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Effect of verapamil on cephaloridine nephrotoxicity in the rabbit
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
( 1990)
103,383-388
Effect of Verapamil on Cephaloridine
Nephrotoxicity
in the Rabbit
MARCC.BROWNING Department q/Pediatrics, Bowman Gray School ofMedicine, Uhke Forest UniverJit~~. 300 South Hawthorne Road, Winston-Salem. North Carolina 27103
Received Februarv IO. I989; acceptedJantrar~~ 17. I990 Effect of Verapamil on Cephaloridine Nephrotoxicity in the Rabbit. BROWNING. M. C. (1990). Toxicol. .4pp/. Pharmaco/. 103,383-388. Cephaloridine produces proximal tubular necrosis in the rabbit kidney. Calcium channel blockers have ameliorated tissue injury due to toxic and ischemic insults. To determine whether renal damage caused by cephaloridine could be modified by pretreatment with verapamil, groups of rabbits were given cephaloridine, 100 mg/ kg SC.90 min after administration of verapamil. 200 pg/kg iv. Histologic scoring of the extent of proximal tubular necrosis 48 hr later demonstrated increased necrosis in the group receiving verapamil plus cephaloridine. Verapamil pretreatment increased the concentration ofcephaloridine in the renal cortex at 0.5 hr, but did not alter the peak concentration (2 hr after the dose) or cortical concentrations at I or 3 hr. Assay of total calcium content in cortical mitochondria 2 hr after cephaloridine showed that verapamil pretreatment abolished the increased accumulation following cephaloridine administration. We conclude that verapamil does not protect renal proximal tubular cells from the toxic effect of cephaloridine, and that verapamil prevents the cephaloridine-induced uptake of calcium by cortical mitochondria. (c 1990 Academic Press. Inc.
Cephaloridine produces acute dose-dependent necrosis of the proximal tubule of the kidney of several species (Atkinson et al., 1966). Toxicity in the kidney depends on uptake of cephaloridine by the organic anion transport system of the proximal tubule and is directly related to the cortical concentration of the antibiotic (Tune et al., 1977). Although the physiologic processes that target the proximal tubule for cephaloridine-induced injury are well understood, the cellular mechanisms of cephaloridine nephrotoxicity remain unclear. Over the last several years, abnormal intracellular calcium homeostasis has been implicated as a common step in the lethal processes of several toxic compounds (Farber, 1982). These studies were performed to determine whether verapamil, a calcium channel blocker, ameliorates the extent of proximal tubular necrosis, as it has other models of acute renal failure (Burke et al., 1984; Lee et al., 1987; Sumpio et al., 1987).
We also examined calcium accumulation in the renal cortex and cortical mitochondria after a toxic dose of cephaloridine with and without pretreatment with verapamil. METHODS .4nirnclis. Female New Zealand white rabbits weighing 2-3 kg were used in all experiments. Drug administration. Both cephaloridine and verapamil were dissolved in 0.9% NaCI, in concentrations of 100 and 2 mg/ml. respectively. Verapamil. 200 fig/kg. was injected intravenously 90 min before cephaloridine. 100 mg/kg. or vehicle. Cephaloridine was injected subcutaneously in all but the pharmacokinetic study in which it was administered either subcutaneously or intravenously to separate groups of rabbits. Preparation of tissue /br his/ologic scoring. Rabbits were killed by decapitation 48 hr after injection ofcephaloridine. The kidneys were removed and a coronal section from the midportion of the kidney was placed in buffered formalin for fixation. After staining with hematoxylin and eosin. proximal tubular necrosis was assessed at 250X magnification in every other field around the cir383
004 1-008X/90 $3.00
384
MARC C. BROWNING
cumference of the cortex (15-20 fields per kidney). The extent of necrosis was determined by counting each of 9 points on a 3 X 3 grid that overlay a necrotic cell. The grid was oriented with one side parallel to and just below the renal capsule. Approximately two-thirds of the width ofthe cortex was included in the grid. The superficial cortex is most sensitive to cephaloridine nephrotoxicity (Silverblatt et al., 1970). The results are expressed as the average score per field. Preparation qftissuefi,r measurement ofcalcium. Rabbits were killed 2 hr after injection of cephaloridine. The kidneys were promptly removed and decapsulated and the renal cortex was dissected from the red medulla. The cortical fragments were placed in chilled solution A containing 270 mM sucrose. I mM EGTA, 5 mM Tris, and 0.5% bovine serum albumin, pH 7.4. The cortex was diced and rinsed twice in the same solution. Several small fragments of cortex totalling 100-200 mg wet wt were blotted dry, weighed, and frozen for later determination of protein and calcium. The bulk of the cortical tissue was diluted to 50 ml with solution A and ground in a glass-Teflon homogenizer. The mitochondria were isolated by differential centrifugation using solution A without BSA to resuspend the mitochondrial pellet. An aliquot of the mitochondrial pellet was frozen for determination of protein and calcium. Assa?a. Calcium: The concentration of calcium in tissue was determined by the acid extraction method of Tew et al. (198 I), using a Perkin-Elmer atomic absorption spectrophotometer. Protein: The method of Bradford (Bradford, 1976) was used with bovine serum albumin as standard. Cephaloridine: Tissue concentrations of cephaloridine were determined using the fluorometric assaydescribed by Tune (1972). Statistical treatment. The results of tissue calcium measurements were examined by analysis of variance with least-squares correction for multiple comparisons. Histologic scores were analyzed by the Wilcoxon ranked sum test. Ap < 0.05 is considered significant. Results are presented as means -+ standard error.
