215
Toxicology Letters, 53 (1990) 215-217 Elsevier
TOXLET
02421
Biochemical mechanisms of cephaloridine nephrotoxicity in suspensions of isolated rabbit proximal tubules
Glenn F. Rush and G. Douglas Ponsler Lilly Research Laboratories,
Key words: Cephaloridine;
Eli Lilly and Co.. Greenfield, IN (U.S.A.)
Nephrotoxicity;
Lipid peroxidation;
Mitochondrial
injury; Isolated
proximal
tubules
It is well established that cephaloridine (CLD) can cause renal dysfunction, characterized by decreased glomerular filtration rate, glycosuria, enzymuria and proteinuria in both humans and laboratory animals. These changes are often accompanied histologically by widespread necrosis of the S2 segment of the proximal tubule [l]. Nephrotoxicity is not unique to CLD and is often observed during the preclinical development of other, new /3-lactam antibiotics. Thus, these compounds, as a class, have gained the reputation for being nephrotoxic. There has been considerable progress made on the elucidation of the biochemical mechanisms of B-lactam-induced nephrotoxicity. Studies with CLD have indicated that perioxidative decomposition of membrane lipids may be a key event leading to renal injury. Kuo et al. [2] have presented data suggesting that the pyridinium ring of CLD can undergo redox cycling producing oxygen free radicals leading to glutathione (GSH) depletion, malondialdehyde (MDA) formation and renal necrosis. In contrast, Tune and colleagues have suggested that the nephrotoxic cephalosporins can acylate and inactivate the mitochondrial transporters for anionic substrate uptake and thus limit the entry of succinate into the inner mitochondrial compartment [3]. Although these two theories provide an explanation for many of the renal biochemical changes that can occur following administration of nephrotoxic cephalosporins, there are some questions left unresolved. For example, studies examining lipid peroxidation as a mechanism of CLD nephrotoxicity have been conducted alAddress for correspondence:
Glenn
F. Rush,
Lilly Research
Laboratories,
Eli Lilly and Co., Greenfield
IN 46140. U.S.A.
037%4274/90/$3.50
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
216
most exclusively in rats while mit~hondri~ injury has been evaluated p~marily in rabbits (the most susceptible species). Furthermore, the role of lipid peroxidation in the mechanism of in vivo nephrotoxicity of cephalosporins other than CLD is not clear. Similarly, the in vivo consequences of cephalosporin-induced mitochondrial dysfunction are also unknown since the kidney may derive the majority of its metabolic energy in vivo from fatty-acid oxidation and circulating acetoacetic acid, neither of which would be expected to be influenced by changes in the mitochondrial anionic transporter. The present experiments were designed to determine if isolated renal tubules from rabbits could serve as a suitable model for cephalosporin-induced cellular injury in vitro and to determine the relationships between mitochondrial and peroxidative injury as mechanisms of /%lactam nephrotoxicity. Renal proximal tubules were prepared from female New Zealand white rabbits (N 2 kg) by using magnetic iron oxide to remove the glomeruli. Tubules were then dispersed into a modified Krebs-bicarbonate buffer containing 5.0 mM glucose, 4.0 mM lactate and 5.0 mM butyrate and oxygenated at 37°C in a rotary shaker bath. Reduced and oxidized GSH were measured as described by Griffith [4]. Lactate dehydrogena~ (LDH), MDA and cellular ATP were measured as described by Rush et al. [5]. Exposure of isolated proximal tubules to CLD caused dose-dependent cell injury reflected by an increase in the leakage of LDH and MDA formation (IC5e of approx. 1 mM) and GSH depletion (ICse of approx. 0.1 mM). The onset of renal cell injury (LDH leakage) did not occur until after 60-90 min of incubation, whereas GSH depletion and MDA formation occurred within 30 min, clearly preceding the onset of cell injury. CLD was cytotoxic only in isolated renal tubules that were initially GSHdepleted; inclusion of 1.OmM glutamate, cystine and glycine to the incubation media to supporf GSH synthesis protected tubules from cell damage. CLD did not cause any changes in rabbit tubule, antimycin-insensitive respiration. Menadione, which was used 8s a positive control, resulted in a 2-fold increase in antimycin-insensitive respiration. CLD is accumulated in renal tissue in vivo by a probenecid-sensitive anionic transporter on the basolateral membrane. In isolated tubules, 2.0 mM probenecid completely prevented CLD-induced MDA formation and cell injury and attenuated the depletion of GSH. The acute (3 h) injury to isolated renal tubules could also be prevented by the antioxidant DPPD which blocked CLD-induced MDA formation and LDH leakage. DPPD had no effect on depletion of tubule GSH caused by CLD. In contrast, inhibition of MDA formation with DPPD had no effect on CLD-induced renal tubule injury after 8 h of incubation. Incubation of renal tubules with CLD also caused a dose-dependent decrease in cellular ATP content and cellular respiration; however, these changes did not precede, but rather were coincidental with, the onset of cell injury. These data demonstrate that CLD was capable of causing acute cell injury in a model of isolated renal proximal tubules. The mechanism of this injury probably involved lipid peroxidation. Intracellular accumulation of CLD was important in this
