UF researchers show arteries can serve as highway to route new genes to kidneys

University of Florida scientists studying rats have hit on a new way to deliver genes to the kidneys through the renal arteries, the circulatory equivalent of a modern superhighway system. Researchers have found they can inject a virus modified to transport DNA directly through the kidney's largest vessel, speeding genes through the complex organ to a specific site — though not the area they first targeted.

Instead, the cellular cargo winds up at an unexpected yet potentially beneficial destination, a part of the kidney containing cells that control reabsorption of important chemicals from filtered blood or help maintain proper blood pH.

The experimental approach is opening up new avenues for research and — if eventually proved effective in people — could someday help doctors prevent organ rejection after kidney transplantation and treat patients with diabetes or other diseases that scar or damage the kidneys, said nephrologist Anupam Agarwal, M.D., an associate professor of medicine at UF's College of Medicine.

“In the kidney it’s hard to target a gene because the kidney has more than 30 different cell types, each with a specific function,” Agarwal said. “Different kidney disorders involve different parts of the kidney, so if you pick a disease you want to treat, you have to target that particular cell.”

Little research has been performed using the virus in kidneys, and a previous study conducted elsewhere showed that injecting it directly into kidney tissue had little effect except in the area the needle penetrated. Because the kidneys receive one-quarter of the heart's blood output, UF researchers reasoned the renal arteries might provide an ideal route for distributing the virus throughout the organ.

In the study, UF researchers tried to send a genetically altered form of the apparently harmless adeno-associated virus to cells that line the kidney's network of blood vessels, a first step toward developing therapies to protect transplanted kidneys from forming damaging scar tissue.

But the virus transmitted its payload — copies of a gene that triggers production of green fluorescent protein — to two cell groups located in the kidney tubules, tiny tubes that help filter blood, separating waste products from chemicals the body still needs. When the cells produced the protein, a process known as gene expression, researchers could then determine where the gene had been delivered by using antibodies to stain the protein, making it visible.

The results were published in the April issue of the Journal of the American Society of Nephrology and were featured in the issue's cover story.

“We were lucky to get this expression, it worked out really well,” said Agarwal, the study's principal investigator in its latter stages. “We’re going to go with it, and in the meantime, we have alternate strategies to get back to where we actually wanted to be.”

UF scientists modified the adeno-associated virus to contain copies of the green fluorescent protein gene, then injected the solution into the left renal arteries of seven male rats. Blood flow in and out of the left kidney was interrupted for 45 minutes so the virus could disperse, then the rats were allowed to recover from the surgical procedure, said microsurgery expert Sifeng Chen, M.D., an assistant scientist in UF’s College of Medicine who, along with Agarwal, was primary author of the article. A control group of seven rats underwent a nearly identical procedure but received saline solution. Researchers examined kidneys from rats in both groups after two, four and six weeks.

In the experimental group, the left kidneys showed protein expression in two cell types — proximal tubule cells that put needed chemicals such as sodium, phosphorous and potassium back into filtered blood, and intercalated cells, which help maintain proper blood pH. Very little expression was found after two weeks, but after six weeks it was significant. No expression was found in the right kidneys or other organs of the experimental group or anywhere in the control group.

Researchers believe rats in the experimental group might have continued to produce the protein indefinitely, Agarwal said. Another study is under way to investigate long-term gene expression in this model system.

Agarwal said an enzyme called heme oxygenase-1 might prevent scar formation in kidneys, and he hopes to investigate whether the gene controlling the enzyme’s production could be delivered to this organ. If the gene were expressed strongly in the kidney, enough of the enzyme might be produced to protect nearby blood vessels from scarring.

Funding for the current study was provided by the National Institutes of Health and through a five-year, $10.6 million grant from the Juvenile Diabetes Research Foundation International awarded to UF and the University of Miami to establish the JDRF Gene Therapy Center for the Prevention of Diabetes and Its Complications, said Mark Atkinson, Ph.D., the Sebastian family eminent scholar for diabetes research at UF's College of Medicine and the center's director.

Because diabetes patients can require multiple kidney transplants, methods of prolonging transplant survival are of great interest, said Atkinson, a co-author of the study.

“The average survival of a transplanted kidney in a diabetic patient is about seven years,” he said. “With gene therapy we may be able to prolong transplant survival, and if we achieved that goal, with fewer people needing to be retransplanted, there would be more organs to go around.”

The finding that intercalated cells expressed the fluorescent protein surprised researchers, because previous kidney gene therapy studies had not indicated this cell group would respond, said C. Craig Tisher, M.D., dean of UF’s College of Medicine and the study’s original principal investigator.

“It was particularly interesting to us because we’ve been working on understanding the structure and function of these intercalated cells for the last decade or more,” said Tisher, an internationally known nephrologist who together with UF colleague and study co-author Kirsten Madsen, M.D., Ph.D., a UF associate professor of medicine, and Jill Verlander Reed, D.V.M., a UF associate scientist in the College of Medicine, was the first to describe two types of intercalated cells in the kidney.

The finding could lead to therapies for diseases involving intercalated cells, such as renal tubular acidosis, which causes increased blood acidity and calcium loss that can lead to bone deformities and kidney stones, he said.

“As so often occurs, as you get into a project new opportunities present themselves, based on the data that you develop,” Tisher said. “This now presents the opportunity to target some of those cell types we thought might be very difficult to approach, and I think the opportunities are pretty significant.”