Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease Rafiou Agoro 1 , Intawat Nookaew 2 , Megan L. Noonan 1 , Yamil G. Marambio 1 , Sheng Liu 1 , Wennan Chang 1,3 , Hongyu Gao 1,4 , Lainey M. Hibbard 1 , Corinne E. Metzger 5 , Daniel Horan 5 , William R. Thompson 5 , Xiaoling Xuei 1 , Yunlong Liu 1,4 , Chi Zhang 1,3 , Alexander G. Robling 5 , Lynda F. Bonewald 5,6 , Jun Wan 1 and Kenneth E. White 1,7 * 1 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, United States, 2 Department of Biomedical Informatics, University of Arkansas for Medical Sciences, Little Rock, United States, 3 Department of Electrical and Computer Engineering, Purdue University, Indianapolis, IN, United States, 4 Center for Medical Genomics, Indiana University School of Medicine, Indianapolis, IN, United States, 5 Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN, United States, 6 Indiana Center for Musculoskeletal Health, Indiana University, Indianapolis, IN, United States, 7 Department of Medicine/Nephrology, Indiana University School of Medicine, Indianapolis, IN, United States Introduction: Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. Methods: To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9 + mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Results: Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. Conclusion: In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized. KEYWORDS bone, scRNAseq, osteoblasts, osteocytes, chronic kidney disease Frontiers in Endocrinology frontiersin.org 01 OPEN ACCESS EDITED BY Ryan C. Riddle, University of Maryland, Baltimore, United States REVIEWED BY Alexander Rauch, University of Southern Denmark, Denmark Satoru Otsuru, University of Maryland, Baltimore, United States *CORRESPONDENCE Kenneth E. White kenewhit@iu.edu SPECIALTY SECTION This article was submitted to Bone Research, a section of the journal Frontiers in Endocrinology RECEIVED 06 October 2022 ACCEPTED 04 January 2023 PUBLISHED 26 January 2023 CITATION Agoro R, Nookaew I, Noonan ML, Marambio YG, Liu S, Chang W, Gao H, Hibbard LM, Metzger CE, Horan D, Thompson WR, Xuei X, Liu Y, Zhang C, Robling AG, Bonewald LF, Wan J and White KE (2023) Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease. Front. Endocrinol. 14:1063083. doi: 10.3389/fendo.2023.1063083 COPYRIGHT © 2023 Agoro, Nookaew, Noonan, Marambio, Liu, Chang, Gao, Hibbard, Metzger, Horan, Thompson, Xuei, Liu, Zhang, Robling, Bonewald, Wan and White. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 26 January 2023 DOI 10.3389/fendo.2023.1063083