RESULTS Eflect of verapamil pretreatment on proximal tubular necrosis. Cephaloridine alone produced mild proximal tubular necrosis in the superficial cortex whereas cephaloridine given 90 min after verapamil produced significantly more widespread necrosis of proximal tubular epithelial cells (histologic scores for proximal tubular necrosis: cephaloridine, 0.9 ? 0.3, N = 12; and verapamil-cephaloridine, 2.1 +- 0.4, N = 12) (Figs. 1 and 2). No effect on the cortical histology could be ap-
preciated when verapamil was administered alone (N = 4, Fig. 1). Eflect of verapamilpretreatment on cortical concentration of cephaloridine. In the rabbits pretreated with verapamil, the cortical concentrations of cephaloridine were significantly higher 30 min after subcutaneous injection than those in the cephaloridine alone group (cephaloridine, 680 + 48; verapamilcephaloridine, 1193 f 97; p = 0.015; n = 34). However, cortical concentrations at the three later times were not statistically different between the two groups and the peak levels in the cortices were not higher in the group pretreated with verapamil (cephaloridine, 60, 120, and 180 min, 1885 + 290,2378 + 367, and 925 + 169 pg/g wet cortex, respectively; verapamil-cephaloridine, 60, 120, and 180 min, 1568 f 165, 1535 + 242, and 1162 + 383 pg/g wet cortex, respectively) (Fig. 3). The difference in cortical levels 30 min after cephaloridine is most probably the result of mobilization from the site of injection since intravenous administration of cephaloridine did not affect cortical levels at any of the three intervals in rabbits pretreated with verapamil (cephaloridine, 30, 60, and 120 min, 3247 * 177, 3445 -t 324, and 1132 + 167 pg/g wet cortex; verapamil-cephaloridine, 30, 60, and 120 min, 3536 f 285, 2704 + 265, and 1013 + 66 pg/g wet cortex, n = 3-6). Verapamil did not significantly alter assay results when cephaloridine was added to cortical homogenate from animals receiving verapamil30 min before decapitation. Calcium content of renal cortex and cortical mitochondria. The calcium content of cortical mitochondria was significantly increased in the cephaloridine group compared to each of the others (control, 32.6 f 5.3; verapamil, 27.6 f 3.3; cephaloridine, 70.0 f 14.9; and verapamil-cephaloridine, 32.3 f 4.62 nmol/mg protein; n = 6 for each group) (Fig. 4). The calcium contents of the cortex in each of the four groups were not significantly different from one another (control, 25.5 f 4.3; verapamil, 27.1 f 3.8; cephalori-
VERAPAMIL
FIG. I. Histology of the renal cortex idine, 100 mg/kg SC. (B) Cephaloridine.
IN
CEPHALORIDINE
385
NEPHROTOXICTTY
2 daysafter dru rg treatment. Renal capsule is at the top. (A) Cephalor100 mg/kg SC:. 90 min after verapamil, 200 rg/kg iv.
dine, 3 1.6 f. 3.1; and verapamil-cephaloridine, 37.1 + 4.3 nmol/mg protein) (Fig. 4). DISCUSSION Calcium channel blockers have been found to ameliorate the renal injury produced by
FIG. 2. Cephaloridine nephrotoxicity 2 days after administration of the drugs. For scoring procedure. see Methods. (*)I, i 0.05 vs cephaloridine group. Verapamil. N = 4; cephaloridine. N = 12: verapamil-cephaloridine. N= 12.
gentamicin (Lee et al., 1987) and transient &hernia (Burke et al., 1984; Arnold et al.. 1985; Goldfarb et al., 1983: Weinberg et al., 1984; Garthoff et al., 1987). Lee and coworkers (1987) found that nitrendipine markedly improved the functional and morphologic consequences of chronic adminis-
FIG. 3. Cephaloridine rum after subcutaneous il-pretreated animals: cephaloridine alone
concentrations in cortex and seinjection. Solid circles. verapamopen circles. animals receiving
386
MARC C. BROWNING
CORTEX
MITOCHONDRIA
FIG. 4. Calcium content of renal cortex and cortical mitochondria obtained from each of four groups of rabbits 3.5 hr after administration of verapamil or vehicle. (*)p < 0.05 vs other mitochondria. N = 6 for each group.
