Hu Bo, Wu Hao-Ran, Ma Zhi-Yong, Wu Zhuan-Chang, Lu Ying-Mei, Shi Guo-Wei
Department of Urology, Shanghai 5th People's Hospital Affiliated to Fudan University, Shanghai, 200240, China.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
J Huazhong Univ Sci Technolog Med Sci. 2014 Jun;34(3):376-381. doi: 10.1007/s11596-014-1286-0. Epub 2014 Jun 18.
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.
维生素K环氧化物还原酶复合体亚基1（VKORC1）是维生素K循环的限速酶，在尿路结石患者的肾脏中显著下调。本研究寻找直接证据来确定VKORC1对草酸钙（CaOx）晶体形成的抑制活性。在VKORC1过表达实验中，将HK - 2细胞用pFLAG - CMV - 7.1 - VKORC1质粒转染作为pFLAG - CMV - 7.1 - VKORC1转染组，或用pFLAG - CMV - 7.1质粒转染作为pFLAG - CMV - 7.1对照组。在VKORC1敲低实验中，将HK - 2细胞用PGPU6/GFP/Neo - VKORC1shRNA - 2转染作为PGPU6/GFP/Neo - VKORC1shRNA - 2转染组，或用PGPU6/GFP/Neo - shRNA - NC质粒转染作为PGPU6/GFP/Neo - shRNA - NC对照组。通过实时定量PCR和蛋白质印迹法检测HK - 2细胞中VKORC1的表达。在激光扫描共聚焦显微镜下观察CaOx晶体形成情况。发现pFLAG - CMV - 7.1 - VKORC1转染组中VKORC1 mRNA和蛋白质的表达水平显著高于pFLAG - CMV - 7.1对照组（P<0.01）。在激光扫描共聚焦显微镜下，在荧光标记的一水草酸钙（COM）晶体培养基中孵育48小时后，pFLAG - CMV - 7.1 - VKORC1转染组HK - 2细胞中每视野（100×）CaOx晶体数量为14±4个，pFLAG - CMV - 7.1对照组为26±5个。与pFLAG - CMV - 7.1对照组相比，pFLAG - CMV - 7.1 - VKORC1转染组中CaOx晶体聚集和形成量显著减少（P<0.05）。PGPU6/GFP/Neo - VKORC1shRNA - 2转染组中VKORC1 mRNA和蛋白质的表达水平显著低于PGPU6/GFP/Neo - shRNA - NC对照组（P<0.05）。在激光扫描共聚焦显微镜下，在荧光标记的COM晶体培养基中孵育后，PGPU6/GFP/Neo - VKORC1shRNA - 2转染组HK - 2细胞中每视野（100×）CaOx晶体数量为65±11个，PGPU6/GFP/Neo - shRNA - NC对照组为24±其6个。与PGPU6/GFP/Neo - shRNA - NC对照组相比，PGPU6/GFP/Neo - VKORC1shRNA - 2转染组中CaOx晶体聚集和形成量显著增加（P<0.05）。这些发现表明VKORC1蛋白可抑制CaOx盐结晶、黏附及聚集。本研究将有助于我们理解结晶与上皮细胞相互作用及CaOx形成的机制。
