A digitized fluorescence imaging study of intracellular free calcium, mitochondrial integrity and cytotoxicity in rat renal cells exposed to ionomycin, a calcium ionophore

Abstract

The objective of this study was to explore the role of extracellular Ca 2+ and mitochondrial integrity in ionomycin-induced cytotoxicity in primary cultures of rat kidney cortical epithelial cells using dignitized fluorescence imaging (DFI), which is a powerful tool for continuously observing the dynamic intracellular biochemistry of single living cells. Using DFI, intracellular free calcium ion concentration ([Ca 2+] i), mitochondrial membrane potential and loss of cell viability in individual rat renal cortical epithelial cells were examined temporally by fura-2, rhodamine 123 (Rh-123) and propidium iodide (PI), respectively. Images were taken within 10 min after exposure to 5 and 10 μM ionomycin. These three parameters, [Ca 2+] i, mitochondrial membrane potential and cell viability, were also measured in populations of cells by a multiwell fluorescence scanner with fluo-3, Rh-123 and PI, respectively. Cytotoxicity was also assessed by two colorimetric cytotoxicity tests (LDH leakage and mitochondrial MTT reduction). Using DFI, the fluorescence scanner and the colorimetric cytotoxicity tests, we found that exposure of primary cultures of rat kidney cortical epithelial cells to high concentrations of ionomycin (5 and 10 μM) caused a rapid and sustained rise in [Ca 2+] i, which preceded dissipation of the mitochondrial membrane potential and loss of cell viability and that chelation of extracellular Ca 2+ with EGTA attenuated these responses. We demonstrated the value of using DFI to continuously observe the dynamic intracellular biochemistry of single living cells by establishing a sequence of elevated [Ca 2+] i, dissipation of mitochondrial membrane potential and cytotoxicity. We conclude that a combination of the influx of extracellular Ca 2+ and loss of mitochondrial integrity may be responsible for the cytotoxicity observed in individual renal cells and populations of renal cells after treatment with ionomycin.