Fig. 5. PKD cysts form via expansion of outwards-facing epithelium.

a Confocal immunofluorescence images of cilia (acetylated α-tubulin, abbreviated AcT) and tight junctions (ZO-1) in proximal tubules (LTL) of PKD and non-PKD organoids, as well as in PKD cyst lining epithelial cells. Dashed arrow indicates how line scans were drawn. Images are representative of three independent experiments. b ZO1 and AcT intensity profiles in cysts vs. organoids. Line scans were drawn through cilia from lumen to exterior of structures. (mean ± s.e.m. from n = 5 line scans pooled from three organoids or cysts per condition from three independent experiments). c Fluorescent images of stromal markers in PKD organoids compared to human kidney tissue from a female patient 50 years of age with autosomal dominant PKD. Scale bars 20 µm. d Fluorescent images of cysts after having been overlaid with collagen. Images are representative of three independent experiments. e Z-stack confocal images of early (day 30) PKD organoid cyst in adherent culture. Zoom shows boxed region. White arrow indicates a podocyte cluster continuous with the peripheral epithelium. Images are representative of three independent experiments. f Close-up image showing peripheral epithelium of control (non-PKD) organoid in adherent culture. Yellow arrowhead indicates region of epithelial invagination. Images are representative of three independent experiments. g Phase contrast time-lapse images showing formation of PKD cysts from non-cystic structures in adherent cultures. Red arrows indicates tubular structures internal to the peripheral cyst. Images are representative of three independent experiments. h Schematic model of absorptive cyst expansion in organoids. Fluid flow (blue arrows) is absorbed into outwards-facing proximal tubular epithelium, which generates internal pressure that drives expansion and stretching of the epithelium (red arrows). A simplified organoid lacking podocytes or multiple nephron branches is shown for clarity.