Maintaining a positive calcium balance is vital for bone mineralization during postnatal development. We delineate transcellular calcium absorption pathways in the jejunum and ileum, which are present only early in life and contribute to a positive calcium balance. BACKGROUND & AIMS: Intestinal Ca 2þ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca 2þ absorption have been defined in adults after peak bone mass is obtained, but they are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca 2þ absorption during this critical period. METHODS: Expression of small intestinal and renal calcium transport genes was assessed by using quantitative polymerase chain reaction. Net calcium flux across small intestinal segments was measured in Ussing chambers, including after pharmacologic inhibition or genetic manipulation of TRPV6 or Ca v 1.3 calcium channels. Femurs were analyzed by using micro-computed tomography and histology. RESULTS: Net TRPV6-mediated Ca 2þ flux across the duodenum was absent in pre-weaned (P14) mice but present after weaning. In contrast, we found significant transcellular Ca 2þ absorption in the jejunum at 2 weeks but not 2 months of age. Net jejunal Ca 2þ absorption observed at P14 was not present in either Trpv6 mutant (D541A) mice or Ca v 1.3 knockout mice. We observed significant nifedipine-sensitive transcellular absorption across the ileum at P14 but not 2 months. Ca v 1.3 knockout pups exhibited delayed bone mineral accrual, compensatory nifedipine-insensitive Ca 2þ absorption in the ileum, and increased expression of renal Ca 2þ reabsorption mediators at P14. Moreover, weaning pups at 2 weeks reduced jejunal and ileal Ca v 1.3 expression. CONCLUSIONS: We have detailed novel pathways contributing to transcellular Ca 2þ transport across the distal small intestine of mice during development, highlighting the complexity of the multiple mechanisms involved in achieving a positive Ca 2þ balance early in life.

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Objectives Intestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults, after peak bone mass has been reached, but are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption across the small intestine which facilitate a positive calcium balance during growth. Methods We used wildtype, Cav1.3 knockout and Trpv6 mutant mice. Expression of small intestinal and renal calcium transport genes was assessed using quantitative PCR. Net transcellular 45-calcium flux across intestinal segments was measured in Ussing chambers. Femurs we analyzed using micro-CT and histology. Results Significant TRPV6 mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but occurred post-weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum and ileum at P14 but not 2 months. TRPV6 and Cav1.3 are necessary for this jejunal absorption and Cav1.3 appears to mediate absorption across the ileum although compensation is present in knockout pups. Knockout of Cav1.3 induces a compensatory increase in renal Ca2+ reabsorption in P14 mice although these pups have increased growth plate thickness suggesting delayed bone mineralization. Conclusions This work provides molecular details of how the small intestine facilitates increased demand for Ca2+ early in life to meet the requirements of growth and highlights the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance. Funding Sources This work is funded by grants from the Women and Children's Health Research Institute, supported by the Stollery Children's Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology. MRB is supported by a Vanier Canada Graduate Scholarship, Alberta Innovates Clinician Fellowship and an NSERC Michael Smith Foreign Study Supplement. H. Dimke is funded by the Danish Medical Research Council. Work at UdS was funded by Deutsche Forschungsgemeinschaft (DFG) by IRTG1830 (to JE, VF), Sonderforschungsbereich (SFB) 894 (to JE, PW) and SFB TRR152 (to VF).

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