Cephaloridine-induced biochemical changes and cytotoxicity in suspensions of rabbit isolated proximal tubules.
Cephalosporin antibiotics, such as cephaloridine (Cld), are known to be nephrotoxic in vivo and in vitro. In vivo, Cld causes proximal tubule necrosis in rabbits which is preceded by glutathione (GSH) depletion and, under certain conditions, inhibition of mitochondrial function. In vitro, Cld causes GSH depletion, lipid peroxidation, and inhibition of rat kidney slice organic ion uptake. The present investigations were designed to evaluate the temporal relationships of the biochemical "lesions" caused by Cld to the onset of lethal cell injury in suspensions of isolated rabbit proximal tubules. Cld was cytotoxic to suspensions of rabbit proximal tubules (EC50 = 1.10 +/- 0.33 mM) in the absence of amino acids (to support GSH synthesis). In this model, Cld also caused GSH and ATP depletion, lipid peroxidation (malondialdehyde formation), and inhibition of tubule respiration. Probenecid prevented Cld accumulation, tubule injury, ATP depletion, and lipid peroxidation and markedly attenuated the GSH depletion. Addition of glycine, cystine, and glutamate to the incubation buffer to support GSH synthesis decreased the tubule accumulation of Cld (due solely to the presence of glutamate) and blocked Cld-induced tubule lethality, lipid peroxidation, ATP depletion, and GSH depletion. Glycine or glutamate alone had no effect on Cld-induced cytotoxicity, whereas cystine was cytoprotective. Buthionine sulfoximine partially reversed the amino acid protection against Cld-induced tubule injury. Thus amino acid-induced protection of tubules from Cld cytotoxicity was due to the combination of a high intracellular GSH content and cytoprotection by cystine. The antioxidant N-N'-diphenyl-p-phenylenediamine (DPPD) blocked tubule injury, ATP depletion, and lipid peroxidation but had no effect on Cld-induced GSH depletion when tubules were incubated for 3 hr. However, when incubations were allowed to run for up to 8 hr, DPPD had no effect on Cld cytotoxicity, despite continued inhibition of lipid peroxidation. These data demonstrate that Cld-induced tubule injury in short-term (3 hr) incubations in vitro occurs by a mechanism probably involving lipid peroxidation and occurs only in the absence of amino acids to support GSH synthesis. Inhibition of tubule respiration and ATP depletion could not clearly be causally linked to the onset of cell death in this model. The mechanism of the peroxidation-independent Cld toxicity in tubules incubated for 8 hr or longer is not known at this time.
Rush
G F
GF
Lilly Research Laboratories, Eli Lilly and Co., Greenfield, Indiana.
Ponsler
G D
GD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002511:Cephalosporins / Q000493:pharmacokinetics / Q000633:toxicity*; D005260:Female; D005978:Glutathione / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D010655:Phenylenediamines / Q000494:pharmacology; D011817:Rabbits
D000596:Amino Acids; D000975:Antioxidants; D002511:Cephalosporins; D010655:Phenylenediamines; D000255:Adenosine Triphosphate; C004845:N,N'-diphenyl-4-phenylenediamine; D007770:L-Lactate Dehydrogenase; D005978:Glutathione