Spatial detection and consequences of nonrenal calcitriol production as assessed by targeted mass spectrometry imaging
Mark B. Meyer, Seong Min Lee, Shannon R. Cichanski, Diego F. Cobice, J. Wesley Pike
Mark B. Meyer, Seong Min Lee, Shannon R. Cichanski, Diego F. Cobice, J. Wesley Pike
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Research Article Endocrinology

Spatial detection and consequences of nonrenal calcitriol production as assessed by targeted mass spectrometry imaging

Abstract

The immune benefits of vitamin D3 supplementation beyond calcium and phosphate maintenance are highly clinically debated. Kidney expression of CYP27B1 is the source of endocrine, circulating 1,25(OH)2D3 (active form of vitamin D) that maintains serum calcium and phosphate. 1,25(OH)2D3 may also be made by the CYP27B1 enzyme in nonrenal cells, like immune cells, in a process driven by cellular availability of 25(OH)D3 and inflammation. Due to the endocrine nature of 1,25(OH)2D3 in circulation, it is difficult to discern between these 2 sources. We recently created a regulatory deletion model of Cyp27b1 (M1/M21-DIKO) where mice have normal inflammatory-regulated Cyp27b1 expression in nonrenal tissues (unlike global Cyp27b1-KO) but no expression within the kidney. Here, utilizing on-tissue chemical derivatization and matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), we investigated the distribution of 1,25(OH)2D3 and 25(OH)D3 in the kidney, liver, spleen, and thymus. MALDI-MSI demonstrated increased 1,25(OH)2D3 in nonrenal tissues such as the spleen after vitamin D3 supplementation in M1/M21-DIKO mice. Additionally, from this, we found increased Il4 and decreased Tnfa in the spleen after vitamin D3 supplementation. Taken together, these data demonstrate nonrenal production of 1,25(OH)2D3 in vivo and provide a consequence of vitamin D3 supplementation and nonrenal 1,25(OH)2D3 production in cytokine changes.

Authors

Mark B. Meyer, Seong Min Lee, Shannon R. Cichanski, Diego F. Cobice, J. Wesley Pike

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Figure 4

Detection of nonrenal 1,25(OH)2D3 production after vitamin D3 supplementation.

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Detection of nonrenal 1,25(OH)2D3 production after vitamin D3 supplement...
(A and B) Plasma levels of 25(OH)D3 (A, 25D, ng/mL) and 1,25(OH)2D3 (B, 1,25D, pg/mL) as detected by LC-MS/MS in the WT littermates, Cyp27b1-KO (C27-KO), M1/M21-DIKO (DIKO), M1/M21-DIKO 12-week rescue diet followed by 4 weeks of 0 IU vitamin D diet (12wR-4w 0 IU), and M1/M21-DIKO 12-week rescue diet followed by 4 weeks of 20 IU vitamin D diet (12wR-4w 20 IU). One-way ANOVA with multiple comparison Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (C–F) MSI was performed and displayed for kidney (C), liver (D), spleen (E), and thymus (F) in the M1/M21-DIKO 12wR-4w 0 IU and M1/M21-DIKO 12wR-4w 20 IU. Signal intensity is depicted by parula color scale for semiquantitative assessment on the scale shown (additional images in Supplemental Figure 2). (G and H) The relative quantitation of biological triplicates is shown for the 25(OH)D3 (G, ng/g) and 1,25(OH)2D3 (H, pg/g) for each tissue (all values in Supplemental Figure 1). Data points that fall below the lower limits of quantitation (<LLOQ) but above the LLOD are shown in red. n = 3 for all tissues. One-way ANOVA (genotypes within tissues) with multiple comparison Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (I) H&E staining of thymus section with red and white pulp indicated. Overlaid data from spleen 25D and 1,25D panels from E.