Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium

Abstract

Renal vitamin D receptor (VDR) is required for 1,25-dihydroxyvitamin D3-[1,25(OH)2D3]-induced renal reabsorption of calcium and for 1,25(OH)2D3-induced 1,25(OH)2D3 24-hydroxylase. The long-term effect of vitamin D and dietary calcium on the expression of renal VDR was examined in the nonobese diabetic mouse. Vitamin D-deficient and vitamin D-replete mice were maintained on diets containing 0.02%, 0.25%, 0.47%, and 1.20% calcium with or without 50 ng of 1,25(OH)2D3 per day. Vitamin D-replete mice on a 1.20% calcium diet had renal VDR levels of 165 fmol/mg protein. Calcium restriction caused renal VDR levels to decrease to <30 fmol/mg protein in vitamin D-deficient mice and to approximately 80 fmol/mg protein in vitamin D-replete mice. When dietary calcium was present, 50 ng of 1,25(OH)2D3 elevated the VDR levels 2- to 10-fold, depending on vitamin D status and the level of calcium. In the absence of either vitamin D or calcium, the VDR mRNA was expressed at a basal level. 1,25(OH)2D3 supplementation caused relative VDR mRNA to increase 8- to 10-fold in the vitamin D-deficient mouse when dietary calcium was available. This increase was completely absent in the calcium-restricted mice. This in vivo study demonstrates that 1,25(OH)2D3 and calcium are both required for renal VDR mRNA expression above a basal level, furthering our understanding of the complex regulation of renal VDR by 1,25(OH)2D3 and calcium.

Fig. 1.

Regulation of renal VDR by calcium and 1,25(OH)₂D₃. Mice were fed a purified diet that contained 0.02%, 0.25%, 0.47%, or 1.20% calcium with or without 50 ng of 1,25(OH)₂D₃ per mouse per day. One kidney per mouse was harvested into isotonic buffer, whole-cell extract was prepared, and VDR content was determined by ELISA and standardized to total protein, as described in Materials and Methods. (A) Vitamin D-deficient mice, housed under incandescent lighting without dietary intake of vitamin D₃.(B) Vitamin D-replete mice, housed under fluorescent lighting and receiving dietary supplementation of vitamin D₃. Each group contained four to six mice. The values represent the mean ± SEM. *, significance at P ≤ 0.01.

Fig. 2.

Regulation of renal VDR mRNA by calcium and 1,25(OH)₂D₃. Mice were fed a purified diet that contained 0.02%, 0.25%, 0.47%, or 1.20% calcium with or without 50 ng of 1,25(OH)₂D₃ per mouse per day. One kidney per mouse was harvested into Tri reagent and total RNA was prepared. After reverse transcription, VDR mRNA was quantified by means of real-time PCR and standardized to β-actin mRNA. Values are expressed as relative mRNA with a maximal value of one. (A) Vitamin D-deficient mice, housed under incandescent lighting without dietary intake of vitamin D₃. (B) Vitamin D-replete mice, housed under fluorescent lighting and receiving dietary supplementation of vitamin D₃. Each group contained four mice. The values represent the mean ± SEM. *, significance at P ≤ 0.01.

Fig. 3.

Regulation of renal 1α-hydroxylase mRNA by calcium and 1,25(OH)₂D₃. Mice were fed a purified diet that contained 0.02%, 0.25%, 0.47%, or 1.20% calcium with or without 50 ng of 1,25(OH)₂D₃ per mouse per day. One kidney per mouse was harvested into Tri reagent, and total RNA was prepared. After reverse transcription, 1α-hydroxylase mRNA was quantified by means of real-time PCR and standardized to β-actin mRNA. Values are expressed as relative mRNA with a maximal value of one. (A) Vitamin D-deficient mice, housed under incandescent lighting without dietary intake of vitamin D₃.(B) Vitamin D-replete mice, housed under fluorescent lighting and receiving dietary supplementation of vitamin D₃. Each group contained four mice. The values represent the mean ± SEM. *, significance at P ≤ 0.01.

Fig. 4.

Regulation of renal 24-hydroxylase mRNA by calcium and 1,25(OH)₂D₃. Mice were fed a purified diet that contained 0.02%, 0.25%, 0.47%, or 1.20% calcium with or without 50 ng of 1,25(OH)₂D₃ per mouse per day. One kidney per mouse was harvested into Tri reagent, and total RNA was prepared. After reverse transcription, 24-hydroxylase mRNA was quantified by means of real-time PCR and standardized to β-actin mRNA. Values are expressed as relative mRNA with a maximal value of one. (A) Vitamin D-deficient mice, housed under incandescent lighting without dietary intake of vitamin D₃.(B) Vitamin D-replete mice, housed under fluorescent lighting and receiving dietary supplementation of vitamin D₃. Each group contained four mice. The values represent the mean ± SEM. ND, not detectable. *, significance at P ≤ 0.01.