A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis
Sung-Hee Yoon, … , Michael Mannstadt, Marc N. Wein
Sung-Hee Yoon, … , Michael Mannstadt, Marc N. Wein
Published March 2, 2023
Citation Information: J Clin Invest. 2023;133(9):e163627. https://doi.org/10.1172/JCI163627.
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Research Article Bone biology Nephrology

A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis

Abstract

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell–derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor–inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease–mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

Authors

Sung-Hee Yoon, Mark B. Meyer, Carlos Arevalo, Murat Tekguc, Chengcheng Zhang, Jialiang S. Wang, Christian D. Castro Andrade, Katelyn Strauss, Tadatoshi Sato, Nancy A. Benkusky, Seong Min Lee, Rebecca Berdeaux, Marc Foretz, Thomas B. Sundberg, Ramnik J. Xavier, Charles H. Adelmann, Daniel J. Brooks, Anthony Anselmo, Ruslan I. Sadreyev, Ivy A. Rosales, David E. Fisher, Navin Gupta, Ryuji Morizane, Anna Greka, J. Wesley Pike, Michael Mannstadt, Marc N. Wein

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Figure 7

CRTC2 plays a crucial role in Cyp27b1 transcriptional control downstream of PTH/SIK signaling.

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CRTC2 plays a crucial role in Cyp27b1 transcriptional control downstream...
(A) Kidney organoids were treated with DMSO, PTH (242 nM), or SK-124 (10 μM) for 3 hours and immunostained to show CRTC2 nuclear translocation in Lotus Tetragonolobus Lectin–positive (LTL-positive) proximal tubules. Images on the top and bottom are identical with the exception that images on the lower row are shown without the LTL channel (LTL for proximal tubule; CDH1 for distal tubule; podocalyxin [PODXL] for podocytes; Sytox Blue as the nuclear dye). Scale bars: 50 μm. Quantification is shown in the histogram on the right, with P values from 1-way ANOVA followed by Tukey’s post hoc test. (B) Opossum kidney (OK) cells were treated for the indicated times with PTH (242 nM), YKL-05-099 (10 μM), and SK-124 (20 μM), followed by total and phospho-CRTC2 S275 immunoblotting. (C) Top: Mice were treated with PTH (230 ng/g, 30 min, i.p.) or vehicle followed by ChIP-Seq for CRTC2 or phospho-CREB (n = 3 per treatment). Bottom: Control and inducible/global SIK1/2/3 TKO mice (n = 3 per genotype) (see Figure 4) were treated with tamoxifen and sacrificed 48 hours later followed by kidney ChIP-Seq. Quantification of peak density is shown on the right for the various M21 enhancer subpeaks, along with the M1 enhancer and the Cyp27b1 promoter (CP). Fold change (versus vehicle) or peak density is shown, with asterisks indicating inducible binding of the indicated protein (CRTC2 on the top, pCREB on the bottom) at each site. (D) CRTC2 ChIP-qPCR using M1 enhancer primers was performed on kidneys from mice treated for 30 minutes as indicated. Recovered DNA relative to input samples is shown. (E) Representative images of Px459 and CRTC2-KO organoids show the development of nephron segments including proximal tubules by LTL staining. (MEIS for stromal cells; CDH1 for distal tubule; CD146 for vasculature; Sytox-Blue is for nucleic acid, staining all the cells.) Scale bars: 123.5 μm. (F and G) Cyp27b1 expression and 1,25-vitamin D secretion after SIK inhibitor treatment with YKL-05-099 (10 μM) and SK-124 (20 μM) in PX459 and CRTC2-KO kidney organoids are shown. Blue dots in G indicate the baseline 0-hour control for both YKL-05-099 and SK-124 drugs.