Kidney Week

Abstract: TH-PO528

25(OH)D₃ Alone Stimulates Expression of 1,25(OH)₂D₃-Responsive Genes, Calbindin-D9K, and Megalin in mpkDCT Cells Where the Cyp27b1 Gene Was Deleted by CRISPR-Cas9

Session Information

• Bone and Mineral Metabolism: Basic
  November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Bone and Mineral Metabolism

• 401 Bone and Mineral Metabolism: Basic

Authors

• Kikuyama, Takahiro, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Takahiro Kikuyama,

• Susa, Takao, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Takao Susa,

• Tamamori-Adachi, Mimi, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Mimi Tamamori-Adachi,

• Iizuka, Masayoshi, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Masayoshi Iizuka,

• Uchida, Shunya, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Shunya Uchida,

• Okazaki, Tomoki, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Tomoki Okazaki,

• Shibata, Shigeru, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Shigeru Shibata,

Background

During vitamin D synthesis, 25(OH)D₃ (25D3) is converted to the active form, 1,25(OH)₂D₃ (1,25D3), by CYP27B1 in the proximal tubule of the kidney, then the latter exerts its function via vitamin D3 receptor (VDR). In the kidney, the 1,25D3-VDR axis is responsible for the increase in calcium reabsorption. Recently, it has been reported that 25D3 has bioactivity in certain tissues derived from Cyp27b1 knockout mice. On the other hand, others reported that 25D3-fed Cyp27b1 knockout mice produced normal level of 1,25D3 in the plasma. Such 1,25D3 might have been brought about by the other unknown enzyme(s) and it would be difficult to prove the direct cell-intrinsic activity of 25D3 (but not 1,25D3) using the Cyp27b1 knockout mouse models.

Methods

We used CRISPR-Cas9 system to knock out Cyp27b1 gene in the mouse kidney distal tubule cell line, mpkDCT cells. We then investigated the 25D3 function in these cells.

Results

Cyp27b1 knockout mpkDCT cells did not produce any measurable 1,25D3 after 25D3 administration to the cells. We found that 10^-7 M of 25D3 translocated VDR into the nucleus and promoted expression of the positive control Cyp24a1 gene in the Cyp27b1 knockout mpkDCT cells. Microarray analysis also revealed that the exhaustive target gene profiles of 25D3 showed results similar to those of 1,25D3. Subsequently, we confirmed that 25D3 induced the expression of calcium reabsorption-related genes, Calbindin D9K (S100g) and Megalin (Lrp2), in the way analogous to 1,25D3.

Conclusion

In this study, we showed that a high dose(s) of 25D3 exerted vitamin D3 activity directly in the kidney cells without the aid of CYP27B1 function. We surmise that the ability to induce VDR target genes may provide a novel benefit with 25D3 in certain tissues. In the case of decreased CYP27B1 activity in advanced CKD, high concentrations of 25D3 may have a promising effect to maintain the serum calcium level.