Effects of thymol on Ca ²⁺ homeostasis and apoptosis in MDCK renal tubular cells

Thymol is a natural essential oil present in many plants and has many different effects in various cell types. However, the effect of thymol on the physiology of Madin-Darby canine kidney (MDCK) renal tubular cells is unknown. The action of the phytochemical thymol on cytosolic Ca ²⁺ concentrations ([Ca ²⁺ ]i) and apoptosis in MDCK renal tubular cells was explored. Fura-2, a Ca ²⁺ -sensitive fluorescent dye, was used to assess [Ca ²⁺ i. Thymol at concentrations of 200-500 μM caused a [Ca ²⁺ ]i rise in a concentration-dependent manner. Removal of extracellular Ca ²⁺ partially reduced the effects of thymol. Thymol-induced Ca ²⁺ entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In a Ca ²⁺ -free medium, treatment with the endoplasmic reticulum Ca ²⁺ pump inhibitor thapsigargin inhibited thymol-induced [Ca ²⁺ ]i increases. Treatment with thymol also inhibited thapsigargin-induced [Ca ²⁺ i rise. Thymol killed cells at concentrations of 300-500 μM in a concentrationdependent fashion. Chelating cytosolic Ca ²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent thymol cytotoxicity. Thymol (400 and 500 μM) induced apoptosis detected by using Annexin V/propidium iodide staining. At 400 or 500 μM, thymol increased levels of reactive oxygen species (ROS). Together, in MDCK cells, thymol induced a [Ca ²⁺ ]i rise by inducing Ca ²⁺ release from the endoplasmic reticulum and Ca ²⁺ entry via protein kinase C-sensitive store-operated Ca ²⁺ channels. Our data suggest that thymol-induced apoptosis might involve ROS production.