Characterization of a primary cell culture model of the avian renal proximal tubule

Abstract

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. γ-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 Ω ⋅ cm2), lumen-negative potentials, and baseline currents of 6–12 μA/cm2 (with or without 5 mM glucose). Both α-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.

Diclofenac toxicity in Gyps vulture is associated with decreased uric acid excretion and not renal portal vasoconstriction

“…After seven days of incubation, the culture medium was replaced with the HBSS transport medium as previously described (Ichida et al, 2003, Sutterlan and Laverty, 1998and Waugh, 2006. The reaction was started by adding the drug into the basal well.…”

The Interface Between Generating Renal Tubules and a Polyester Fleece in Comparison to the Interstitium of the Developing Kidney

“…4 Due to the spatial microarchitecture of the kidney experiments are frequently performed applying three-dimensional culture experiments in combination with primary cells or cell lines. 2,30 Normally the cells are coated by extracellular matrix proteins such as collagen or Matrigel Ò . Applying serum-containing medium the cells migrate within the coat of extracellular matrix proteins, proliferate and aggregate to form cysts and tubules.…”

PTH stimulates a Cl−-dependent and EIPA-sensitive current in chick proximal tubule cells in culture

“…We have recently developed a primary cell culture model of the avian (chick) PT using methods similar to those developed for rabbit and rat (45). Cells grown as confluent monolayers on permeable membrane filters become highly polarized and exhibit transepithelial transport, measurable by classic electrophysiological methods.…”