title: A primary culture of mouse proximal tubular cells, established on collagen-coated membranes.
abstract: A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immunoblotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and gamma-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by Na(+) similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures.
authors.affiliation: Laboratory of Cell Physiology, Hasselt University, Agoralaan, Gebouw D, B-3590 Diepenbeek, Belgium. sara.terryn@uhasselt.be
authors.forename: Sara
authors.initials: S
authors.lastname: Terryn
authors.forename: François
authors.initials: F
authors.lastname: Jouret
authors.forename: Frank
authors.initials: F
authors.lastname: Vandenabeele
authors.forename: Inge
authors.initials: I
authors.lastname: Smolders
authors.forename: Marjan
authors.initials: M
authors.lastname: Moreels
authors.forename: Olivier
authors.initials: O
authors.lastname: Devuyst
authors.forename: Paul
authors.initials: P
authors.lastname: Steels
authors.forename: Emmy
authors.initials: E
authors.lastname: Van Kerkhove
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D003584:Cytological Techniques; D004285:Dogs; D004594:Electrophysiology; D004847:Epithelial Cells / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D007150:Immunohistochemistry; D007672:Kidney Cortex / Q000648:ultrastructure; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D008566:Membranes; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission; D012964:Sodium / Q000502:physiology; D005723:gamma-Glutamyltransferase / Q000378:metabolism
chemical_list: D003470:Culture Media; D003094:Collagen; D012964:Sodium; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D005947:Glucose