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. 2022 Dec;44(1):1426-1442.
doi: 10.1080/0886022X.2022.2114370.

Sclerostin is involved in osteogenic transdifferentiation of vascular smooth muscle cells in chronic kidney disease-associated vascular calcification with non-canonical Wnt signaling

Qiong Xiao  1   2   3   4 , Yun Tang  1   2   3 , Haojun Luo  1   2   3   5 , Sipei Chen  1   2   3 , Rong Chen  1   2   3 , Zhe Yan  6 , Lei Pu  1   2   3 , Li Wang  1   2   3 , Guisen Li  1   2   3 , Yi Li  1   2   3
Affiliations

Sclerostin is involved in osteogenic transdifferentiation of vascular smooth muscle cells in chronic kidney disease-associated vascular calcification with non-canonical Wnt signaling

Qiong Xiao et al. Ren Fail. 2022 Dec.

Abstract

Vascular calcification is prominent in patients with chronic kidney disease (CKD) and is a strong predictor of cardiovascular mortality in the CKD population. However, the mechanism underlying CKD-associated vascular calcification remains unclear. To identify potential therapeutic targets, a 5/6 nephrectomy rat model was established by feeding of a high-phosphorous diet as the CKD group and compared with sham group rats at 4 and 16 weeks. Sequencing analyses of the rat aorta revealed 643 upregulated and 1023 downregulated genes at 4 weeks, as well as 899 upregulated and 1185 downregulated genes at 16 weeks in the CKD group compared to the sham group. Bioinformatics analyses suggested that SOST (which encodes sclerostin) and Wnt signaling are involved in CKD-associated vascular calcification. Furthermore, protein-protein interactions analysis revealed interactions between SOST, WNT5A, and WNT5B, that involved runt-related transcription factor 2 (RUNX2) and transgelin (TAGLN). SOST was increased in CKD-associated vascular calcification following reduction of the Wnt signaling, including WNT5A and WNT5B, both in vivo and in vitro. TargetScan was used to predict the microRNAs (miRNAs) targeting WNT5A and WNT5B. The expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, and miR-3568 were significantly reduced, whereas that of miR-742-3p was significantly increased in calcified rat aortic vascular smooth muscle cells (VSMCs). In CKD rat aortas, the expression of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, miR-742-3p, and miR-22-5p were significantly reduced at both 4 and 16 weeks. Altogether, owing to several assessments, potentially diagnostic and prognostic biomarkers for improving common CKD diagnostic tools were identified in this study. Abbreviations: BUN: blood urea nitrogen; CKD: chronic kidney disease; CKD-MBD: chronic kidney disease-mineral bone disorder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GO: the Gene Ontology; HE: hematoxylin-eosin; HRP: horseradish peroxidase; KEGG: Kyoto Encyclopedia of Genes and Genomes; MiRNAs: microRNAs; PAS: periodic acid-Schiff; RUNX2: runt-related transcription factor 2; SCr: serum creatinine; STRING: the Search Tool for the Retrieval of Interacting Genes/Proteins; TAGLN: transgelin; VSMC: vascular smooth muscle cell.

