Studies On The Role And Mechanism Of VKORC1 In CaOx Formation

[Objective]:We detected the effects of various doses of fluorescence-labeled COM crystals on HK-2 cells so as to find the appropriate dose of CaOx, which eliminates its influence on the subsequent experiment. Through overexpression VKORC1, we observed VKORC1 protein expression on the influence of adhesion and forming ability of CaOx. We examined knockdown VKORC1 protein on the effect of the ability of CaOx crystals formation and inferred VKORC1 protein on the effect of the formation of CaOx. It can provide a new direction for further research and new thought on the formation of CaOx.[Methods]:HK-2 cells were seeded on 6-well culture plates and the cells were incubated with fluorescence-labeled COM crystals mixed with culture medium for 48h. The level of apoptosis in the cells was assayed using an Annexin V-FITC Apoptosis Detection Kit. HK-2 cells were transfected with either pFLAG-CMV-7.1-VKORC1 or pFLAG-CMV-7.1 vector.Through overexpression VKORC1, we used RT-PCR detection mRNA and westblot detection protein level and confocal laser scanning microscope observed the difference of crystal formation in two different groups (pFLAG-CMV-7.1-VKORC1 as a positive control and pFLAG-CMV-7.1 as a negative control). HK-2 cells were transfected with either pGPU6/GFP/Neo-VKORC1 shRNAs or pGPU6/GFP/Neo-shRNA-NC vector. We used RT-PCR detection mRNA at 24,48,72h and screened the best effect plasmid. We acquired the stabilized expressed cell lines, then westblot detection protein level and confocal laser scanning microscope observed the difference of crystal formation in two different groups (pGPU6/GFP/Neo-VKORC1shRNA2 pGPU6/GFP/Neo-shRNA-NC).[Results]:We took different dozes of fluorescence-labeled COM crystal:50ug/ml, 100ug/ml,400ug/ml,800ug/ml. Percentage of total cell apoptosis= [(number of both apoptotic and necrotic cells/number of all cells)* 100%]:50ug/ml group was 6.30±0.25;100ug/ml group was 7.0±0.64;400ug/ml group was 10.7±0.44; 800ug/ml group was 17.2±0.29; mock group was 5.8±0.4.50ug/ml group and 100ug/ml group shown no statistical significance compared to mock group (P>0.05),whereas 400ug /ml,800ug/ml was statistically significant (P<0.05). HK-2 cells were respectively transfected with pFLAG-CMV-7.1-VKORC1 or pFLAG-CMV-7.1 vector, and we used RT-PCR detection mRNA at 24,48 h. We found that mRNA expression in pFLAG-CMV-7.1-VKORC1 group was significant difference compared with pFLAG-CMV-7.1 group (P<0.001). Western blotting analysis shown that protein expression in experiment group was higher than the control group. HK-2 cells were transfected with either pFLAG-CMV-7.1-VKORC1 or pFLAG-CMV-7.1 plasmid, and mixed with fluorescence-labeled COM crystal medium culture for 48 h. The number of calcium oxalate crystal in pCMV-7.1-VKORC1 group is 14±4 per field(x100) under laser-scanning confocal microscopy, whereas the number in the control group is 26±4 per field(x100). The experiment group was statistically significant compared to control group (P<0.05). HK-2 cells were respectively transfected pGPU6/GFP/Neo-VKORC1 shRNAs and pGPU6/GFP/Neo-shRNA-NC plasmids, and we used RT-PCR detection mRNA at 24,48,72 h. We found that pGPU6/GFP/Neo-VKORC1-shRNA2 obviously inhibited VKORC1 mRNA expression. We acquired the stabilized pGPU6/GFP/Neo-VKORC1-shRNA2 and pGPU6/GFP/Neo-shRNA-NC cell lines. The number of calcium oxalate crystal in pGPU6/GFP/Neo-VKORC1-shRNA2 group is 65±11 per field(x100) under laser-scanning confocal microscopy, whereas the number in the pGPU6/GFP/Neo-shRNA-NC group is 24±8 per field(x100). The pGPU6/GFP/Neo-VKORC1-shRNA2 group was statistically significant compared to pGPU6/GFP/Neo-shRNA-NC group (P<0.05)[Conclusions]:Low concentration of calcium oxalate crystal on cell apoptosis or death shown no obvious difference compared with the control group. More than 400 ug/ml calcium oxalate salt crystals will produce damage to cells.2. Overexpression and knockdown VKORC1 expression, we found that VKORC1 protein inhibited calcium oxalate salt crystallization adhesion and aggregation. This research better understand the mechanism of the interaction of crystallization and epithelial cells and the formation of CaOx, which provides a new method and insight in stone formation mechanism.