ABSTRACT
Urinary prothrombin fragment 1 (UPTF1) is a potent inhibitor of urinary stone formation.UPTF1 exerts such inhibitory effect by effective γ-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 VKORC 1 in renal tissues were determined by using immunohistochemistry,immunofluorescence microscopy,Western blotting and SYBR Green Ⅰreal-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 VKORC1was located in the cytoplasm of renal tubular epithelial cells.The expression of VKORC 1 in the urolithiasis group was significantly lower than that in the other two control groups (P＜0.05).Moreover,the 3'- and 5'-UTR sequence of the VKORC 1 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 upstream of 5'-UTR end in the urolithiasis group.It was concluded that the decreased expression of VKORC1 may contribute to the development of calcium oxalate urolithiasis in the kidney.
ABSTRACT
To investigate the exon mutation of vitamin K-dependent gamma-glutamyl carboxylase (GGCX or VKDC) in patients with calcium oxalate urolithasis, renal cortex and peripheral blood sam-ples were obtained from severe hydronephrosis patients (with or without calculi), and renal tumor pa-tients undergoing nephrectomy. GGCX mutations in all 15 exons were examined in 44 patients with calcium oxalate urolithiasis (COU) by polymerase chain reaction (PCR) and denatured high pressure liquid chromatography (DHPLC), and confirmed by sequencing. Mutation was not found in all COU samples compared to the controls. These data demonstrated that functional GGCX mutations in all 15 exons do not occur in most COU patients. It was suggested that there may be no significant association between the low activity and mutation of GGCX in COU.
ABSTRACT
Summary: The expression of calcium epithelium TRPV5, alcium binding protein Calbindin-D28k and Na+/Ca2+ exchanger NCX1 was detected in renal distal convoluted tubule, and their effects on urine calcium reabsorption and the possible pathogenic mechanism in idiopathic hypercalciuria (IH) were investigated. Genetic hypercalciuric stone-forming (GHS) rats were chosen as animal models to study urine calcium reabsorption and IH. The cognate female and male rats that had maximal urine calcium were matched to breed next generation. Twelve GHS rats and 12 normal control (NC) SD rats were selected. Western blot and real time quantitative PCR were used to detect the protein and gene expression of TRPV5, Calbindin-D28k and NCX1 respectively. The expression levels of TRPV5 protein and mRNA in GHS rats were significantly lower than in NC rats (P<0.05). Western blot revealed that the expression levels of Caibindin-D28k in GHS rats and NC rats were 0.49±0.02 and 0.20±0.01 respectively, with the difference being significant between them (P<0.05). By using real time quantitative PCR, it was found that there was no significant difference in Calbindin-28k mRNA expression levels between GHS rats and NC rats (P0.05). There was no significant difference in the NCX1 expression between GHS rats and NC rats (P0.05). It was suggested that TRPV5 and Caibindin-D28k might play an important role in urine calcium reabsorption and IH, but they differently contributed to the pathogenesis: The down-regulation of TRPV5 decreases urine calcium reabsorption, directly leading to loss of the urine calcium and resulting in hypercalciuria, and the increased Calbindin-D28k expression could relieve, neutralize and decrease intracellular Ca2+ concentration to maintain calcium balance. NCX1 is not the key protein in urine calcium reabsorption.