Water does not flow across the tight junctions of MDCK cell epithelium

Abstract

Although it has been known for decades that the tight junctions of fluid-transporting epithelia are leaky to ions, it has not been possible to determine directly whether significant transjunctional water movement also occurs. An optical microscopic technique was developed for the direct visualization of the flow velocity profiles within the lateral intercellular spaces of a fluid-absorptive, cultured renal epithelium (MDCK) and used to determine the velocity of the fluid flow across the tight junction. The flow velocity within the lateral intercellular spaces fell to near zero adjacent to the tight junction, showing that significant transjunctional flow did not occur, even when transepithelial fluid movement was augmented by imposition of osmotic gradients.

Figure 1

(Upper) A confocal optical section approximately halfway through the MDCK cell epithelium showing the LIS filled with 70,000 M_r fluorescein dextran before (Left) and after (Right) digital deconvolution. Scale bar indicates 2 μm. (Lower) Mean ± SEM width of uniform regions of the LIS of seven MDCK cells are shown as a function of the focal distance from the TJ. Optical sections, obtained at 0.32-μm focus intervals, were digitally deblurred (Cell-Scan) by using an experimentally determined point-spread function as described in Materials and Methods.

Figure 2

Intensity (mean ± SEM) of the fluorescence in digitized confocal images of 70,000 M_r fluorescein dextran along the length of the LIS of MDCK cells is expressed as the logarithm of concentration multiplied by the dextran diffusion coefficient in the LIS. All intensities were corrected for differences in gain of the photomultiplier tube in the confocal microscope as well as for variations in the intensity of the laser light used for excitation. (a) The profile obtained under control conditions (n = 51) during perfusion of bicarbonate-buffered Ringer’s solution at 37°C. (b) The effect of a 28 mOsm osmotic gradient (n = 7) created by adding 28 mM PEG (3,000 M_r) to the apical perfusate. (c) The intensity profile obtained when 50 mM PEG (n = 8) was added to the apical perfusate. (d) The effect of 50 mM PEG (n = 8) in the basal perfusate.

Figure 3

The rate of volume flow along the LIS (mean ± SEM) calculated from Figs. 1 and 2 is shown. Absorptive flows are denoted as positive and secretory flows as negative. (a) The isotonic condition. (b) The flow with 28 mM PEG in the apical perfusate. (c) The flow with 50 mM PEG in the apical perfusate. (d) The flow with 50 mM PEG in the basal perfusate. The predicted values of volume flow at the TJ (mean ± SEM from Table 1) are indicated as the data point at zero on the x axis.

Figure 4

(a) The fluorescence intensity profile for BCECF in the LIS in the absence of an imposed osmotic gradient (n = 20). (b) The mean ± SEM volume flow rate in the LIS when the apical perfusate was 50 mOsm hyperosmotic and the epithelium had been treated with 10 μM Sp-cAMPS to increase the permeability of the TJ (n = 14). Note that the ordinate scale differs from that in Fig. 3.