The full-length calcium-sensing receptor dampens the calcemic response to 1alpha,25(OH)2 vitamin D3 in vivo independently of parathyroid hormone

Abstract

1Alpha,25(OH)(2) vitamin D(3) [1,25(OH)(2)D(3)] increases serum Ca(2+) concentration in vivo, an action counteracted by activation of the Ca(2+)-sensing receptor (CaSR), which decreases parathyroid hormone (PTH) secretion and increases renal Ca(2+) excretion. Relatively little is known of the role the CaSR plays in this response through its potentially direct actions in kidney, gut, and bone independently of PTH. We report PTH-independent roles of the CaSR in modulating the response to exogenous 1,25(OH)(2)D(3) in mice with targeted disruption of both the CaSR and PTH genes (C(-)P(-)) compared with that in mice with disruption of the PTH gene alone (C(+)P(-)) or wild-type mice (C(+)P(+)). After intraperitoneal injection of 0.5 ng/g body wt 1,25(OH)(2)D(3), peak calcemic responses were observed at 24 h in all three genotypes in association with 1) a greater increase in serum Ca(2+) in C(-)P(-) mice than in the other genotypes on a Ca(2+)-replete diet that was attenuated by a Ca(2+)-deficient diet and pamidronate, 2) increased urinary Ca(2+)-to-creatinine ratios (UCa/Cr) in the C(+)P(-) and C(+)P(+) mice but a lowered ratio in the C(-)P(-) mice on a Ca(2+)-replete diet, and 3) no increase in calcitonin (CT) secretion in the C(+)P(+) and C(+)P(-) mice and a small increase in the C(-)P(-) mice. PTH deficiency had the anticipated effects on the expression of key genes involved in Ca(2+) transport at baseline in the duodenum and kidney, and injection of 1,25(OH)(2)D(3) increased gene expression 8 h later. However, the changes in the genes evaluated did not fully explain the differences in serum Ca(2+) seen among the genotypes. In conclusion, mice lacking the full-length CaSR have increased sensitivity to the calcemic action of 1,25(OH)(2)D(3) in the setting of PTH deficiency. This is principally from enhanced 1,25(OH)(2)D(3)-mediated gut Ca(2+) absorption and decreased renal Ca(2+) excretion, without any differences in bone-related release of Ca(2+) or CT secretion among the three genotypes that could explain the differences in their calcemic responses.

Fig. 1.

Pharmacokinetic profile of serum 1α,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] levels at baseline as well as 1 and 24 h after a single intraperitoneal (ip) dose of 1,25(OH)₂D₃ in 3 genotypes of mice: Ca²⁺-sensing receptor (CaSR)^+/+parathyroid hormone (PTH)^−/− (C⁺P⁻ mice), CaSR^−/−PTH^−/− (C⁻P⁻ mice), or wild-type CaSR^+/+PTH^+/+ (C⁺P⁺ mice). Data are means ± SE (n = 8–12/genotype). ○P < 0.02, ANOVA, baseline comparisons across genotypes. •P > 0.05, baseline comparison, C⁺P⁻ vs. C⁻P⁻. ⧫P = 0.07 across-group comparisons at 1 h postinjection. ##P > 0.05 within-genotype comparison of baseline and 24-h 1,25(OH)₂D₃ levels. *P = 0.045, within-group comparison of baseline and 24-h 1,25(OH)₂D₃ levels.

Fig. 2.

Effect of a single ip dose (0.5 ng/g body wt) of 1,25(OH)₂D₃ in mice fed a Ca²⁺-replete diet and plain water on serum Ca²⁺ concentrations (conc) over 72 h. Data are means ± SE (n = 8–12/genotype). *P = 0.001, C⁻P⁻ vs. C⁺P⁻ at 24 h.

Fig. 3.

