Calcifying Nanoparticles Induces Cellapoptosis And Autophagy Mediated By ROS-JNK Signaling Pathways

Objective: It has been confirmed that calcifying nanoparticles(CNPs)play an important role in the formation of kidney stones,but its precise mechanism is still not clear yet.This paper aimed to investigate the mechanism and the regulatory pathway of kidney stones formation induced by CNPs.Methods: CNPs were cultured and isolated from midstream urine of patients with kidney stone.Transmission electron microscope(TEM),scanning electron microscopy(SEM),Electrophoresis & Brownian motion video analysis were used to observe the morphology and electrical characteristics of CNPs;the principal elements and fetuin-A in CNPs were respectively analyzed by Energy-Dispersive X-ray microanalysis(EDX)and Western blotting(WB).CNPs in Randall,s plaque(RP)of patients with renal calculi was detected by TEM.Reactive oxygen species(ROS),mitochondrial membrane potential(MMP)and apoptosis rate were evaluated by flow cytometric analysis.Autophagy and apoptosis of HK-2 cells were detected by TEM,confocal microscopy and flow cytometry.Ultrastructure change of HK-2 cells after intervention were showedby TEM.Western blotting was used to analysis the changes of protein level in cell.Three groups were included: CNPs 0h,CNPs 12 h and CNPs 72 h.Results: 1.CNPs can be cultured from the midstream urine of the patients with calcium oxalate stone,The characteristic of the CNPs is consistent with the classical description by various methods.The particle size of CNPs was about130 nm,and the CNPs was found to have negative potential.CNPs contains fetal globulin-A(Fetuin-A).The morphology of the cultured CNPs was observed in the ultrathin sections was so similarity to the renal papilla calcification.2.The results of CCK-8 assay showed that the cell viability of HK-2 decreased with the time after CNPs intervention.The results of flow cytometry showed that CNPs could induce the production of ROS,and decreased the mitochondrial membrane potential of HK-2.3.We observed that the early stage of CNPs intervention in HK-2 could be autophagy by Ad-mRFP-GFP-LC3 adenovirus transfection.Cell apoptosis was detected by flow cytometry.Cells of ultrathin section transmission electron microscopy images showed that HK-2 by phagocytosis CNPs endocytosis,intracellular CNPs encapsulated in vesicles,mitochondria swelling,and can be observed in autophagy,apoptosis and cell necrosis.4.After CNPs intervention,the expression of Bcl-2 protein was decreased,while the expression of Bax was significantly increased,the expression of LC3-B and Beclin-1 were significantly increased,and the expression of p-JNK/JNK was increased(12h),but p-p38/p38 was not.Conclusion: 1.calcium oxalate kidney stones in the middle of the urine can be cultured in CNPs,midstream urine culture can be used as a method of urinary tract infection CNPs;CNPs can enter into HK-2 cells by endocytosis vesicles,and the negative potential of CNPs may play an important role in endocytosis;2.CNPs is a crystal protein complex,the proliferation of CNP is akind of protein as the carrier of calcium and phosphorus deposition bionic behavior;3.CNPs can induce the production of intracellular ROS by acting on mitochondria;4.Overexpression of ROS can lead to autophagy and apoptosis of HK-2,which can lead to cell necrosis;5.CNPs plays an important role in the formation of renal papilla calcification in calcium oxalate kidney stones,and the persistent calcium and phosphorus deposition of CNPs is the basis of the formation of calcium phosphate core in the plaque;6.ROS-JNK pathway plays a key role in the regulation of CNPs induced HK-2 cell injury.