The Mendelian disorders of abnormal calcium homeostasis that are caused by CaR mutations are all characterized by an altered response to Ca2+o by the parathyroid gland and kidneys. However, CaR is expressed in multiple tissues in addition to the parathyroid glands and kidney. It has been unclear what contribution, if any, CaR mutations in these tissues make to the overall phenotypes of CaR–/– mice and the human disorders FHH and NSHPT. Furthermore, it has been difficult to determine which abnormalities caused by inherited defects in CaR are direct effects of the CaR alterations and which are secondary to hypercalcemia and hyperparathyroidism.
In this study, we developed a CaR-deficient mouse model that survives into adulthood. By eliminating PTH, we separated the detrimental effects of hyperparathyroidism and severe hypercalcemia from the direct effects of CaR deficiency. In order to compare the effects of CaR deficiency rather than the compound effects of two mutant genes, we made all of our comparisons between Pth–/–CaR–/– mice and PTH-deficient control sex-matched littermates (as opposed to wild-type mice). The Pth–/–CaR–/– mice were viable and fertile, although females had difficulty maintaining pregnancies and nursing pups when not on a calcium-enriched diet. Our results show that the major phenotypic features characteristic of CaR–/– mice are the consequence of impaired suppression of PTH secretion and the resulting elevation in serum calcium levels and not the direct effect of CaR deficiency. This work further supports the hypothesis that the primary role of CaR is to suppress PTH secretion.
All of the Pth–/–CaR–/–
mice had grossly enlarged parathyroid glands. Ca2+o
inhibits parathyroid cell proliferation. However, through studies of cyclin D1 transgenic mice, Imanishi et al. have shown that parathyroid hyperplasia can occur independently of altered Ca2+o
-mediated control of PTH secretion (34
). Our finding that CaR-deficient mice show parathyroid hyperplasia in the absence of PTH demonstrates that parathyroid gland hyperplasia also does not depend on the overproduction of PTH per se. The enlarged parathyroid glands also observed in some of the control mice may be secondary to sustained hypocalcemia during the fetal period. Calcium is actively transported across the placenta from mother to fetus, resulting in a higher fetal calcium concentration (35
). The parathyroid glands of a fetus in a calcium-stressed Pth–/–
mother would likely be exposed to a calcium level below normal, leading to secondary parathyroid hyperplasia.
Despite the fact that CaR-deficient mice are viable in the absence of PTH, our results indicate a role for CaR in maintaining calcium homeostasis independent of PTH. The serum calcium levels of PTH-deficient mice are much less tightly regulated in the absence of CaR. The large amount of variability between clinically significant high and low measured serum calcium values suggests that serum calcium levels in Pth–/–CaR–/– mice fluctuate widely. Similarly, urinary calcium excretion varied widely in mice lacking CaR. The mechanism by which CaR helps to maintain serum calcium levels within a narrow range in the absence of PTH is unclear, but this model should be useful for more detailed studies of CaR function in intestine, kidney, bone, and other homeostatic tissues. Our work indicates that CaR is necessary for the tight regulation of serum calcium levels independent of its effect on PTH secretion and that CaR is involved in fine tuning renal calcium excretion in a PTH-independent manner.
The trend in the Pth–/–CaR–/–
animals was towards hypercalcemia and hypercalciuria. We therefore examined the expression levels of two calcium entry channels involved in the maintenance of calcium homeostasis, CaT1 (30
) and CaT2/ECaC (31
). CaT1 is expressed throughout the human gastrointestinal tract and mediates intestinal calcium absorption (30
). CaT2/ECaC is kidney specific and localized to the apical membrane of the distal convoluted tubule (32
). CaT1 and CaT2/ECaC levels in the kidney and duodenum were increased in the Pth–/–CaR–/–
mice, although the trend was less consistent in the duodenum than in the kidney. The increases in calcium transporter expression in the kidney and duodenum may have an impact on calcium balance and contribute to the greater variability in renal calcium excretion and serum calcium concentrations observed in the CaR-deficient mice.
We observed no detrimental bone abnormalities in Pth–/–CaR–/–
mice. However, our measurements found that female Pth–/–CaR–/–
mice have elevated BMD in the spine. Since multiple hypotheses were tested in the course of the various studies described in this paper, we acknowledge the possibility that this result may represent a “false positive,” particularly given the P
value of 0.043, which is just under the generally accepted value of 0.05. However, this finding is consistent with complementary findings from other studies. Genetic factors have been shown to account for about 50–85% of the variation in bone mass, and CaR
has been examined as a possible susceptibility locus for osteoporosis. An A986S CaR
polymorphism has been reported to have an effect on serum calcium, circulating PTH concentrations, and BMD in healthy Caucasian girls (40
). (A second group found no significant difference in the distribution of the A986S polymorphism between groups of postmenopausal and healthy control women and concluded that CaR had no effect on BMD ). Other investigators have shown that high Ca2+o
stimulates CaR in chondrogenic cell lines, leading to increased production of mineral matrix (43
We have demonstrated that the lethal CaR-deficient phenotype is dependent on PTH. In the absence of PTH, serum calcium levels and urine calcium excretion are much less tightly regulated in CaR-deficient mice than in mice with CaR. These mice provide a model for more detailed examination of the role of CaR in a wide variety of physiological functions that cannot be addressed by studying mice deficient only in CaR. These findings also have implications for understanding the role of CaR in regulating bone function independently of its effect on PTH secretion.