Glenville P. Jones

Description:Vitamin D metabolism is controlled through the kidney mitochondrial P450 enzymes 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1) that activate and degrade the endocrine vitamin D hormone (1,25(OH)2D3), respectively. We recently demonstrated that extrarenal cells can make 1,25(OH)2D3 with adequate vitamin D supplementation by targeted mass spectrometry imaging in our Cyp27b1 kidney enhancer deletion mouse model that lacks circulating 1,25(OH)2D3 (M1/M21-DIKO mouse). Based on these observations, we selectively deleted Cyp27b1 (Cyp27b1fl/fl) from the mouse kidney using the Six2- and Pax8-cre drivers that target tubule and nephron development to see if we could recapitulate the remarkable phenotype of the M1/M21-DIKO mice. While Six2-cre/Cyp27b1fl/fl mice had a mild phenotype, Pax8-cre/Cyp27b1fl/fl mice had a marked elevation of parathyroid hormone and a reduction in bone mineral density. The vitamin D metabolic profile in the Pax8-cre/Cyp27b1fl/fl clearly indicated a dysfunction in the CYP24A1 enzyme with reductions in 24,25(OH)2D3 and 25(OH)D3-26,23-lactone with an accompanying elevation of 25(OH)D3. However, despite these compensatory reductions in CYP24A1 derived metabolites and apparent deletion of Cyp27b1 in the kidney, the 1,25(OH)2D3 levels were not changed from wildtype in either mouse. Like 24,25(OH)2D3, the 1,24,25(OH)3D3 levels were also reduced. These data highlight the robust homeostatic mechanisms to salvage 1,25(OH)2D3, point towards potential compensatory mechanisms of 1,25(OH)2D3 production from non-kidney tissues, and reinforce the utility of the M1/M21-DIKO model as a non-global deletion of Cyp27b1 with reductions in serum 1,25(OH)2D3 to be used to understand the complexity of vitamin D metabolism in health and inflammatory disease.

Description:Liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as the latest technology to be used to assay the metabolites of vitamin D. The method uses molecular mass as a detection technique after straightforward extraction and chromatography steps. LC-MS/MS assay provides a level of accuracy and reproducibility not seen before with other methods and is beginning to rival antibody-based methods in terms of sensitivity and convenience. Methods for detection of underivatized and DMEQ-TAD derivatized vitamin D metabolites are evaluated. Sensitivity is improved by 10-100 fold with derivatization and allows for the simultaneous assay of multiple vitamin D metabolites, a process termed vitamin D metabolite profiling. Clinical and research applications of vitamin D metabolite profiling are discussed.

Description:Vitamin D receptor (VDR)-mediated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent gene expression is compromised in the VDR null mouse. The biological consequences include: hypocalcemia, hypophosphatemia, elevated parathyroid hormone (PTH) and 1,25(OH)2D3, and consequential skeletal abnormalities. CYP24A1 is a cytochrome P450 enzyme that is involved in the side chain oxidation and destruction of both 1,25(OH)2D3 and 25-hydroxyvitamin D3 (25-OH-D3). In the current studies, we used liquid chromatography-tandem mass spectrometry technology to compare the metabolic profiles of VDR null mice fed either a normal or a calcium and phosphate-enriched rescue diet and to assess the consequence of transgenic expression of either mouse or human VDR genes in the same background. Serum 1,25(OH)2D3 levels in VDR null mice on normal chow were highly elevated (>3000 pg/mL) coincident with undetectable levels of catabolites such as 24,25-(OH)2D3 and 25-OH-D3-26,23-lactone normally observed in wild-type mice. The rescue diet corrected serum Ca(++), PTH, and 1,25(OH)2D3 values and restored basal expression of Cyp24a1 as evidenced by both renal expression of Cyp24a1 and detection of 24,25-(OH)2D3 and the 25-OH-D3-26,23-lactone. Unexpectedly, this diet also resulted in supranormal levels of 3-epi-24,25-(OH)2D3 and 3-epi-25-OH-D3-26,23-lactone. The reappearance of serum 24,25-(OH)2D3 and renal Cyp24a1 expression after rescue suggests that basal levels of Cyp24a1 may be repressed by high PTH. Introduction of transgenes for either mouse or human VDR also normalized vitamin D metabolism in VDR null mice, whereas this metabolic pattern was unaffected by a transgene encoding a ligand binding-deficient mutant (L233S) human VDR. We conclude that liquid chromatography-tandem mass spectrometry-based metabolic profiling is an ideal analytical method to study mouse models with alterations in calcium/phosphate homeostasis.

Description:The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH)2D3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1 We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH)2D3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease.

Description:Pharmacologists have been interested in vitamin D since its metabolism was elucidated in the early 1970s. Despite the synthesis of thousands of vitamin D analogues in the hope of separating its calcemic and anti-proliferative properties, few molecules have reached the market for use in the treatment of clinical conditions from psoriasis to chronic kidney disease. This review discusses vitamin D drugs, recently developed or still under development, for use in various diseases, but in particular bone disease. In the process we explore the mechanisms postulated to explain the action of these vitamin D analogues including action through the vitamin D receptor, action through other receptors e.g. FAM57B2 and dual action on transcriptional processes.