tration of toxic doses of gentamicin. A variety of calcium channel blockers have been found to reduce functional and morphologic evidence of damage in ischemic models of acute renal failure. Burke et al. (1984) in a careful study of norepinephrine-induced acute renal failure, showed functional, histologic, and metabolic improvement when verapamil was infused after norepinephrine. In contrast, our experiments demonstrate increased proximal tubular necrosis in rabbits pretreated with verapamil despite the finding of normal total calcium in the mitochondria isolated from rabbits pretreated with verapamil. Sumpio et al. (1987) also showed that calcium uptake by cortical mitochondria could be dissociated from aspects of the toxic process. In an isolated, perfused kidney model of cyclosporine toxicity, they found that verapamil pretreatment prevented calcium accumulation in cortical mitochondria but did not prevent tubular proteinuria or depletion of ATP. Insofar as calcium channel blockers may prevent toxicity by reducing calcium influx into cells of the kidney, others have provided evidence that calcium may not be the final common pathway to irreversible cell injury in some toxic insults. Cronin and Newman ( 1985) found that the increased calcium content of kidneys 8 days after beginning gentamicin therapy was not significantly affected by treatment with thyroxine, which nevertheless prevented the functional consequences. Similarly, thyroxine did not prevent
calcium accumulation in the cortex in uranyl nitrate-induced renal failure despite reducing the fall in creatinine clearance (Cronin et al., 1986). Troyer et al. (1982) reported that death of LLCPKl cells exposed to mellitin was not prevented by a calcium-free medium. Because cortical levels of cephaloridine correlate strongly with nephrotoxicity (Tune et al., 1977) and data from experiments with cortical slices suggested that verapamil could increase the tissue concentration of compounds transported by the organic anion transport system (Matsushima and Gemba, 1982), we measured the in vivo concentration of the antibiotic in the cortex of control and verapamil-pretreated animals. Verapamil transiently increased cephaloridine concentrations in the cortex at 30 min compared to control rabbits but thereafter cortical levels of the antibiotic in the verapamil-pretreated rabbits were not statistically different from those receiving cephaloridine alone. These data do not readily explain the increased toxicity in verapamil-pretreated animals. Because calcium channels sensitive to verapamil have not been demonstrated in the proximal tubule, we examined calcium accumulation by cortical mitochondria. We selected mitochondria for study because in vivo they are sequestered from the high calcium concentration of the extracellular fluid which could confound interpretation of calcium content in whole cortex, they take up calcium avidly, and they are sensitive to changes in intracellular calcium activity (Mandel and Murphy, 1984). Cephaloridine produced a significant increase in the calcium content of cortical mitochondria 2 hr after administration of the antibiotic. Pretreatment with verapamil completely prevented the increase in mitochondrial calcium content. The mitochondrial calcium accumulation probably reflects in vivo mitochondrial calcium status since (1) mitochondria were prepared in the presence of EGTA, which limits calcium uptake during isolation (Weinberg and Humes, 1985). (2) they were exposed to similar cal-
VERAPAMIL
IN
CEPHALORIDINE
NEPHROTOXICITY
387
cium concentrations during isolation (Fig. 4), sary component in the irreversible injury not favoring uptake by the organelles from produced by cephaloridine but rather an epithe group receiving cephaloridine alone, and phenomenon which is a marker of the (3) calcium channel blockers do not prevent administration of a mildly toxic dose of cephcalcium uptake by mitochondria (Burke er aloridine. We speculate that calcium influx al., 1984: Vaghy et al., 1982), and would not into the cytosol and mitochondria is not a reprotect the verapamil-cephaloridine-treated quirement for the lethal effect of cephalorimitochondria from in vitro calcium accumudine. lation. However, this experiment looked at mitochondrial calcium at only one time REFERENCES point and cannot distinguish ionized from total calcium. Free calcium in the mitochonARNOLD, P. E., LUML.ERTGUL, D., BURKE, 7. J.. AND dria may yet be important in the toxic proSCHRIER, R. W. (1985). In vitro versus in vivo mitochondrial calcium loading in ischemic acute renal failcess. ure. Amrr. J. Ph.vsio1.248, F845-F850. Despite verapamil’s dissociation of calATKINSON, R. M.. CURRIE, J. P., DAVIS. B.. PRATT cium accumulation in cortical mitochondria D. A. H., SHARPE, H. M.. AND TOMICH, E. G. (1966). from the toxic process, the calcium content Acute toxicity of cephaloridine, an antibiotic derived of cortical mitochondria was a sensitive and from cephalosporin C. T~~.CcoJ. :l.up,pl.Pharnwol8. 