Decreased Expression of Vitamin K Epoxide Reductase Complex Subunit 1 in Kidney of Patients with Calcium Oxalate Urolithiasis

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.

Cutaneous allergic reaction to subcutaneous vitamin K_(1):A case report and review of literature

BACKGROUND Vitamin K1(phytomenadione)is a fat-soluble naturally occurring vitamin that is widely used to treat certain coagulation disorders.Adverse cutaneous reactions to vitamin K1 can occur;however,owing to its low incidence and considerable variability in presentation and morphology,its diagnosis can be easily overlooked.Managing these reactions may be challenging for patients and clinicians.Therefore,reviewing the adverse cutaneous reactions to vitamin K1 is important.CASE SUMMARY Here we report the case of a 50-year-old woman with no pre-existing hepatic disease who developed a cutaneous allergic reaction to subcutaneous vitamin K1 that presented as localized eczematous plaques at the vitamin K1 injection site.The eruption developed within 5 d of the injection and persisted for 32 mo despite treatment with topical and intralesional steroids.Eczema was diagnosed based on the results of the pathological examination,immunohistochemical staining,and a skin biopsy.The patient was advised to take herbal medicines orally twice daily.After treatment and follow-up,the patient’s eczematous urticarial plaques improved and her condition stabilized.CONCLUSION Here we present the first case of a cutaneous allergic reaction to subcutaneous vitamin K1 that was successfully treated with Chinese medicine.

Involvement of VKORC1 in the Inhibition of Calcium Oxalate Crystal Formation in HK-2 Cells

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

Fabrication of soy film with in-situ mineralized bioactive glass as a functional food for bone health

Delivery of nutrients through the oral route is considered to be the most admissible and preferred path as it follows the same natural process of food consumption in the body.To provide nutrition to the bone for overall good bone health,the present work aimed to fabricate soy films as a functional food for bone nutrition.The film formed was fortified with an in-situ mineralized bioactive glass (BG) network containing essential minerals required for good bone health.Also,vitamin K1 was supplemented into the films.Nutritional analysis depicted that the films are a rich source of protein containing about 67% of protein per 100 g having energy calories of 330 Kcal/100 g and essential micronutrients required for bones.The rheological studies depict viscoelastic hydrogel film-like properties of the functional soy-based film.The tensile strength of the said film is about 3.46 ± 0.17 MPa.The in-situ mineralization of the BG network in the soy protein matrix is responsible for the increased tensile strength and swelling properties of the functional film.The presence of essential oils in the film imparts antimicrobial properties to the film.Hence these soy films can be served as nutritional functional food having antimicrobial activity.