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Updated: Aug 10, 2025
Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice
Published on: May 2, 2025
Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease
Rafiou Agoro1, Intawat Nookaew2, Megan L Noonan1
1Department of Medical and Molecular Genetics, 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.
Insights
This study defines distinct bone cell populations using single-cell RNA sequencing. It reveals molecular defects in chronic kidney disease (CKD) bone cells, suggesting earlier and broader bone pathology than previously known.
Area of Science:
- Bone Biology
- Single-cell Genomics
- Skeletal Diseases
Background:
- The precise molecular mechanisms driving bone dysfunction in aging, osteoporosis, and chronic kidney disease (CKD) are poorly understood due to limitations in single-cell resolution.
- Understanding these mechanisms is crucial for developing effective treatments for bone diseases.
Purpose of the Study:
- To define distinct osteoblast and osteocyte populations at the single-cell level.
- To identify molecular defects in bone cells associated with CKD.
- To investigate the early skeletal pathology in a mouse model of CKD.
Main Methods:
- Single-cell RNA sequencing (scRNAseq) and flow cytometry were performed on mouse long bone cells.
- Clustering analysis identified specific gene expression profiles for osteoblast precursors, mature osteoblasts, and osteocytes.
- Integrative analyses including ATAC-seq and single-cell flux estimation analysis (scFEA) were used for validation and functional prediction.
- A mouse model of CKD was utilized to assess gene expression changes in osteolineage cells.
Main Results:
- Distinct transcriptional signatures were identified for osteoblast precursors (Tnc, Mmp13, Spp1), mature osteoblasts (Smpd3, Col1a1, Col11a1), and osteocytes (Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, Phex).
- Trajectory analysis predicted osteoblast-to-osteocyte transitions linked to metabolic shifts.
- In the CKD mouse model, gene expression was disturbed across all osteolineage stages even before significant skeletal alterations were apparent.
Conclusions:
- This study successfully defined distinct osteoblast and osteocyte populations at the single-cell level.
- Molecular defects were identified in multiple bone cell populations in a CKD mouse model.
- The findings suggest that bone pathology in CKD may be more widespread and initiate earlier than previously recognized.
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