Dynamic Compartmentalisation of Intracellular Sodium in Collecting Duct Cells

Abstract

In Principal cells (PC) of the cortical collecting duct (CCD), the highly regulated and co‐ordinated reabsorption of sodium occurs through the epithelial sodium channel (ENaC) at the apical membrane and Na/K ATPase at the basolateral membrane. However, it is not known how sodium ions (Na+) are transported across the cell and if it is a regulated mechanism. We investigated trans‐cellular transport in the mouse collecting duct cell line, mCCDcl1 cells using a fluorescent sodium dye, Corona Green AM. The dye was found to accumulate in fluid‐filled domes forming between the single cell layer and the plastic dish, indicating that the cells are polarised and that Na+ ions are transported across and concentrated beneath the principal cells. Dye uptake was stimulated by aldosterone, blocked by amiloride (an ENaC inhibiter), and basolateral transport was prevented by ouabain (an Na/K ATPase blocker) thus validating the dye’s apparently faithful replication of sodium transport across the cells. Live cells exhibited a consistent pattern of sodium‐containing vesicles, of various sizes, surrounded by cytoskeleton and lipid membrane. While the smallest vesicles (~0.5µm) co‐stained with lysotracker, larger vesicles (up to 6.4µm) did not co‐stain with either lysosomal‐ or mitochondrial‐specific dyes and appeared to have internal structure, suggesting that they were multivesicular bodies. Time‐lapse confocal imaging of cells cultured on a 3D polyHYPE scaffold and subsequent movies of intracellular dynamics showed a subset of these multivesicular bodies release or take up sodium dye in a controlled manner, over the course of a few minutes. Our novel data reveal intracellular sodium compartmentalisation, which may offer new insights into intracellular sodium dynamics, possibly in response to stress, in the collecting duct.