In vitro metabolism of 25-hydroxyvitamin D₃ by isolated rat kidney cells

Cells were dispersed from rat kidney after enzymatic digestion of the extracellular matrix. When the cells were suspended in a serum-free medium and incubated with ³H-labeled 25-hydroxyvitamin D₃ (25-OH-D₃) several polar metabolites, including 1,25-(OH)₂[³H]D₃ and 24,25-(OH)₂[³H]D₃ were produced. The specific activities of the 25-OH-D₃:1- and 24-hydroxylases in isolated rat kidney cells were 10-100 times greater than in avian kidney homogenates. The rates of production of 1,25-(OH)₂D₃ and 24, 25-(OH)₂D₃ were linear over a wide range in cell densities (0.65-5.0x10⁶ cells per ml) and substrate concentrations (3.5-70nM). The rate of production of 24,25-(OH)₂[³H]D₃ from 25-OH-[³H]D₃ by cells isolated from rats fed control diet was linear with time for up to 30 min, while the synthesis of 1,25-(OH)₂[³H]D₃ was linear for over 90 min. The specific activity of the 25-OH-D₃:1-hydroxylase was increased in kidney cells from vitamin D-deficient rats (11.5 fmol/min per 10⁶ cells) as well as calcium-deficient rats (8.1 fmol/min per 10⁶ cells) when compared to cells from rat fed the control diet (2.0 fmol/min per 10⁶ cells). Also, the specific activity of the 25-OH-D₃:24-hydroxylase was reduced in cells from the vitamin D-deficient rats (<0.2 fmol/min per 10⁶ cells) and calcium-deficient rats (5.1 fmol/min per 10⁶ cells) compared to the controls (15.2 fmol/min per 10⁶ cells). On the basis of these results, as well as previous in vivo studies, we conclude that the metabolism of 25-OH-D₃ by freshly isolated rat kidney cells reflects the in vivo activities of the renal vitamin D-metabolizing enzymes and may prove useful as an assay.