Characterization of a primary cell culture model of the avian renal proximal tubule.
Methods have been developed for producing functional, transporting monolayers of avian proximal tubule (PT) cells. A highly homogenous fraction of PT fragments was prepared by enzymatic digestion (collagenase + Dispase) of chick (3- to 5-day-old) kidneys, followed by Percoll gradient centrifugation. The PT fraction was enriched in glucose-6-phosphatase, a proximal enzyme marker, and reduced in specific activity of hexokinase, a distal marker. PT fragments were grown to confluence in serum-free media on collagen-coated permeable filter supports. Electron microscopy of confluent monolayers revealed numerous microvilli and mitochondria, central cilia, and tight junctions, all characteristic of PT cells. gamma-Glutamyltranspeptidase, a proximal brush-border enzyme, showed threefold higher activity on apical than on basolateral sides of the monolayer. The electrophysiological characteristics of monolayers were investigated by voltage-clamp techniques. Monolayers displayed low transepithelial resistances (40-60 Omega . cm2), lumen-negative potentials, and baseline currents of 6-12 microA/cm2 (with or without 5 mM glucose). Both alpha-methyl-D-glucose (2 mM), a nonmetabolizable hexose, and phenylalanine (2 mM) significantly stimulated short-circuit current when added to the mucosal side of glucose-free monolayers. Phloridzin, a specific inhibitor of Na+-coupled glucose transport, significantly inhibited short-circuit current, as did 10(-5) M amiloride. Monolayers also expressed net secretory transport of urate. This cell culture preparation may provide a useful working model for the study of avian PT transport.
Sutterlin
G G
GG
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA.
Laverty
G
G
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002923:Cilia / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects; D008757:Methylglucosides / Q000494:pharmacology; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008871:Microvilli / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure; D008928:Mitochondria / Q000648:ultrastructure; D018408:Patch-Clamp Techniques; D010649:Phenylalanine / Q000494:pharmacology; D010695:Phlorhizin / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000032:analysis
D008757:Methylglucosides; D010649:Phenylalanine; C027020:methylglucoside; D000584:Amiloride; D010695:Phlorhizin; D005723:gamma-Glutamyltransferase; D006593:Hexokinase; D005952:Glucose-6-Phosphatase