Na+ transport and impedance properties of cultured renal (A6 and 2F3) epithelia

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Abstract
Previous impedance analysis studies of intact epithelia have been complicated by the presence of connective tissue or smooth muscle. We now report the first application of this method to cultured epithelial monolayers. Impedance analysis was used as a nondestructive method for deducing quantitative morphometric parameters for epithelia grown from the renal cell line A6, and its subclonal cell line 2F3. The subclonal 2F3 cell line was chosen for comparison to A6 because of its inherently higher Na+ transport rate. In agreement with previous results, 2F3 epithelia showed significantly higher amiloride-sensitive short-circuit currents (I sc) than A6 epithelia (44±2 and 27±2 μA/cm2, respectively). However, transepithelial conductances (G T) were similar for the two epithelia (0.62±0.04 mS/cm2 for 2F3 and 0.57±0.04 mS/cm2 for A6) because of reciprocal differences in cellular (G c) and paracellular (G j) conductances. Significantly lower G j and higher G c values were observed for 2F3 epithelia than A6 (G j = 0.23±0.02 and 0.33 ± 0.04 mS/ cm2 and G c = 0.39±0.16 and 0.26±0.10 mS/cm2, respectively). Nonetheless, the cellular driving force for Na+ transport (E c) and the amount of transcellular Na+ current under open-circuit conditions (I c) were similar for the two epithelia. Three different morphologically-based equivalent circuit models were derived to assess epithelial impedance properties: a distributed model which takes into account the resistance of the lateral intercellular space and two models (the “dual-layer” and “access resistance” models), which corrected for impedance of small fluid-filled projections of the basal membrane into the underlying filter support. Although the data could be fitted by the distributed model, the estimated value for the ratio of apical to basolateral membrane resistances was unreasonably large. In contrast, the other models provided statistically superior fits and reasonable estimates of the membrane resistance ratio. The dual-layer model and access resistance models also provided similar estimates of apical and basolateral membrane conductances and capacitances. In addition, both models provided new information concerning the conductance and area of the basolateral protrusions. Estimates of the apical membrane conductance were significantly higher for 2F3 (0.79±0.23 mS/cm2) than A6 epithelia (0.37±0.07 mS/cm2), but no significant difference could be detected for apical membrane capacitances (1.4±0.04 and 1.2±0.1 μF/cm2 for 2F3 and A6, respectively) or basolateral membrane conductances (3.48±1.67 and 2.95±0.40 mS/cm2). The similar basolateral membrane properties for the two epithelia may be explained by their comparable transcellular Na+ currents under open-circuit conditions. We conclude that impedance analysis can be a highly useful and noninvasive method for deducing the membrane properties of cultured epithelial monolayers. The difference between A6 and 2F3 epithelia with respect to apical membrane Na+ conductance and paracellular conductance may provide a useful tool for assessing the regulation of Na+ transport and tight junctional proteins.