Simulated acidosis does not impair 1,25-dihydroxyvitamin D3 production by cultured kidney cells

Abstract

Cultured mouse kidney cells grown in serum-free medium were used to assess the metabolism of 25-hydroxyvitamin D3 in the presence of simulated metabolic acidosis. Kidney epithelial cells isolated from 4-6 week old mice were grown to confluence in a defined serum-free medium at pH 7.4. The confluent monolayers were incubated with tritiated 25-hydroxyvitamin D3 for 6 hours, the samples were extracted, and vitamin D metabolites were separated and quantitated by high pressure liquid chromatography (HPLC). The pH of the incubation medium was set at 6.9, 7.1, 7.4, or 7.7 by adjusting the bicarbonate concentration, using chloride as the balancing anion at constant Pco2. When pH was altered at the beginning of the 6 hour assay, production of 1,25-dihydroxyvitamin D3 was the same at each pH. More prolonged pH perturbation for a total of 30 hours likewise had no influence on 1,25-dihydroxyvitamin D3 production. These results confirm that intact mammalian kidney cells in serum-free culture possess an active 25-hydroxyvitamin D3-1-hydroxylase and that the activity of the enzyme is unaffected by pH over the range 6.8-7.7. In experiments where acidosis has been shown to alter 1,25-dihydroxyvitamin D3 production, the mechanism was probably indirect.