Transcellular Water Transport and Stability of Expression in Aquaporin 1-Transfected LLC-PK1Cells in the Development of a Portable Bioartificial Renal Tubule Device

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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-PK1 cells, rat AQP1-transfected cells had high transcellular osmotic water permeability. The expression of rat AQP1 mRNA (ratio of AQP1 to β-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 LLCPK1 cells, the protein was localized mainly to the basolateral side, but also the apical side, of the plasma membrane. Wild-type LLC-PK1 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.