| Objective:In the course of establishing rat kidney calculus model by calcified nanoparticles.By measuring the levels of Beclin-1,LC-3 proteins and the number of autophagosomes in kidney tissue also Tamm-Horsfall protein in the urineof differentgroups.Studying pathological changes of renal tissue of different time groups and calcium salt crystal formation.Those provide us the new way to discussing the mechanism of kidney calculus formation induced by CNPs preliminarily.Methods:CNPs were cultured with stones taken from patients with kidney stones and made into CNPs suspension.48 SD rats were randomly divided into experimental group(group A)and blank control group(group B).The rats in the experimental group were injected with CNPs by tail vein and the same amount of 0.9%sterile saline in the control group.The urine of rats was collected after injectionat 3h,6h,12 h,24h,1w,2w,4w and 8w.At the same time,killed the rats and taken the kidney tissue.Measuring the levels of THP in urine by ELISA and the expression ofBeclin-1,LC-3 proteins in the kidney tissues of rats by immunohistochemistry and Western-blotting methods.Through the transmission electron microscopy we can detection the number and morphology of autophagosomes in different time groups.Studying pathological changes of the cells and the formation of the calcium saltscrystal by HE staining.Results:Western-blottingshowed:The expression of Beclin-1,LC-3 in the renal tissue of the experimental group was significantly higher than that of the control group(P<0.05)in the experimental group after injection of CNPs,and the expression of Beclin-1 and LC-3 increased with time,reached the highest value at 24 h,and then decreased gradually with time(P<0.05).Immunohistochemical showed: Beclin-1,LC-3 in the kidney tissue overexpressed in renal tubular epithelial cells,a small amount of expression in the glomerular.The integral optical density(IOD)of the expression of Beclin-1 andLC-3 in the renal tissue of the experimental group after injection of CNPs was significantly higher than that of the control group(P<0.05).The expression of Beclin-1 and LC-3 in the experimental group increased with time and reached the highest value at 24h(Beclin-1:15.33±0.42/Hp;LC-3:9.33±0.12/Hp),andgradually weakened with time(P<0.05).The results of transmission electron microscopy showed:Autophagosomes was mainly located in renal tubular epithelial cells,the phagocytosed CNPs were observed in the autophagosome in the experimental group.Compared with the number of autophagosomes(1.17±0.35/Hp)in group 3h,the number of autophagosomes before 24 h group(1.47±0.31/Hp)increased slowly(P>0.05),after the 24 h group(2.83±0.32/Hp)increased significantly.During the observation period,the number of autophagosomes(4.70±0.51/Hp)reachedthe max(P<0.05)at 8th weekend,while the control group had a small amount of autophagosomes formation.The correlation between thenumber of autophagosomes andcalcium salt crystals score byPearson correlation,the correlation coefficientr=0.902(P<0.05).ELISA:Compared with the control group,the levels of THP in the urine of the experimental group werehigher than those in the control group significantly(P<0.05).Compared with the THP content in the experimental group at 3h(165.89±2.23pg/mL),theTHP before 24h(166.03±3.02pg/mL)increased slowly and no statistically significant(P>0.05).After 24 h,the THP content increased significantly.During the observation period,the content of 8th THP(177.11±1.76pg/mL)reached the peak(P<0.05).At last we analyzed the correlation coefficient between the THP content and the Pearson correlation of the calcium salt crystals,the correlation coefficient r=0.843(P<0.05).Finally HE staining showed:The edema of renal tubular epithelial cells in the experimental group increased with the prolongation of time.At the time of 24 h,the epithelium was vacuolated,and some of the kidney was observed at 1w to 8w tubular epithelial cells were granule-like degeneration,calcium salt crystals formed in the renal tubular lumen.But there was no obvious pathological change in glomeruli and no clear pathological change in the control group.Compared with the 6h group of calcium salt crystals(1score),the calcium salt crystals score(2score)increased slowlyin the experimental group before 1w.After 2w,the calcium salt crystals increased(3.0score).The highest score(6.7score)(P<0.05)at 8th weekend during observation period.Conclusion:Calcified nanoparticles can damage the renal tubular epithelial cells,so that cells have significant acute inflammatory changes,accompanied by a sharp increase in autophagy,to the late with the cell inflammatory response gradually reduced,the corresponding level of autophagy protein weakened,calcium salt crystals formed in renal tubular lumen.Autophagy and renal tubular epithelial cell injury in parallel.The autophagic activity of the calcified nanoparticles was induced by calcified nanoparticles,and the number of autophagosomes increased gradually.The autophagosomes were positively correlated with the formation of calcium crystals.The content of Tamm-Horsfall protein in the urine of the rats after infected with calcified nanoparticles gradually increased,and the calcium salt crystal score was positively correlated with the content of Tamm-Horsfall protein. |
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