Calcifying Nanoparticles Induce Cytotoxicity Mediated by ROS-JNK Signaling Pathways

Urolithiasis. 2019 Apr;47(2):125-135. doi: 10.1007/s00240-018-1048-8. Epub 2018 Mar 6.

Abstract

Calcifying nanoparticles (CNPs) play an important role in kidney stone formation, but the mechanism(s) are unclear. CNPs were isolated and cultured from midstream urine of patients with kidney stones. CNP morphology and characteristics were examined by electron microscopy and electrophoresis analysis. Chemical composition was analyzed using energy-dispersive X-ray microanalysis and Western blotting. Human renal proximal convoluted tubule cell (HK-2) cultures were exposed to CNPs for 0, 12 and 72 h, and production of reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis levels were evaluated. CNPs isolated from patients showed classical morphology, the size range of CNPs were 15-500 nm and negative charge; they were found to contain fetuin-A. Exposure of HK-2 cells to CNPs induced ROS production, decreased mitochondrial membrane potential and decreased cell viability. Transmission electron microscopy showed that CNPs can enter the cell by phagocytosis, and micrographs revealed signs of apoptosis and autophagy. CNPs increased the proportion of apoptotic cells, down-regulated Bcl-2 expression and up-regulated Bax expression. CNPs also up-regulated expression of LC3-B, Beclin-1and p-JNK.CNPs are phagocytosed by HK-2 cells, leading to autophagy, apoptosis and ROS production, in part through activation of JNK signaling pathways. ROS and JNK pathways may contribute to CNP-induced cell injury and kidney stone formation.

Keywords: Calcifying nanoparticles; Cytotoxicity; HK-2 cells; Kidney stone; ROS.

MeSH terms

  • Apoptosis
  • Calcifying Nanoparticles / metabolism*
  • Calcifying Nanoparticles / urine
  • Cell Line
  • Down-Regulation
  • Epithelial Cells / pathology
  • Epithelial Cells / ultrastructure
  • Humans
  • Kidney Calculi / etiology*
  • Kidney Calculi / surgery
  • Kidney Calculi / urine
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / pathology*
  • Kidney Tubules, Proximal / ultrastructure
  • MAP Kinase Signaling System*
  • Membrane Potential, Mitochondrial
  • Microscopy, Electron, Transmission
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Reactive Oxygen Species / metabolism*
  • Up-Regulation
  • bcl-2-Associated X Protein / metabolism

Substances

  • BAX protein, human
  • BCL2 protein, human
  • Calcifying Nanoparticles
  • Proto-Oncogene Proteins c-bcl-2
  • Reactive Oxygen Species
  • bcl-2-Associated X Protein