Development of artificial kidney hybridized with human organic anion transporter expressed cells

1.人有机阴离子转运蛋白1（hOAT 1）基底外侧稳定表达细胞系的建立用哺乳动物表达载体将人有机阴离子转运蛋白1（hOAT 1）转染中国仓鼠卵巢细胞系（CHO）、猪肾上皮细胞系（LLC-PK 1）和人胚肾细胞系（HEK）。将转染的HEK细胞从培养皿中分离。转染的CHO和LLC-PK 1细胞具有对氨基马尿酸（PAH）的弱转运活性，PAH是hOAT 1的典型底物。将hOAT 1基因转染到转ts-SV 40-T抗原基因小鼠的肾S2段细胞系中，获得了高转运活性的hOAT 1基因。表达hOAT 1的S2细胞具有较高的PAH转运活性（Km：73.1±7.0μM，Vmax：1736± 19 pmol/mg/min），但不表达单向转运。建立了表达hOAT 1的MDCK细胞系.人有机阴离子转运蛋白1（hOAT 1）稳定表达细胞系的克隆用免疫组织化学方法研究了hOAT 3和hOAT 4的细胞内定位。hOAT 3表达于人肾近曲小管的基底面，hOAT 4表达于人肾近曲小管的管腔面。hOAT 4也具有PAH转运体活性，因此，它是PAH在近曲小管腔膜上的外排转运体的候选者。hOAT 1-hOAT 4共表达细胞系正在建立。

1.Establishment of basolateral human organic anion transporter (hOAT1) stable expressing cell linesBasolateral human organic anion transporter (hOAT1) was transfected with mammalian expression vector into the Chinese hamster ovary cell line (CHO), the swine renal epithelial cell line (LLC-PK1), and the human embryonic kidney cell line (HEK). Transfected HEK cells were detached from dishes. Transfected CHO and LLC-PK1 cells had weak transport activity of p-aminohippurate (PAH), typical substrate of hOAT1. Transfected LLC-PK1 cells expressed unidirectional transport.For high transport activity, hOAT1 gene was transfected in mouse kidney S2 segment cell lines derived from ts-SV40-T antigen gene harboring transgenic mouse. hOAT1 expressing S2 cells has high transport activity of PAH (Km : 73.1±7.0μM, Vmax : 1736±19pmol/mg/min), but they didn't express unidirectional transport.For unidirectional transport characteristics, hOAT1 gene was transfected in MDCK cells, which keep the epithelial polarity. hOAT1 expressing MDCK cells has established.2. Molecular Cloning of luminal basolateral human organic anion transporter (hOAT1) stable expressing cell linesIntracellular localizations of hOAT3 and hOAT4, members of OAT family, were investigated by immunohistochemistry. hOAT3 was expressed on the basal surface of proximal tubule of human kidney, and hOAT4 was expressed on the luminal surface of proximal tubule of human kidney. hOAT4 had also PAH transprt activity, therefore, it is a candidate of efflux transporter of PAH at the luminal membrane of proximal tubule. hOAT1-hOAT4 co-expressing cell line are establishing.