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Department of Biological Sciences, University of Delaware, Newark, Delaware 19716
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. 
-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
· cm2),
lumen-negative potentials, and baseline currents of 6-12
µA/cm2 (with or without 5 mM
glucose). Both
-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.
electrophysiology; electron microscopy; sodium-glucose luminal transporter; amiloride; urate
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