211
model as cytotoxicity could be blocked with probenecid. However, the profile of the ‘short-term’ (< 4 h) CLD toxicity in this model did not duplicate the events that occurred in vivo. For example, in vivo renal GSH content was reduced by only approximately 40% and recovered by 3-4 h and cell necrosis was not observed in rabbits until approximately 10-16 h following CLD exposure [I]. In the present model, proximal tubule damage was observed within 90 min only tubules partially depleted of GSH and there was essentially complete GSH depletion by 30 min which lasted the duration of the experiment. Therefore, it is not clear at this point if this in vitro model for cephalosporin nephrotoxicity accurately represents events occurring in vivo. No evidence for redox cycling of CLD resulting in the production of reactive oxygen species could be found in this model as there were no increases in antimycin-insensitive tubule respiration. Mitochondrial toxicity and ATP depletion were probably not involved as a mechanism of CLD toxicity in 3 h incubations as these changes were more likely a result of cell death rather than a cause. The mechanism of CLD cytotoxicity in 8 h incubations is unknown at this time, but may represent more accurately the CLD-induced biochemical changes occurring in vivo. REFERENCES I Silverblatt, F., Turck, M. and Bulger, R. (1970) N~hrotoxicity due to CLD: a light and electron-microscopic study in rabbits. J. infect. Dis. 122,33-44. 2 Kuo, C-H., Maita, K., Sleight, SD. and Hook, J.B. (1983) Lipid peroxidation: a possible mechanism of CLD-induced nephrotoxicity. Toxicol. Appl. Pharmacol. 67,78&88. 3 Tune, B.M., Sibley, R.K., and Hsu, C.Y. (1988) The mitochondrial respiratory toxicity of cephalosporin antibiotics. An inhitibory effect on substrate uptake. J. Pharmacol. Exp. Ther. 245,10.54-1059. 4 Griffith, O.W. (1980) Determination of GSH and GSH disulfide using GSH reductase and 2-vinyl pyridine. Anal. Biochem. 1%,207-212. 5 Rush, G.F., Ripple, M. and Chenery, R. (1985) Mechanism of oxmetidine (SK&F 92994) cytotoxicity in isolated rat hepatocytes. J. Pharmacol. Exp. Ther. 233, 741-756.
Toxicology Letters, 53 (1990) 215-217 Elsevier
TOXLET
02421
Biochemical mechanisms of cephaloridine nephrotoxicity in suspensions of isolated rabbit proximal tubules
Glenn F. Rush and G. Douglas Ponsler Lilly Research Laboratories,
Key words: Cephaloridine;
Eli Lilly and Co.. Greenfield, IN (U.S.A.)
Nephrotoxicity;
Lipid peroxidation;
Mitochondrial
injury; Isolated
proximal
tubules
It is well established that cephaloridine (CLD) can cause renal dysfunction, characterized by decreased glomerular filtration rate, glycosuria, enzymuria and proteinuria in both humans and laboratory animals. These changes are often accompanied histologically by widespread necrosis of the S2 segment of the proximal tubule [l]. Nephrotoxicity is not unique to CLD and is often observed during the preclinical development of other, new /3-lactam antibiotics. Thus, these compounds, as a class, have gained the reputation for being nephrotoxic. There has been considerable progress made on the elucidation of the biochemical mechanisms of B-lactam-induced nephrotoxicity. Studies with CLD have indicated that perioxidative decomposition of membrane lipids may be a key event leading to renal injury. Kuo et al. [2] have presented data suggesting that the pyridinium ring of CLD can undergo redox cycling producing oxygen free radicals leading to glutathione (GSH) depletion, malondialdehyde (MDA) formation and renal necrosis. In contrast, Tune and colleagues have suggested that the nephrotoxic cephalosporins can acylate and inactivate the mitochondrial transporters for anionic substrate uptake and thus limit the entry of succinate into the inner mitochondrial compartment [3]. Although these two theories provide an explanation for many of the renal biochemical changes that can occur following administration of nephrotoxic cephalosporins, there are some questions left unresolved. For example, studies examining lipid peroxidation as a mechanism of CLD nephrotoxicity have been conducted alAddress for correspondence:
Glenn
F. Rush,
Lilly Research
Laboratories,
Eli Lilly and Co., Greenfield
IN 46140. U.S.A.
