Study Of The Relationship Between Idiopathic Hypocitraturia And Vitamin D Receptor

Objective:(1) To investigate the relationship between FokI vitamin D receptor (VDR), TaqI vitamin D receptor, BsmI vitamin D receptor and ApaI vitamin D receptor polymorphism and idiopathic hypocitraturia respectively, and to evaluate it's clinical significance. (2) To produce and filter an effectual SiRNA to interfere the expression of VDR gene, which can contribute to the follow-up studies of the relationship between VDR and idiopathic hypocitraturia. (3) To investigate the relationship between vitamin D receptor (VDR) and the regulation of expression of Na+/dicarboxylate cotransporter (NaDC1), in order to uncover the pathogenesis of idiopathic hypocitraturia.Method:(1) 81 volunteers were divided into 2 groups:50 volunteers with normal urine citrate (N); 31 volunteers with idiopathic hypocitraturia (L). The polymorphisms of FokI vitamin D receptor, TaqI vitamin D receptor, BsmI vitamin D receptor and ApaI vitamin D receptor were detected using PCR-RFLP technique and correlations were analyzed among these 2 groups each other, respectively. (2) According to the VDR gene sequences displayed in the GeneBank, designed and produced three pairs of SiRNA, then transfected them into three groups of renal proximal convoluted tubules epithelial cells by Lipofectamine 2000, respectively. And then test the expression level of VDR gene before and after the transfection with Real-Time PCR and Western-Blot technique respectively. (3) According to the VDR gene sequences displayed in the GeneBank, designed and produced an effectual SiRNA, then transfected it into the renal proximal convoluted tubules epithelial cells. And test the expression level of NaDC1 before and after the transfection with Real-Time PCR and Western-Blot technique.Result:(1) We found a different distribution (P< 0.05) of FokI VDR, TaqI VDR and BsmI VDR genotypes in L compared with N, but VDR-ApaI is not. The prevalence of ff VDR, TT VDR and bb VDR genotypes in L were significantly higher than in N, and the average urinary 24-h citrate excretion in ff (1.91±1.03mmol/24h) VDR, TT (1.90±0.96mmol/24h) VDR and bb (1.97±0.90mmol/24h) VDR genotypes people were significantly lower than those in FF (2.67±1.02 mmol/24h)/Ff (2.55±0.95 mmol/24h) VDR, Tt (2.58±0.98mmol/24h)/tt (2.72±1.05mmol/24h) VDR and BB (3.41±0.52mmol/24h)/Bb (2.67±1.05mmol/24h) VDR genotypes people (P<0.05) respectively. (2) After transfected the three pairs of SiRNA into VDR gene of the renal proximal convoluted tubules epithelial cells respectively, we found the expression level of VDR gene of the cells were all declined than before in the three groups. And there was most significant difference in the expression level of VDR gene before and after the interference in one of the three groups, P<0.05. But the othe two groups were not as obvious difference as it. (3) After transfected the effectual SiRNA into the renal proximal convoluted tubules epithelial cells to interfere the expression of VDR gene, we found there was significant difference in the expression level of NaDCl before and after the interference, P<0.05. And the expression level of NaDC1 of the cells was declined obviously than before.Conclusion:(1) These results point to a genetic association between FokI/TaqI/BsmI VDR polymorphisms and idiopathic hypocitraturia, not as ApaI VDR. The genotypes ff of FokI VDR, TT of TaqI VDR and bb of BsmI VDR are hoped to become the heredity marks of idiopathic hypocitraturia. (2) The result showed that there was one pair of SiRNA, which was one of the three pairs of SiRNA designed and produced according to the VDR gene sequences displayed in the GeneBank, could be transfected into VDR gene of the renal proximal convoluted tubules epithelial cells to interfere the expression of VDR most effectively. The interference led to the expression level of VDR declined obviously than before, which was benefit to conduct the follow-up studies of the relationship between VDR and idiopathic hypocitraturia. (3) The result point to an obvious correlation between VDR receptor and NaDC1. While VDR express decline, NaDC1 also express decline. VDR may participate in the expression regulation of NaDC1.