May 1, 2006 -- Researchers seeking insights into kidney failure in human infants have located the source of a 30-year-old mystery mutation that causes similar problems in a mouse line.
Scientists have known of the mouse line's naturally occurring mutation since the early 1970s. Researchers at Washington University School of Medicine in St. Louis are the first to identify the mutated gene, allowing them to determine the mutation's effects and the origins of the disease.
"The gene codes for a protein that moves water across membranes, and we showed that the mutated form of the protein doesn't get properly distributed in the urinary system," says senior author Feng Chen, Ph.D., assistant professor of medicine. "If something similar happens in human disorders, one way to treat such diseases would be to redirect the protein to its appropriate location."
Results from Chen and his colleagues are published online in the Proceedings of the National Academy of Sciences.
Ultrasonic scans reveal fetal hydronephrosis, or enlargement of the kidney, in approximately 1 in every 100 human fetuses. The condition results from impairment of passage of urine from the kidney to the bladder. About 20 percent of those cases lead to clinical complications that, if left untreated, can include kidney failure and death.
Scientists suspect a variety of environmental and genetic factors contribute to fetal hydronephrosis. However, they frequently cannot tie it to any causative factors, making it difficult to determine how normal molecular and cellular processes in kidney development break down in infants affected by the condition.
Researchers at The Jackson Laboratory first noted the mouse line's mutation in the early 1970s when some of the mice developed enlarged bellies and died within three to four weeks of their birth. Scientists initially assumed that the mutation was causing a form of polycystic kidney disease, but a follow-up study more than a decade later showed that passage of urine from the kidney was blocked, and the condition was renamed congenital progressive hydronephrosis.
With assistance from the mouse genome sequence and Li Ding, Ph.D., a research instructor at the Genome Sequencing Center at Washington University, Chen and his colleagues showed that the protein mutated in the mice is aquaporin 2 (aqp2), which belongs to an important family of proteins that channel water across membranes.
"We knew that the aqp2 protein is found in the principal cells of the collecting duct, the final stretch of the kidney filtration machinery," Chen says. "Water and some other useful components are reabsorbed from the urine here. The concentrated urine is then passed on to the bladder and other downstream parts of the urinary system."
When scientists used an antibody to identify where aqp2 is in the kidneys of the mutant mice, they saw that distribution of the protein was changed. Normally aqp2 is concentrated on the side of collection duct cells that faces the urine, where aqp2 can extract water from the waste stream for recirculation in the body. In the mutant mice, though, aqp2 was scattered around the collection duct cells.
"The protein is still there, but it's not in the right place," Chen says. "To make sure this was the cause and not just a result of the problem, we analyzed the sequence of the aqp2 gene from the mutant mice, comparing it to the gene from other normal mice, and found a single base pair had changed."
The change swaps the amino acid serine for the amino acid leucine at a key position in the protein. Serine can undergo phosphorylation, a form of chemical modification frequently used in biological processes; leucine cannot. This change apparently disrupts the processes that otherwise produce a normal distribution pattern for the protein.
Chen compares the machinery that transfers water from the kidney to a plumbing system. With aqp2 unable to reabsorb water, he says, that results in up to 30 times more urine being dumped into the downstream pipes.
"Eventually, you overwhelm the plumbing system, and it gets backed up to an upstream location: the kidney," Chen says.
Chen plans follow-up studies of aqp2 to determine how important various parts of the protein are to its proper distribution in the kidney collecting duct epithelium. His lab also is looking for other genetic factors that contribute to renal diseases.
Reference: McDill BW, Li S-Z, Kovach PA, Ding L, and Chen F. Congenital progressive hydronephrosis (cph) is caused by an S256L mutation in aquaporin-2 that affects its phosphorylation and apical membrane accumulation. Proceedings of the National Academy of Sciences, online edition.
Funding from the Department of Internal Medicine/Renal Division, the March of Dimes, and the National Institutes of Health supported this research.
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