The in Vitro Metabolism of 25-Hydroxycholecalciferol to 1,25-Dihydroxycholecalciferol by Chick Renal Tubules

Abstract

Abstract Free cell suspensions of renal tubules obtained from the rachitic chicken were observed to be capable of the rapid and sustained metabolism of 25-hydroxycholecalciferol. Among the products of the in vitro metabolism of 25-hydroxycholecalciferol was a metabolite identified to be identical to 1,25-dihydroxycholecalciferol, the cholecalciferol (vitamin D3) metabolite known to be most active for the initiation of intestinal calcium transport. Unlike the results reported for kidney homogenates, the renal tubule metabolism results in the production of at least one major metabolic product other than 1,25-dihydroxycholecalciferol. Further-more the renal tubule preparation was observed to be able to rapidly metabolize 1,25-dihydroxycholecalciferol to other as yet unidentified metabolites. The initial rates of consumption of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol were estimated to be, respectively, 4.5 and 27.9 fmoles per min per mg of renal tubule protein. The rates of renal tubule RNA and protein synthesis were observed to be linear for 15 and 180 min, respectively. The short interval for linear RNA synthesis appeared to be due to precursor pool equilibration as nonlinear RNA synthesis could be demonstrated during at least a 45-min incubation interval. The complete inhibition of RNA synthesis by actinomycin D or protein synthesis by puromycin did not diminish either the rate of 25-hydroxycholecalciferol consumption or the amount of 1,25-dihydroxycholecalciferol produced by renal tubules. This result was obtained even after the renal tubules had been incubated for 3 hours in the presence of the metabolic inhibitors prior to the initiation of 25-hydroxycholecalciferol metabolism. The rate of α-ketoglutarate consumption by the chick renal tubules was observed to be sensitive to changes in extracellular calcium ion concentration in a manner analogous to that reported for rat renal tubules. Additionally the chick renal tubules showed a marked elevation in cyclic 3',5'-adenosine monophosphate content after stimulation by parathyroid hormone. Identical changes in extracellular calcium ion concentration or stimulation by parathyroid hormone had no effect on the metabolism of 25-hydroxycholecalciferol or production of 1,25-dihydroxycholecalciferol as measured after 30 min of incubation. It was concluded that the ability of renal tubules to metabolize 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol is not dependent upon their ability to synthesize either RNA or proteins and that the renal metabolism of 25-hydroxycholecalciferol is not subject to regulation by extracellular calcium ion concentration. Additionally parathyroid hormone was observed to have no regulatory effect upon the metabolism of 25-hydroxycholecalciferol by renal tubules isolated from rachitic chickens.