Age-related alterations in calbindin-D_28K induction by 1, 25-dihydroxy vitamin D₃ in primary cultures of rat renal tubule cells

In vivo studies have indicated that renal calbin-din-D_28K protein and mRNA levels decrease in adult and old rats, and this decrease parallels the age-associated decline in serum 1, 25-dihydroxyvitamin D₃ [1, 25-(OH)₂D₃] levels. However, diminished renal responsiveness to 1, 25-(OH)₂D₃ with advancing age could also contribute to decreased calbindin-D_28K expression. To study renal responsiveness with age, primary cell cultures were established from the kidney cortices of young (1 month old), adult (10-12 months old), and old (20-24 months old) rats. Cells were incubated in medium K-1 containing 2% fetal calf serum. Calbindin-D_28K protein levels were determined by Western blot and enzyme-linked immunosorbent assay. In young animals, the levels of calbindin-D28K declined from 12.1 ± 1.3 μg/mg protein in the intact kidney to 1.6 ± 0.07 μg/mg protein in cells that had been cultured for 3 days in the absence of 1, 25-(OH)₂D₃. This sharp decline in calbindin-D_28K protein concentration moderated by days 6-8. The continuous presence of 10-7 M 1, 25-(OH)₂D₃ in the medium did not abolish the decline. The low levels of calbindin-D_28K in the cells cultured in the absence of 1, 25-(OH)₂D₃ provided an excellent experimental system in which to compare the response of the cells to 1, 25-(OH)₂D₃ between age groups. In cultured cells treated with 1, 25-(OH)₂D₃ for 72 h, calbindin-D_28K induction was greater in cells from adult and old animals compared to cells from young animals. The ratios of calbindin-D_28K content (with vitamin D/without vitamin D) were 2.2 ± 0.2, 4.7 ± 0.5, and 7.1 ± 1.5 for young, adult, and old cells, respectively. These studies suggested that the observed in vivo decrease in renal calbindin-D_28K with age is primarily due to the lowered circulating 1, 25-(OH)₂D₃.