Effects of dietary calcium restriction on 1,25-dihydroxyvitamin D₃ net synthesis by rat proximal tubules

We measured in vitro 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) production by kidney proximal tubules prepared by Percoll density centrifugation from male and female rats. 1,25(OH)₂D₃ in tubule extracts was determined by a sensitive and specific radioreceptor assay. Ingestion of diets adequate in vitamin D₃ and containing either normal calcium (1.2% Ca, NC), reduced calcium (0.6% Ca, RCD) or low calcium (0.002% Ca, LCD) increased 1,25(OH)₂D₃ net synthesis (for male rats, NC vs. RCD vs. LCD 1.8 ± 0.1 SEM vs. 9 ± 2 vs. 17 ± 2 pmol/mg protein/ 20 min; P < 0.05 for all comparisons). At either level of reduced calcium intake, tubules from male rats produced more 1,25(OH)₂D₃ than tubules from females. Serum 1,25(OH)₂D₃ and tubule cyclic adenosine monophosphate (cAMP) content rose in parallel with progressive dietary calcium restriction, and males had higher circulating 1,25(OH)₂D₃ and tubule cAMP content than females at each level of reduced calcium intake. l-Epinephrine (10^-4 mol/L), in vitro, increased tubule accumulation of 1,25(OH)₂D₃ and cAMP. Yohimbine, an α₂-receptor antagonist, blocked this response, whereas prazosin was without effect. Increased 1,25(OH)₂D₃ net synthesis by tubules from male vs. female rats partly explains the higher serum levels and enhanced mineral conservation demonstrated previously in male rats. Preparation of proximal tubules from vitamin D-replete rats permits studies in vitro of 1,25(OH)₂D₃ production and regulation under more physiologic conditions in which parathyroid hormone, inorganic phosphorus, and calcium may be varied independently.