Bland R, Zehnder D, Hughes S V, Ronco P M, Stewart P M, Hewison M
Division of Medical Sciences, The University of Birmingham, Queen Elizabeth Hospital, Birmingham, England, United Kingdom.
Kidney Int. 2001 Oct;60(4):1277-86. doi: 10.1046/j.1523-1755.2001.00966.x.
Recent studies have shown that renal expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1alpha-OHase expression and activity in a human cortical collecting duct cell line (HCD).
Expression of 1alpha-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.
HCD cells expressed mRNA and protein for 1alpha-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 1alpha-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 1alpha-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.
These 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 1alpha-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron.
最近的研究表明，25-羟维生素D3-1α-羟化酶（1α-OHase）在肾脏中的表达并不局限于近端小管。为了研究这种表达的意义，我们对人皮质集合管细胞系（HCD）中1α-OHase表达和活性的调节进行了特征分析。
通过逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹分析评估1α-OHase mRNA和蛋白质的表达。以25-羟维生素D3为底物定量酶活性；然后通过薄层色谱法测定其转化为1,25-二羟维生素D3 [1,25(OH)2D3]和24,25-二羟维生素D3的情况。
HCD细胞表达1α-OHase的mRNA和蛋白质。然而，基础1,25(OH)2D3的产生低于近端小管HKC-8细胞中的水平。在这两种细胞系中，福斯可林、甲状旁腺激素和低钙培养基均可增加1,25(OH)2D3的合成。相反，用1,25(OH)2D3本身处理会降低1α-OHase活性。这种作用在HCD细胞中更为明显，HCD细胞中24-羟化酶活性水平也显著更高。在用脂多糖孵育后的HCD细胞中观察到1α-OHase活性的最显著诱导，这与CD14和Toll样受体4的mRNA表达一致。
这些结果突出了肾单位更远端区域细胞合成1,25(OH)2D3的能力。然而，HCD细胞中1α-OHase更敏感的反馈调节和免疫诱导表明1,25(OH)2D3在远端肾单位产生中具有更局部化的作用。
