Effects of Mannitol Or Furosemide Diuresis on the Nephrotoxicity and Physiological Dispositionof Cis-Dichlorodiammineplatinum-(Ll) in Rats1

Home , Nephrotoxicity

(CANCER RESEARCH 39, 1269-1278, April 1979] 0008-5472/79/0039-0000$02.00 Effects of Mannitol or Furosemide Diuresis on the Nephrotoxicity and Physiological Dispositionof cis-Dichlorodiammineplatinum-(ll) in Rats1

Martin F. Pera, Jr.,2 Bernard C. Zook, and Harold C. Harder3

Departments of Pharmacology (M. F. P., H. C. H.J and Pathology (B. C. Z.J, The George Washington UniversityMedical Center, Washington, D.C. 20037, and Departmentof Pharmacology,OralRobertsUniversity,Tulsa,Oklahoma,74171(H.C. HJ

ABSTRACT platinum. However, a mannitol-induced diuresis may re duce the duration of tubular necroses. It is possible that' Although furosemide and mannitol have been reported to while tubular necrosis might be related to cumulative plati protect against cis-dichlorodiammineplatinum(ll) (COOP)- num uptake in the kidney, the reduction of platinum con induced nephrotoxicity, the possibility that these diuretics centration in the tubular fluid caused by the diuretics might might act in different ways to alter the physiological dispo account for the protection of renal function. sition) and in vivo antitumor activity of COOP has not been adequately assessed. Therefore, we compared the effects INTRODUCTION of furosemide (12.5 mg/kg i.p. with 6.0 ml 0.9% NaCI solution) and mannitol (300 mg in 3.0 ml 0.45% NaCI COOP4 is an inorganic coordination complex which dis solution as a 30-mm i.v. infusion) on the nephrotoxicity and plays activity against a variety of experimental and human physiological disposition of COOP (6 mg/kg i.v.) in male neoplasms (5, 14, 15, 25). However, administration of the F344 rats. Serial histopathological evaluation of kidneys compound at therapeutic doses produces renal toxicity in indicated that an approximately equivalent degree of proxi rats, dogs, monkeys, and humans (8, 12, 14, 26, 32). mal tubular necrosis occurred in rats given COOP alone or Clinical interest in CODP has increased considerably since with either furosemide or mannitol 1 to 4 days after drug the publication of several reports indicating that induction administration. Thereafter, a trend developed toward less of diuresis prior to COOP administration to cancer patients persistence of damage in the mannitol groups and progres ameliorated the renal damage caused by the drug (3, 13, sion of tubular injury in furosemide-treated rats compared 20). However, the possibility that various diuretics might act to COOP alone (5 to 10 days after drug administration). in different ways to alter the physiological disposition, However, renal function, assessed by measurement of toxicities, and the in vivo antitumor action of CDDP has not blood urea nitrogen, estimation of glomerular filtration rate, been adequately assessed. Therefore, we have undertaken and p-aminohippurate clearance was partially protected in to investigate the toxicological, pharmacokinetic, and ther rats given either diuretic. apeutic aspects of the interaction between COOP and di The administration of furosemide or mannitol did not uretic drugs. In this paper, we evaluate the effects of markedly affect the triphasic plasma decay of platinum. A furosemide- and mannitol-induced diuresis on CDDP renal similar uptake of platinum into spleen and small intestine toxicity and physiological disposition in rats. In an accom was seen at early time points (2 mm to 2 hr) in groups given panying report (23), we describe effects of diuretics on COOP alone or with either diuretic. Kidney platinum content acute lethality, gastrointestinal and hematopoietic toxicity, in rats receiving mannitol was similar to that of animals and antitumor activity of COOP. receiving COOP alone. Kidneys of furosemide-treated ani Cvitkovic et a!. (7) found that coadministration of COOP mals contained higher levels of platinum than did kidneys with mannitol or large doses of i.v. fluids prevented the rise of rats given COOP alone or with mannitol at 1, 2, 24, and in BUN and serum creatinine which occurs following COOP 96 hr after treatment. Total 24-hr urinary excretion of administration at toxic doses in dogs. These investigators platinum was decreased, although not significantly, by both also presented some data to suggest that the level of diuretics. Furosemide or mannitol diuresis resulted in a platinum in serum after COOP was similar in dogs given significant decrease in platinum concentration in 0- to 24- mannitol or prehydration. The data provided no comparison hr urine samples. Thus, under these conditions the partial with dogs given COOP alone. The same study stated that protection of renal function observed with diuretics is not the mannitol and prehydration treatments did not alter total the result of increased excretion of platinum in urine, faster urinary clearance of platinum, although apparent increases plasma clearance of platinum, or decreased renal levels of in the recovery of platinum in urine were reported in prehydration and mannitol groups. I Supported by Grant Cl-107 from the American Cancer Society and by Grant CA-02978fromthe NationalCancerInstitute, Departmentof Health, Ward et a!. showed that the male F344 rat is sensitive to Education,and Welfare.A portion of this work was presentedpreviouslyin the nephrotoxic effects of CODP (32) and that renal dam abstractform (24). age, evidenced by elevation of BUN and necrosis of cells in 2 From a dissertation to be presented to the Graduate School of Arts and Sciences, The George Washington University, in partial fulfillment of the the proximal tubule, could be ameliorated by administration requirements for the Ph.D. degree. 3 To whom all correspondence should be addressed at Oral Roberts 4 The abbreviations used are: CDDP, cis-dichlorodiammineplatinum(lI); University. Tulsa, Okla. 74171. BUN,blood urea nitrogen; DTPA,diethylenetriaminepentaacetate;PAH,p ReceivedJuly24, 1978;acceptedJanuary3, 1979. aminohippurate; [3HJPAH,p-[glycyI-2-3H@aminohippuric acid.

