Regulation of vitamin D-1α-hydroxylase in a human cortical collecting duct cell line

Abstract

Regulation of vitamin D-1α-hydroxylase in a human cortical collecting duct cell line.BackgroundRecent studies have shown that renal expression of 25-hydroxyvitamin D3-1α-hydroxylase (1α-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1α-OHase expression and activity in a human cortical collecting duct cell line (HCD).MethodsExpression of 1α-OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D3 as the substrate; conversion to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 was then determined by thin-layer chromatography.ResultsHCD cells expressed mRNA and protein for 1α-OHase. However, basal 1,25(OH)2D3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)2D3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)2D3 itself decreased 1α-OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1α-OHase activity was observed in the HCD cells following incubation with lipopolysaccharide, which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4.ConclusionsThese results highlight the capacity for synthesis of 1,25(OH)2D3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1α-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron