A nonclassical vitamin D receptor pathway suppresses renal fibrosis
Ichiaki Ito, … , Kazuo Nagasawa, Junn Yanagisawa
Ichiaki Ito, … , Kazuo Nagasawa, Junn Yanagisawa
Published October 25, 2013
Citation Information: J Clin Invest. 2013;123(11):4579-4594. https://doi.org/10.1172/JCI67804.
View: Text | PDF
Research Article

A nonclassical vitamin D receptor pathway suppresses renal fibrosis

Abstract

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β–SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3–bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-β–SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-β–SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β–SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-β–SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β–SMAD signal transduction. Since TGF-β–SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.

Authors

Ichiaki Ito, Tsuyoshi Waku, Masato Aoki, Rumi Abe, Yu Nagai, Tatsuya Watanabe, Yuka Nakajima, Ichiro Ohkido, Keitaro Yokoyama, Hiroyuki Miyachi, Toshiyuki Shimizu, Akiko Murayama, Hiroyuki Kishimoto, Kazuo Nagasawa, Junn Yanagisawa

×

Figure 8

DLAM-iPr and DLAM-4P suppress tubulointerstitial sclerosis without inducing hypercalcemia in UUO mice.

Options: View larger image (or click on image) Download as PowerPoint
DLAM-iPr and DLAM-4P suppress tubulointerstitial sclerosis without induc...
(A) DLAM-iPr and DLAM-4P suppress the expression of profibrotic genes. Cyp24a1, Serpine1, Acta2, and Col1a1 mRNA levels were determined by qPCR using RNA isolated from sham-operated (n = 4) or UUO kidneys of mice treated with vehicle (n = 7), 1,25(OH)2D3 (n = 8), DLAM-iPr (n = 9), or DLAM-4P (n = 9). (B) Western blot analysis of SMAD3, pSMAD3, PAI-1, α-SMA, and β-actin in UUO and sham-operated mouse kidneys. Quantification of PAI-1 protein levels (n = 3 per treatment group; see Methods) is also shown. (C) DLAM-iPr and DLAM-4P inhibited SMAD3 recruitment to the Serpine1 promoter in the kidneys. Kidney samples from UUO or sham-operated mice were subjected to ChIP assay with control IgG or anti-SMAD3 antibodies. Immunoprecipitated DNA was examined using qPCR with primers specific for the Serpine1 promoter. Samples were normalized to input DNA level (n = 3 per group). (D) Representative photomicrographs of Masson’s trichrome staining (top row, whole kidney; second row, magnified view of boxed region, enlarged ×4.37), FSP-1– (third row), and α-SMA–specific immunofluorescence staining (bottom row) of kidney sections from sham-operated or UUO mice injected with vehicle, 1,25(OH)2D3, DLAM-iPr, or DLAM-4P. Scale bars: 1.0 mm (top row); 200 μm (second row); 20 μm (bottom 2 rows). (E) Fibrotic areas (n = 3 per group), FSP-1–positive cells (n = 12 per group), and α-SMA–positive areas (n = 8 per group) in stained kidney sections. (F) Serum calcium concentration. *P < 0.05; **P < 0.01; ***P < 0.001.