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of furosemide prior to the platinum drug on an acute or 5 to 6 weeks of age, the animals were separated into 4 chronic basis (33, 34). In contrast to the results of Cvitkovic treatment groups. The first group received 0.5 ml of 0.9% et al. (7)withmannitolindogs,the reportof Wardet al. (34) NaCI solution i.v. All i.v. injections were made into the mentioned unpublished work indicating that furosemide lateral tail vein using a 26-gauge x 0.5-inch needle. The treatmentofratsresultedinincreasedlevelsofplatinumin second group received COOP (6 mg/kg i.v.), the highest blood and kidney. We therefore extended the studies of dose of COOP that can be administered to these rats these groups to provide a more detailed comparison of the without producing acute lethal toxicity. The third group effects of furosemide and mannitol on the nephrotoxicity received furosemide (12.5 mg/kg i.p.) in 3.0 ml 0.9% NaCI and physiological disposition of COOP in the rat. solution 30 mm prior to COOP (6 mg/kg i.v.); 4 hr after Since data on BUN levels and renal histopathology did COOP administration, these animals were given 3.0 ml 0.9% not always agree in our experiments (24), we evaluated NaCI solution i.p. to reduce the possibility of excessive fluid additional parameters of renal function in our studies. or electrolyte derangement. The last group received man Administration of other nephrotoxic compounds, such as nitol infusions, performed after animals were lightly anes mercuric chloride and uranyl acetate, causes a decrease in thetized with pentobarbital (25 to 30 mg/kg i.p.) to facilitate glomerularfiltration (9, 11). Therefore, we used [â€œln]OTPA insertion of the 27-gauge x 0.5-inch needle of an infusion blood clearance to estimate the effects of COOP alone or catheter set (EZ-Set Infusion Set; Deseret Pharmaceutical with diuretics on glomerulan filtration rate. Because other Co., Sandy, Utah) into the lateral tail vein. The rats were agents causing proximal tubular necrosis, such as paraquat placed in restraining devices with their tails secured. After or uranyl acetate, depress the organic acid secretion system the i.v. line was primed with 0.5 ml heparinized mannitol in vivo (10, 11) and because the pars recta of the proximal solution, an infusion of 10% mannitol in 0.45% NaCI solu tubule, the site of organic acid secretion (31), is apparently tion was begun at the rate of 0.10 mI/mm using a Harvard most sensitive to COOP-induced damage in the rat (32), we Infusion Pump Model 975 (Harvard Apparatus Co. Inc., measured clearance of PAH as a further indication of Millis, Mass.). Following 25 mm of mannitol infusion, COOP nephron function. In other experiments, we studied the (6 mg/kg) was injected as a bolus into the freely flowing i.v. effects of furosemide and mannitol on the physiological line. The infusion was continued for an additional 5 mm so disposition of COOP by monitoring plasma, urine, kidney, that rats received a total of 300 mg mannitol in 3.0 ml 0.45% intestine, and spleen levels of platinum, the active center of NaCI solution. COOP, at various time points. Through these measure Collectionof PlasmaTissue, and Urine Samples. Four ments, we hoped to determine if diuretics were causing series of experiments were performed to study BUN levels, major alterations in the disposition of COOP, a compound kidney pathology, and platinum disposition. In Experiment which is eliminated predominantly by the kidneys. 1, ratsin each treatmentgroup were housed in plastic cages. Blood samples (150 to 200 @tI)wereobtained from MATERiALS AND METHODS the netroorbital sinus using hepaninized Natelson capillary tubes 1 and 2 hr after COOP administration; these samples Chemicals. COOPwas obtainedfrom the Drug Liaison were spun down in a Bninkmann Model 3200 Micro Centri and Distribution Section, Division of Cancer Treatment, fuge (Brinkmann Instruments, Inc., Westbury, N. V.) to National Cancer Institute, NIH, Bethesda, Md. and diluted obtain plasma, which was later analyzed for platinum con to 1 mg/mI in 0.9% NaCI solution immediately prior to use. tent. Four days later, the animals were anesthetized with Furosemide, a gift of Hoescht-Roussel Pharmaceuticals, ether, and blood was removed from the abdominal aorta Sommerville, N. J., was diluted so as to yield a dose of 12.5 using a heparinized syringe. After centnifugation, the mg/kg in 3.0 ml sterile 0.9% NaCI solution. Mannitol, plasma was analyzed for platinum content and BUN. Fol obtained from Sigma Chemical Co., St. Louis, Mo., was lowing exsanguination of the rat, the left ventricle was prepared as a 10% solution in 0.45% sterile NaCI solution. cannulated, and the tissues were slowly perfused with 0.9% [111InJOTPAwas purchased from Diagnostic Isotopes, Inc., NaCI solution. One kidney was fixed in 10% buffered for Bloomfield, N. J. and diluted in 0.9% NaCI solution to an maim and sectioned at 5 to 6 @m,andthe sections were activity of 20 @Ci/mlat the time of injection. [3H]PAH was stained with hematoxylin-eosin and periodic acid-Schiff obtained at a specific activity of 1.26 Ci/mmol from New stains.Thecontralateralkidneywas analyzedforplatinum England Nuclear, Boston, Mass. The compound was pre content. pared as a 10 pCi/mI solution in 0.9% NaCI solution. Chloral Experiment 2 was performed in a similar fashion, except hydrate was obtained from Sigma Chemical Co. and diluted that blood samples were obtained prior to COOP adminis to 75 mg/kg in 0.9% NaCI solution. Pentobanbital was a tration and at 3 hr, and 3-, 4-, and 5-day time points product of Veterinary Laboratories Inc., Lanexa, Kans. thereafter by retroorbital sinus bleeding. At 7 days, animals Drug Treatment. Male F344 rats weighing75 to 100 g were sacrificed, and terminal blood and kidney samples were obtained from Microbiological Associates, Bethesda, were obtained as described above. The 0-, 3-, 4-, 5-, and 7- Md . or Charles River Breeding Laboratories, Wilmington, day plasma samples were analyzed for BUN; 3-hr samples Mass. and fed a diet of standard lab chow (Wayne Lab were analyzed for platinum, and the kidneys were prepared Blox, Allied Mills, Chicago, Ill.) and water ad !ibitum. They for platinum analysis and histopathology as described were housed in groups of 5 to 6 animals in plastic cages above. with corncob bedding (Experiments 1 and 2), stainless steel In Experiment 3, rats were housed in plastic metabolic wire bottom cages (renal function study and Experiment 4), cages (Maryland Plastics, Inc., New York, N. Y.). After an or individually in plastic metabolic cages (Experiment 3). At adjustment period of 2 days, the rats were given the de