398-406. early indicator of in vivo cephaloridine adBURKE, T. J.. ARNOLD. P. E.. GORDON. J. A., BULCXR. ministration. The majority of studies examR. E., DOBYAN. D. C., AND SCHRIER. R. W. (1984). ining the metabolic bases of cephaloridine Protective effect of intrarenal calcium membrane toxicity have used doses considerably larger blockers before or after renal ischemia. J. <‘/in IIIthan those used in our experiments. The ear~~1.74, 1830- I84 I. liest biochemical alteration produced by a CRONIN. R. E., BROWN. D. M.. AND SIMONSEN, K. (1986). Protection by thyroxine in nephrotoxic acute toxic dose of cephaloridine in viuo is the fall renal failure. Amer. J. Physiol.251, of reduced glutathione content in the renal CRONIN. R. E.. AND NEWMAN, J. A. F408-F416. (1985). Protective cortex reported by Kuo and Hook (1982) 1 hr effect of thyroxine but not parathyroidectomy on genafter injection of 250 mg/kg SC.Morphologic tamicin nephrotoxicity. .Jnwr J. Physid248, F333F339. alterations have also been found 1 hr after 200 mg/kg im (Silverblatt et al., 1970). In- FARBER. J. L. ( 1982). Membrane injury and calcium homeostasis in the pathogenesis of coagulative necrosis. creased content of conjugated dienes in the Luh. IrnX51.47, 114-123. renal cortex of rats (Kuo et al., 1983) and the GARTHOFF, 8.. HIRTH. C.. FEDERMANN. A.. KAZDA. S.. reduction in succinate-supported mitochonAND STASCH. J-P. Renal effects of 1,4-dihydropyridines in animal models of hypertension and renal faildrial oxidative phosphorylation (Tune et al., ure. (I 987). J. (irrdiovusc~. Pharmad.9, Suppl. I SX1979) in rabbits have been demonstrated 2 hr Sl3. after administration of 2000 and 200 mg/kg, GOI.DFARB, D., IAINA. A., SERBAN, I., GAVENM), S.. respectively. KAPULER, S., AND EUAHOU. H. E. (1983). Benetictal In summary, (I) pretreatment with veraeffect of verapamil in ischemic acute renal failure in the rat. Pm,. Sock. E-\-p Bid. Afed.172, 389-392. pamil augmented, rather than ameliorated, HUMES. H. D.. HUN-I’, D. A.. AND WHITE. M. D. (1987). the proximal tubular necrosis produced by a Direct toxic effect of the radiocontrast agent diatrimildly toxic dose of cephaloridine, (2) calzoate on renal proximal tubule cells. :Imrpr .I P/r!y cium accumulated in mitochondria isolated ~1.252, F246-F255. from renal cortex of rabbits within 2 hr ofthat JONFS, T. W.. WALLIN. A., THOR. H.. GERDES. R (i.. ORMSTAD, K.. ANDORRENIUS, S. (1986). The mechamildly toxic dose, and (3) that mitochondrial nism of pentachlorobutadienyl-glutathione nephroaccumulation of calcium was abolished by toxicity studied with isolated rat renal epithelial cells. pretreatment with verapamil. We conclude Arch. Biochrm. Biophj:s.251, 504-5 13. that an increase in the total calcium content Ku~, C.-H.. AND HOOK, J. B. (1982). Depletion of renal of renal cortical mitochondria is not a necesglutathione content and nephrotoxicity of cephalori-
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C. BROWNING
dine in rabbits, rats, and mice. Toxicol. Appl. Pharmacol.63,292-302. Kuo, C.-H., MAITA, K., SLEIGHT, S. D., AND HOOK, J. B. (1983). Lipid peroxidation: A possible mechanism of cephaloridine-induced nephrotoxicity. Toxicol. Appl. Pharmacol.67,78-88.
KURTZ, I. (1987). Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule. J. Clin. Invesf.80, 928-935. LEE, S. M., PATTISON, M. E., AND MICHAEL, U. F. (I 987). Nitrendipine protects against aminoglycoside nephrotoxicity in the rat. J. Cardiovasc. Pharmacol. 9, Suppl. 1, S65. NAGINENI, C. N., MISRA, B. L., LEE, D. B. N., AND YANAGAWA, N. (1987). Cyclosporine A- Calcium channels interaction: A possible mechanism for nephrotoxicity. Transplantation Proc.19, 1358-I 362. SILVERBLATT, F., TURCK, M., AND BULGER, R. (1970). Nephrotoxicity due to cephaloridine: A light- and electron-microscopic study in rabbits. J. Infect Dis.122, 33-44. SUMPIO, B. E., BAUE, A. E.. ANDCHAUDRY, I. H. (1987). Alleviation of cyclosporine nephrotoxicity with verapamil and ATP-MgC12. Ann. Surg.206,655-660. TEW, W. P., MALIS, C. D., AND WALKER, W. G. (1981). A rapid extraction technique for atomic absorption determinations of kidney calcium. Anal. Biochem.112, 346-350. TROYER, D., KREISBERG, M., AND VENKATACHALAM. M. (1982). Role of calcium (CA++) in plasma membrane blebbing and cell death in a kidney epithelial cell line (LL-CPKI) after toxin. Kidney Int.21,207(A).
