Fig. 1. Evaluation of renin-inhibiting activity of Vitamin D analogs in cell culture. (A) Vitamin D analogs listed in Table 1 (compounds 1–9) were used to treat As4.1-hVDR cells at 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M for 24 h. Renin mRNA levels were determined by Northern blot analyses. Each compound is indicated by its code. Note Gemini compounds RO-27-5646 and RO-27-2310 have inhibitory activity comparable to that of 1,25(OH)₂D₃. (B) Luciferase reporter assays, confirming the potent activity of RO-27-2310 and RO-27-5646 to suppress renin transcriptional activity. As4.1-hVDR cells were transfected with pGL3-basic and pGL-4.1-Luc, and treated with ethanol (E), 10⁻⁸ M of 1,25(OH)₂D₃ (1,25D) or 10⁻⁸ M of different analogs RO-27-5157, RO-27-2310, RO-50-5371 and RO-27-5646 as indicated.

Fig. 2. Evaluation of the potency of Gemini compounds to suppress renin expression. (A) Quantitative data obtained from Northern blot analyses. Gemini compounds listed in Table 1 (compounds 10–20) were used to treat As4.1-hVDR cells at 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M for 24 h. Levels of the renin mRNA transcript were determined by Northern blot analyses. Shown are the quantitative data obtained at 10⁻¹⁰ and 10⁻⁹ M of the analogs. Each compound is indicated by its code. E, ethanol-treated control; 1,25D, 1,25(OH)₂D₃. Note the super-potency of compounds RO-4383586, RO-4383582, RO-4383562, RO-4383561 and BXL-01-0002. (B) Representative luciferase reporter assays. As4.1-hVDR cells were transfected with pG3-basic and pGL-4.1-Luc, and treated with ethanol (E), 1,25(OH)₂D₃ or different analogs as indicated by their code at the dose of 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M.

Fig. 3. Evaluation of the activity of Vitamin D analogs to stimulate 25-hydroxyvitamin D 24-hydroxylase expression. As4.1-hVDR cells were treated with ethanol (E), 1,25(OH)₂D₃ (1,25D) or different analogs at the dose of 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M for 24 h. The RNA samples were analyzed by Northern blotting with ³²P-labeled CYP24 cDNA probe. Shown are data of representative analog compounds. Note the induction by 1,25(OH)₂D₃ is relatively weak compared with other analogs.

Fig. 4. Evaluation of calcemic and renin-inhibiting activities of Gemini compound RO-27-5646 in mice. (A) Blood ionized calcium levels. Normal CD-1 mice (n = 5) were treated with vehicle, 1,25(OH)₂D₃ (1,25D) or RO-27-5646 at a daily dose of 1.5 μg/kg body weight for 1 week. Arrow indicates that two out of five 1,25D-treated mice died at day 6. Note the low calcemic effect of RO-27-5646. (B) Serum intact PTH levels at the end of the treatment. (C) Renin mRNA levels in the kidneys after treatment, determined by Northern blot analysis. ^*P < 0.01 vs. vehicle-treated control.

Fig. 5. Inhibition of steady state renin mRNA levels in mice treated with Gemini compound RO-27-5646. Normal CD-1 mice (n = 4–6) were treated with RO-27-5646 for 1 week at a daily dose of 4.5 μg/kg (A) or 12 μg/kg body weight (B), and renin mRNA levels in the kidneys were determined by Northern blot analysis after the treatment. Each lane represents one individual animal. As a positive control for the analog's action, marked calbindin-D9k stimulation was clearly seen in the kidney. The quantitative data were presented below the Northern blot panels. Note 54 ± 8 and 76 ± 9% reduction in renin mRNA levels after treatment with the two doses of RO-27-5646, respectively. ^*P < 0.01 vs. vehicle-treated control. The P value was calculated based on all the vehicle-treated values vs. the RO-27-5646-treated values in each experiment.

Vitamin D analog compounds used in this study