MITOCHONDRIAL CA2-CELLS BY CYTOTOXIC CONCENTRATIONS OF CYCLOSPORINE -A DIGITIZED FLUORESCENCE IMAGING STUDY( OVERLOAD IN PRIMARY CULTURES OF RAT RENAL CORTICAL EPITHELIAL)

Cyclosporine (CsA) has been reported to disrupt Ca2+ efflux from mitoc hondria, which suggests that CsA interference with Ca2+ homeostasis ma y be related to its nephrotoxicity. Therefore, the purpose of this stu dy was (1) to determine intracellular free Ca2+ concentration ([Ca2+]i ) and mitochondrial free Ca2+ concentration ([Ca2+]m) after primary cu ltures of rat renal cortical epithelial cells were exposed to cytotoxi c concentrations of CsA; and (2) to explore the role of disruption of intracellular and mitochondrial Ca2+ homeostasis in CsA-induced cytoto xicity. [Ca2+]i in single kidney cells was examined by digitized fluor escence imaging (DFI) of the Ca2+ fluorescent probe, fura-2, and [Ca2]m in single cells was observed by DFI of fura-2 entrapped in mitochon dria after selective permeabilization of plasma membrane and other non -mitochondrial organelles by digitonin. Mitochondrial membrane potenti al (Delta Psi) in single kidney cells was examined by rhodamine 123 (R h-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, dis sipation of Delta Psi and depletion of ATP in a dose- and time-depende nt manner. The elevation of [Ca2+]i and [Ca2+]m and depletion of ATP p receded CsA-induced cytotoxicity in kidney cells as measured by lactat e dehydrogenase (LDH) leakage. We conclude that CsA-induced alteration s in mitochondrial Ca2+ homeostasis and a subsequent loss of energy su pply may play a key role in CsA-induced cytotoxicity in primary cultur es of rat renal cortical epithelial cells.