Serum-free culture of Japanese quail kidney cells regulation of vitamin D metabolism

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 10⁶ cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25-30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)₂D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)₃D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)₂D-3 decreased 1,25(OH)₂D-3 synthesis, and increased both 24,25(OH)₂D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)₂D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.