TUNE, B. M. (1972). Effect of organic acid transport inhibitors on renal cortical uptake and proximal tubular toxicity of cephaloridine. J. Pharmacol. Exp. Ther. 181,250-256. TUNE, B. M., Wu, K. Y., FRAVERT, D., AND HOLTZMAN, D. (1979). Effect of cephaloridine on respiration by renal cortical mitochondria. J. Pharmacol. Exp. Ther.210,98-100.
TUNE, B. M., Wu, K. Y., AND KEMPSON, R. L. (1977). Inhibition of transport and prevention of toxicity of cephaloridine in the kidney. Dose-responsiveness of the rabbit and the guinea pig to probenecid. J. Pharmacol. Exp. Ther.202,466-47 1. VAGHY, P. L., JOHNSON, J. D., MATLIB, M. A., WANG, T.. AND SCHWARTZ, A. (1982). Selective inhibition of Na+-induced Ca2+ release from heart mitochondria by diltiazem and certain other Ca2+ antagonist drugs. J. Biol. Chem.257,6000-6002.
WEIBERG, J. M., HARDING, P. G.. AND HUMES, H. D. (1983). Alterations in renal cortex cation homeostasis during mercuric chloride and gentamicin nephrotoxicity. Exp. Mol. Pathol.39,43-60. WEINBERG, J. M., AND HUMES, H. D. (1985). Calcium transport and inner mitochondrial membrane damage in renal cortical mitochondria. Amer. J. PhvsioI.248, F876-F889. WEINBERG, J. M., HUNT, D., AND HUMES, H. D. (1984). Effects of verapamil on in vitro ischemic injury to isolated rabbit proximal tubules. Kidney I&.25,239. WILSON, P. D., AND SCHRIER, R. W. (1986). Nephron segment and calcium as determinants of anoxic death in renal cultures. Kidney Int. 29, 1172- 1179.
AND
APPLIED
PHARMACOLOGY
( 1990)
103,383-388
Effect of Verapamil on Cephaloridine
Nephrotoxicity
in the Rabbit
MARCC.BROWNING Department q/Pediatrics, Bowman Gray School ofMedicine, Uhke Forest UniverJit~~. 300 South Hawthorne Road, Winston-Salem. North Carolina 27103
Received Februarv IO. I989; acceptedJantrar~~ 17. I990 Effect of Verapamil on Cephaloridine Nephrotoxicity in the Rabbit. BROWNING. M. C. (1990). Toxicol. .4pp/. Pharmaco/. 103,383-388. Cephaloridine produces proximal tubular necrosis in the rabbit kidney. Calcium channel blockers have ameliorated tissue injury due to toxic and ischemic insults. To determine whether renal damage caused by cephaloridine could be modified by pretreatment with verapamil, groups of rabbits were given cephaloridine, 100 mg/ kg SC.90 min after administration of verapamil. 200 pg/kg iv. Histologic scoring of the extent of proximal tubular necrosis 48 hr later demonstrated increased necrosis in the group receiving verapamil plus cephaloridine. Verapamil pretreatment increased the concentration ofcephaloridine in the renal cortex at 0.5 hr, but did not alter the peak concentration (2 hr after the dose) or cortical concentrations at I or 3 hr. Assay of total calcium content in cortical mitochondria 2 hr after cephaloridine showed that verapamil pretreatment abolished the increased accumulation following cephaloridine administration. We conclude that verapamil does not protect renal proximal tubular cells from the toxic effect of cephaloridine, and that verapamil prevents the cephaloridine-induced uptake of calcium by cortical mitochondria. (c 1990 Academic Press. Inc.
Cephaloridine produces acute dose-dependent necrosis of the proximal tubule of the kidney of several species (Atkinson et al., 1966). Toxicity in the kidney depends on uptake of cephaloridine by the organic anion transport system of the proximal tubule and is directly related to the cortical concentration of the antibiotic (Tune et al., 1977). Although the physiologic processes that target the proximal tubule for cephaloridine-induced injury are well understood, the cellular mechanisms of cephaloridine nephrotoxicity remain unclear. Over the last several years, abnormal intracellular calcium homeostasis has been implicated as a common step in the lethal processes of several toxic compounds (Farber, 1982). These studies were performed to determine whether verapamil, a calcium channel blocker, ameliorates the extent of proximal tubular necrosis, as it has other models of acute renal failure (Burke et al., 1984; Lee et al., 1987; Sumpio et al., 1987).
