 days after receipt from the breeder and were kept on clay chip bedding. The diet consisted of purina rat chow and water ad libi:um.

Cisplatin and transplatin were purchased from Sigma Co., St. Louis, MO. All other chemicals were of analytical grade.

Animal studies The mice were given a single i.p. injection of 10 mg/kg of either cisplatin or

transplatin. Control mice were given an equal volume of 0.9°70 saline by i.p. injection. Blood samples were obtained from each animal every 24 h for 4 days. The animals were sacrificed by cervical dislocation and the kidneys were removed and placed on ice. One kidney was fixed in glutaraldehyde and processed for electron microscopy and slices of kidney were fixed in 2°70 glularaldehyde buffered in 0.1 M sodium CaCodylate buffer at pH 7.4, postfixed in 1°70 osmium tetroxide in the same buffer, dehydrated in ethanol and embedded in Spurr 's resin [13] while the other was used to prepare mitochondria for biochemical analysis from every animal. Blood was collected from the retro-orbital plexus of the eye. Blood samples were used for analysis of blood urea nitrogen (BUN) and creatinine by autoanalyser to confirm the occurrence of nephrotoxicity induced

by cisplatin treatment.

Cell cultures Cultured human fibroblast cells were grown on 12-mm round glass coverslips

[14]. Cells were incubated with cisplatin 5/ag/ml for 24 h in Dulbecco's modified Eagle's medium. Then they were incubated with Rhodamine 123 (10 tag/ml) for 30 min and washed 3 times and examined by epifluorescent illumination at 546 nm. Photographs were taken by using Kodak Ektachrome 400 film with the automatic exposure control of the microscope set at ASA 6300 and 546 nm exci-

tation.

Mitochondrial studies Mitochondria from kidneys were prepared by differential centrifugation

technique [15] and were purified by Ficoll gradient Preparat ion [16]. Cytochrome

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C-oxidase, a mitochondrial enzyme was measured as a biochemical endpoint using a spectrophotometric technique [17]. Respiration in mitochondria was measured to assess the functional parameter of control vs. treated animal kidney mitochondria by the modified technique of Chance and Williams [18]. A Clark microelectrode (bore = 0.5 cm) with a minimum effective operating volume of 0.5 ml was used. Respiration was assayed polarographically at 30°C in a medium containing 0.25 M sucrose, 15 mM KCI, 1 mM EDTA, 5 mM MgCI 2, 10 mM phosphate buffer and 50 mM T r i - - H C ! buffer (pH 7.4). Succinate (10 mM) was used as the substrate and ADP (85--300 nmol) was added. Carbonyl cyaide m- chlorophenyl hydrazone (M-CCCP) is a potent uncoupler, that is characterized by its ability to release state 4 respiration in the absence of phosphate acceptor. Its mode of uncoupling is different than known inhibitors such as oligomycin [19]. In this study M-CCCP (70--100 nmol) was added. Protein concentration was determined by the Lowry method [20].

Statistical analysis Data were analyzed using an unpaired two-tailed student 's t-test. A probability

level of P < 0.05 was selected as indicating statistical significance [21].

Results

Assessment o f renal function Blood urea nitrogen and blood creatinine are an adequate indicator of

impaired renal function. As shown in Table I, both BUN and creatinine levels are significantly elevated on days 3 and 4 following a single dose of 10 mg/kg of cisplatin. These levels are indicative of renal dysfunction. However, with higher doses of 20 mg /kg and 40 mg/kg , the animals became sick on day 2 or day 1 and died. Due to these observations, we examined kidneys from animals treated with 10 mg/kg only. Furthermore, 20 mg/kg of transplatin did not show any sign of toxicity. This confirms that only the cis stereoisomer is nephrotoxic in mice. Single doses of 5 mg /kg cisplatin did not show any sign of toxicity either,

TABLE I

EVALUATION OF BUN AND CREATININE IN CIS-PLATINUM TREATED MICE

Days Blood urea nitrogen (mg/dl) Creatinine (mg/dl)

Control Cisplatin Control Cisplatin

Day 1 28 ± 10 24 _ 17 1.5 _ 0.8 1.4 ± 0.5 Day 2 37 ± 8 44 _ 10 2.0 _ 0.5 1.9 __. 0.9

Day 3 35 - 11 105 +__ 9* 1.4 ± 0.3 12.7 _ 1.2"

D a y 4 33 ± 15 228 ± 18" I.I ± 0.6 22.4 ± 1.8'

C57BL/6J male mice (20--25 g) were injected i.p. with a single dose of either saline or cisplatin (10 mg/kg) and blood was collected every day from the tail. BUN and creatinine were analyzed by a clini-

cal autoanalyzer. Each determination is a mean of 6 animals __ S.E.M. *P < 0.05.