037%4274/90/$3.50
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
216
most exclusively in rats while mit~hondri~ injury has been evaluated p~marily in rabbits (the most susceptible species). Furthermore, the role of lipid peroxidation in the mechanism of in vivo nephrotoxicity of cephalosporins other than CLD is not clear. Similarly, the in vivo consequences of cephalosporin-induced mitochondrial dysfunction are also unknown since the kidney may derive the majority of its metabolic energy in vivo from fatty-acid oxidation and circulating acetoacetic acid, neither of which would be expected to be influenced by changes in the mitochondrial anionic transporter. The present experiments were designed to determine if isolated renal tubules from rabbits could serve as a suitable model for cephalosporin-induced cellular injury in vitro and to determine the relationships between mitochondrial and peroxidative injury as mechanisms of /%lactam nephrotoxicity. Renal proximal tubules were prepared from female New Zealand white rabbits (N 2 kg) by using magnetic iron oxide to remove the glomeruli. Tubules were then dispersed into a modified Krebs-bicarbonate buffer containing 5.0 mM glucose, 4.0 mM lactate and 5.0 mM butyrate and oxygenated at 37°C in a rotary shaker bath. Reduced and oxidized GSH were measured as described by Griffith [4]. Lactate dehydrogena~ (LDH), MDA and cellular ATP were measured as described by Rush et al. [5]. Exposure of isolated proximal tubules to CLD caused dose-dependent cell injury reflected by an increase in the leakage of LDH and MDA formation (IC5e of approx. 1 mM) and GSH depletion (ICse of approx. 0.1 mM). The onset of renal cell injury (LDH leakage) did not occur until after 60-90 min of incubation, whereas GSH depletion and MDA formation occurred within 30 min, clearly preceding the onset of cell injury. CLD was cytotoxic only in isolated renal tubules that were initially GSHdepleted; inclusion of 1.OmM glutamate, cystine and glycine to the incubation media to supporf GSH synthesis protected tubules from cell damage. CLD did not cause any changes in rabbit tubule, antimycin-insensitive respiration. Menadione, which was used 8s a positive control, resulted in a 2-fold increase in antimycin-insensitive respiration. CLD is accumulated in renal tissue in vivo by a probenecid-sensitive anionic transporter on the basolateral membrane. In isolated tubules, 2.0 mM probenecid completely prevented CLD-induced MDA formation and cell injury and attenuated the depletion of GSH. The acute (3 h) injury to isolated renal tubules could also be prevented by the antioxidant DPPD which blocked CLD-induced MDA formation and LDH leakage. DPPD had no effect on depletion of tubule GSH caused by CLD. In contrast, inhibition of MDA formation with DPPD had no effect on CLD-induced renal tubule injury after 8 h of incubation. Incubation of renal tubules with CLD also caused a dose-dependent decrease in cellular ATP content and cellular respiration; however, these changes did not precede, but rather were coincidental with, the onset of cell injury. These data demonstrate that CLD was capable of causing acute cell injury in a model of isolated renal proximal tubules. The mechanism of this injury probably involved lipid peroxidation. Intracellular accumulation of CLD was important in this
211
model as cytotoxicity could be blocked with probenecid. However, the profile of the ‘short-term’ (< 4 h) CLD toxicity in this model did not duplicate the events that occurred in vivo. For example, in vivo renal GSH content was reduced by only approximately 40% and recovered by 3-4 h and cell necrosis was not observed in rabbits until approximately 10-16 h following CLD exposure [I]. In the present model, proximal tubule damage was observed within 90 min only tubules partially depleted of GSH and there was essentially complete GSH depletion by 30 min which lasted the duration of the experiment. Therefore, it is not clear at this point if this in vitro model for cephalosporin nephrotoxicity accurately represents events occurring in vivo. No evidence for redox cycling of CLD resulting in the production of reactive oxygen species could be found in this model as there were no increases in antimycin-insensitive tubule respiration. Mitochondrial toxicity and ATP depletion were probably not involved as a mechanism of CLD toxicity in 3 h incubations as these changes were more likely a result of cell death rather than a cause. The mechanism of CLD cytotoxicity in 8 h incubations is unknown at this time, but may represent more accurately the CLD-induced biochemical changes occurring in vivo. REFERENCES I Silverblatt, F., Turck, M. and Bulger, R. (1970) N~hrotoxicity due to CLD: a light and electron-microscopic study in rabbits. J. infect. Dis. 122,33-44. 2 Kuo, C-H., Maita, K., Sleight, SD. and Hook, J.B. (1983) Lipid peroxidation: a possible mechanism of CLD-induced nephrotoxicity. Toxicol. Appl. Pharmacol. 67,78&88. 3 Tune, B.M., Sibley, R.K., and Hsu, C.Y. (1988) The mitochondrial respiratory toxicity of cephalosporin antibiotics. An inhitibory effect on substrate uptake. J. Pharmacol. Exp. Ther. 245,10.54-1059. 4 Griffith, O.W. (1980) Determination of GSH and GSH disulfide using GSH reductase and 2-vinyl pyridine. Anal. Biochem. 1%,207-212. 5 Rush, G.F., Ripple, M. and Chenery, R. (1985) Mechanism of oxmetidine (SK&F 92994) cytotoxicity in isolated rat hepatocytes. J. Pharmacol. Exp. Ther. 233, 741-756.
