Study On The Damage Of Human Calcium-Oxalate-Nanocrystal On Renal Tubular Epithelial Cells

Objective: Nephrolithiasis is a highly prevalent disease of the urogenital system,Its existence brings great psychological and physiological burden to people.It has been reported that the injury of renal tubular epithelial(HK-2)cells is closely related to the formation of kidney stones.Therefore,we simulated the initial stage of stone formation and verified the mechanism of renal tubular epithelial damage caused by calcium oxalate(Ca Ox)nanocrystals.Methods: The Ca Ox stones were collected from patients with kidney stones undergoing surgery.The composition of Ca Ox stones was identified by Fourier-transform infrared spectroscopy(FT-IR),the relative contents of each component were analyzed by X-ray photoelectron spectroscopy(XPS)and energy-dispersive spectroscopy(EDS),The Ca Ox stones were ground into nanoscale particles by high-energy planetary ball milling.And X-ray diffraction(XRD)is used to determine the crystal structure.Then,the morphology of Ca Ox nanocrystals were observed by transmission electron microscopy(TEM)and scanning electron microscopy(SEM).The particle size of Ca Ox nanocrystals was analyzed by dynamic light scattering(DLS),and the thermal stability of the crystal was determined by Thermal gravimetric analysis(TGA).After that,the potential mechanism of Ca Ox nanocrystals acting on HK-2 cells was studied by cell culture technique,and It was repeatedly verified by Bio-Electron Microscopy,Western Blotting Analysis,Flow cytometry and Autophagic Marker Dye Staining to explore its exact mechanism.Results: The physical and chemical identification results showed that the main component of the calculus is COM,COD,and carbonate apatite,and the atomic components were C(38.49%),O(47.46%),Ca(11.75%),and P(2.12%).Further,the average particle diameters of the nanocrystals were confirmed by DLS analyses to be 339 nm in deionized water(DIW)and 460 nm in Dulbecco's modified Eagle's medium(DMEM)plus 10% fetal bovine serum.Meanwhile,TGA of Ca Ox nanocrystals showed a 10.1 wt% loss below 220 °C due to dehydration of COM and COD,and an additional 15 wt% loss between 400 and 550 °C.Subsequently,a weight loss of about 30 wt% above 550 °C.The results of cell biology experiments showed that Ca Ox nanocrystals present cytotoxicity to HK-2 cells,furthermore,Ca Ox nanocrystals elicit authentic autophagy by enhancing autophagosomes formation.Moreover,crosstalk between autophagy and apoptosis contributes to Ca Ox nanocrystals-Induced HK-2 cell deathConclusion: Ca Ox nanocrystals,isolated and prepared from clinical samples of patients with renal calcium oxalate stones,could significantly reduce HK-2 cell viability in dose and time-dependent manners.Meanwhile,Ca Ox nanocrystals were illustrated to induce complete autophagy flux,whose inhibition by the autophagic specific chemical inhibitor Wort or CQ obviously attenuated Ca Ox nanocrystal-elicited cytotoxicity,strongly suggesting that Ca Ox nanocrystals induced prodeath autophagy.Meanwhile,Ca Ox nanocrystals were also demonstrated to elicit HK-2 cell apoptosis,and the crosstalk exploration revealed that autophagy was an essential signaling pathway participating in apoptosis modulation.Taken together,our findings demonstrate the role of autophagy in Ca Ox nanocrystals triggered cytotoxicity and raise the possibility that autophagy inhibition might effectively reduce Ca Ox nanocrystal-elicited kidney injury,which effort would be meaningful to design a promising strategy for nephrolithiasis prevention and therapy.