3D-CCD: Supplemental Figures and Video

<p>The cortical collecting duct (CCD) of the mammalian kidney plays a major role in the maintenance of total body electrolyte, acid/base and fluid homeostasis by tubular reabsorption and excretion. The mammalian CCD is heterogeneous, comprised of Na+-absorbing principal cells (PCs) and acid-base transporting intercalated cells (ICs). Perturbations in luminal flow rate alter hydrodynamic forces to which these cells in the cylindrical tubules are exposed. However, most studies of tubular ion transport have been performed in cell monolayers grown on or epithelial sheets affixed to a flat support, since analysis of transepithelial transport in native tubules by in vitro microperfusion requires considerable expertise. Here, we report on the generation and characterization of an in vitro, perfusable 3D kidney CCD model (3D CCD), in which immortalized mouse PC-like mpkCCD cells are seeded within a cylindrical channel embedded within an engineered extracellular matrix and subjected to luminal fluid flow. We find that a tight epithelial barrier composed of differentiated and polarized PCs forms within 1 week. Immunofluorescence microscopy reveals the apical epithelial Na⁺&nbsp;channel ENaC and basolateral Na⁺/K⁺-ATPase. On cessation of luminal flow, benzamil-inhibitable cell doming is observed within these 3D CCDs consistent with the presence of ENaC-mediated Na+&nbsp;absorption. Our 3D CCD provides a geometrically and microphysiologically relevant platform for studying the development and physiology of renal tubule segments.</p>