By Leslie Mertz
Dr. Churchill is trying to determine if genetic differences in the kidneys are the cause or consequence of high blood pressure.
A lab in Wayne State University’s medical school is the only one in the world that can successfully transplant the fingernail-sized kidney of a rat, and it has held that distinction for years. What is now exciting the lab’s scientists is the blending of gene transfer possibilities with the intricate transplanting technique, and the promises the alliance holds for learning about the genetics behind different types of kidney dysfunction, and about an individual’s susceptibility or resistance to damage from high blood pressure.
In one of two elegant experimental protocols, the researchers remove one kidney of a rat and replace it with a kidney that differs by just one part of one chromosome. They then monitor the animal’s blood pressure, applying a setup that gives far more precise readings than the blood pressure cuff commonly used in a doctor’s office.
"It’s like doing a gene transplant into the kidney. We do it by actually transplanting the kidney," said Paul Churchill, PhD, professor of physiology. "The two genetically different kidneys are seeing the same blood, the same concentrations of hormones and everything else you can think of." This was important because so many factors can also affect blood pressure, such as an individual’s metabolism, diet or stress level. "The question is: Are the two kidneys behaving the same? If not, the only way you can explain it is a genetic difference. So it’s pretty exciting."
The project is called kidney specific gene transfer, and involves collaborators from the United States and abroad. Funding comes from the National Institutes of Health, the Grant Agency of the Czech Republic and the PECO Program of the European Commission.
"The animals that we’re working with are called congenic strains of rats," Dr. Churchill said. "They were created by taking spontaneously hypertensive rats, or SHRs, and breeding them with inbred brown Norway rats, which have normal blood pressures. Actually the brown Norway rats are very stubbornly normotensive – they refuse to get high blood pressure; you can’t give them high blood pressure."
Using DNA fingerprinting and backbreeding methods, researchers from the Czech Republic and from the University of California-San Francisco, created nearly three dozen strains of rats that are like the spontaneously hypertensive rat except for a certain region on one chromosome, and that region came from a brown Norway rat. "Each of the 32 strains have slightly different blood pressures, so these chromosome regions are affecting blood pressure," Dr. Churchill explained. "He sends them to us, and we do kidney transplants between these strains." The rats also have the same major histocompatibility complex, so they don’t have to worry about kidney rejection complications.
The transplant technique itself is unique to the Wayne State lab. Monique Churchill, research assistant, does the kidney transplants. "She’s the only one in the world who has done it right. No one else can get the kidneys to function normally. She can. She does a lot of little things different." Dr. Churchill added, "Her transplanted kidney – you can’t tell it from the other kidney. It’s absolutely amazing."
They create an animal that is entirely SHR, except for a part of one chromosome in a single kidney. This method will tell them whether gene expression in the kidney is responsible for the blood pressure difference between the animals. "Why that’s exciting is that I would say it’s been at least 100 years that people have known that the kidney one way or another affects blood pressure." For example, he said, people with an impeded blood flow to the kidney, perhaps through a narrowing of the renal artery, are hypertensive.
Speculation abounds about what causes high blood pressure, Dr. Churchill said, and whether differences in the kidney are the cause or the consequence of high blood pressure. "Nobody is really sure. Well, this (work) is going to answer that."
They will also use the same kidney specific gene transfer protocol to determine how much genetic differences may affect the regulation of blood pressure. "Nobody would care what your blood pressure was if your blood vessels could stand it. What’s bad about high blood pressure is some of the smallest blood vessels, the capillaries, cannot withstand high blood pressure, and if they’re exposed to it, they blow out."
When a physician peers into the eye of a patient with a penlight, he or she is actually conducting a test for high blood pressure, he said. "Your retina is one of the only places in your body where you can directly see capillaries." Burst capillaries in the eyes, a telltale sign of high blood pressure, can cause blindness. In the brain, it can cause stroke; in the heart, heart attack; and in the kidney, kidney failure.
"There are genetic differences there, too," he said. "African-American hypertensives are 15-20 times more likely to develop end-stage renal disease (a severe form) as a result of high blood pressure than Caucasian Americans. If you read the literature it looks like it’s not a difference in how high the blood pressure is or how long it’s been high, it’s genetic."
He has seen the same thing with the rats. "The SHR has an enormous blood pressure, but it can take it. It has the same life span as a regular rat. Another strain of rat, called the stroke-prone SHR, doesn’t have a blood pressure even quite as high as the SHR, but it strokes out from vascular damage." Fortunately for his research, Dr. Churchill said, the brown Norway rats, which have normal blood pressure, are "enormously susceptible to hypertensive damage." That makes sense, he noted. From an evolutionary standpoint, the brown Norway rat would have experienced no selective advantage in carrying a tolerance for a condition it didn’t have.
Armed with this information about susceptibility, Dr. Churchill’s lab tried transplanting a brown Norway rat’s kidney into an SHR congenic that carried the major histocompatibility complex of the brown Norway rat. "Now you’ve got an animal that’s got one SHR kidney and one brown Norway kidney. They’re exposed to the same blood pressure, the same hormones, everything. When you look at them after, say, six weeks to see if there’s differential kidney damage, it’s like black and white. The brown Norway kidney is shot to hell and the SHR kidney is normal." Photographs of the two kidneys show that all of the normal structure in the brown Norway kidney was obliterated. This finding became the lead article in an issue of the Journal of Clinical Investigation.
"What that brings up, then, is that now we can actually map the genes that are responsible for hypertensive damage, because we’ve got 32 different strains, and they have varying pieces of chromosomes from this susceptible strain that’s been transplanted into them. We can create rats that have genetically different kidneys and be very selective about it: is it part of chromosome 15 or chromosome 1?"
They have already learned that the susceptibility to damage is multigenic, and that a region on chromosome 1 is responsible for about 10 percent of the total difference between the hypertensive and normal rats. He speculated that at least five or six genes are involved, and he is currently seeking grant funding to determine which ones.
"If you start fantasizing about this, and we have, once you figure out the genes, you can do the gene therapy," he said. "Right now, you’ve got people who have high blood pressure and are susceptible to damage, and the only way you can treat them is to lower their blood pressure. What if you could give them something that would just make them immune to the effect of it?"
Dr. Churchill is particularly pleased that this research involves so many people from other disciplines. In addition to the scientists who developed the strains, others at Loyola University Medical Center in Chicago, examine the kidneys. "What’s also exciting about this sort of marriage of different skills is we can answer any number of questions. For example, in diabetes, people usually die of end-stage renal disease, and some people are more susceptible than others," he said. "We can use the same paradigm, and create an animal with genetically different kidneys. We can give the animal diabetes and track it down to what chromosome is causing the susceptibility, and where it is on that chromosome."
Of the interdisciplinary work, he added, "I feel we’re sort of a cog in the wheel. They couldn’t do without us and we can’t do without them."