The Effect And Mechanism Of Deacetylase SIRT3 In The Formation Of Renal Calcium Oxalate Stones

Objective The aim of this study was to find changes of deacetylase SIRT3 in renal tubular epithelial cells stimulated by calcium oxalate crystals via animal and cell experiments,and the impact of it on oxidative stress(OS)and apoptosis,which would illustrate the effect and mechanism of deacetylase SIRT3 in the formation of calcium oxalate stones.Methods Calcium oxalate crystal mouse models were constructed,and experimental group and control group were designed,in which Von Kossa staining was used to observe the deposition of black calcium salts in the kidneys.Gene expression profiling of the two group kidney tissues were conducted,with the help of literature review,to find out Sirt3 as the key gene of differential expression and testify it with real-time PCR and Western blot.Immunohistochemistry was used to detect the level of SIRT3,HE stainning and TUNEL were applied to detect apoptosis,and reactive oxygen species(ROS)was detected in the kidney tissues of both groups mice.ELISA analysis was conducted to show levels of creatinine,BUN and NGAL in serum and urine of both groups mice.Calcium-oxalate crystal cell models(TCMK-1)were constructed,in which 0 ?g/ml,10 ?g/ml,100 ?g/ml and 1000 ?g/ml calcium oxalate monohydrate(COM)of different concentrations were added into the medium for 24 hr,48 hr and 72 hr.Flow cytometry was utilised to analyse the apoptosis and necrosis rates of cells.ROS was detected and Western blot was used to find changes of SIRT3 in different set of cells.Plasmid overexpression of SIRT3 experiment in cells was carried out and control cells were designed,in which 1000 ?g/ml COM were added into the medium for 72 hr.ROS was detected and flow cytometry was utilised to analyse the apoptosis and necrosis rates of cells.Results Calcium oxalate crystal mouse model was successfully constructed and renal crystal deposition was observed in all 12 mice which were injected with glyoxylate.The gene expression profiling showed there were up to 2425 differential genes between model mice and the control group,and real-time PCR showed down regulation of Sirt3,Nlrp6 and up regulation of Il 1beta,Nf?b 1,Stat3,Il 6 in calcium oxalate crystal mouse model;at the same time,Sirt3 was found out in the mitochondria related gene expression profiling.Consequent Western blot testified lower expression of protein SIRT3 in the kidneys of model mice compared with the control group.Immunohistochemistry showed lower expression of SIRT3 in the renal tubular epithelial cells of model mice compared with the control group.Injuries and apoptosis changes were found in the cortex and medulla of model mice kidneys in HE stainning.TUNEL showed mean apoptosis cells per vision under high power microscope were more in model mice than the control group,in the renal cortex(45.6±10.2 vs.12.4±1.6,p<0.001)and medulla(16.8±3.4 vs.4.6±1.8,p<0.001)respectively.Western blot testified lower expression of protein SIRT3 in the kidneys of model mice compared with the control group,accompanied by higher expression of apoptosis prompting protein Bax and lower expression of apoptosis inhibiting protein Bcl-2.Significantly higher reactive oxygen species(ROS)in the kidneys of model mice was detected compared with the control group(41530 RFU vs.16440 RFU,p<0.001).Compared with the control group,serum creatinine(80.45±31.73 vs.29.72±16.48 ?mol/L,p<0.05)and urine creatinine(184.00±116.41 vs.61.38±44.07 ?mol/L,p<0.05)were both higher in model mice revealed by renal function tests.Compared with the control group,serum BUN(30.46±9.44 vs.5.89±2.38 mmol/L,p<0.001)and urine BUN(57.65±21.61 vs.37.76±8.07 ?mol/L,p<0.001)were both higher in model mice revealed by renal function tests.Though there was not significant difference in serum NGAL(273.45±17.45 vs.275.92±30.53 mmol/L,p>0.05)between the two groups,higher urine NGAL(239.54±19.42 vs.121.52±34.61 ?mol/L,p<0.001)was foud in the model mice.In the calcium oxalate crystal cell(TCMK-1)model,increase of apoptosis,necrosis and ROS induced by calcium oxalate crystals were concentration and time dependent.Decrease of SIRT3 induced by calcium oxalate crystals was concentration and time dependent too.After plasmid overexpression of SIRT3,apoptosis(8.6±0.2 % vs.5.3±0.4 %,p<0.001),necrosis(18.2±0.6 % vs.14.7±0.8 %,p<0.05)and ROS(1373 RFU vs.530 RFU,p<0.05)were decreased in TCMK-1 cells compared to the control goups.Conclusions SIRT3 is downregulated by calcium oxalate crystals,thus promoting OS,apoptosis and necrosis of renal tubular epithelial cells,which further results in deposition of calcium oxalate crystals in the kidney.