Low permeabilities of MDCK cell monolayers: a model barrier epithelium

Abstract

Barrier epithelia such as the renal collecting duct (in the absence of antidiuretic hormone) and thick ascending limb, as well as the stomach and mammalian bladder, exhibit extremely low permeabilities to water and small nonelectrolytes. A cell culture model of such epithelia is needed to determine how the structure of barrier apical membranes reduce permeability and how such membranes may be generated and maintained. In the present studies, the transepithelial electrical resistance and isotopic water and urea fluxes were measured for Madin-Darby canine kidney (MDCK) type I and type II cells, as well as type I cells expressing the mucin protein, MUC1, in their apical membranes. Although earlier studies had found the unstirred layer effects too great to permit measurement of transepithelial permeabilities, use of ultrathin semipermeable supports in this study overcame this difficulty. Apical membrane diffusive water permeabilities were 1.8 +/- 0.4 x 10(-4) cm/s and 3.5 +/- 0.5 x 10(-4) cm/s in MDCK type I and type II cells, respectively, at 20 degrees C. Urea permeability in type I cells at the same temperature was 6.0 +/- 0.9 x 10(-6) cm/s. These values resemble those of other barrier epithelial apical membranes, either isolated or in intact epithelia, and the water permeability values are far below those of other epithelial cells in culture. Transfection of MDCK type I cells with the major human urinary epithelial mucin, MUC1, led to abundant expression of the fully glycosylated form of the protein on immunoblots, and flow cytometry revealed that virtually all the cells expressed the protein. However, MUC1 had no effect on water or urea permeabilities. In conclusion, MDCK cells grown on semipermeable supports form a model barrier epithelium. Abundant expression of mucins does not alter the permeability properties of these cells.

Reconstituting the Barrier Properties of a Water-tight Epithelial Membrane by Design of Leaflet-specific Liposomes

“…From Equation 1 we can calculate a single leaflet permeability from the exofacial liposomes and a single leaflet permeability for the cytoplasmic liposomes (both symmetric bilayers) and combine them to arrive at a theoretical permeability for an MDCK apical membrane. Performing this calculation for water permeability yields a value of 4.6 ϫ 10 Ϫ4 cm/s, which is in good agreement with published permeabilities for MDCK type 1 apical membranes, which range from 1.8 ϫ 10 Ϫ4 cm/s to 10 ϫ 10 Ϫ4 cm/s (17,(27)(28)(29). Water Permeability of Modified Exofacial Liposomes-One of the more striking features of the bilayer asymmetry observed in certain barrier membranes is the highly concentrated localization of sphingolipids in the exofacial leaflet (13).…”

“…Therefore, our reconstituted membrane system has a urea permeability approximately 1 order of magnitude lower than that seen for some other barrier membranes. We have, in addition, previously measured the diffusive urea permeability of MDCK cells to be 6.0 ϫ 10 Ϫ6 cm/s (17). This is approximately 1 order of magnitude higher than is seen for other barrier epithelia, and 2 orders of magnitude higher than our model liposomes would predict for this membrane.…”

“…We, and others have previously shown that MDCK type 1 cells grown on permeable supports exhibit the permeability properties of a barrier epithelium (17). Because the lipid structure of the MDCK cell apical membrane is well defined, we aimed to reconstitute the low permeability properties of the MDCK type 1 cell, in an effort to understand the determinants that contribute significantly to this membrane's barrier properties (17).…”

Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia

“…In highly differentiated cultures from normal airways, transepithelial osmotic permeability ranged between 168.6 ± 24.3 µm/s (NaCl) and 220 ± 9.8 µm/s (raffinose). By comparison, these HBE P f T values are more than tenfold higher than those for water flow across relatively water-impermeable epithelia, e.g., MDCK cell cultures and unstimulated vasopressin-responsive epithelia (12)(13)(14)(15)(16). The P f T values for HBE compare favorably with values measured in epithelia that exhibit isosmotic absorption, e.g., gallbladder (20,21) and stimulated vasopressin-responsive epithelia (16).…”

“…For these studies, we interfaced well-dif-Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia ferentiated cultures of normal and CF bronchial epithelia that contain both lumen facing columnar cells and basement membrane facing basal cells with a confocal microscopy-based system that simultaneously measures bulk liquid flow and epithelial cell height during osmotic challenges. Our goals were to (a) measure osmotic permeabilities in normal airway epithelia and to compare these values to a renal epithelial cell line with a reported low water permeability (12)(13)(14)(15)(16); (b) test whether the apical membrane exhibited evidence of water channels and their putative mercury (Hg) sensitivity; and (c) compare P f of normal and CF airway epithelia. Finally, the ability to determine simultaneously transepithelial water flow and cell height during anisosmotic solution exposure permitted independent analyses of the time courses of the changes in cell volume and transepithelial water flow.…”