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however, multiple low doses of cisplatin also caused nephrotoxicity (Data not shown). This situation is analogous to cancer patients being administered low- dose cisplatin for a prolonged period.

Morphology o f cisplatin-treated kidney The administration of 10 mg/kg body weight of cisplatin resulted in

morphological alterations in the S-3 segment of the proximal tubule. The pathologic changes were rarely noticed on days 1 and 2 after cisplatin administration. On days 3 and 4, changes in loss of brush border, pyknotic nuclei, increased cytoplasmic vesicles were all observed as described in the rat [6,221. In this manuscript we concentrated on analyzing changes observed in the mitochondria prior to damage of the brush border. We hypothesized that alteration or damaged mitochondria would result in a multitude of effects due to loss of energy required to maintain structural integrity. In Fig. 1, it is evident that drastic changes in mitochondria occur on day 4 following cisplatin administration. Using the Bolender technique [23] for stereological analysis, we observed a 40% decrease in the number of mitochondria per nuclei as compared to control, and a 54°70 increase on average in surface area of mitochondria (Table II). The increase in surface area could be the result of swelling of mitochondria or due to fusion of mitochondria. On days 1 and 2, only 10--20070 decrease in mitochondria was observed (Data not shown).

Changes in Rhodamine 123 fluorescence We used HF-172 cells to observe if any changes occurred in Rhodamine 123

fluorescence following cisplatin treatment. In Figs. 2(a), (b), and (c), we observed a gradual change in Rhodamine 123 fluorescence. In Fig. 2(a), cells prior to cisplatin exposure demonstrate a discrete distribution of fluorescence that is typical of mitochondria. However, 12 h following cisplatin treatment we observed a more diffuse pattern with some distal projections disappearing, and 24 h after treatment, Rhodamine 123 fluorescence is observed only around the nucleus. At this stage, the cells were still viable according to the Trypan blue exclusion test. The HF-172 cell line is suitable to demonstrate an in vitro effect of a chemical on mitochondrial function. These results corroborate morphologic data obtained from mouse kidneys. Furthermore it reveals a gross time-course relationship with damage. These data are reproducible in vero cells (Green monkey kidney cells). The major reason for using HF-172 cell line instead of freshly cultured mouse kidney cell was due to the known characterization of this cell line for mitochondrial distribution in live cells with Rhodamine 123. [141

Biochemical evidence o f renal mitochondrial damage Mitochondrial protein concentration in control and cisplatin-treated animals

were similar. However, respiration rates of mitochondria obtained from cisplatin- treated mice kidney were at least 35% lower as compared to control. Similarly, one of the main proteins, cytochrome C-oxidase, involved in the respiratory chain, was also decreased by 36°70 as shown in Table II1. The change in respiration was not due to uncoupling of mitochondria because in the presence of

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m

Fig. i. Electronmicrographs obtained from S-3 segment of the proximal tubule of the mouse kidney. BB, Brush border; M, mitochondria; N, nucleus; bar represents I tam; (I) saline control; (2) cisplatin- treated (10 mg/kg) 4 days after treatment. (9000 × ).

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TABLE I1

MORPHOMETRIC ANALYSIS

Treatment No. of mitochondria Average area of mitochondria

Saline 90 ± 10 109 ± 34 Cisplatin 10 mg/kg 54 ± 15" 171 __. 34*

Data expressed as means __. S.E. The data were obtained by analyzing at least 15 micrographs for each group. *P < 0.05.

uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) a similar difference between control and animal-treated renal mitochondria was obtained.

Discussion

Cisplatin-induced nephrotoxicity represents a life-threatening complication and is a major limiting factor in its widespread use as an antitumor agent [24,251.

