Endocrine Abstracts

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), is synthesised by the enzyme 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase). The principal site of 1alpha-hydroxylase activity is in the proximal tubule of the kidney. In order to investigate renal vitamin D metabolism we have used a human proximal tubule cell line, HKC-8. We have previously shown that synthesis of 1,25D3 activity in these cells is sensitive to the concentration of calcium and Northern and Western blot analyses have demonstrated the presence of calcium sensing receptor (CaR) mRNA and protein. The aim of this study was to investigate whether changes in the expression of the CaR modified 1alpha-hydroxylase activity. HKC-8 cells were transiently transfected with human CaR (in pcDNA3.1) in the forward or reverse orientation, which allowed for either over- or under-expression of the CaR. Following overnight transfection, cells were transferred to serum-free defined medium for 24 hours. Changes in CaR expression were confirmed by RT-PCR. 1alpha- and 24-hydroxylase activity was assessed by incubating the cells with 3.75nM 3H-25(OH)D3 for 4 hours. The resulting vitamin D metabolites were separated by TLC and analysed using a Bioscan System 200 imaging scanner. Data are reported as production of 1,25D3 (1alpha-hydroxylase activity) or 24,25D3 (24-hydroxylase activity). All results were compared with cells transfected with empty vector. In cells which over-expressed the CaR 1,25D3 production was significantly lower than control cells (200 vs 520 fmoles/mg protein/hour; p<0.05). Conversely in cells under-expressing the CaR (CaR/pcDNA3.1 in the reverse orientation) 1alpha-hydroxylase activity was significantly higher compared with controls cells (1500 vs 520 fmoles/mg protein/hour; p<0.05). No significant changes in 24-hydroxylase activity were observed. Preliminary data also demonstrated inhibition of 1alpha-hydroxylase activity in HKC-8 cells following treatment with a CaR agonist. This data indicates that the direct regulation of renal 1alpha-hydroxylase by extra-cellular calcium occurs via a CaR-mediated pathway.