Endosomes from Kidney Collecting Tubule Cells Contain the Vasopressin-sensitive Water Channel

Title: Endosomes from Kidney Collecting Tubule Cells Contain the Vasopressin-sensitive Water Channel
Authors: Verkman, Alan S.; Lencer, Wayne I.; Brown, Dennis; Ausiello, M.D., Dennis A.
Publisher: Nature
Date Published: May 19, 1988
Reference Number: 427
The mechanism by which vasopressin rapidly and dramatically increases the water permeability of target epithelial cell membranes is thought to involve a cycle of exo- and endocytosis during which vesicles carrying 'water channels' are successively inserted into, and removed from the apical plasma membrane of epithelial cells. Clusters of intramembranous particles, visible by freeze-fracture electron microscopy and presumed to represent water channels, appear on apical membranes in parallel with increased transepithelial water flow. In the collecting duct, these clusters are located in clathrin-coated pits which are subsequently internalized. There has been no direct evidence, however, that subcellular membranes in vasopressin-sensitive epithelia contain functional water channels. In this report, we have used fluorophores that are sensitive to volume and do not pass through membranes to label and to measure directly the osmotic water permeability of endocytosed vesicles isolated from renal papilla. We present direct evidence that vasopressin induces the appearance of a population of endocytic vesicles whose limiting membranes contain water channels.

This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Until 1988 there was scientific consensus, though no direct evidence, that the increase in water permeability of the apical membrane of the principal cells of the kidney collecting duct was due to molecular structures called water channels responding to the presence of the antidiuretic hormone, vasopressin (VP).
Research suggested that water-transporting proteins (the water channels) waited in little sacs called vesicles inside the cell just beneath the apex of the cell membrane. When VP was present, the water channel-bearing vesicles traveled to the apex of the membrane and fused with it. When the vesicles fused with the membrane the water channels inserted themselves into the membranes, and acted as channels through which relatively large volumes of water could travel. When VP became absent from the collecting duct cell, the water channel-bearing vesicle, with the water channel inside, would separate from the channel and return to just beneath the membrane.

Verkman, et al., provided direct evidence that the appearance and insertion of the water channels is the direct effect of the presence of VP. Taking a group of rats that had congenital diabetes insipidus (DI), the researchers dehydrated them by restricting them from water for a period of hours. After this period the researchers infused one group of rats with both a special dye and VP, and another group with just the dye. They killed the rats 15 minutes after the infusion and examined their kidney tissue. The dye serves as a measuring device because it travels to and collects in the cavity of the kidney collecting duct. From there it is taken up inside the kidney collecting duct cells by endocytosis, the uptake by a cell of material outside it when the cell membrane enfolds the material - in this case, the dye.

The researchers examined the cell vesicles with the dye entrapped inside to measure their degree of osmotic water permeability. As water moves out of the vesicles in which it was trapped it increases the concentration of the dye. This reduces the intensity of the fluorescence in the dye. The time it takes for the fluorescence intensity to diminish serves as a measure of water permeability. The more quickly the intensity decreases, the greater the cells' water permeability.

The researchers found the tissue with both VP and the dye had a much greater water permeability than the tissue with just the dye. This measure, along with a measure of the energy required to activate water transport across the cell membrane, and the water permeability coefficient, pointed to the existence of VP-induced water channels in the vesicles.

The authors performed control experiments that demonstrated that the appearance of the water channels in the vesicles was a direct effect of VP and not an indirect effect of urine osmolality, the concentration of the dye they used, vascular changes or the structure of the vesicles.