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scnibed drug treatments. Four, 8, and 12 mm after CDDP toxicity described in an accompanying paper (23). These administration, blood samples were obtained from the kidneys were processed and examined in the same fashion, retroorbital sinus. At 24 hr, the animals were sacrificed, and and we report the results here for purposes of confirmation. blood and kidney samples were obtained as described Platinum Assay, Determinationof Radioactivity, and above. Plasma samples from each time point and one BUN Determination. Platinum was measured by flameless kidney from each rat were analyzed for platinum content. atomic absorption as described previously (22). â€œInwas The contralateral kidney was prepared for histopathological counted in a Model 1195 Automatic â€˜yCounting System evaluation. Urine output and water intake volumes for the (Amersham/Searle, Arlington Heights, III.) using a window 24-hr period were determined, and an aliquot of urine from 125 to 300 keV. Plasma samples of [3H]PAH were collected under mineral oil was removed for platinum anal counted in 10 ml of Biofluor aqueous scintillation fluid (New ysis. England Nuclear, Boston, Mass.) using a Beckman LS-255 In Experiment4, ratshoused in stainlesssteel,wire Liquid Scintillation Counter (Beckman Instruments, Inc., bottom cages were given COOP alone or with diuretics. At Fullerton, Calif.) and external standard quench calibration. 2- and 15-mm and 1- and 2-hr time points, the animals were BUN was determined by colorimetry using the diacetylmo sacrificed, and kidneys, spleens, and sections of proximal noxime reagent (6). duodenum were removed and rinsed in 150 mM NaCI Statistical Analysis. Results were analyzed first by single solution. Approximately 500 mg of each tissue was homog factor analysis of variance; if a significant difference be enized in 10 ml ice-cold 150 mM NaCI solution. Two aliquots tween means was indicated, comparisons of means were were removed; one for determinations of platinum and a carried out using the Student-Newman-Keuls multiple range second for measurement of protein by the method of Lowry test (35). The histopathological ratings were evaluated us et al. (18). Results were then expressed in terms of ng of ing nonparametric statistics, first by the Kruskai-Wallis test platinum per mg protein. and then by the Mann-Whitney U test with error rate set on Renal Function Experiments. To estimate glomerular a pen experiment basis (16). Differences were considered filtration rates, we measured clearance of [â€œln]DTPA(4, significant at the level ofp < 0.05. 28) using a single injection clearance technique (1, 2). Three days after drug treatment, rats (housed in stainless RESULTS steel, wire-bottom cages) were anesthetized with chloral hydrate (300 mg/kg i.p.) and given injections of 10 pCi of Body Weight and 24-Hr Fluid Balance. Chart 1 shows [1111n]DTPA i.v. Blood samples (100 @l)weneobtained at 5, results from Experiment 2, where the nadir of weight loss in 10, 15, 20, 40, 50, 60, and 70 mm by retroorbital puncture, treated rats occurred on Day 5 (5 days after administration) and at the final point, the rats were sacrificed. After plasma with body weights 88.8 Â±3.4%, 83.2 Â±1.9%, and 89.8 Â± was obtained and counted as described below, a biphasic 4.5% (S.E.) of Day 0 weights in rats given COOP, COOP with plasma decay curve was plotted and clearance calculated furosemide, and COOP with mannitol, respectively. By Oay using the formula 7, all rats were gaining weight. No significant differences in weight loss among treatment groups were noted, although C â€”dose x b1b2 furosemide-treated rats consistently lost more weight. The @ Ab2 Bb 120 where C is clearance in mI/mm, dose is total dpm adminis tered, b1 is slope of slow terminal component, b2 is slope of 0 @ early rapid component, A is intercept of slow terminal 110 component, and B is intercept of early rapid component. z 0 Clearance of PAH was estimated 3 days after drug treatment U 0 using an i.v. dose of 15 @tCiof[3H-PAH] per rat and a single >. 100 4 injection technique similar to that described above for 0 [â€œln]DTPA(2). 0 Renal Histopathology.Stainedsectionsof kidneysfrom @90 Experiments 1, 2, and 3 were studied under light micros I.. copy and assigned a score which was a relative index of the I @ degree of tubular necrosis on a scale of 0 to 5 (0, no 80 necrosis; 5, extensive necrosis). The extent of tubular 0 epithelial regeneration was graded in a similar fashion. The 0 @ sections were evaluated on at least 2 different occasions; in 70 no case did the score vary more than one grade between examinations. When variance occurred, the 2 scores were 0 averaged. The observer scoring the slides was unaware of the code identifying the treatment group to which each TIME POST TREATMENT IDAYSI specimen belonged . Because histopathological results Chart 1. Effect of COOP (6 mg/kg iv.) alone or with diuretics on body from this set of experiments did not correlate with renal weight. Data are from Experiment 2. Values are mean weights relative to Day 0 Â±SE. , versus time after drug injection in days, with n = 6 for each group. function studies, we also studied kidneys from rats given 0, control; â€¢,COOP;A, COOP with furosemide; â€¢,COOPwith mannitol. identical treatments in a later experiment on bone marrow There are no significant differences among treatment groups.

