Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: Determination of water permeability changes of renal principal cells

The water channel aquaporin-2 (AQP2), a key component of the antidiuretic m achinery in the kidney, is rapidly regulated by the antidiuretic hormone va sopressin. The hormone exerts its action by inducing a translocation of AQP 2 from intracellular vesicles to the cell membrane. This step requires the elevation of intracellular cyclic AMP. We describe here a new method, laser scanning reflection microscopy (LSRM), suitable for determining cellular o smotic water permeability coefficient changes in primary cultured inner med ullary collecting duct (IMCD) cells. The recording of vertical-reflection-m ode x-z-scan section areas of unstained, living IMCD cells proved useful an d valid for the investigation of osmotic water permeability changes. The ti me-dependent increases of reflection-mode x-z-scan section areas of swellin g cells were fitted to a single-exponential equation. The analysis of the t ime constants of these processes indicates a twofold increase in osmotic wa ter permeability of IMCD cells after treatment of the cells both with forsk olin, a cyclic AMP-elevating agent, and with Clostridium difficile toxin B, an inhibitor of Rho proteins that leads to depolymerization of F-actin-con taining stress fibers. This indicates that both agents lead to the function al insertion of AQP2 into the cell membrane. Thus, we have established a ne w functional assay for the study of the regulation of the water permeabilit y at the cellular level.