Effect of luminal flow on doming of mpkCCD cells in a 3D perfusable kidney cortical collecting duct model

Abstract

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, composed 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 three-dimensional 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 wk. Immunofluorescence microscopy reveals the apical epithelial Na⁺ 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⁺ absorption. Our 3D CCD provides a geometrically and microphysiologically relevant platform for studying the development and physiology of renal tubule segments.

Keywords: 3D cortical collecting duct model; ENaC; cell physiology; fluid shear stress; principal cell.

Fig. 1.

Schematic illustrations of the kidney chip taken after completion of each fabrication step (A–I) used to create the 3D CCD model. ECM, extracellular matrix. [Printed with permission from ©Mount Sinai Health System.]

Fig. 2.

Epithelialization of the 3D CCD. A: an empty channel. B: mpkCCD cells in the channel immediately after cell seeding. C: cell growth 1 day after cell seeding. D: epithelialized channel after 7 days (differential interference contrast microscopy at ×10. Scale bar = 100 μm). E and F: cell growth in the 3D CCD after 7 days imaged at different focal planes to demonstrate the three dimensionality of the cell growth around the circumference of the channel (phase contrast microscopy at ×20. Scale bar = 100 μm).

Fig. 3.

Localization of structural proteins in the 3D CCD. A and B: transmission electron microscopy of mpkCCD cells in the 3D CCD demonstrating apical microvilli (black arrow), adherent junction (white arrow), and basal infoldings (blue arrow). Scale bar = 800 nm. C: strong apical F-actin localization in mpkCCD cells grown in the 3D CCD. Scale bar = 100 μm. D: 3D reconstruction revealing a curved epithelial monolayer as identified by F-actin staining. Scale bar = 100 μm. E: cells grown in the 3D CCD had immunodetectable cilia. Scale bar = 10 μm. F: cells deposit collagen along the basal surface onto the extracellular matrix (ECM) as identified by linear blue staining with Masson’s trichrome and visualized by light microscopy. Scale bar = 20 μm.

Fig. 4.

Immunolocalization of relevant transport proteins in the 3D CCD. The 3D CCD expressed apical epithelial Na⁺ channel (ENaCα) (A), ENaCβ (B), large conductance BKα (C), aquaporin 2 (AQP2) (D), and basolateral Na⁺/K⁺-ATPase (E), consistent with native mammalian principal cells. ECM, extracellular matrix.

Fig. 5.

Epithelial integrity assessed with 3- to 5-kDa FITC-dextran over 1 h. A: representative phase-contrast and widefield fluorescent images. Scale bar = 100 μm. B: histogram of mean fluorescence across the channel cross section showing tight barrier function to perfused 3- to 5-kDa FITC-dextran over 1 h. C: the diffusional permeability of the FITC-dextran was lower in epithelialized versus empty channels. Individual data points as well as means ± SE are shown. *P = 0.01 versus “empty.”

Fig. 6.

Analysis of possible mechanisms underlying cell doming. A: mpkCCD cells grown on glass as 2-dimensional (2D) monolayers under static conditions formed domes spontaneously upon reaching cell confluence while cells grown on 2D ECM under static conditions did not (phase-contrast microscopy at ×10). Scale bar = 100 μm. B: in the 3D CCD, dome formation occurred in the absence of flow but did not occur with exposure to flow. Scale bar = 100 μm. C: schematic comparing the control to the drainage 3D CCD. D: the percentage of 3D CCDs with domes was not different among groups 1 h after stopping flow. E: the number of domes/mm 3D CCD was lower with 1 μM benzamil (BZ) compared with the control and drainage 3D CCDs. *P < 0.01 vs. control. F: the mean dome volume was lower with 1 μM BZ or a drain vs. control 3D CCDs. *P < 0.01 vs. control. Individual data points as well as means ± SE are shown in E and F. [Printed with permission from ©Mount Sinai Health System.]