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inhibition of weight gain seen in control animals during Days 3 to 5 may be due to the repeated bleeding from the retroorbital sinus, a procedure which induces a degree of stress (21). In a later experiment reported in the following 100 paper where no bleeding was performed, it was found that recovery of body weight in mannitol-treated rats signifi cantly outpaced that from other treatment groups during this period (23). Chart 2 from Experiment 3 indicates fluid output (as urine volume) and fluid intake (as water consumption plus injec tions or infusions) for the 24-hr period following drug administration . Mannitol and furosemide groups excreted significantly greater urine volumes (p < 0.05 by the multiple a, E range test) compared to COOP or 0.9% NaCI solution z treated control groups. The apparent positive fluid balances seen are due to evaporation of urine and the fact that insensible fluid loss is not measured. However, since the net fluid balance appears similar for the groups, it is unlikely that diuretic-treated animals were significantly de hydrated due to increased urine output. Evaluation of fluid balance over the short period immediately following COOP administration proved difficult because of variable urine output in animals not given diuretics. However, in other studies, we were able to show that hematocnit values and plasma chloride concentrations were within control limits at the time of COOP administration. Renal Toxicity:BUN Measurements.Serial BUN deter minations from Experiments 1 and 2 are shown in Chart 3. A dose of COOP (6 mg/kg i.v.) produced significant eleva TIME POST TREATMENT IDAYSI tion of BUN above control levels to a peak of 98.8 Â±24.0 Chart 3. Effect of COOP (6 mg/kg iv.) alone or with diuretics on BUN mg/dl on Day 5, followed by incomplete recovery toward levels. Data are from Experiments 1 and 2. Values are mean BUN in mg/dl Â± control levels on Day 7. Furosemide and mannitol groups SE. versustime in daysafter drug injection,with n = 6 exceptas noted in parentheses.0, control; C, COOPalone; A, COOPwith furosemide;U, had BUN levels that were elevated, although not signifi COOPwith mannitol. @,valuessignificantly larger than other groups; p < 0.05.

cantly, above control; the BUN levels in these rats were significantly below those in rats given COOP alone (COOP > furosemide, mannitol, or control on Days 3, 4, 5, and 7; p < 0.05 by the multiple range test). RenalHistopathology.AftertreatmentwithCOOP(6 mg/ kg i.V.) alone or with diuretics, a mild degree of early E degenerative changes was observed on Day 1. The lesions â€˜U were limited to epithelial cells of the straight proximal tubules in the outer stripe of the outer zone of the medulla. -I 0 The changes consisted of early pyknosis and cytoplasmic > vacuolation with focal loss of the brush border and intralu Â£ minalhyalinecasts.LesionsonDay 3 were more advanced, consisting of pyknosis, karyorrhexis, karyolysis, and cyto 0 plasmic eosinophilia with partial loss of the brush border. Necrotic epithelial cells were sloughed into tubular lumina. In addition to the straight proximal tubules, a few convo luted tubular cells were affected to a lesser degree. The necrotizing changes were more advanced by Day 4 and even more marked by Day 7. Many necrotic epithelial cells CONTROL CDDP COOP CDDP dotted with chromatin debris were contained in the lumina WITH WITH of the straight proximal tubules (Figs. 1 to 4). Lesions FUROSEMIDE MANNITOL outside of the outer stripe were much less marked, but Chart 2. Effect of COOP (6 mg/kg iv.) alone or with diuretics on 24-hr necrotic tubular cells were observed in the proximal tubules fluid balance. Data are from Experiment 3. Values for water intake and urine output are mean Â±S.E. with n = 6, except as noted in parentheses. @,urine in medullary rays and especially in tubules adjacent to the outputs significantly greater than control or COOPalone; p < 0.05. capsule at the top of medullary rays. By Day 10, the

