The calcium-sensing receptor (CaSR) defends against hypercalcemia independently of its regulation of parathyroid hormone secretion

Abstract

The calcium-sensing receptor (CaSR) controls parathyroid hormone (PTH) secretion, which, in turn, via direct and indirect actions on kidney, bone, and intestine, maintains a normal extracellular ionized calcium concentration (Ca(2+)(o)). There is less understanding of the CaSR's homeostatic importance outside of the parathyroid gland. We have employed single and double knockout mouse models, namely mice lacking PTH alone (CaSR(+/+) PTH(-/-), referred to as C(+)P(-)), lacking both CaSR and PTH (CaSR(-/-) PTH(-/-), C(-)P(-)) or wild-type (CaSR(+/+) PTH(+/+), C(+)P(+)) mice to study CaSR-specific functions without confounding CaSR-mediated changes in PTH. The mice received three hypercalcemic challenges: an oral Ca(2+) load, injection or constant infusion of PTH via osmotic pump, or a phosphate-deficient diet. C(-)P(-) mice show increased susceptibility to developing hypercalcemia with all three challenges compared with the other two genotypes, whereas C(+)P(-) mice defend against hypercalcemia similarly to C(+)P(+) mice. Reduced renal Ca(2+) clearance contributes to the intolerance of the C(-)P(-) mice to Ca(2+) loads, as they excrete less Ca(2+) at any given Ca(2+)(o) than the other two genotypes, confirming the CaSR's direct role in regulating renal Ca(2+) handling. In addition, C(+)P(+) and C(+)P(-), but not C(-)P(-), mice showed increases in serum calcitonin (CT) levels during hypercalcemia. The level of 1,25(OH)(2)D(3) in C(-)P(-) mice, in contrast, was similar to those in C(+)P(-) and C(+)P(+) mice during an oral Ca(2+) load, indicating that increased 1,25(OH)(2)D(3) production cannot account for the oral Ca(2+)-induced hypercalcemia in the C(-)P(-) mice. Thus, CaSR-stimulated PTH release serves as a "floor" to defend against hypocalcemia. In contrast, high-Ca(2+)(o)-induced inhibition of PTH is not required for a robust defense against hypercalcemia, at least in mice, whereas high-Ca(2+)(o)-stimulated, CaSR-mediated CT secretion and renal Ca(2+) excretion, and perhaps other factors, serve as a "ceiling" to limit hypercalcemia resulting from various types of hypercalcemic challenges.

Fig. 1.

Serum Ca²⁺ concentration in wild-type (C⁺P⁺), parathyroid hormone (PTH) knockout (C⁺P⁻), and PTH and calcium-sensing receptor (CaSR) double-knockout (C⁻P⁻) mice as a function of Ca²⁺ in the drinking water. All mice received chow with 0.8% Ca²⁺ throughout the study. After 1 wk on 0% Ca²⁺ water, blood and urine samples were obtained, and 1% Ca²⁺ (as CaCl₂) was added to the water. After 1 wk on 1% Ca²⁺ water, blood and urine samples were obtained, and Ca²⁺ in the water was changed to 2%. Blood and urine samples were obtained a third time 1 wk later. The serum Ca²⁺ concentration of the C⁻P⁻ mice was significantly higher than in the C⁺P⁻ mice with both 1% and 2% Ca²⁺ water and significantly higher than that of the C⁺P⁺ mice with 2% Ca²⁺ water (means ± SE, P < 0.01, n = 12–16 in each genotype).

Fig. 2.

Serum 1,25(OH)₂D₃ levels in C⁺P⁺, C⁺P⁻, and C⁻P⁻ mice receiving 0 or 2% Ca²⁺ water. Mice were fed normal chow and 0% Ca²⁺ water for 1 wk, and blood was drawn for measurement of serum levels of 1,25(OH)₂D₃ as described in materials and methods. Mice then received normal chow and 2% Ca²⁺ water for 1 wk, and measurements of 1,25(OH)₂D₃ were repeated. The level of 1,25(OH)₂D₃ in the C⁺P⁺ mice on 0% Ca²⁺ water was significantly higher than that in either C⁺P⁻ or C⁻P⁻ mice. Values of 1,25(OH)₂D₃ did not differ significantly among the 3 genotypes when receiving 2% Ca²⁺ in the drinking water. (means ± SE, n = 8–16 in each genotype).

