RNA-seq analysis of extracellular vesicles from hyperphosphatemia-stimulated endothelial cells provides insight into the mechanism underlying vascular calcification

BMC Nephrol. 2022 May 21;23(1):192. doi: 10.1186/s12882-022-02823-6.


Background: Hyperphosphatemia (HP) is associated with vascular calcification (VC) in chronic kidney disease (CKD). However, relationship between HP-induced-endothelial extracellular vesicles (HP-EC-EVs) and VC is unclear, and miR expression in HP-EC-EVs has not been determined.

Methods: We isolated HP-EC-EVs from endothelial cells with HP and observed that HP-EC-EVs were up-taken by vascular smooth muscle cells (VSMCs). HP-EC-EVs inducing calcium deposition was characterized by Alizarin Red S, colourimetric analysis and ALP activity. To investigate the mechanism of HP-EC-EVs-induced VSMC calcification, RNA-sequencing for HP-EC-EVs was performed.

Results: We first demonstrated that HP-EC-EVs induced VSMC calcification in vitro. RNA-seq analysis of HP-EC-EVs illustrated that one known miR (hsa-miR-3182) was statistically up-regulated and twelve miRs were significantly down-regulated, which was verified by qRT-PCR. We predicted 58,209 and 74,469 target genes for those down- and up-regulated miRs respectively through miRDB, miRWalk and miRanda databases. GO terms showed that down- and up-regulated targets were mostly enriched in calcium-dependent cell-cell adhesion via plama membrane cell-adhesion molecules (GO:0,016,338, BP) and cell adhesion (GO:0,007,155, BP), plasma membrane (GO:0,005,886, CC), and metal ion binding (GO:0,046,914, MF) and ATP binding (GO:0,005,524, MF) respectively. Top-20 pathways by KEGG analysis included calcium signaling pathway, cAMP signaling pathway, and ABC transporters, which were closely related to VC.

Conclusion: Our results indicated that those significantly altered miRs, which were packaged in HP-EC-EVs, may play an important role in VC by regulating related pathways. It may provide novel insight into the mechanism of CKD calcification.

Keywords: Extracellular vesicles (EVs); High-throughput sequencing (HTS); Hyperphosphatemia; Vascular calcification.

MeSH terms

  • Calcium / metabolism
  • Cells, Cultured
  • Endothelial Cells / metabolism
  • Extracellular Vesicles* / metabolism
  • Humans
  • Hyperphosphatemia* / genetics
  • Hyperphosphatemia* / metabolism
  • MicroRNAs* / genetics
  • MicroRNAs* / metabolism
  • Muscle, Smooth, Vascular
  • Myocytes, Smooth Muscle / metabolism
  • Renal Insufficiency, Chronic* / genetics
  • Renal Insufficiency, Chronic* / metabolism
  • Sequence Analysis, RNA
  • Vascular Calcification* / genetics
  • Vascular Calcification* / metabolism


  • MicroRNAs
  • Calcium