Urinary prothrombin fragment 1 （UPTFl） is a potent inhibitor of urinary stone formation. UPTF1 exerts such inhibitory effect by effective 7-carboxylation in which vitamin K epoxide reductase complex subunit 1 （VKO...Urinary prothrombin fragment 1 （UPTFl） is a potent inhibitor of urinary stone formation. UPTF1 exerts such inhibitory effect by effective 7-carboxylation in which vitamin K epoxide reductase complex subunit 1 （VKORC1）, the rate-limiting enzyme, is involved. This study examined the correlation between VKORC1 expression and calcium oxalate urolithiasis. The renal cortex samples were obtained from patients undergoing nephrectomy and then divided into 3 groups： urolithiasis group, control group A [hydronephrosis-without-stone （HWS） group], control group B （normal control group）, The localization and expression of VKORC1 in renal tissues were determined by using immunohistochemistry, immunofluorescence microscopy, Western blotting and SYBR Green I real-time reverse-transcription PCR. The rapid amplification of cDNA ends （RACE） were conducted to obtain the 3＇- and 5＇-untranslated region （UTR） of VKORC1. The results showed that VKORC1 was located in the cytoplasm of renal tubular epithelial cells. The expression of VKORC1 in the uro- lithiasis group was significantly lower than that in the other two control groups （P〈0.05）. Moreover, the 3＇- and 5＇-UTR sequence of the VKORC1 gene was successfully cloned. No insertion or deletion was found in the 3＇- and 5＇-UTR. However, a 171-bp new base sequence was discovered in the up- stream of 5＇-UTR end in the urolithiasis group. It was concluded that the decreased expression of VKORC 1 may contribute to the development of calcium oxalate urolithiasis in the kidney.展开更多
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 fo...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-VKORClshRNA-2 as a PGPU6/GFP/Neo-VKORClshRNA-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-VKORC 1 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-VKORC 1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group （P〈0.05）. The expression levels of VKORC 1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC 1 shRNA-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 （100x） in the PGPU6/GFP/Neo-VKORC 1 shRNA-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-VKORClshRNA-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 VKORC 1 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. Key words： calcium oxalate crystals; kidney stone; vitamin K epoxide reduetase complex subunit 1; laser-scanning confocal microscopy展开更多
基金supported by a grant from the National Natural Science Foundation of China(No.30901482)
文摘Urinary prothrombin fragment 1 （UPTFl） is a potent inhibitor of urinary stone formation. UPTF1 exerts such inhibitory effect by effective 7-carboxylation in which vitamin K epoxide reductase complex subunit 1 （VKORC1）, the rate-limiting enzyme, is involved. This study examined the correlation between VKORC1 expression and calcium oxalate urolithiasis. The renal cortex samples were obtained from patients undergoing nephrectomy and then divided into 3 groups： urolithiasis group, control group A [hydronephrosis-without-stone （HWS） group], control group B （normal control group）, The localization and expression of VKORC1 in renal tissues were determined by using immunohistochemistry, immunofluorescence microscopy, Western blotting and SYBR Green I real-time reverse-transcription PCR. The rapid amplification of cDNA ends （RACE） were conducted to obtain the 3＇- and 5＇-untranslated region （UTR） of VKORC1. The results showed that VKORC1 was located in the cytoplasm of renal tubular epithelial cells. The expression of VKORC1 in the uro- lithiasis group was significantly lower than that in the other two control groups （P〈0.05）. Moreover, the 3＇- and 5＇-UTR sequence of the VKORC1 gene was successfully cloned. No insertion or deletion was found in the 3＇- and 5＇-UTR. However, a 171-bp new base sequence was discovered in the up- stream of 5＇-UTR end in the urolithiasis group. It was concluded that the decreased expression of VKORC 1 may contribute to the development of calcium oxalate urolithiasis in the kidney.
基金supported by grants from the Scientific Research Starting Foundation for Young Scientist from ShanghaiMedical College of Fudan University(No.11L-33)theShanghai Municipal Key Specialist Construction Projects(No.ZK2012A22)
文摘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-VKORClshRNA-2 as a PGPU6/GFP/Neo-VKORClshRNA-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-VKORC 1 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-VKORC 1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group （P〈0.05）. The expression levels of VKORC 1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC 1 shRNA-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 （100x） in the PGPU6/GFP/Neo-VKORC 1 shRNA-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-VKORClshRNA-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 VKORC 1 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. Key words： calcium oxalate crystals; kidney stone; vitamin K epoxide reduetase complex subunit 1; laser-scanning confocal microscopy
