Mechanisms of cyclosporine A toxicity in defined cultures of renal tubule epithelia: a role for cysteine proteases.
The mechanisms of toxicity of cyclosporine A (CsA) were studied in primary cultures of individually microdissected rabbit and human renal tubules of proximal and distal regions of the nephron. A direct toxic effect of CsA on renal tubule epithelia was demonstrated using nigrosine uptake and LDH release as indicators of cell death. Proximal convoluted tubules (PCT) and proximal straight tubules (PST) were shown to be highly sensitive, while thick ascending limbs of Henle (TAL) were much less sensitive and cortical collecting tubules (CCT) relatively resistant. The effects of CsA were time and dose dependent over the range 50 ng/ml to 100 micrograms/ml. Protection against CsA-induced PST cell death was afforded by reduction in extracellular calcium levels in the media or addition of the calcium entry antagonists: verapamil, nifedipine or diltiazem. In addition, treatment with the cysteine protease inhibitor, E64, attenuated CsA-induced cell damage. A role for the lysosomal cysteine proteases (cathepsins), however, was ruled out on the basis of identical activity levels in all cell types; no beneficial effects of lysosomal enzyme depletion and no evidence of lysosomal rupture prior to death. By contrast, a role for the cytoplasmic, calcium-dependent cysteine protease calpain was suggested since activity levels were significantly higher in PST than CCT cultures and were inducible by CsA.
Wilson
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Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Hartz
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D000818:Animals; D002118:Calcium / Q000502:physiology; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000009:adverse effects* / Q000493:pharmacokinetics; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004110:Diltiazem / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007674:Kidney Diseases / Q000139:chemically induced; D007684:Kidney Tubules / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009543:Nifedipine / Q000494:pharmacology; D011817:Rabbits; D013997:Time Factors; D014700:Verapamil / Q000494:pharmacology
D015853:Cysteine Proteinase Inhibitors; D016572:Cyclosporine; D014700:Verapamil; D003546:Cysteine Endopeptidases; D004110:Diltiazem; D007930:Leucine; D009543:Nifedipine; C024974:E 64; D002118:Calcium