Keywords: Chronic kidney disease; Wnt signaling pathway; microRNA; sclerostin; vascular calcification; vascular smooth muscle cells.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Technology roadmap to identify potential targets in chronic kidney disease (CKD)-associated vascular calcification, including the procedure used to establish the animal model, sequencing and bioinformatics analyses, and validation of potential target molecules in vivo and in vitro. SOST: encoding sclerostin; VSMCs: vascular smooth muscle cells.
Figure 2.
Figure 2.
5/6 nephrectomy with high-phosphorus diet induces chronic kidney disease (CKD)-associated vascular calcification in rats. a) Serum biochemical measurements. Serum creatinine (SCr) and blood urea nitrogen (BUN) in the CKD and sham groups at 4 and 16 weeks. #p < 0.05, ## p < 0.01, 4 vs. 16 weeks within the same treatment; + p < 0.05, ++ p < 0.01, CKD vs. Sham at the same time point; n ≥ 6 in each group. b) Hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining of rat kidneys from the CKD and sham groups at 4 and 16 weeks. c) Alizarin red S staining of rat aortas and quantification in the CKD and sham groups at 4 and 16 weeks. Arrows indicate the arterial medial calcification areas. The microscopic magnification of rat kidney is 400×, and those of the rat vessel are 200× and 800×. Scale bars = 20 and 100 μm.
Figure 3.
Figure 3.
Bioinformatics analyses. a) Between the chronic kidney disease (CKD) and sham groups at 4 weeks, volcano plot analysis of differentially expressed genes, Log2FC > ± 1 and p < 0.05. Clustering analysis of principal significant genes. The experiment was conducted in triplicate and the mean value was determined. The minimum fold change was ± 2 (p < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. b) Between the CKD and sham groups at 16 weeks, volcano plot analysis, clustering analysis, and KEGG pathway analysis. c) Enrichment analysis of the protein-protein interaction network.
Figure 4.
Figure 4.
Representative immunohistochemical staining of sclerostin, WNT5A, WNT5B, RUNX2, and TAGLN in rat aortas between the CKD and sham groups at 16 weeks and quantification. Arrows indicate positive areas. The microscopic magnification of rat vessel is 800×. Scale bar = 20 μm.
Figure 5.
Figure 5.
High phosphate induced calcification in rat aortic VSMCs. a) Original images following alizarin red S staining of rat aortic VSMCs treated with high phosphate medium for 7 d and quantification. b) Expression levels of RUNX2 and TAGLN in rat aortic VSMCs between the calcification (CAL) and the non-calcification (CTRL) groups based on immunoblotting. *p < 0.05, **p < 0.01, n = 3 in each group.
Figure 6.
Figure 6.
Differential expression of sclerostin, WNT5A, and WNT5B in vitro between the calcification (CAL) and the non-calcification (CTRL) groups. a) Expression levels of SOST (encoding sclerostin), WNT5A, and WNT5B in rat aortic VSMCs determined using real-time PCR. b) Expression levels of sclerostin, WNT5A, and WNT5B in rat aortic VSMCs determined using immunoblotting. *p < 0.05, ** p < 0.01, n ≥ 3 in each group.
Figure 7.
Figure 7.
miRNA prediction of WNT5A and WNT5B and validation of miRNAs in rat aortic VSMCs and rat aortic tissue. a) TargetScan was used to predict miRNAs targeting WNT5A and WNT5B. Green dots indicate downregulated genes and yellow dots indicate an inconsistent trend. b) Expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, and miR-742-3p between the calcification (CAL) and the non-calcification (CTRL) groups based on real-time PCR. c) Expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, and miR-742-3p in rat aortas between the CKD and sham groups at 4 and 16 weeks based on real-time PCR. *p < 0.05, **p < 0.01, n ≥ 3 in each group.
Figure 8.
Figure 8.
Interaction between sclerostin and WNT5A/WNT5B may be involved in CKD-associated vascular calcification via miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, and miR-742-3p. The osteochondrogenic transdifferentiation of VSMC phenotype is depicted by switching of the contractile phenotype of VSMC into the osteoblastic-like cell phenotype/synthetic phenotype in CKD. The arrow colors indicate the following: orange, upregulated; blue, downregulated; green, promoted; and red, prevented.
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References

    1. Global, regional, and national burden of chronic kidney disease, 1990–2017: a systematic analysis for the global burden of disease study 2017. Lancet. 2020;395(10225):709–733. - PMC - PubMed
    1. Zhang L, Wang F, Wang L, et al. . Prevalence of chronic kidney disease in China: a cross-sectional survey. Lancet. 2012;379(9818):815–822. - PubMed
    1. Liu ZH, Yu XQ, Yang JW, et al. . Prevalence and risk factors for vascular calcification in Chinese patients receiving dialysis: baseline results from a prospective cohort study. Curr Med Res Opin. 2018;34(8):1491–1500. - PubMed
    1. Vervloet M, Cozzolino M.. Vascular calcification in chronic kidney disease: different bricks in the wall? Kidney Int. 2017;91(4):808–817. - PubMed
    1. Chen W, Melamed ML.. Vascular calcification in predialysis CKD: common and deadly. Clin J Am Soc Nephrol. 2015;10(4):551–553. - PMC - PubMed

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Grants and funding

This work was supported by the National Natural Science Foundation of China [U21A20349, 81700607, 8170742, 81800613, 81970641, and 82070690]; PHD Foundation of Sichuan Academy of Sciences & Sichuan Provincial People’s Hospital [2015BS05]; Fundamental Research Funds for the Central Universities from UESTC [ZYGX2019J105]; Key R&D projects in Sichuan Province [2019YFS0538, 2021YFS0372 and 2021YFS0370]; Department of Science and Technology of Sichuan Province [2020ZYD034]; Foundation of Health and Family Planning Commission in Sichuan [16PJ424]; Foundation of Sichuan Medical Association [S20071]; and Zhongguancun Nephrology & Blood Purification Innovation Alliance [NBPIA20QC0102].