We also examined calcium accumulation in the renal cortex and cortical mitochondria after a toxic dose of cephaloridine with and without pretreatment with verapamil. METHODS .4nirnclis. Female New Zealand white rabbits weighing 2-3 kg were used in all experiments. Drug administration. Both cephaloridine and verapamil were dissolved in 0.9% NaCI, in concentrations of 100 and 2 mg/ml. respectively. Verapamil. 200 fig/kg. was injected intravenously 90 min before cephaloridine. 100 mg/kg. or vehicle. Cephaloridine was injected subcutaneously in all but the pharmacokinetic study in which it was administered either subcutaneously or intravenously to separate groups of rabbits. Preparation of tissue /br his/ologic scoring. Rabbits were killed by decapitation 48 hr after injection ofcephaloridine. The kidneys were removed and a coronal section from the midportion of the kidney was placed in buffered formalin for fixation. After staining with hematoxylin and eosin. proximal tubular necrosis was assessed at 250X magnification in every other field around the cir383
004 1-008X/90 $3.00
384
MARC C. BROWNING
cumference of the cortex (15-20 fields per kidney). The extent of necrosis was determined by counting each of 9 points on a 3 X 3 grid that overlay a necrotic cell. The grid was oriented with one side parallel to and just below the renal capsule. Approximately two-thirds of the width ofthe cortex was included in the grid. The superficial cortex is most sensitive to cephaloridine nephrotoxicity (Silverblatt et al., 1970). The results are expressed as the average score per field. Preparation qftissuefi,r measurement ofcalcium. Rabbits were killed 2 hr after injection of cephaloridine. The kidneys were promptly removed and decapsulated and the renal cortex was dissected from the red medulla. The cortical fragments were placed in chilled solution A containing 270 mM sucrose. I mM EGTA, 5 mM Tris, and 0.5% bovine serum albumin, pH 7.4. The cortex was diced and rinsed twice in the same solution. Several small fragments of cortex totalling 100-200 mg wet wt were blotted dry, weighed, and frozen for later determination of protein and calcium. The bulk of the cortical tissue was diluted to 50 ml with solution A and ground in a glass-Teflon homogenizer. The mitochondria were isolated by differential centrifugation using solution A without BSA to resuspend the mitochondrial pellet. An aliquot of the mitochondrial pellet was frozen for determination of protein and calcium. Assa?a. Calcium: The concentration of calcium in tissue was determined by the acid extraction method of Tew et al. (198 I), using a Perkin-Elmer atomic absorption spectrophotometer. Protein: The method of Bradford (Bradford, 1976) was used with bovine serum albumin as standard. Cephaloridine: Tissue concentrations of cephaloridine were determined using the fluorometric assaydescribed by Tune (1972). Statistical treatment. The results of tissue calcium measurements were examined by analysis of variance with least-squares correction for multiple comparisons. Histologic scores were analyzed by the Wilcoxon ranked sum test. Ap < 0.05 is considered significant. Results are presented as means -+ standard error.
RESULTS Eflect of verapamil pretreatment on proximal tubular necrosis. Cephaloridine alone produced mild proximal tubular necrosis in the superficial cortex whereas cephaloridine given 90 min after verapamil produced significantly more widespread necrosis of proximal tubular epithelial cells (histologic scores for proximal tubular necrosis: cephaloridine, 0.9 ? 0.3, N = 12; and verapamil-cephaloridine, 2.1 +- 0.4, N = 12) (Figs. 1 and 2). No effect on the cortical histology could be ap-
preciated when verapamil was administered alone (N = 4, Fig. 1). Eflect of verapamilpretreatment on cortical concentration of cephaloridine. In the rabbits pretreated with verapamil, the cortical concentrations of cephaloridine were significantly higher 30 min after subcutaneous injection than those in the cephaloridine alone group (cephaloridine, 680 + 48; verapamilcephaloridine, 1193 f 97; p = 0.015; n = 34). However, cortical concentrations at the three later times were not statistically different between the two groups and the peak levels in the cortices were not higher in the group pretreated with verapamil (cephaloridine, 60, 120, and 180 min, 1885 + 290,2378 + 367, and 925 + 169 pg/g wet cortex, respectively; verapamil-cephaloridine, 60, 120, and 180 min, 1568 f 165, 1535 + 242, and 1162 + 383 pg/g wet cortex, respectively) (Fig. 3). The difference in cortical levels 30 min after cephaloridine is most probably the result of mobilization from the site of injection since intravenous administration of cephaloridine did not affect cortical levels at any of the three intervals in rabbits pretreated with verapamil (cephaloridine, 30, 60, and 120 min, 3247 * 177, 3445 -t 324, and 1132 + 167 pg/g wet cortex; verapamil-cephaloridine, 30, 60, and 120 min, 3536 f 285, 2704 + 265, and 1013 + 66 pg/g wet cortex, n = 3-6). Verapamil did not significantly alter assay results when cephaloridine was added to cortical homogenate from animals receiving verapamil30 min before decapitation. Calcium content of renal cortex and cortical mitochondria. The calcium content of cortical mitochondria was significantly increased in the cephaloridine group compared to each of the others (control, 32.6 f 5.3; verapamil, 27.6 f 3.3; cephaloridine, 70.0 f 14.9; and verapamil-cephaloridine, 32.3 f 4.62 nmol/mg protein; n = 6 for each group) (Fig. 4). The calcium contents of the cortex in each of the four groups were not significantly different from one another (control, 25.5 f 4.3; verapamil, 27.1 f 3.8; cephalori-
VERAPAMIL
FIG. I. Histology of the renal cortex idine, 100 mg/kg SC. (B) Cephaloridine.
