Apical membrane permeability of MDCK cells

Area of Science:

• Cell Biology
• Biophysics
• Renal Physiology

Background:

• Madin-Darby canine kidney (MDCK) C12 cells are a widely used model for epithelial research.
• Understanding apical membrane permeability is crucial for epithelial transport studies.
• Previous methods for measuring membrane permeability often used isolated vesicles, not intact cells.

Purpose of the Study:

• To determine the osmotic water permeability (Pf) and nonelectrolyte permeability of the apical membrane of MDCK C12 cell cysts.
• To develop and validate a novel method for measuring membrane permeability in intact epithelial cells.
• To investigate the influence of cell number and specific substances on membrane permeability.

Main Methods:

• Cultured MDCK C12 cells as apical-out cysts, with the apical membrane exposed to the medium.
• Applied instantaneous osmotic challenges to cysts and recorded volume changes via automated image analysis.
• Calculated Pf and solute permeabilities from volume change rates and fitted data to determine kinetic parameters.

Main Results:

• A precise average Pf of 2.4 +/- 0.1 micron/s was determined for the MDCK apical membrane.
• Amphotericin B increased Pf, while forskolin had no significant effect.
• Urea permeability was negligible, but acetamide and formamide permeabilities correlated with their oil-water partition coefficients.
• Paracellular water flow was found to be insignificant in multi-cell cysts.

Conclusions:

• The apical-out MDCK cyst model combined with automated image analysis provides a highly precise method for determining epithelial apical membrane permeability.
• The measured Pf and solute permeabilities are consistent with those of water-tight epithelial apical membranes.
• This technique offers a valuable tool for investigating epithelial transport mechanisms in a more physiologically relevant context.