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necrotizing changes were much less marked, but lumina of affected tubules were dilated. Tubular dilation first ob served on Day 7 was much more marked by Day 10 and involved tubules in the outer stripe and, to a lesser degree, 0 tubules in medullary rays. Distal tubules (even in the outer U stripe) and collecting tubules, as well as glomeruli were S 0 largely unaffected except an occasional pyknotic nucleus. K a Regenerative changes were first observed in occasional S outer stripe proximal tubules on Day 3; they were much more obvious by Day 4 and continued to involve more tubules by Days 4 through 10. The regenerative cells had large nuclei with prominent chromatin granules, nucleoli, and extensive basophilic cytoplasm (Figs. 2 to 4). Such TIME POST TREATMENTIDAYSI regenerating epithelial cells were often seen flattened be Chart 4. Effect of diuretics on the degree of proximal tubular necrosis tween tubular basement membranes and dying proximal after COOP(6 mg/kg iv.) Dataare from Experiments1, 2, and 3, and one additional experiment. Values are indices of proximal tubular necrosis tubular epithelia. As the necrotic epithelia sloughed in the versustime in daysafterdrug injectionon a scalefrom 0 = no necrosisto 5 lumina, the large regenerating cells took their places (Fig. = extensive necrosis. Individual ratings for each rat as well as median ratings 4). Some of the regenerative cells underwent karyorrhexis are shown. Symbols for individual ratings: oâ€¢,COOPonly; E@A,COOPwith furosemide; DU, COOP with mannitol (0, t@,and 0 and â€¢,A, and U are and death; a few appeared to die during mitosis. from 2 separate series of experiments). Median ratings: C, COOP only; A, Chart 4 plots the course of the indices of necrosis from COOPwithfurosemide;U, COOPwithmannitol. individual rats, as well as the median indices, as a function of time for the 3 treatment groups. Treatment with diuretics Table 1 did not have a noticeable effect on the site of necrotizing EffectrateRats of CDDPalone or with diuretics on glomerular filtration changes within the kidney. There were no significant differ COOP(6were given injections of 0.5 ml 0.9% NaCI solution or glomerularfiltrationmg/kg iv.) alone or with diuretics. Three days later, ences in the degree of tubular necrosis on Days 1, 3, or 4. clearanceusingrate was estimated from [â€œln]DTPA blood Thereafter, particularly on Days 5 and 7, there was a trend a single injection technique (1 , 2).[1 towards longer persistence of necrotizing processes in @@ clearanceTreatment ln]OTPA furosemide-pretreated rats, and a tendency for mannitol (ml/min/loogbodywt)Control n pretreated animals to display less damage compared to rats Â±COOP 6 0.926 given COOP alone (Figs. 2 to 4). Analysis of variance 0.00629k'COOP 6 0.0752Â± showeda differenceinthe3 groupsonDays5 and 7 (p< 8bCOOPwith furosemide 5 0.529 Â±0.11 0.05 by the Kruskal-Wallis test), but the data were insuffi with mannitol 5 0.294 Â± cient to allow a localization of the difference within groups a Values are mean Â±S.E. determined 3 days posttreatment. (per experiment, p > 0.05 by the Mann-Whitney U test). The b Control > COOP plus furosemide > COOP plus mannitol > time course of appearance of regenerative lesions tended COOP;p < 0.05. to be accelerated in mannitol-treated rats and to lag some what in rats given furosemide. As necrotizing lesions sub Table 2 sided on Days 7 and 10 in rats receiving COOP with clearanceRatsEffect of CDDPalone or with diuretics on PAH mannitol, there were also relatively fewer regenerating cells COOP(6were given injections of 0.5 ml 0.9%NaCIsolution or PAHclearancemg/kg i.v.) alone or with diuretics. Three days later, compared to the other treatment groups. Because the radiolabeledcompoundwas estimatedfrom blood clearanceof the histopathological evaluation of kidneys from Experiments using a single injection technique (2).PAH 1, 2, and 3 was in conflict with the observed protection clearanceTreatment against BUN elevation, another set of kidneys from an wt)Control n (ml/min/100 g body experiment in which rats were given identical drug treat 0190a.bCOOP 5 2.59 Â± ments (23) was also evaluated histopathologically. The 0â€¢084bCOOP 5 0.530 Â± above description and Chart 4 incorporate results from this 0.225@@COOPwith furosemide 5 1.59 Â± latter study. with mannitol 5 1.57 Â±0.186@@ GlomerularFiltrationand PAHClearance. BecauseBUN a Values are mean Â±SE. determined 3 days after treatment. measurements and histopathological results did not come b Control > COOP with furosemide or COOP with mannitol > late, additional parameters of renal function were meas CODP;p <0.05. ured. Glomerular filtration, estimated from plasma clear ance of [â€œln]DTPA, was markedly depressed 3 days after than that of untreated controls (COOP < furosemide = COOP (6 mg/kg) (Table 1). Rats given furosemide or man mannitol < control; p < 0.05 by the multiple range test). nitol with COOP had glomerular filtration rates which were Thus, diuresis as induced in these studies partially pro significantly higher than those in animals given COOP alone tected renal function, as measured by these parameters. but also significantly lower than those of untreated controls Effect of Diuresison the Dispositionof Platinum.The (COOP < mannitol < furosemide < control; p < 0.05 by the plasma decay of platinum following a dose of COOP (6 mg/ multiple range test). Clearance of PAH (Table 2) was also kg) is illustrated in Chart 5, a composite curve constructed significantly depressed by COOP. Again, diuretic treatment from Experiments 1, 2, and 3. Tniphasic decay was ob significantly lessened the severity of the depression, but the served, with half-lives of 8.8 and 49 mm and 1.7 days for a, PAH clearance rate in diuretic-treated rats was still lower 13,and y phases in animals given COOP only. Neither

APRIL 1979 1273

z â€˜U 0 a.a, E a. U) 4 0 0 S z 4 4 z

TIME POST TREATMENT (MIN)

TIME POST TREATMENT ml.) Chart 5. Effect of diuretics on plasma decay of platinum after COOP (6 mg/kg iv.) Composite curves constructed from Experiments 1, 2, and 3. z Valuesare mean plasmaplatinum concentrationsin pg/mI Â±SE. versus â€˜U time in mm (lowerpanel)or hr (upperpanel)with n = 6 exceptas noted in 0 parentheses.Upperpanel, continuationof lower panel on a different time a. scale with 180-mm points replotted. â€¢,COOPalone; A, COOPwith furosem E ide; U, COOPwith mannitol. @,valuessignificantly greater than COOPalone, p < 0.05. a. Cl) 4 diuretic had marked effects on the rate of plasma decay. C, 0 Significantly higher plasma platinum levels during the sec z 4 ond phase of plasma decay were observed in furosemide z treated rats compared to rats receiving COOP only for 2- and 3-hr time points (p < 0.05 by the multiple range test). Mannitol treatment did not alter plasma platinum levels TIME POST TREATMENT(MIN) significantly. The lower plasma platinum levels seen in this group at 4, 8, and 12 mm may be the result of an expansion of the central compartment by rapid infusion of hypertonic solute. Chart 6 shows results from studies of platinum levels at early time points following administration of COOP in spleen, small intestine, and kidney, all of which are suscep z tible to COOP toxicity. Significantly lower spleen platinum â€˜U levels were measured in both furosemide and mannitol 0 groups than in spleens from rats receiving COOP alone 2 a. mm after COOP (p < 0.05 by multiple range test). Thereaf E ten, no significant differences were seen in platinum content a: among the 3 treatment groups in spleen or in small intestine Cl) at any time points. In contrast, kidneys from mannitol 4 treated rats had significantly lower platinum levels than did C,0 those of the other groups 2 mm after COOP administration. z 4 At 15 mm post-COOP injection, there were no differences z among the groups. However, by 60 and 120 mm, kidneys from funosemide-treated rats had significantly higher plati num content than did kidneys from rats given either COOP alone or COOP plus mannitol. This latter finding is consist ent with more extensive studies at later time points (below). On the basis of data on the amount of platinum associated TIME POST TREATMENT(MIN) with acid-insoluble material, it appears that the slow rate of Chart 6. Effect of diuretics on spleen (a), small intestine (b), and kidney (c) platinumcontentat earlytime pointsafterCOOP(6mg/kg i.v.). Dataare loss of platinum from the above tissues was the result of from Experiment 4. Values are mean tissue platinum concentrations in ng/ rapid binding to macromoleculam components and that the mg tissue protein Â±SE. versus time in mm, with n = 3 except as noted in diuretics did not affect this process. However, much addi parentheses. â€¢,COOP alone; A, COOP with furosemide, U, COOP with mannitol. @,valuessignificantly higher than those of all other treatment @ tional work is required to substantiate this conclusion. groups. values significantly lower than those of all other treatment Table 3 shows kidney platinum levels at 24 and 96 hr groups (p < 0.05 by multiple range test).

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post-drug administration . Kidneys from furosemide-treated recovered in 0 to 24 hr urine samples. Both furosemide and animals contained significantly more platinum at 24 hr than mannitol tended to decrease, although not significantly, the did those from mannitol-treated rats (p < 0.05 by multiple amount of platinum excreted in urine over a 24-hr period. range test). At 96 hr, kidneys from the funosemide group The 2 diuretics did cause a significant drop in the concen contained more platinum than did those from animals given trationofplatinumin0-to24-hoururinesamples,shown in COOP on COOP plus mannitol (p < 0.05 by the multiple Chart 7b (COOP < furosemide = mannitol; p < 0.05 by the range test). Mannitol-treated animals did not differ signifi multiple mangetest). Thus diuresis did not cause a more cantly from those given COOP alone in terms of kidney rapid plasma clearance, lower tissue platinum levels, or platinum content 24 on 96 hr after drug administration. increased urinary excretion of platinum but resulted in a In Chart 7, and the cumulative 24-hr urinary excretion of drop in urinary platinum concentration. platinum and the 24-hr urine platinum concentration are plotted, respectively. Roughly 40% of injected platinum was DISCUSSION

