Regulation of vitamin D-1 alpha-hydroxylase in a human cortical collectingduct cell line

Background. Recent studies have shown that renal expression of 25-hydroxyvi tamin D-3-1 alpha -hydroxylase (1 alpha -OHase) is not restricted to proxim al tubules. To investigate the significance of this expression, we characte rized the regulation of 1 alpha -OHase expression and activity in a human c ortical collecting duct cell line (HCD). Methods. Expression of 1 alpha -OHase mRNA and protein was assessed by reve rse transcription-polymerase chain reaction (RT-PCR) and Western blot analy ses. Enzyme activity was quantified using 25-hydroxyvitamin D-3 as the subs trate; conversion to 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] and 24,25-d ihydroxyvitamin D-3 was then determined by thin-layer chromatography. Results. HCD cells expressed mRNA and protein for 1 alpha -OHase. However, basal 1,25(OH)(2)D-3 production was lower than that observed in proximal tu bule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)(2)D-3 was incre ased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)(2)D-3 itself decreased 1 alpha -OHase activity. Th is effect was more pronounced in HCD cells, which also demonstrated signifi cantly higher levels of 24-hydroxylase activity. The most striking inductio n of 1 alpha -OHase activity was observed in the HCD cells following incuba tion with lipopolysaccharide. which was coincident with the expression of m RNA for both CD14 and Toll-like receptor 4. Conclusions. These results highlight the capacity for synthesis of 1.25(OH) (2)D-3 in cells from more distal areas of the nephron. However, more sensit ive feedback regulation and immune induction of 1 alpha -OHase in the HCD c ells suggest a more localized role for 1,25(OH)(2)D-3 production in the dis tal nephron.