The Research Of Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) In The Kidneys Of Patients With Calcium Oxalate Calculi

PartⅠThe expression of vitamin K epoxide reductase complex subunit 1(VKORC1) in the kidneys of patients with calcium oxalate calculiObjectives To study the expression of vitamin K epoxide reductase complex subunit 1 in the kidneys of patients with calcium oxalate calculi and investigate the relationship between the VKORC1 and the formation of calcium oxalate calculi.Methods The renal cortex samples were obtained from 155 patients undergoing nephrectomy from November 2004 to April 2010 in Tongji Hospital and divided into 3 groups:(1) stone group; (2) control group A (hydronephrosis-without-stone group, HWS); and (3) control group B (normal control group). The localization and expression of VKORC1 in renal tissues were determined by using immunohisto-chemical analysis, immunofluorescence microscopy, western blot and SYBR Green I real-time reverse-transcription PCR.Results VKORC1 were not only found in the stone group but also in the control groups, mainly in the cytoplasm of renal tubular epithelial cells. The expression of VKORC1 proteins in the stone group (0.327±0.055) were weaker than the other 2 control groups (control group A 0.913±0.090, control group B 0.983±0.117, P<0.05). There were no significant differences of the expression of VKORC1 proteins between the control groups (P>0.05). VKORC1 mRNA expression were significantly lower in the stone patients than that in the normal control group A and B by using the real-time reverse-transcription PCR (fold:6.063±5.275,26.324±10.632 and 27.167±14.248, P<0.05). Moreover, the expression in the HWS group (control group A) were similar with the normal control group (control group B) and there were no significant statistical difference (P >0.05).Conclusions The expression of vitamin K epoxide reductase complex subunit 1(VKORC1) are confined mainly to the cytoplasm of renal tubular epithelial cells and lower in the kidney tissues of patients with calcium oxalate calculi. Through vitamin K circle, VKORC1 may be involved in the formation of calcium oxalate calculi. PartⅡThe expression and identification of splicing isoforms of vitamin K epoxide reductase gene in the kidneys of patients with calcium oxalate calculiObjective To analyze the splicing isoforms of vitamin K epoxide reductase complex subunit 1 (VKORC1) in the kidney tissues of patients with calcium oxalate calculi.Methods The renal cortex samples were obtained from 65 patients with calcium oxalate calculi, who undergoing nephrectomy from November 2004 to April 2010 in Tongji Hospital. Application of VKORC1 in kidney by the RT-PCR, then the PCR products were purified, the obtained VKORC1 gene splicing isoforms were analyzed by clone sequencing.Results VKORCl was successfully amplified in kidney tissues of patients with calcium oxalate calculi and get two splice isoforms. With 3% agarose gel electrophoresis showed that PCR amplification products of the two purposes, a treaty band of about 500bp was named V1, the other about 400bp was named V2. After sequencing, the two bands differed 110bp, the remaining sequences were identical.Conclusion VKORC1 splicing isoforms are found in the kidney tissues of patients with calcium oxalate calculi. Part III The amplification and identification of the 3'-untranslated region (UTR) and 5'-untranslated region (UTR) of vitamin K epoxide reductase complex subunit 1 (VKORC1) in the kidney tissues of patients with calcium oxalate calculiObjective To amplify and identify the 3'-untranslated region (UTR) and 5'-untranslated region (UTR) of vitamin K epoxide reductase complex subunit 1 (VKORCl) in the kidney tissues of patients with calcium oxalate calculi. Analyze the relationship between the molecular variation of 3'-UTR and 5'-UTR of VKORC1 and the formation of calcium oxalate calculi.Methods The total RNA isolated from 65 samples of kidney tissues were harvested from urolithic patients. cDNA fragment was synthesized by reversely transcribing total RNA. The rapid amplification of cDNA ends (RACE) were applied to obtain the 3'and 5'-untranslated region (UTR) of VKORC1. The products were purified and sequenced. Comparative and bioinformatic analyses of obtained VKORC1 gene nucleotide sequence were carried out online and analyzed by using the Sequencher software. Sequence comparison were conducted through database searches using the BLAST program.Results The 3'-UTR and 5'-UTR sequence of VKORC1 gene were successfully cloned. 3'RACE and 5'RACE generated a 299-bp fragment and a 422-bp fragment respectively. By alignment and assembling of these sequences, the full-length cDNA sequence of obtained VKORC1 was deduced and confirmed by sequencing. The full-length cDNA of obtained VKORC1 was 1174-bp, and it contained a 492-bp open reading frame (ORF) with a 3'UTR of 279-bp downstream of the stop codon and a 5'UTR of 397-bp upstream of the start codon. Comparison and analysis of the obtained nucleotide sequence, no insertion or deletion was found in the 3'and 5'UTR, however,171 bp new base sequence was discovered in the upstream of 5'UTR end. Conclusion There is no significant correlation between the 3'-untranslated region of VKORC1 and decreasing expression of VKORC1 in the renal tissues of patients with calcium oxalate calculi. Further studies are required to confirm the impacts of the 171bp new base in 5'UTR to the activity of VKORC1.