Mitochondrial Ca2+ overload in primary cultures of rat renal cortical epithelial cells by cytotoxic concentrations of cyclosporine: a digitized fluorescence imaging study.
Cyclosporine (CsA) has been reported to disrupt Ca2+ efflux from mitochondria, which suggests that CsA interference with Ca2+ homeostasis may be related to its nephrotoxicity. Therefore, the purpose of this study was (1) to determine intracellular free Ca2+ concentration ([Ca2+]i) and mitochondrial free Ca2+ concentration ([Ca2+]m) after primary cultures of rat renal cortical epithelial cells were exposed to cytotoxic concentrations of CsA; and (2) to explore the role of disruption of intracellular and mitochondrial Ca2+ homeostasis in CsA-induced cytotoxicity. [Ca2+]i in single kidney cells was examined by digitized fluorescence imaging (DFI) of the Ca2+ fluorescent probe, fura-2, and [Ca2+]m in single cells was observed by DFI of fura-2 entrapped in mitochondria after selective permeabilization of plasma membrane and other non-mitochondrial organelles by digitonin. Mitochondrial membrane potential (delta psi) in single kidney cells was examined by rhodamine 123 (Rh-123) with DFI. Intracellular ATP in kidney cells was determined by a HPLC method. CsA resulted in an elevation in [Ca2+]i and [Ca2+]m, dissipation of delta psi and depletion of ATP in a dose- and time-dependent manner. The elevation of [Ca2+]i and [Ca2+]m and depletion of ATP preceded CsA-induced cytotoxicity in kidney cells as measured by lactate dehydrogenase (LDH) leakage. We conclude that CsA-induced alterations in mitochondrial Ca2+ homeostasis and a subsequent loss of energy supply may play a key role in CsA-induced cytotoxicity in primary cultures of rat renal cortical epithelial cells.
Jiang
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Division of Pharmacology & Toxicology, College of Pharmacy, University of Texas, Austin 78703.
Acosta
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D016572:Cyclosporine / Q000633:toxicity*; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects; D016257:Fura-2; D006706:Homeostasis / Q000187:drug effects*; D007424:Intracellular Fluid; D007672:Kidney Cortex / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
D016572:Cyclosporine; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2