Boi‐Ogi‐To, a Traditional Japanese Kampo Medicine, Promotes Cellular Excretion of Chloride and Water by Activating Volume‐Sensitive Outwardly Rectifying Anion Channels

The Japanese Kampo medicine Boi-ogi-to (BOT) is known as an effective therapeutic agent for edema and nephrosis by promoting the excretion of excess body fluids. Despite its empirical effectiveness, scientific evidence supporting its effectiveness remains limited. In this study, we conducted a retrospective study of the effects of BOT administration on the blood test values of patients before and after taking the drug to attempt translational research between basic science and daily clinical practice by focusing on the molecular mechanism of action of BOT in vitro. We found that blood sodium and chloride levels are higher after taking BOT by analyzing the clinical test values before and after taking the drug from 28 patients attending Akita University Hospital. In this light, we measured the cell volume of human embryonic kidney HEK293T cells in vitro in order to investigate the possibility that BOT induces Cl^- excretion and cell volume reduction. BOT showed concentration-dependent cell volume reduction with an EC₅₀ of 686 μg/mL. The volume reduction effect was suppressed by the Cl^- channel inhibitors DIDS and DCPIB. Furthermore, patch-clamp studies showed that BOT-activated Cl^- currents exhibit outward rectification and time-dependent inactivation upon depolarization. These biophysical properties of BOT-induced Cl^- currents correspond to those of volume-sensitive outward rectifier (VSOR) anion channels. The Cl^- currents activated by the administration of BOT were inhibited by applying DIDS, DCPIB, and siRNA targeting the gene of LRRC8A, a core component of the VSOR channel, as well as in LRRC8-deficient cells. Additionally, BOT-induced Cl^- currents were restored by coexpression of LRRC8A/C in LRRC8-deficient cells. Also, BOT was found to translocate LRRC8A proteins to the plasma membrane. These results demonstrated that BOT activates LRRC8-containing VSOR channels by delivering LRRC8A to the plasma membrane and induces Cl^- release, thereby promoting water excretion.