Intestinal Cyp24a1 regulates vitamin D locally independent of systemic regulation by renal Cyp24a1 in mice
Michaela A.A. Fuchs, … , Tomokazu Souma, Myles Wolf
Michaela A.A. Fuchs, … , Tomokazu Souma, Myles Wolf
Published December 17, 2024
Citation Information: J Clin Invest. 2025;135(4):e179882. https://doi.org/10.1172/JCI179882.
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Research Article Metabolism Nephrology

Intestinal Cyp24a1 regulates vitamin D locally independent of systemic regulation by renal Cyp24a1 in mice

Abstract

Vitamin D regulates mineral homeostasis. The most biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D), is synthesized by CYP27B1 from 25-dihydroxyvitamin D (25D) and is inactivated by CYP24A1. Human monogenic diseases and genome-wide association studies support a critical role for CYP24A1 in regulation of mineral homeostasis, but little is known about its tissue-specific effects. Here, we describe the responses of mice with inducible global deletion, kidney-specific, and intestine-specific deletion of Cyp24a1 to dietary calcium challenge and chronic kidney disease (CKD). Global and kidney-specific Cyp24a1 deletion caused similar syndromes of systemic vitamin D intoxication: elevated circulating 1,25D, 25D, and fibroblast growth factor 23 (FGF23), activation of vitamin D target genes in the kidney and intestine, hypercalcemia, and suppressed parathyroid hormone (PTH). In contrast, mice with intestine-specific Cyp24a1 deletion demonstrated activation of vitamin D target genes exclusively in the intestine, despite no changes in systemic vitamin D levels. In response to a high calcium diet, PTH was suppressed, despite normal serum calcium. In mice with CKD, intestinal Cyp24a1 deletion decreased PTH and FGF23 without precipitating hypercalcemia. These results implicate kidney CYP24A1 in systemic vitamin D regulation while independent local effects of intestinal CYP24A1 could be targeted to treat secondary hyperparathyroidism in CKD.

Authors

Michaela A.A. Fuchs, Alexander Grabner, Melody Shi, Susan L. Murray, Emily J. Burke, Nejla Latic, Venkataramana Thiriveedi, Jatin Roper, Shintaro Ide, Koki Abe, Hiroki Kitai, Tomokazu Souma, Myles Wolf

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Figure 3

Validation of tissue specificity and loss of function after Cyp24a1 deletion.

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Validation of tissue specificity and loss of function after Cyp24a1 dele...
Homozygous Cyp24fl/fl mice were crossed with different Cre-drivers to induce global (Ubc-CreERT2), kidney-specific (Six2-Cre), or intestine-specific (Villin-CreERT2) deletion of Cyp24a1; Cre-negative littermates served as controls. Recombination efficiency was evaluated 6 hours after calcitriol injection, 10 μg/kg. (A) In situ hybridization using a probe specific to exon 5 of Cyp24a1 confirmed successful deletion from most kidney cells in UbcCreERT2Cyp24fl/fl and Six2CreCyp24fl/fl mice, but not VillinCreERT2Cyp24fl/fl mice. (B) Evaluation of the small intestines confirmed successful deletion of Cyp24a1 in UbcCreERT2Cyp24fl/fl and VillinCreERT2Cyp24fl/fl mice, but not Six2CreCyp24fl/fl mice. (C) Kidneys of Rosa26tdTomato controls and VillinCreERT2Rosa26tdTomato mice demonstrated no red fluorescence after tamoxifen treatment, while intestinal epithelia showed robust red tdTomato fluorescence, confirming intestinal specificity of Villin-CreERT2. (D) Primary cultures of proximal tubule cells derived from kidney-specific Six2CreCyp24fl/fl mice and controls were used to evaluate Cyp24a1 function in vitro. Levels of 24,25D increased significantly in cultures of WT cells after 12 hours of treatment with 7nM 25D (grey), whereas there was no change in 24,25D levels in cultures of proximal tubule cells derived from Six2CreCyp24fl/fl mice (red). (E) UbcCreERT2Cyp24fl/fl mice received 2 calcitriol injections, 2.5 μg/kg, 48 hours apart 1 week after the last tamoxifen injection. At baseline, UbcCreERT2Cyp24fl/fl mice had higher 1,25D and FGF23 levels, consistent with impaired Cyp24a1 function. Calcitriol injection increased serum calcium, 1,25D, and FGF23 levels in all mice, but exacerbated increases in UbcCreERT2Cyp24fl/fl mice versus controls, indicating failure of these mice to metabolize exogenous calcitriol. n ≥ 4 for all measurements. Results are mean ± SEM. Statistical significance was calculated using mixed effect analysis with Holm-Šidák’s correction. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001. Representative images from ≥ 4 animals of each strain that were evaluated. Scale bars: 50 μm.