Investigation of effect of tectorigenin (O-methylated isoflavone) on Ca2+ signal transduction and cytotoxic responses in canine renal tubular cells

Abstract

Tectorigenin, a traditional Chinese medicine, is isolated from the flower of plants such as Pueraria thomsonii Benth. It is an O-methylated isoflavone, a type of flavonoid. Previous studies have shown that tectorigenin evoked various physiological responses in different models, but the effect of tectorigenin on cytosolic-free Ca2+ levels ([Ca2+]i) and cytotoxicity in renal tubular cells is unknown. Our research explored if tectorigenin changed Ca2+ signal transduction and viability in Madin–Darby Canine Kidney (MDCK) renal tubular cells. [Ca2+]i in suspended cells were measured by applying the fluorescent Ca2+-sensitive probe fura-2. Viability was explored by using water-soluble tetrazolium-1 as a fluorescent dye. Tectorigenin at concentrations of 5–50 μM induced [Ca2+]i rises. Ca2+ removal reduced the signal by approximately 20%. Tectorigenin (50 μM) induced Mn2+ influx suggesting of Ca2+ entry. Tectorigenin-induced Ca2+ entry was inhibited by 10% by three inhibitors of store-operated Ca2+ channels, namely, nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited 83% of tectorigenin-evoked [Ca2+]i rises. Conversely, treatment with tectorigenin abolished thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C with U73122 inhibited 50% of tectorigenin-induced [Ca2+]i rises. Tectorigenin at concentrations between 10 and 60 μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis (2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl did not reverse tectorigenin's cytotoxicity. Our data suggest that, in MDCK cells, tectorigenin evoked [Ca2+]i rises and induced cell death that was not associated with [Ca2+]i rises. Therefore, tectorigenin may be a Ca2+-independent cytotoxic agent for kidney cells.

Keywords

• ca2+ signal transduction
• madin-darby canine kidney
• tectorigenin
• viability