Fig. 3.

Serum calcitonin (CT) levels in C⁺P⁺, C⁺P⁻, and C⁻P⁻ mice as a function in serum Ca²⁺ concentration. Mice were maintained on standard chow and 0% Ca²⁺ water for 1 wk, 1% Ca²⁺ water for 1 wk and finally, 2% Ca²⁺ water for a 3rd wk. Serum samples were obtained at the end of each of the 3 wk, and levels of Ca²⁺ and CT were determined as described in materials and methods. Data are plotted as serum Ca²⁺ concentration in any given serum sample vs. the CT concentration in that sample. Trend lines represent C⁺P⁻ and C⁺P⁺ (solid) and C⁻P⁻ (dotted).

Fig. 4.

Serum Ca²⁺ concentrations in C⁺P⁺, C⁺P⁻, and C⁻P⁻ mice receiving 2% Ca²⁺ water as a function of time after receiving an injection of CT. Mice were maintained on normal chow and 2% Ca²⁺ water for 1 wk. They then received an SC injection of salmon CT (150 ng/g body wt), and serum samples were obtained for determination of serum Ca²⁺ concentration as before at times shown. The maximal decrement in the absolute level of serum Ca²⁺ concentration after the CT injection in C⁻P⁻ mice was greater than in C⁺P⁺ or C⁺P⁻ mice (P < 0.05).

Fig. 5.

Urine Ca²⁺-to-creatinine ratio as a function of serum Ca²⁺ concentration in C⁺P⁺, C⁺P⁻, and C⁻P⁻ mice maintained on 0, 1, and then 2% Ca²⁺ water. Mice were initially maintained on normal chow and 0% Ca²⁺ water for 1 wk. Blood and spot urine specimens were collected on the 7th day. The drinking water was then changed to 1% Ca²⁺ for 7 days, and blood and urine specimens again obtained on day 7. Finally, the water was changed to 2% Ca²⁺ for 7 days, and urine and blood collections were again obtained on day 7. Serum Ca²⁺ and urine Ca²⁺ and creatinine were measured as in materials and methods, and results are plotted as serum Ca²⁺ concentration vs. corresponding Ca²⁺-to-creatinine ratio. Urine Ca²⁺ levels in C⁻P⁻ mice receiving 0% Ca²⁺ water were significantly lower than those in C⁺P⁻ mice under the same conditions (0.33 ± 0.038 and 0.17 ± 0.03, respectively, P < 0.001). The urine Ca²⁺ concentration in C⁺P⁻ and C⁻P⁻ mice with serum Ca²⁺ concentrations between 8 and 10 mg/dl also differed significantly (3.6 ± 0.27 vs. 1.6 ± 0.25 mg/dl, respectively, P < 0.01). It was not possible to compare urinary Ca²⁺ concentration in C⁺P⁺ and C⁺P⁻genotypes at higher Ca²⁺ concentrations because of the difficulty in raising serum Ca²⁺ concentration above 10 mg/dl in C⁺P⁻ (and C⁺P⁺) mice (means ± SE, n = 8–12 in each genotype). Trend lines represent C⁺P⁻ (solid) and C⁻P⁻ (dotted).

Fig. 6.

Urinary deoxypyridinolines (DPD; A) and serum osteocalcin (OC) concentrations (B) in C⁺P⁺, C⁺P⁻, and C⁻P⁻ mice receiving 0 or 2% Ca²⁺ water. Mice were maintained for 1 wk on each concentration of Ca²⁺ in the water, and serum and urine specimens were obtained on the last day of each of the 2 wk. Urinary DPD and creatinine as well as serum OC were measured as in materials and methods. There were no significant differences in DPD levels of C⁻P⁻ and C⁺P⁻ mice on 0 or 2% Ca²⁺ water, and changes in DPD within genotypes also did not change significantly when calcium in the drinking water was increased from 0 to 2%. Serum OC levels were not significantly different among the 3 genotypes when receiving 0% Ca²⁺ water. OC levels for C⁺P⁺ and C⁺P⁻ mice dropped significantly (P < 0.05) when mice received 2% Ca²⁺ water; however, OC levels for C⁻P⁻ mice showed no significant decline when mice received 2% Ca²⁺ water (P > 0.5) (means ± SE, n = 8–12 in each genotype for both DPD and OC).

