Association Between Idiopathic Hypocitraturia In The Yunnan Bai Population And Vitamin D Receptor Gene Polymorphisms And The Expression Of VDR And NaDC1 In HK-2 Cells

Objective(s):To investigate the association between idiopathic hypocitraturia in Yunnan bai population and rs7975232、rs2228570、rs731236 and rs1544410 vitamin D receptor gene polymorphisms and the expression of vitamin D receptor (VDR) and sodium-dicarboxylate cotransporter protein 1 (NaDCl) in human renal proximal tubule epithelial cell (HK-2 cells).Methods:Enroll 500 cases of patients of Yunnan bai population with urinary calculi diagnosed by urinary CT plain scan, kub before operation and intranperative record about lithotripsy and 200 cases of controls of Yunnan bai population with none urinary calculi diagnosed by urinary tract CT plain scan under physical examination and take their fasting venous blood specimens and urine specimens from October 2013 to December 2015. Enroll 200 idiopathic hypocitraturia patients of Yunnan bai population and 120 controls of Yunnan bai population with normal urinary citrate, detect the rs7975232、rs2228570、rs731236 and rs1544410 vitamin D receptor gene polymorphisms by Sanger sequence and analysis the association between the four sites talk above and idiopathic hypocitraturia in Yunnan bai population. Culture HK-2 cells under the cultivation condition of HK-2 cells. The apoptosis of HK-2 cells cultured was detected by Flow Cytometry, and the expression of VDR and NaDCl in HK-2 cells was detected by immunocytochemical SP method.Results:The mean urinary citrate content of 200 idiopathic hypocitraturia patients of Yunnan bai population is (239.58±38.66)mg/24h and that of 120 controls is (593.65±130.33)mg/24h, the differences of which has the statistical significance(P<0.05). The differences of the practical frequency and expected frequency of the two SNP sites has the statistical significance in the hard-weinberg equilibrium(P>0.05).Under the comparation of patients and controls, each genotype frequency differences of the VDR gene SNP rs7975232 and rs2228570 has the statistical significance (P< 0.05) and the rs7975232 genotype AA frequency of patients is more than that of controls, so as the rs2228570 genotype TT. The urinary citrate content of rs7975232 genotype AA which is (365.71±195.23) mg/24h,is significantly lower than that of genotype CC which is [(507.67±197.42) mg/24h] (t=4.366, P<0.05)and genotype CA which is [(485.55±180.15) mg/24h] (t=3.963, P<0.05), and the differences have the statistical significance. The urinary citrate content of rs2228570 genotype TT which is (363.92±185.43) mg/24h, is significantly lower than that of genotype CC which is [(506.47±198.02) mg/24h] (t=6.276, P<0.05)and genotype CT which is [(485.95±190.95) mg/24h] (t=4.709, P<0.05), and the differences have the statistical significance. The culture of HK-2 cells:The state of HK-2 cells was recorded by taking pictures; Flow cytometry: HK-2 cells apoptosis rate of flow tube number 1-4 were respectively 6.87%,6.80%, 8.31% and 4.274%; Immunocytochemistry:The expression of NaDCl in HK-2 cells: the cell membrane of HK-2 cells and NaDC1 positive control (human embryonic kidney 293 cells, HEK293 cells) were stained. While cell membrane of NaDCl negative control was not stained. The expression of VDR in HK-2 cells:the nuclear membrane of HK-2 cells and VDR positive control (human lung cancer cell line A549 cells) were stained. While nuclear membrane of VDR negative control was not stained.Conclusion(s):Idiopathic hypocitraturia in Yunnan bai population has the association with rs7975232 and rs2228570 vitamin D receptor gene polymorphisms, and the genotype AA of VDR gene SNP rs7975232 and the genotype TT of VDR gene SNP rs2228570 may be the candidate gene of idiopathic hypocitraturia in Yunnan bai population. The apoptosis rate of the HK-2 cells cultured was relatively low, and the cells were in good condition. VDR and NaDC1 were respectively expressed in the nuclear membrane and cell membrane of HK-2 cells. Both VDR and NaDC1 are associated with the occurrence and development of hypocitraturia. We proved the expression of VDR and NaDC1 in HK-2 cells and suggested that VDR may be involved in regulating the activity or expression of NaDC1. The experiment laid the foundation for the next step that we will detecte the expression of NaDC1 by interfering the expression of VDR and study the etiology of hypocitraturia.