IN
CEPHALORIDINE
385
NEPHROTOXICTTY
2 daysafter dru rg treatment. Renal capsule is at the top. (A) Cephalor100 mg/kg SC:. 90 min after verapamil, 200 rg/kg iv.
dine, 3 1.6 f. 3.1; and verapamil-cephaloridine, 37.1 + 4.3 nmol/mg protein) (Fig. 4). DISCUSSION Calcium channel blockers have been found to ameliorate the renal injury produced by
FIG. 2. Cephaloridine nephrotoxicity 2 days after administration of the drugs. For scoring procedure. see Methods. (*)I, i 0.05 vs cephaloridine group. Verapamil. N = 4; cephaloridine. N = 12: verapamil-cephaloridine. N= 12.
gentamicin (Lee et al., 1987) and transient &hernia (Burke et al., 1984; Arnold et al.. 1985; Goldfarb et al., 1983: Weinberg et al., 1984; Garthoff et al., 1987). Lee and coworkers (1987) found that nitrendipine markedly improved the functional and morphologic consequences of chronic adminis-
FIG. 3. Cephaloridine rum after subcutaneous il-pretreated animals: cephaloridine alone
concentrations in cortex and seinjection. Solid circles. verapamopen circles. animals receiving
386
MARC C. BROWNING
CORTEX
MITOCHONDRIA
FIG. 4. Calcium content of renal cortex and cortical mitochondria obtained from each of four groups of rabbits 3.5 hr after administration of verapamil or vehicle. (*)p < 0.05 vs other mitochondria. N = 6 for each group.
tration of toxic doses of gentamicin. A variety of calcium channel blockers have been found to reduce functional and morphologic evidence of damage in ischemic models of acute renal failure. Burke et al. (1984) in a careful study of norepinephrine-induced acute renal failure, showed functional, histologic, and metabolic improvement when verapamil was infused after norepinephrine. In contrast, our experiments demonstrate increased proximal tubular necrosis in rabbits pretreated with verapamil despite the finding of normal total calcium in the mitochondria isolated from rabbits pretreated with verapamil. Sumpio et al. (1987) also showed that calcium uptake by cortical mitochondria could be dissociated from aspects of the toxic process. In an isolated, perfused kidney model of cyclosporine toxicity, they found that verapamil pretreatment prevented calcium accumulation in cortical mitochondria but did not prevent tubular proteinuria or depletion of ATP. Insofar as calcium channel blockers may prevent toxicity by reducing calcium influx into cells of the kidney, others have provided evidence that calcium may not be the final common pathway to irreversible cell injury in some toxic insults. Cronin and Newman ( 1985) found that the increased calcium content of kidneys 8 days after beginning gentamicin therapy was not significantly affected by treatment with thyroxine, which nevertheless prevented the functional consequences. Similarly, thyroxine did not prevent
calcium accumulation in the cortex in uranyl nitrate-induced renal failure despite reducing the fall in creatinine clearance (Cronin et al., 1986). Troyer et al. (1982) reported that death of LLCPKl cells exposed to mellitin was not prevented by a calcium-free medium. Because cortical levels of cephaloridine correlate strongly with nephrotoxicity (Tune et al., 1977) and data from experiments with cortical slices suggested that verapamil could increase the tissue concentration of compounds transported by the organic anion transport system (Matsushima and Gemba, 1982), we measured the in vivo concentration of the antibiotic in the cortex of control and verapamil-pretreated animals. Verapamil transiently increased cephaloridine concentrations in the cortex at 30 min compared to control rabbits but thereafter cortical levels of the antibiotic in the verapamil-pretreated rabbits were not statistically different from those receiving cephaloridine alone. These data do not readily explain the increased toxicity in verapamil-pretreated animals. Because calcium channels sensitive to verapamil have not been demonstrated in the proximal tubule, we examined calcium accumulation by cortical mitochondria. We selected mitochondria for study because in vivo they are sequestered from the high calcium concentration of the extracellular fluid which could confound interpretation of calcium content in whole cortex, they take up calcium avidly, and they are sensitive to changes in intracellular calcium activity (Mandel and Murphy, 1984). Cephaloridine produced a significant increase in the calcium content of cortical mitochondria 2 hr after administration of the antibiotic. Pretreatment with verapamil completely prevented the increase in mitochondrial calcium content. The mitochondrial calcium accumulation probably reflects in vivo mitochondrial calcium status since (1) mitochondria were prepared in the presence of EGTA, which limits calcium uptake during isolation (Weinberg and Humes, 1985). (2) they were exposed to similar cal-
