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Involvement of VKORC1 in the inhibition of calcium oxalate crystal formation in HK-2 cells

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Summary

The vitamin K epoxide reductase complex subunit 1 (VKORC1), the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate (CaOx) crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORC1shRNA-2 as a PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC1 transfection group than in the pFLAG-CMV-7.1 control group (P<0.01). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate (COM) crystal medium for 48 h was 14±4 per field (100×) in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field (100×) in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group (P<0.05). The expression levels of VKORC1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field (100×) in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group and 24±6 per field (100×) in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). These findings suggested that the VKORC1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx.

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Authors and Affiliations

  1. Department of Urology, Shanghai 5th People’s Hospital Affiliated to Fudan University, Shanghai, 200240, China

    Bo Hu  (胡 波), Hao-ran Wu  (吴浩然) & Guo-wei Shi  (施国伟)

  2. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China

    Zhi-yong Ma  (马志永), Zhuan-chang Wu  (武专昌) & Ying-mei Lu  (陆莹梅)

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  1. Bo Hu  (胡 波)
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  2. Hao-ran Wu  (吴浩然)
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  3. Zhi-yong Ma  (马志永)
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  4. Zhuan-chang Wu  (武专昌)
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  5. Ying-mei Lu  (陆莹梅)
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  6. Guo-wei Shi  (施国伟)
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Corresponding author

Correspondence to Guo-wei Shi  (施国伟).

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Both authors contributed equally to this work.

This project was supported by grants from the Scientific Research Starting Foundation for Young Scientist from Shanghai Medical College of Fudan University (No. 11L-33) and the Shanghai Municipal Key Specialist Construction Projects (No. ZK2012A22).

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Hu, B., Wu, Hr., Ma, Zy. et al. Involvement of VKORC1 in the inhibition of calcium oxalate crystal formation in HK-2 cells. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 34, 376–381 (2014). https://doi.org/10.1007/s11596-014-1286-0

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  • DOI: https://doi.org/10.1007/s11596-014-1286-0

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