The Effect Of VKDC/VKOR On Formation Of Calcium Oxalate Calculus And Its Molecular Mechanism

PartⅠActivity and expression of vitamin K dependentgamma-glutamyl carboxylase in patients with calcium oxalateurolithiasisObjectives To explore the activity and expression of vitamin K dependentgamma-glutamyl carboxylase (GGCX or VKDC) in patients with calcium oxalateurolithiasis and evaluate the correlation between the VKDC and the formation of calciumoxalate calculus.Methods Specimens of renal tissues were harvested from urolithic patients, controltissues were obtained from renal tumor patients performing nephrectomy and non-urolithicpatients. The carboxylase activity was detected by the isotope labeled carboxylatic reactionin vitro and immunohistochemical technique was established to study the location ofVKDC in renal tissues. We also detected the differential expression of VKDC in renaltissues in surgical tissue specimens with Western blot.Results VKDC was located in the cytoplasm of renal tubular epithelial cell and theactivity in urolithic group was significantly decreased compared with that of controls(P＜0.05). The expression of protein in urolithic group (27.64±0.29) was weaker than theother two control groups (55.22±0.36 and 53.78±0.33), P＜0.05.Conclusions The activity and expression of VKDC are decreased in renal tissues of patients with calcium oxalate urolithiasis, and VKDC may be involved in the formation ofcalcium oxalate calculus. PartⅡVariation of gamma-glutamyl carboxylase gene3'-untranslated region in patients with urolithiasisObjectives: To get the 3'-untranslated region of gamma-glutamyl carboxylase (GGCX)gene and analyze the relationship between 3'UTR of GGCX and urolithiasis.Methods: Specimens of renal tissues were harvested from urolithic patients, controltissues were obtained from renal tumor patients performing nephrectomy and non-urolithicpatients. The 3'UTR of GGCX sequence was amplified by rapid amplification of cDNAends (RACE). The fragment was purified to construct the corresponding recombinantplasid, subsequently the plasid was sequenced to analyse and BLAST with NCBInucleotide data bank.Results: Although the specific fragments were not quite clear after the first nestedPCR reaction, about 1000 bp fragment was obtained in second nested PCR amplification,and inserted into pGEM-T vector. The recombinant plasmid was transformed into JM 109cells, and then sequenced. We didn't find insertion or deletion in this region.Conclusions: We succeed to clone the 3'UTR sequence of GGCX by RACE. but wecouldn't conclude that there are some relationship between 3'UTR of GGCX andurolithiasis. PartⅢThe expression of vitamin K epoxide reductase complexsubunit 1 (VKORC1) in patients with urolithiasisObjective: To stud the expression of vitamin K epoxide reductase complex subunit 1(VKORC1) in patients with urolithiasis and evaluate the correlation between the quantityof VKORC1 and the formation of calcium oxalate calculus.Methods: Forty-four specimens of renal tissues were harvested from urolithic patients,control tissues were harvested from non-urolithic patients (6 specimens) and renal tumorpatients performing nephrectomy (21 specimens). Immunohistochemical technique wasestablished to study the location of VKORC1 in renal tissues. Western blot technique wasused to detect the differential expression of VKORC1 protein in renal tissues.Results: VKORC1 protein located in the cytoplasm of renal tubular epithelial cell.The protein expression of VKORC1 in urolithic group (122.33±11.34) was weaker than theother two control groups (262.16±7.33 and 286.71±9.82), P＜0.05.Conclusion: The low expression reductase in the calcium oxalate urolithic patientsmay play an important role by vitamin K circle in the formation of calcium oxalateurolithiasis.