Gene cloning,subcellular localization and expression in kidney tissue of human ASBT protein

AIM:To compare the cDNA sequences of ASBT (apical Na+/bile acid cotransporter) gene cloned from human kidney and intestine tissues, and to determine subcellular localization of ASBT in renal tubular epithelial cells and expression site of ASBT in human kidney tissue.METHODS:The total RNA was extracted from human kidney and intestine tissues. The full-length cDNA gene of ASBT was amplified by RT-PCR technique using primers with 8-peptide FLAG tag, sequenced and inserted into eukaryotic expression vector to construct ASBT protein expression vector, which was then transfected into swine renal tubular epithelial cell line LLC-PK1. The subcellular localization of ASBT protein was determined by immunofluorescence staining and laser confocal microscope. Immunohistochemical analysis was used to observe expression position of ASBT in kidney tissue.RESULTS:The results revealed that the sequence of kidney ASBT cDNA gene was identical to that of human intestine ASBT gene. Western blotting analysis indicated that ASBT protein was correctly expressed in LLC-PK1 cells. Confocal scanning analysis showed that ASBT protein was mainly localized at the cellular plasma membrane, consistent with the predicting result obtained by bioinformatics. The results of immunohistochemistry revealed that ASBT was expressed at brush border membrane of proximal renal tubular cells, but not expressed in distal tubule and renal interstitium.CONCLUSION:The gene sequence of kidney ASBT is the same as that of intestine ASBT and ASBT protein is expressed at the lumen (apical) membrane of proximal renal tubular epithelial cells.