Table 3 Cvitkovic et al. (7) showed that induction of mannitol Effect of diuretics on kidney levels of platinum after 6 mg/kg iv. diunesis prevented the increase in BUN and serum creati Rats were given injections of COOP(6 mg/kg iv.) alone or in nine caused by COOP administration to dogs. Ward et a!. combination with diuretics and sacrificed 24 or 96 hr later. Kidneys (34), on the basis of blood chemistry data and histopathol were removed,and platinum content was determined by flameless ogy, reported that when furosemide was administered to atomic absorption spectrometry(22).Dataare from Experiments1 F344 rats prior to COOP, there was a decrease in the acute and 3. renal toxicity of the platinum compound, although toxicity tissue24 (@.tg/gwet weight of still occurred. Furthermore, the same investigators showed hrnCOOPTreatmentPlatinum hrn96 that when COOP was administered in low weekly doses (3.0 mg/kg i.v.) for periods of 5 to 6 weeks, the renal failure that ultimately developed in rats given COOP alone was not COOPwithfuro 12.1 Â±0.93c 5 885Ã·0598b 6 semide observed in rats receiving furosemide with each course COOPwith man 6.27 Â±1.38k'6 65,44Â±0.4l8@@6.17 Â±0.8l6@@6 6 (33). We shall restrict our comments to acute toxicity after nitolg.64Â±1.10a a single, high, nonlethal dose because it is possible that the a Values are mean Â±S.E. pathogenesis of chronic renal failure induced by COOP is b COOP with furosemide > COOP with mannitol or COOP; p < different from that seen after acute intoxication. We chose 0.05. to study a high dose in light of current clinical trends C COOP with furosemide > COOP with mannitol; p < 0.05. towards escalation of COOP dosage in combination with diuretics(3,13,25). Our data on BUN levels confirm previous work. Two A B additional parameters were studied to confirm that renal function was protected in rats. Glomerular filtration rate was shown to be depressed markedly 3 days after COOP administration. Similar findings have been reported for 150 30 r P other nephrotoxic compounds, such as mercuric chloride a. or umanyl nitrate (9, 11). Diuretic treatment partially pre U. vented this decrease in filtration. Clearance of PAH was 0 E EJ CDDP z a, also greatly diminished 3 days after COOP administration. 0@ CDDP WITH FUROSEMIDE The cells of the straight portion of the proximal tubule, the z predominant site of COOP toxicity in the rat (32), are @ .@ 100 0 20 @- CDDP WITH I- responsible for organic acid secretion (31), and PAH clear 4 MAN NITOL I. ance is depressed in vivo by other compounds that damage a, z â€˜U the proximal tubule (10, 11). Rats given diuretics with COOP U z cleared PAH faster than did rats given COOP alone. This 0 U faster clearance could be the result of improved function of a. PAH-secreting cells on improved renal hemodynamics. We >-.. 50 â€˜U believe that it is important to point out that although these 4 z -I parameters of renal function were partially protected and although BUN levels in diuretic-treated rats were not sign if C., icantly higher than controls, both glomerular filtration and PAH clearance were significantly lower in diuretic-treated groups compared to untreated controls. Thus BUN level is 1001 a reliable but relatively insensitive indicator of filtration Chart 7. Effect of diuretics on cumulative urinary excretion (a) and con centration (b) of platinum in urine during a 0- to 24-hr collection period after function. When Dentino et a!. (8) studied the long-term COOP(6mg/kg iv.). Dataarefrom Experiment3. Valuesaremeanrecovery effect of COOP without diuretics on human renal function, of platinum in urine, expressed as g.tgplatinum per 24 hr per 100 g body they also concluded that moderate increases in BUN or weight Â±SE. (a) or mean concentration of platinum in 24-hr urine sample. @.tg/mlÂ±S.E. (b) with n = 6 except as noted in parentheses. @,values plasma creatinine levels may be accompanied by substan significantly lower than COOP alone; p < 0.05. tial decrements in glomerular filtration nate. We emphasize

APRIL 1979 1275

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. M. F. Pera,Jr. et a!. that the protection of function by diuretics in our studies pharmacological significance. A greater degree of persist was partial, not complete. ent histological damage in the furosemide group was asso Despite protection of renal function, the diuretic-treated ciated with a higher amount of platinum in the kidneys of rats still exhibited tubular necrosis. The trend was toward these animals. The protection of renal function by both longer persistence of histological damage in the fumosemide diuretics was accompanied by a drop in the urinary piati group and faster recovery in rats given mannitol relative to num concentration, a phenomenon also observed in dogs rats given COOP alone. Our results here disagree with those with mannitol (7). of Ward et a!. (34), who reported histological protection Two recent reviews have discussed current concepts with furosemide on an acute basis. Theme are 2 possible concerning the pathogenesis of acute renal failure and the reasons for the discrepancy. First, Ward et a!. reported possible association between tubular necrosis and depres histological data on rats given a slightly lower dose of sion of filtration mate. It has been hypothesized that the COOP (4.8 versus 6.0 mg/kg i.v. in the present work). depression in filtration rate might be associated with back Second, Ward et a!. reported histological data only for leak of normally nonreabsombed substances through dam kidneys studied 3 days after drug exposure. Inspection of aged epithelium, obstruction of tubules by debris, persist Chart 4 shows that in our study on Day 3 the median ent vasoconstriction in the afferent arteriole secondary to necrosis index for matsgiven fumosemide was in fact slightly activation of the intmamenalmenin-angiotensis system, or a but insignificantly lower than that for rats given COOP direct effect on the glomerulus to decrease the glomerular alone. Later, on Days 4, 5, 7, and 10, the median necrosis capillary ultrafiltration coefficient (17, 29). Backleak of index for the furosemide group exceeded those of the other nonreabsorbed substances through damaged epithelium is groups. In Chart 3, it may be seen that BUN levels also unlikely to account for our results, since the damage to the peaked later in furosemide-treated mats. Larger studies tubule was similar in the diuretic-treated animals that ex would be required to statistically verify the differences in hibited improved filtration. However, any of the other path the degree of necrosis between the groups. Nonetheless, ophysiological alterations might be triggered by high tubu the persistence of the COOP-induced lesions in fumosemide lamlevels of toxins such as COOP, and dilution of the urine treated rats 9bserved in 2 experiments was disturbing, as could ameliorate the ensuing decrease in renal function. was the tendency of this group to lose more weight. Further studies are required to investigate these possibili Fumosemide is a potent diuretic agent with toxicities that ties, but we mention them to illustrate that the diluting overlap those of COOP. This diuretic drug has been shown effect of diuretics might reduce functional impairment of to produce ototoxicity in humans (27), and it was recently the kidney without markedly altering the course of tubular demonstrated that at high doses the compound can un necrosis. dergo a process of metabolic activation in animals to The concentration of platinum in plasma and intestine reactive intermediates which bind covalently to macromol was not altered by diuretic administration, nor did the ecules, resulting in proximal tubular necrosis (19). Unless diuretics increase the total urinary excretion of platinum. further study proves that themeis any advantage to the use Cvitkovic et a!. (7) reported that mannitol diumesis did not of furosemide, these data suggest that fumosemide should affect serum platinum levels in dogs, although a direct be contraindicated for use in prevention of COOP-induced comparison of dogs given COOP alone to mannitol-tmeated nephmotoxicity. It would, therefore, seem prudent to use dogs was not provided. Another report mentioned unpub other compounds for prevention of COOP-induced renal lished findings of increased blood levels of platinum with failureinhumans. fumosemide in mats(34). Although we have noted a tendency The contradiction between renal structure and renal func toward higher plasma platinum levels in fumosemide-treated tion measurements observed in these studies is not unpnec rats, particularly in their later phases of plasma decay, the edented. OiBonaetal. (9) compared chronically salt-loaded effect was not marked. Our studies on intestine and all but matsgiven HgCI2 to rats given HgCl2 alone and found less the 2-mm time point on spleen showed no difference in depression of glomerular filtration and less elevation of uptake of platinum into these tissues, consistent with the BUN in the 0.9% NaCI solution-loaded animals, despite plasma clearance data. No significant alterations in urinary equivalent degrees of proximal tubular necrosis. Further recovery of platinum were observed with furosemide or more, Thiel et a!. (30) found that pretreatment with chronic mannitol, similar to previous reports with mannitol in dogs 0.9% NaCI solution loading plus deoxycorticosterone ace and fumosemide in rats (7, 34). Thus, it seems unlikely that tate, acetazolamide, or furosemide protected mats from mannitol or furosemide diuresis markedly alters the overall mercuric chloride impairment of renal function but did not cumulative disposition of platinum outside of the kidney. If alter the degree of necrosis in the straight portion of the the level of platinum in plasma, spleen, intestine, and urine proximal tubule (30). reflects the disposition of the cytotoxic form(s) of COOP, Our data on the kidney platinum content for the 3 treat the preceding results suggest that combined administration ment groups tend to correlate with the histopathological of diuretics with COOP should reduce renal toxicity without results. Kidneys of mats given COOP alone or with mannitol altering toxicity to host renewing cell populations (gastmoin contained similar amounts of platinum and exhibited a testinal mucosa and bone marrow), and without changing similar degree of necrosis. While the apparent more rapid antitumor activity. The accompanying publication will ad recovery from tubular necrosis in mannitol-treated rats dressthesemattersdirectly. compared to matsgiven COOP alone correlates with sign ifi cantly lower 2-mm kidney platinum levels, further investi REFERENCES gations are required to substantiate and determine its 1. Blaufox, M. 0., and Cohen, A. Single injection clearance of iothalamate