Fig. 7.

Effect of injection with 500 ng/g body wt of hPTH_1-34 on serum Ca²⁺ of C⁺P⁺, C⁺P⁻, and C⁻P⁻ mice. Mice received 0% Ca²⁺ water for 7 days prior to the experiment. Serum was obtained just before PTH injection and then 3, 6, 11, and 24 h later for measurement of serum Ca²⁺ concentration. Serum Ca²⁺ was unchanged in C⁺P⁺ mice. It increased significantly in both C⁺P⁻ and C⁻P⁻ mice, but the increment was greater in C⁻P⁻ mice at 3 (P < 0.1), 6 (P < 0.1), and 11 h (P < 0.5) after injection (means ± SE, n = 4 for each genotype).

Fig. 8.

Effect of infusion with hPTH_1-34 via minipump on serum Ca²⁺ in C⁺P⁻ (circles) and C⁻P⁻ (triangles) mice. Mice were maintained on normal chow and 0% Ca²⁺ water for 1 wk. Osmotic minipumps were then implanted sc, and hPTH_1-34 was infused at 0.02, 0.04, or 0.08 mg·kg⁻¹·day⁻¹ as denoted by solid, shaded, and open symbols, respectively. Serum Ca²⁺ concentrations were determined on days 3 and 6 postimplantation (n = 5 for mice receiving 0.02 and 0.04 mg·kg⁻¹·day⁻¹ and n = 2 for mice receiving 0.08 mg·kg⁻¹·day⁻¹). Mean serum Ca²⁺ for noninfused mice (n = 13–16) after 1 wk on normal chow and 0% Ca²⁺ water, as determined on an independent set of mice, were 6.8 for C⁺P⁻ mice and 6.3 for C⁻P⁻ mice. Serum Ca²⁺ was significantly higher in C⁻P⁻ than in C⁺P⁻ mice infused with 0.04 mg·kg⁻¹·day⁻¹ for either 3 (P < 0.05) or 6 days (P < 0.02). Although serum Ca²⁺ was markedly higher in C⁻P⁻ mice than in C⁺P⁻ mice infused with 0.08 mg·kg⁻¹·day⁻¹, the small number of animals studied precluded statistical analysis.

Fig. 9.

Effect of PTH infusion on urinary Ca²⁺ concentration as a function of serum Ca²⁺ in C⁻P⁻ and C⁺P⁻ mice. Two independent urine samples were obtained on the same days as the serum samples in the experiment shown in Fig. 8, and urine Ca²⁺-to-creatinine ratios are plotted as a function of serum Ca²⁺ concentration. Urine Ca²⁺ values were significantly lower in C⁻P⁻ mice than in C⁺P⁻ mice receiving 0.04 mg·kg⁻¹·day⁻¹ PTH, despite significantly higher serum Ca²⁺ concentrations of C⁻P⁻ mice, demonstrating their relative hypocalciuria. C⁻P⁻ mice exhibited only a modest increase in urinary Ca²⁺ concentration with increasing rates of PTH infusion, in contrast to the marked increase in urinary Ca²⁺ concentration in C⁺P⁻ mice, as serum Ca²⁺ concentration rose in response to PTH infusion. There were significant differences in urinary Ca²⁺ concentration (P < 0.05) between C⁺P⁻ and C⁻P⁻ mice at the 0.02, 0.04, and 0.08 mg·kg⁻¹·day⁻¹ infusion rates when data from days 3 and 6 were pooled. Trend lines represent C⁺P⁻ (solid) and C⁻P⁻ (dotted).

Fig. 10.

Effect of phosphate-deficient diet on serum Ca²⁺ and phosphate concentrations in C⁺P⁺, C⁺P⁻, and C⁻P⁻ mice. Mice were maintained on a nominally phosphate-free diet with 0% Ca²⁺ water for 6 days. Serum was then obtained for measurements of Ca²⁺ and phosphate as described in materials and methods. Serum Ca²⁺ levels differed significantly among all genotypes (P < 0.01) (means SEM, n = 8 in each genotype).