Renal Endocytic Regulation of Vitamin D Metabolism during Maturation and Aging in Laying Hens

Simple Summary Egg-laying hens have a specific and dramatic calcium metabolism to lay eggs with eggshells that are composed of calcium carbonate. Calcium metabolism is mainly regulated by vitamin D3. However, details of the regulatory mechanism of vitamin D3 metabolism have not been clarified. In this study, we found that the 25-hydroxyvitamin D3-binding protein complex is taken up by the endocytic receptors megalin and cubilin in the renal proximal tubules, providing a novel mechanism for the regulation of vitamin D3 metabolism during maturation and aging. These findings may contribute to the prevention of heat stress and age-related deterioration of eggshell quality in egg-laying hens. Abstract Egg-laying hens undergo a specific and dramatic calcium metabolism to lay eggs with eggshells composed of calcium carbonate. Calcium metabolism is mainly regulated by vitamin D3. Although vitamin D3 metabolism is closely related to the deterioration of eggshell quality associated with aging and heat stress, the details of the mechanisms regulating vitamin D3 metabolism are not clear. In mammals, the vitamin D3 metabolite (25(OH)D3) produced in the liver binds to the vitamin binding protein (DBP), is subsequently taken up by renal proximal tubular cells via the endocytic receptors megalin (Meg) and cubilin (CUB), and is metabolized to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Therefore, the present study aimed to examine the expression and localization of Meg and CUB in the kidneys of immature chicks and mature and aged laying hens to prevent eggshell quality deterioration. As a result, we showed that as circulating 1,25(OH)2D3 concentrations increased from 156.0 ± 13.5 pg/mL to 815.5 ± 61.4 pg/mL with maturation in immature chicks, relative expression levels (arbitrary units; AU) of Meg and CUB mRNA in the kidneys of mature hens significantly increased 1.92- and 2.75-fold, respectively, compared to those in immature chicks. On the other hand, the Meg mRNA expression levels of mature hens did not change with age, while CUB mRNA expression levels (1.03 ± 0.11 AU) were significantly decreased compared to mature hens (2.75 ± 0.24 AU). Immunohistochemical observations showed that Meg and CUB proteins were localized to the apical membrane of renal proximal tubular epithelial cells in immature chicks, mature hens, and aged hens, and that DBP protein was observed as granular endosomes in the cytoplasm of proximal tubular cells from the apical membrane to the cell nucleus. Especially in mature hens, the endosomes were larger and more numerous than those in immature chicks. In contrast, in aged hens, DBP-containing endosomes were smaller and limited to the apical cytoplasm. These results indicate that with maturation, the expression of Meg and CUB is promoted in the renal proximal tubules of laying hens, facilitating the uptake of the 25(OH)D3-DBP complex and its conversion to 1,25(OH)2D3, and regulating calcium metabolism in eggshell formation. On the other hand, it is suggested that the age-related decrease in CUB expression suppresses the uptake of the 25(OH)D3-DBP complex in the kidney, resulting in a deterioration of eggshell quality.