Transcellular Water Transport and Stability of Expression in Aquaporin 1-Transfected LLC-PK1Cells in the Development of a Portable Bioartificial Renal Tubule Device
Autor: | Dennis Brown, Masuo Terashima, Johbu Itoh, Takayuki Tokimasa, Akira Saito, Yuji Fujita, Takatosi Kakuta |
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Rok vydání: | 2004 |
Předmět: |
Cell Membrane Permeability
Swine Biological Transport Active Biology Aquaporins Protein Engineering Transfection Immunofluorescence Cell Line chemistry.chemical_compound medicine Animals Tissue Distribution Transcellular Phenol red Messenger RNA Water transport Bioartificial Organs Tissue Engineering medicine.diagnostic_test urogenital system General Engineering Water Water-Electrolyte Balance Recombinant Proteins Cell biology Blot Genetic Enhancement Kidney Tubules Gene Expression Regulation chemistry Aquaporin 1 Feasibility Studies Kidneys Artificial |
Zdroj: | Tissue Engineering. 10:711-722 |
ISSN: | 1557-8690 1076-3279 |
DOI: | 10.1089/1076327041348383 |
Popis: | 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. |
Databáze: | OpenAIRE |
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