title: Transcellular water transport and stability of expression in aquaporin 1-transfected LLC-PK1 cells in the development of a portable bioartificial renal tubule device.
abstract: We investigated a portable bioartificial renal tubule device (BRTD) consisting of renal tubule cells and hollow fibers, to improve the quality of life of patients. It is necessary for a BRTD system to be compact. A compact portable BRTB requires transfection of an appropriate water channel or electrical pump genes in tubular epithelial cells, which should be based on physiological similarities to human kidney function. LLC-PK(1) cells, into which rat kidney aquaporin 1 (AQP1) cDNA was stably transfected, were evaluated for water transport ability. The expression and localization of water AQP1 were examined by Western blotting, RT-PCR, and immunofluorescence. To measure transcellular water permeation, a simple method was applied, using phenol red as a cell-impermeant marker of concentration. In contrast to wild-type LLC-PK(1) cells, rat AQP1-transfected cells had high transcellular osmotic water permeability. The expression of rat AQP1 mRNA (ratio of AQP1 to beta-actin mRNA) and protein bands (a 28-kDa band and a broad, 35- to 45-kDa band) was confirmed to be stably maintained until a population doubling level of 24. In AQP1-transfected LLCPK(1) cells, the protein was localized mainly to the basolateral side, but also the apical side, of the plasma membrane. Wild-type LLC-PK(1) cells were not stained at the plasma membrane. It is possible that enough AQP1-transfected tubule epithelial cells were supplied for a bioartificial renal tubule device.
authors.affiliation: Division of Nephrology and Metabolism, Department of Medicine, Tokai University, Institute of Medical Sciences, Kanagawa, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Fujita
authors.forename: Masuo
authors.initials: M
authors.lastname: Terashima
authors.forename: Takatosi
authors.initials: T
authors.lastname: Kakuta
authors.forename: Johbu
authors.initials: J
authors.lastname: Itoh
authors.forename: Takayuki
authors.initials: T
authors.lastname: Tokimasa
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.forename: Akira
authors.initials: A
authors.lastname: Saito
mesh_terms: D000818:Animals; D020346:Aquaporins / Q000235:genetics* / Q000378:metabolism*; D021522:Bioartificial Organs; D001693:Biological Transport, Active / Q000502:physiology; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology; D005240:Feasibility Studies; D005786:Gene Expression Regulation / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D007684:Kidney Tubules / Q000166:cytology* / Q000502:physiology*; D007688:Kidneys, Artificial; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution; D023822:Tissue Engineering / Q000379:methods; D014162:Transfection / Q000379:methods; D014867:Water / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000502:physiology
chemical_list: D020346:Aquaporins; D011994:Recombinant Proteins; D014867:Water