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cium concentrations during isolation (Fig. 4), sary component in the irreversible injury not favoring uptake by the organelles from produced by cephaloridine but rather an epithe group receiving cephaloridine alone, and phenomenon which is a marker of the (3) calcium channel blockers do not prevent administration of a mildly toxic dose of cephcalcium uptake by mitochondria (Burke er aloridine. We speculate that calcium influx al., 1984: Vaghy et al., 1982), and would not into the cytosol and mitochondria is not a reprotect the verapamil-cephaloridine-treated quirement for the lethal effect of cephalorimitochondria from in vitro calcium accumudine. lation. However, this experiment looked at mitochondrial calcium at only one time REFERENCES point and cannot distinguish ionized from total calcium. Free calcium in the mitochonARNOLD, P. E., LUML.ERTGUL, D., BURKE, 7. J.. AND dria may yet be important in the toxic proSCHRIER, R. W. (1985). In vitro versus in vivo mitochondrial calcium loading in ischemic acute renal failcess. ure. Amrr. J. Ph.vsio1.248, F845-F850. Despite verapamil’s dissociation of calATKINSON, R. M.. CURRIE, J. P., DAVIS. B.. PRATT cium accumulation in cortical mitochondria D. A. H., SHARPE, H. M.. AND TOMICH, E. G. (1966). from the toxic process, the calcium content Acute toxicity of cephaloridine, an antibiotic derived of cortical mitochondria was a sensitive and from cephalosporin C. T~~.CcoJ. :l.up,pl.Pharnwol8. 398-406. early indicator of in vivo cephaloridine adBURKE, T. J.. ARNOLD. P. E.. GORDON. J. A., BULCXR. ministration. The majority of studies examR. E., DOBYAN. D. C., AND SCHRIER. R. W. (1984). ining the metabolic bases of cephaloridine Protective effect of intrarenal calcium membrane toxicity have used doses considerably larger blockers before or after renal ischemia. J. <‘/in IIIthan those used in our experiments. The ear~~1.74, 1830- I84 I. liest biochemical alteration produced by a CRONIN. R. E., BROWN. D. M.. AND SIMONSEN, K. (1986). Protection by thyroxine in nephrotoxic acute toxic dose of cephaloridine in viuo is the fall renal failure. Amer. J. Physiol.251, of reduced glutathione content in the renal CRONIN. R. E.. AND NEWMAN, J. A. F408-F416. (1985). Protective cortex reported by Kuo and Hook (1982) 1 hr effect of thyroxine but not parathyroidectomy on genafter injection of 250 mg/kg SC.Morphologic tamicin nephrotoxicity. .Jnwr J. Physid248, F333F339. alterations have also been found 1 hr after 200 mg/kg im (Silverblatt et al., 1970). In- FARBER. J. L. ( 1982). Membrane injury and calcium homeostasis in the pathogenesis of coagulative necrosis. creased content of conjugated dienes in the Luh. IrnX51.47, 114-123. renal cortex of rats (Kuo et al., 1983) and the GARTHOFF, 8.. HIRTH. C.. FEDERMANN. A.. KAZDA. S.. reduction in succinate-supported mitochonAND STASCH. J-P. Renal effects of 1,4-dihydropyridines in animal models of hypertension and renal faildrial oxidative phosphorylation (Tune et al., ure. (I 987). J. (irrdiovusc~. Pharmad.9, Suppl. I SX1979) in rabbits have been demonstrated 2 hr Sl3. after administration of 2000 and 200 mg/kg, GOI.DFARB, D., IAINA. A., SERBAN, I., GAVENM), S.. respectively. KAPULER, S., AND EUAHOU. H. E. (1983). Benetictal In summary, (I) pretreatment with veraeffect of verapamil in ischemic acute renal failure in the rat. Pm,. Sock. E-\-p Bid. Afed.172, 389-392. pamil augmented, rather than ameliorated, HUMES. H. D.. HUN-I’, D. A.. AND WHITE. M. D. (1987). the proximal tubular necrosis produced by a Direct toxic effect of the radiocontrast agent diatrimildly toxic dose of cephaloridine, (2) calzoate on renal proximal tubule cells. :Imrpr .I P/r!y cium accumulated in mitochondria isolated ~1.252, F246-F255. from renal cortex of rabbits within 2 hr ofthat JONFS, T. W.. WALLIN. A., THOR. H.. GERDES. R (i.. ORMSTAD, K.. ANDORRENIUS, S. (1986). The mechamildly toxic dose, and (3) that mitochondrial nism of pentachlorobutadienyl-glutathione nephroaccumulation of calcium was abolished by toxicity studied with isolated rat renal epithelial cells. pretreatment with verapamil. We conclude Arch. Biochrm. Biophj:s.251, 504-5 13. that an increase in the total calcium content Ku~, C.-H.. AND HOOK, J. B. (1982). Depletion of renal of renal cortical mitochondria is not a necesglutathione content and nephrotoxicity of cephalori-
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