Calcifying nanoparticles induce cytotoxicity mediated by ROS-JNK signaling pathways.

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.

Nanoparticle Tracking Analysis for the Enumeration and Characterization of Mineralo-Organic Nanoparticles in Feline Urine.

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.