Relative sensitivities to repeated ip administration of 1,25(OH)₂D₃ in wild-type and knockout (KO) mice at doses designed to maintain normocalcemia using an alternate-day dosing schedule. C⁺P⁻ mice needed 0.4 ng/g body wt on alternate days to achieve normocalcemia, whereas C⁻P⁻ mice needed only 0.0625 ng/g body wt to achieve normocalcemia. Wild-type mice were able to maintain normocalcemia despite receiving 0.4 ng/g body wt. Arrows represent times of administration of 1,25(OH)₂D₃. Data are means ± SE (n = 6/genotype).

Fig. 4.

Effect of a single dose (0.5 ng/g body wt) of 1,25(OH)₂D₃ in mice fed a Ca²⁺-deplete diet and plain water on serum Ca²⁺ levels over 72 h. Data are means ± SE (n = 8–12/genotype). #P > 0.05, C⁺P⁻ vs. C⁻P⁻ at 24 h.

Fig. 5.

Effect of a single ip dose (0.5 ng/g body wt) of 1,25(OH)₂D₃ in mice fed a Ca²⁺-deplete diet and plain water after 2 wk of pretreatment with pamidronate on serum Ca²⁺ levels over 72 h. Data are means ± SE (n = 8–12/genotype). *P < 0.05, C⁺P⁻ vs. C⁻P⁻.

Fig. 6.

Serum calcitonin (CT) response to a single ip dose (0.5 ng/g body wt) of 1,25(OH)₂D₃ at baseline and 24 h afterwards. Data are means ± SE (n = 8–12/genotype). *P < 0.05, C⁻P⁻ at baseline vs. C⁻P⁻ at 24 h after 1,25(OH)₂D₃ administration.

Fig. 7.

Urinary Ca²⁺ excretion at baseline and 24 h after ip administration of 1,25(OH)₂D₃. Trend lines for urinary Ca²⁺-to-creatine ratio (UCa/Cr) vs. serum Ca²⁺ level are shown for C⁺P⁺ (line A), C⁺P⁻ (line B), and C⁻P⁻ (line C). Note the rightward and downward shift in urinary Ca²⁺ excretion corrected for urinary creatinine as serum Ca²⁺ level increases after 1,25(OH)₂D₃ administration. Data are presented as a scatter plot (n = 8–12/genotype).

Fig. 8.

Expression profiles of transient receptor potential vanilloid channel 5 (TRPV5), vitamin D receptor (VDR), Na⁺/Ca²⁺ exchanger 1 (NCX1), Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), claudin-16, and 1,25-dihydroxy vitamin D₃-24-hydroxylase (CYP24A1) in mouse kidney at baseline (solid bars) on a Ca²⁺-replete diet and plain water and 8 h after ip 1,25(OH)₂D₃ administration (shaded bars). The mRNA quantified by real-time PCR was calculated as a fold-change ratio relative to the mRNA for GAPDH in wild-type (C⁺P⁺) mice. Data are means ± SE (n = 6–8/genotype per condition). *P < 0.05, baseline vs. 8 h within genotypes. Y P < 0.05, baseline vs. 8 h within C⁻P⁻. #P < 0.05, baseline comparison, C⁺P⁺ vs. C⁺P⁻. ⧫ P < 0.05, baseline comparison, C⁻P⁻ vs. C⁺P⁻. ¶P < 0.05, baseline comparison, C⁺P⁺ vs. C⁺P⁻. TP < 0.05; W P < 0.05; G P < 0.05, 8-h comparisons. There were no significant differences for other comparisons for which P values are not given.

Fig. 9.

Expression profile of TRPV6 and calbindin D9k in mouse duodenum at baseline (solid bars) on a Ca²⁺-replete diet and plain water and 8 h after ip 1,25(OH)₂D₃ administration (shaded bars). Expression assays were performed as described in the legend for Fig. 8. Data are means ± SE (n = 6–8/genotype per condition). *P < 0.05, baseline vs. 8 h within genotypes. ¶ P < 0.05, baseline comparison, C⁺P⁺ vs. C⁺P⁻. T P < 0.05; W P < 0.05, 8-h comparisons. There were no significant differences for other comparisons for which P values are not given.