Transcellular water transport and stability of expression in aquaporin 1-transfected LLC-PK1 cells in the development of a portable bioartificial renal tubule device

Tissue Eng. 2004 May-Jun;10(5-6):711-22. doi: 10.1089/1076327041348383.

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.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Animals
  • Aquaporins / genetics*
  • Aquaporins / metabolism*
  • Bioartificial Organs
  • Biological Transport, Active / physiology
  • Cell Line
  • Cell Membrane Permeability / physiology
  • Feasibility Studies
  • Gene Expression Regulation / physiology
  • Genetic Enhancement / methods
  • Kidney Tubules / cytology*
  • Kidney Tubules / physiology*
  • Kidneys, Artificial*
  • Protein Engineering / methods*
  • Recombinant Proteins / metabolism
  • Swine
  • Tissue Distribution
  • Tissue Engineering / methods
  • Transfection / methods
  • Water / metabolism*
  • Water-Electrolyte Balance / physiology

Substances

  • Aquaporins
  • Recombinant Proteins
  • Water