B 9 -

O Fig. 2. Rhodamine 123 fluorescence in HF-172 cells. Cells were grown on coverslips in RPMI. Prior to visualization, cells were incubated with 10 /ag/ml Rhodamine 123 in RPMI for 30 rain. The dye was then washed from the coverslip with three 5-ml changes of media. The cells were examined by epifluorescent illumination at 546 nm (a) control HF-172 cells; (b) HF-172 cells treated with 5 tag/ml cisplatin for 24 h; (c) HF-172 cells treated with 5 tag/ml cisplatin for 48 h.

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'--,1

TABLE III

BIOCHEMICAL ANALYSIS OF RENAL MITOCHONDRIA

Treatment Succino-oxidase activity [Cyt. C-Oxidasel Protein (mg/ml) ~,moles O: /min /mg protein aa~

(605 nm-630 nm)

ADP Stimulated In presence of CCCP

Control 107 _ 6 105 ± 5 110 ± 9 (I.58 ± 0.04 Cisplatin 70 __. 5* 73 _.+ 3" 70 _.+ 7* 0.52 ± 0.05

Mitochondria were prepared from C57BL/6J mouse kidneys 4 days following a single i.p. dose of cisplatin (10 mg/ml), and also from saline-treated mice. Succino-oxidase activity was measured using succinate as substrate with a Clark oxygen electrode. [Cytochrome C-oxidase] was measured from solubilized mitochondrial spectra. Each determination is a mean of 6 animals _ S.E.M. "P < 0.05.

This study indicates that cisplatin toxicity in mice is similar to the toxicity observed in humans and rats. We have postulated a novel mechanism at the cellular level in an attempt to explain nephrotoxicity based on our data. Our results show a good correlation between nephrotoxicity and mitochondrial damage. These biochemical, morphological and functional alterations induced by cisplatin in renal mitochondria provide evidence to support the hypothesis that mitochondria could be the primary target for toxicity. This hypothesis is further supported by experiments in isolated renal tubules that have shown significant inhibition of Na÷-K" ATPase activity as well as Ca2~-Mg 2" ATPase, thus affecting the mitochondrial synthesis of ATP, the Na'-K ÷ pump and the Ca 2. pump [6]. Subcellular accumulation of cisplatin in mitochondria is also supportive of our hypothesis [26]. Recent data has shown change in mitochondrial function in cis- platin-induced tubular damaged kidneys, thus linking renal dysfunction with mitochondrial dysfunction which supports our data [27].

The stereoisomer, transplatin, does not induce nephrotoxicity, however, it decreases sulfhydryl proteins in the kidney and this is evidence that neither platinum atom nor depletion of SH-groups are responsible for cisplatin induced nephrotoxicity. Therefore, the platinum(II) complexes have the additional requirement that the 2 functional groups be in the cis arrangement [28,29].

Currently, there is considerable evidence that DNA is the principal intracellular target of cisplatin. Since there are 2 distinct genomes in a cell, namely mitochondrial and nuclear, we postulate that the mitochondrial DNA damage induced by cisplatin causes nephrotoxicity.

The rationale for postulating that mitochondrial DNA could be the major target for cisplatin is: (a) mitochondrial DNA is less closely associated with protein than nuclear DNA [30] and, hypothetically, would therefore be more accessible to attack by aquated species of cisplatin; (b) DNA repair mechanisms are abundant in the nucleus but sparse in mitochondria [31]; and (c) cisplatin accumulates in higher concentration in mitochondria compared to the nucleus [261.

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Since cisplatin induced nephrotoxicity is a result of prolonged treatment [10,32], it is consistent with our hypothesis because with every dose of cisplatin which a patient receives, a few mitochondria are damaged and over prolonged treatment the number of viable mitochondria decreases and thus cannot sustain a ceil. In addition, the damage of mtDNA is lethal to the organelle because it codes for several critical mitochondriai inner membrane proteins including subunits of cytochrome oxidase and ATPase [32].

Finally, the data obtained with HF-172 cells and Rhodamine 123, a specific mitochondrial dye, are indicative of mitochondrial damage with cisplatin before the cell dies. The effects of cisplatin are observed after 24 or 48 h. This time- course is consistent with turnover of mitochondrial proteins. That is, if mitochondrial DNA is damaged, then denovo synthesis of mitochondrial proteins would not occur and thus degradation of the organelle would result. Further investigations are necessary to determine whether mitochondrial DNA is the ulti- mate target for cisplatin induced nephrotoxicity. These studies are currently being conducted in our laboratory.

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