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

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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.