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I in the rat. Am. J. Physiol., 218: 542-544, 1970. diammine platinum (therapeutic efficacy in previously treated wide 2. Blaufox,M. 0., Guttmann,R.0., andMerrill,J. P.Measurementofrenal spread and recurrent testicular tumors). Proc. Am. Assoc. Cancer Res., function in the rat with single injection clearances. Am. J. Physiol. , 212: 17:243,1976. 629-632,1967. 21. Migdalof, B. H. Methods for obtaining drug time course data from 3. Chary, K. K., Higby, 0. J., Henderson, E. S., and Swinerton, K. 0. Phase individual small laboratory animals: serial microblood sampling and I study of high-dose cis-dichlorodiammineplatinum(ll) with forced di assay. Drug Metab. Rev., 5: 295-310, 1976. uresis. CancerTreat. Rep., 61: 367-370, 1977. 22. Pera,M. F.,andHarder,H.C.Analysisforplatinumin biologicalmaterial 4. Chervu, L. R., Lee, H. B., Goyal, 0., and Blaufox, M. 0. Use ofâ€•Tc-Cu by flameless atomic absorption spectrometry. Clin. Chem., 23: 1245- DTPAcomplexasa renalfunctionagent.J. 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Figs. 1 to 4 were taken from the outer stripe of the outer zone of the renal medulla 7 days after the indicated treatments. The slides were stained with periodic acid-Schiff and photographed at x 400. Fig. 1. Untreated control. Histological grade of necrosis = 0. No lesions are observed. Fig. 2. COOP.Histologicalgradeof necrosis= 3. Straightarrows, moderatedegreeof necrosisindicatedby focal dissolutionof proximaltubular cells. Curved arrow, regenerated cells have large nuclei. Fig. 3. COOPwith furosemide. Histological grade of necrosis = 4. Marked necrosis of straight proximal tubular cells is revealed by pyknosis, cytoplasmic disintegration and sloughing into lumina. Enlarged nuclei (curved arrows) indicate regenerative efforts. The proximal convoluted tubules, with dark-staining brush border (top) and distal tubules (thick arrow) are essentially normal. Fig. 4. COOP with mannitol. Histological grade of necrosis = 1. Mild degree of necrotizing changes is indicated by pyknotic cells and nuclear and cytoplasmic debris, which are forced into lumina by regenerating cells (curved arrows).

APRIL 1979 1277

M. F. Pera, Jr. et a!.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Effects of Mannitol or Furosemide Diuresis on the Nephrotoxicity and Physiological Disposition of cis -Dichlorodiammineplatinum-(II) in Rats

Martin F. Pera, Jr., Bernard C. Zook and Harold C. Harder

Cancer Res 1979;39:1269-1278.

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