Mitochondrial Ca 2+ overload in primary cultures of rat renal cortical epithelial cells by cytotoxic concentrations of cyclosporine: a digitized fluorescence imaging study

Abstract

Cyclosporine (CsA) has been reported to disrupt Ca 2+ efflux from mitochondria, which suggests that CsA interference with Ca 2+ homeostasis may be related to its nephrotoxicity. Therefore, the purpose of this study was (1) to determine intracellular free Ca 2+ concentration ([Ca 2+]i) and mitochondrial free Ca 2+ concentration ([Ca 2+]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 Ca 2+ homeostasis in CsA-induced cytotoxicity. [Ca 2+]i in single kidney cells was examined by digitized fluorescence imaging (DFI) of the Ca 2+ fluorescent probe, fura-2, and [Ca 2+]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 (ΔΨ) 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 [Ca 2+]i and [Ca 2+]m, dissipation of ΔΨ and depletion of ATP in a dose- and time-dependent manner. The elevation of [Ca 2+]i and [Ca 2+]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 Ca 2+ 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.