Novel driving forces for water transport & osmoregulation: carbonate precipitation and osmotic coefficients

Vertebrates, including humans, are made up of about 70% water. Balancing water intake and output is obviously vital for health, but most people don't realise the vast internal movements of water going on all the time within their bodies. For example, the kidney, gut and pancreas collectively transport 8 times our total body water volume in and out of these tissues each day. Understanding the mechanisms these organs use, and how the cells that line them (called epithelia) operates this water transport is therefore important. Despite this importance, the mechanism of water transport is still the subject of much debate. Having said that, for more than 50 years it has been understood that the net transport of water requires salts (especially sodium chloride, or NaCl) to be transported in one particular direction first, to then drive fluid transport (secondarily) in the same direction by a process known as osmosis. Despite this consensus the precise route that water transport takes across epithelia is hotly disputed. For example, does it pass through the cell membranes, via special protein called aquaporins? or does it squeeze between the cells? The novelty of the present proposal lies in the discovery of two new mechanisms for influencing water transport that are conceptually very different to the other current areas of debate. These ideas challenge the established dogma by not relying on salt being transported in the same direction as water, representing a fundamental change in our understanding and providing a novel model for the mechanism of water transport in animal epithelia. The discovery has been made by studying how marine fish drink seawater and process this fluid through the intestine to avoid dehydration. Like humans drinking ordinary fluids, these animals first transport NaCl from the gut into the blood, and water then follows by osmosis. However, marine fish have another trick up their sleeve that maximises their water extraction capability. They secrete a different compound called bicarbonate (same as found in baking soda) into the intestine, in the opposite direction to water absorption. This causes a chemical reaction within the swallowed seawater that causes the high levels of calcium it contains to precipitate as solid, white clumps of calcium carbonate (like limestone). These 'gut rocks' are eventually excreted but the advantage to the fish is to reduce the total dissolved compounds in the gut fluid, which in turn makes it easier to extract water into the blood. We propose to study this novel process further by using 3 different species of marine fish (flounder, tilapia and trout) that produce very different quantities of bicarbonate, and are therefore predicted to have different efficiencies of water absorption. Cold and high pressure also inhibit precipitation, so we will compare water absorption in fish at cold temperature and high pressure (in a barometric chamber). Precipitation of carbonate occurs in human diseases such as kidney and pancreatic stones, so studying this process in fish may help us understand this pathological condition. A second novel process that fish use involves the high levels of magnesium and sulphate in the sea water that they drink. These are not absorbed, and would therefore be expected to get more and more concentrated as swallowed fluid moves down the intestine as water is extracted. This would eventually retard the osmosis of water into the blood. However, magnesium and sulphate are unusual in only having half the potential of other compounds to causes osmosis. It is only because seawater happens to have such high levels of magnesium sulphate that fish extract water so efficiently. This will be explored using samples of gut tissue taken out of the animal and studying its water transport properties in a test tube (in vitro). Many human laxatives use magnesium sulphate (Epsom salts) so this research could reveal insights into how these treatments actually work.

It has long been known that net transport of water by epithelia requires net solute transport in one direction to secondarily drive net fluid transport in the same direction by osmosis, in a process referred to as solute-linked fluid transport. The novelty of the present proposal lies in the recognition of two additional driving forces for water transport that are conceptually different and challenge this dogma by not relying on solute transport in the same direction as water. This proposal concerns none of the previous areas of debate (e.g. aquaporins, molecular water pumps, etc.), but instead focuses upon two novel aspects of solute chemistry that can have a major influence on the driving forces underlying water transport. Specifically, these processes concern solutes that are not traditionally associated with epithelial water transport - the divalent ions, principally calcium, magnesium and sulphate, within imbibed fluids of marine teleost fish and their influence on the crucial process of water absorption by the intestine. The first process concerns the the precipitation of divalent Ca2+ and Mg2+ as insoluble carbonates in the presence of HCO3- ions that marine fish intestines continuously secrete. This removes these osmotically active solutes from solution thus reducing the luminal osmolality and facilitating water absorption. The second arises from the fact that Mg2+ and SO42- ions are poorly-absorbed but have far lower osmotic coefficients (0.58) compared to the ions normally considered in studies of epithelial fluid transport (0.93 for NaCl). These ideas will be explored by comparing in vivo intestinal water transport in 3 species (flounder, tilapia, trout) with different bicarbonate secretion rates, and in flounder at different temperatures and pressures designed to vary CaCO3 solubility and hence precipitation rates. An in vitro gut sac approach will be used to test the influence of osmotic coefficients of non-absorbed solutes on water transport.