Physiological studies in mice demonstrate a surprising role for a kidney protein related to the rhesus factor of red blood cells. Similar research would aid further annotation of mammalian genomes.
The completion of the Human Genome Project spawned a new era of biological research, with many declaring that we have now entered the 'post-genomic' era. This declaration is premature, not least because comprehensive functional annotations are lacking for most of the 20,000-odd protein-coding genes in the human genome1. Without this knowledge, we cannot gain a thorough understanding of human disease. So a crucial task ahead is to discover the in vivo function of each gene product. By investigating the function of the protein encoded by a gene called Rhcg, using classical physiological techniques in mice, Biver and colleagues2 (page 339 of this issue) provide a model for how this goal can be effectively pursued.
We are constantly facing an acid threat. When oxidized in the body, excess dietary proteins produce sulphuric and phosphoric acids, posing the danger of a lethal drop in blood pH. But kidneys prevent our demise by removing these acids through the excretion of hydrogen ions (H+). Most of this H+ is excreted in the urine as ammonium (NH4+), which is made by cells in the proximal tubule of the kidney from the amino acid, glutamine.
From the proximal tubule, a series of NH4+-transport processes, ending with a transport step across the cells of the collecting duct, ensure the transfer of NH4+ to the urine3 (Fig. 1). In the final step, NH4+ is believed to enter the collecting-duct cells by direct transport4, but leaves these cells by the parallel transport of its constituents — H+ and ammonia (NH3) — into the urine5. An ion pump called V-ATPase mediates H+ transport. As for the ammonia, because of its small size (17 daltons), it was believed to diffuse freely across the membranes of collecting-duct epithelial cells into the urine, independently of a transporter protein5. But is this really the case? Biver et al.2 suspected otherwise, and so tested the hypothesis that the protein product of Rhcg is responsible for ammonia transport out of collecting-duct cells.
The protein in question, Rhcg, is related to the rhesus (Rh) antigen protein of red blood cells that is used to determine immunological compatibility before blood transfusions. It had previously been shown6 that the amino-acid sequences of mammalian Rh-family proteins are similar to those of known ammonia-transport proteins of bacteria, fungi, plants and invertebrates. Indeed, when investigators expressed mammalian Rhcg in cells that do not normally express it, they detected transport of ammonia7. But this observation is not a definitive proof for the physiological role of Rhcg at its natural levels in mammalian tissues. What's more, when Rhbg — the gene for another Rh-related protein of the collecting duct — was deleted in mice, no measurable defects were detected in ammonia transport in the kidneys8.
Undaunted by the negative result with Rhbg, Biver et al.2 deleted the Rhcg gene in mice. The authors demonstrate that, contrary to the free-diffusion hypothesis5, Rhcg absence results in a two-thirds reduction in the rate of ammonia movement across the collecting-duct epithelial cells. This indicates that, at most, only one-third of ammonia crosses the lipid membrane by diffusion. So Rhcg seems to function as a membrane channel, allowing the passage of ammonia across the lipid membrane between collecting-duct cells and the urine (Fig. 1). The absence of this channel in the Rhcg-knockout mice results in decreased excretion of ammonia in the urine and a marked drop in blood pH, clearly establishing a role for Rhcg in the regulation of pH in body fluids.
Biver and colleagues also report a reduction in reproductive capability of male Rhcg-knockout mice, associated with altered epididymal function. Like the kidney's collecting duct, the epididymis — a convoluted tube within the testes where sperm maturation occurs — acidifies its lumen using the V-ATPase H+ pump9. Here, however, acidification has a different role: the acidic environment is necessary for sperm maturation and for making sperm cells dormant until they are 're-awoken' by the alkaline environment of the seminal vesicle. In this context, excessive ammonia secretion into the epididymal lumen would be detrimental, prematurely awakening sperm cells. The authors propose that Rhcg in the epididymis could be responsible for scavenging the potentially toxic ammonia from the lumen and transporting it into the cells lining the epididymal lumen where it can be converted to glutamine.
Trying to identify functional differences between knockout and normal mice is technically challenging, in part because of the small size of mice. What helped Biver et al. in their quest was their ability to adapt some classical physiological techniques to the mouse, including measurements of the composition of body fluids, of intracellular pH using fluorescent dyes, and of solute transport in isolated perfused renal tubules. These approaches have been part of the physiological armamentarium for years, but have gradually been displaced by modern molecular and cell-biological techniques. For example, the isolated perfused tubule technique developed10 in the 1960s — like many other intricate physiological methods — has not been used extensively since the 1980s. With expertise in many physiological techniques in decline, Biver and colleagues' work is a testament that such methods are still of enormous value. It will be interesting to see whether physiologists' new role as functional annotators of the genome will spur a resurgence of training in traditional physiological techniques.
Biver and colleagues' data2 resolve a long-standing question in kidney physiology: what is the molecular basis of ammonia transport into the urine? With the identification of Rhcg as the ammonia channel in the collecting duct, researchers can now address questions such as whether and how this channel may be regulated, and what its involvement is in disease processes associated with impaired regulation of blood pH. Reproductive biologists will undoubtedly soon be investigating the specific role of Rhcg in epididymal function and sperm maturation.
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