Establishment of a new murine model of hypercalcemia with anorexia by overexpression of soluble receptor activator of NF-κB ligand using an adenovirus vector.

Hypercalcemia is a significant complication of certain human malignancies that is primarily caused by the release of calcium from bone due to marked bone resorption by osteoclast activation. Osteoclast differentiation and activation is mediated by receptor activator of NF-κB ligand (RANKL). Transgenic mice overexpressing murine soluble RANKL (sRANKL) that we generated previously exhibited severe osteoporosis accompanied with enhanced osteoclastogenesis, but never exhibited hypercalcemia. To analyze the relationship between serum concentration of sRANKL and hypercalcemia and generate a simple and quick hypercalcemia model, an adenovirus vector harboring murine sRANKL cDNA (Ad-sRANKL) was injected i.p. into male C57BL/6 mice. Sera were collected to measure the levels of sRANKL, calcium and biochemical markers of bone turnover. Food intake and body weight were measured every 3 or 4 days. All the mice were killed 2 weeks after the injection, and femurs were collected to measure bone structure and bone mineral density (BMD). Serum sRANKL and calcium increased, peaking on day 7. Food intake and body weight significantly declined on day 7. These results indicated that the mice had anorexia as a symptom of hypercalcemia. Increases in bone resorption and formation markers with a marked decrease in BMD were observed on day 14. These results reflect accelerated bone formation following activation of osteoclasts, indicating coupling between bone formation and resorption. In conclusion, a new murine model of hypercalcemia with anorexia was established by overexpressing sRANKL. This model would be useful for studies of hypercalcemia and coupling between bone formation and resorption.