The wrenches and gaskets of water recycling

Our bodies consist largely of water. Every day we lose a small proportion of it that we replenish through drinking and eating. But at any given time, most of the water in our bodies is obtained by recycling what is already there. A number of diseases are marked by insufficient or excessive water absorption, making the details of the recycling system of intense interest among clinicians and drug developers. Now the lab of Enno Klussmann and Walter Rosenthal at the MDC has helped clarify an important step in the signaling system that regulates the reuptake of water. The group accomplished this by developing a new method for identifying molecular components of the system, in collaboration with Jens von Kries and his colleagues in the Screening Unit of the Leibniz Institute for Molecular Pharmacology (FMP). The new study, published in the April 30 issue of the Journal of the American Society of Nephrology, identifies new molecules that block the transport of the water channel aquaporin-2. Both the findings and the new method offer more general insights into the mechanics of water recycling.

Renal principal cells with aquaporin-2 (blue) in the cell interior (left) and in the plasma membranes (right). The substance discovered by the MDC research group (4-acetyldiphyllin; image 3), prevents the redistribution of aquaporin-2 to the cell membrane. Nuclei are shown in green.

Renal collecting duct cells in the kidney absorb more or less water based on the body’s needs, depending on activity and other factors. The hormone vasopressin plays a key role in these adjustments by stimulating principal cells in the kidney to take in more water. This happens because the hormone triggers water channel proteins called aquaporins to move from the interior of the cells to the surface, where they create pores that specifically allow an entry of water molecules through the plasma membrane.

When healthy renal duct cells sense the hormone, they begin synthesizing a molecule called cAMP. This sets off a cascade of biochemical signaling that eventually reaches aquaporin-2 molecules. The final step is to attach and remove small chemical tags called phosphate groups to specific amino acids that make up the aquaporin-2 protein.

“Such phosphate tags are added to some sites in the protein and removed from others,” Enno says. “Applying a tag at one specific amino acid, serine 256, was thought to be the main trigger for the protein’s migration from the cell interior to the plasma membrane. But what happened at the other sites might be contributing to its transport as well.”

Working with the Screening Unit, a platform jointly operated by the MDC and FMP, the scientists developed a new method of looking for molecules that might inhibit the transport of aquaporin-2. The researchers used cultures of mouse kidney principal cells that produced a human version of aquaporin-2. As a control, they stimulated the cells with a substance that triggers the production of cAMP and normally causes aquaporin-2 to move to the cell surface. Then they applied 17,700 small molecules from the Screening Unit’s “ChemBioNet library” before they stimulated the cells, to try to find something that blocked the redistribution of aquaporin-2.

“We used an antibody that attached itself to aquaporin-2 and could therefore visualize the binding under the microscope,” Enno says. “In each case you could see whether the channel was primarily locked in the cell or moved to the plasma membrane. But imagine trying to examine 17,700 different cell cultures one at a time to find inhibitors – that would have taken years.”

Developing an automated alternative was a long, intensive process. Jana Bogum and members of the Screening Unit team drew on an automatic screening microscope and a computer program that could automatically scan images of the cultures, looking for cases where aquaporin-2 had failed to move. They found 17 potential inhibitors, 14 of which were submitted to a second round of testing in similar cells developed from rats. Five substances passed both tests.

“We took a closer look at two of these compounds in hopes of gaining deeper insights into the mechanisms involved in transporting the channel protein,” Enno says. “One of these, called 4-acetyldiphyllin, or 4AD, was particularly interesting.”

Applying the substance to the cell cultures blocked the transport of the water channel to the cell surface. Aquaporin-2 became stranded in an intracellular compartment called the Golgi apparatus. Surprisingly, 4AD prevented the attachment of phosphate groups to the critical amino acid – serine 256 – in aquaporin-2. But it didn’t affect the tagging of another amino acid, or the removal of a tag from a third site, which are normally associated with the molecule’s transport.

“This supports the idea that a single site – serine 256 – is most crucial to the process of relocation of the aquaporin,” Enno says. “Until now it hasn’t been clear that this tagging at serine 256 is independent of events that affect the three other sites.”

Aquaporin-2 is wrapped in small membrane compartments called vesicles as it moves outward toward the plasma membrane. Normally these compartments contain high levels of acid. 4AD blocks the enzyme that normally acidifies such compartments, possibly eliminating the use of the vesicles as a means of transport. Mechanistically, the work shows for the first time that the activity of the enzyme that acidifies the vesicles controls the tagging of aquaporin-2 and thereby its location.

“This study provides a proof of principle,” Enno says. “First, our automatic screening method successfully identifies new compounds that could eventually be of interest in treating defects in water absorption linked to diseases such as chronic heart failure. Secondly, the effects of these compounds provide new insights into the complex regulation of water channels within renal principal cells.”

- Russ Hodge

Highlight Reference:

Bogum J, Faust D, Zühlke K, Eichhorst J, Moutty MC, Furkert J, Eldahshan A, Neuenschwander M, von Kries JP, Wiesner B, Trimpert C, Deen PM, Valenti G, Rosenthal W, Klussmann E. Small-molecule screening identifies modulators of aquaporin-2 trafficking. J Am Soc Nephrol. 2013 Apr;24(5):744-58.

Full text of the original paper