The Effects Of Human SLC2A9Gene Overexpress On Uric Acid Transport In HK-2Cells

Background: Uric acid nephrolithiasis is common types of urolithiasis, accounts for10%to40%of all kidney stones. Three abnormalities have been strongly associatedwith uric acid kidney stones: persistently low urine pH, hyperuricosuria and low urinevolume. Hyperuricosuria is the most important pathogenic mechanism leading to uricacid stone formation. Urate is excreted by renal proximal tubules (two-third of dailyturnover) and by gastrointestinal tract (one-third of daily turnover). Almast all serumurate is initially filtered by the renal glomeruli but about90%is reabsorbed into theblood by proximal tubule transporters. Recently, a rapid series of studies have foundsignificant correlations between mutations in SLC2A9gene, which codes for GLUT9protein, and uric acid transport.And GLUT9was shown to be a hige-affinity/higr-capacity uric acid. There is strong evidence that SLC2A9gene can mediate rapidurate fluxes, which plays important role in transport urate on kindey. Thus, we plan toconstruct a recombinant lentiviral vector which contains SLC2A9gene. Afterconstructing and packaging lentivirus successfully, we use them to infect HK-2cells,observe the effects of human SLC2A9gene overexpress on uric acid transport inHK-2cells.Objective: To construct lentiviral vector over expressing human SLC2A9gene，andto investigate its effect on uric acid transport function of HK-2cells.Methods: Human SLC2A9gene amplification was used by polymerase chainreaction. Gene amplification products were inserted in the lentiviral vector pLEX, andconstructed lentiviral vector pLEX-SLC2A9. Constructed vector was verified by PCRanalysis and DNA sequencing. Lentiviral vector pLEX-SLC2A9and virus packagingplasmids were cotransfected into293T cells, and the transfected into human kidneyHK-2cells. PCR and western blot was then used to determine the transfecting efficiency of SLC2A9gene, and the effect of SLC2A9gene over expressing on uricacid transport in HK-2cells was observed.Results: PCR analysis and DNA sequencing showed that recombinant lentivirusplasmids pLEX-SLC2A9were construted successfully. In transfected HK-2cells,SLC2A9gene mRNA and protein was over-expressed. Compare to normal HK-2cells,transfected cells have stronger uric acid transport activity.Conclusion: Overexpression of SLC2A9gene can significantly increase the uric acidtransport function of HK-2cells.