One Friday night, as the usual convoy of buses lined up on the Mayo Clinic campus in Rochester, Minn. to whisk weary staff home, a group of scientists were inspired to begin spinning urine samples in the lab centrifuge. What prompted their decision and why the results fomented such excitement, is the latest success story of two research groups from apparently disparate specialties. Their collaboration was galvanized by cilia — single antenna-like structures of epithelial cells that line both the kidney tubules and the bile ducts. Previously ignored as vestigial structures, the Mayo researchers discovered their complicity in the formation of harmful cysts and are investigating new ways to diagnose and treat polycystic kidney and liver disease.

Two research tracks

In 1975, having won a Mayo Clinic young investigators’ scholarship award, Nicholas LaRusso, M.D., was sent to the Rockefeller University in New York, to hone his cell biology skills in the lab of Nobel Laureate Christian de Duve. At the same time, Vicente Torres, M.D., Ph.D., was beginning residencies in internal medicine and nephrology at Mayo Clinic. Dr. Torres had already completed his medical and doctoral degrees in his native Spain and had won several national scholarships, both in Spain and in the United States with which he had completed an additional four years of research training.

Like many Mayo Clinic scientists, Drs. LaRusso and Torres are also physicians whose research is inspired by patient problems. Dr. LaRusso’s recognition began with a disease called primary sclerosing cholangitis, a disease of the bile ducts that many physicians were not convinced existed until he published a landmark paper proving them wrong. He went on to become an expert in the group of diseases referred to as the cholangiopathies, or disorders of the cholangiocytes [ko-LAN-gee-oh-sites] — epithelial cells that line the bile ducts.

“What got me interested in cholangiocytes was seeing patients who had diseases that were primarily affecting these cells,” says Dr. LaRusso. “Because cholangiocytes are required to form the stream of bile and modify its composition, much of our lab’s initial work focused on defining the normal transport and proliferative functions of cholangiocytes.”

Dr. Torres, meanwhile, was earning international recognition both for his research and for his clinical expertise in polycystic kidney disease (PKD). He has played a leading role in the development of large multicenter clinical trials and immediately grasped the significance of a report in the Japanese scientific literature that described a rat, called the PCK rat, which had spontaneously developed PKD. He understood the model’s potential for accelerating PKD research and had it brought to Mayo Clinic.

"We used it to demonstrate how the PKD gene manifests itself clinically," explains Dr. Torres. "That led to a collaboration with Dr. Harris."

Building the research teams

Peter Harris, Ph.D., from the University of Oxford, England had already made landmark discoveries in PKD research. Dr. Torres recruited him to Mayo in 1999.

"Mayo offered me outstanding opportunities to study the interface between genetics and how the disease presents in patients," says Dr. Harris. "Mayo has a huge population of patients with a wide range of disease presentation that had been studied clinically but not genetically, which is my expertise. It also offered everything I needed in terms of core facilities, support, and depth of scientific and medical expertise."

Dr. Harris brought with him fellow molecular biologist Christopher Ward, M.D., Ph.D., a key player in the Oxford discoveries. Dr. Ward has genetically engineered lab animals to produce new animal models of PKD and has developed many reagents for research in the PKD field. The Harris lab’s discoveries include the identification of the major gene for ADPKD (the more common form of PKD) and the gene responsible for ARPKD (the rarer, childhood form of PKD).

The PKD group was later rounded out by a young nephrology trainee, Marie Hogan, M.D., Ph.D., who went to medical school on a scholarship she won after receiving the top high school biology grade in her native Ireland.

“I got Cs in English but I was one of three girls who used to walk to the boys’ school for chemistry class,” remembers Dr. Hogan. “It was a real buzz to see their faces when they couldn’t understand the electron cloud and I had to explain it to them.”

Realizing Dr. Hogan’s potential, Dr. Torres became her mentor and suggested she work with Dr. Harris. They collaborated to map the gene mutated in the animal model of polycystic kidney and liver disease, which led to the Harris group’s identification of the gene (PKHD1) mutated in human ARPKD (Nature Genetics, 2002).

In the meantime, Dr. LaRusso had welcomed Tatyana and Anatoliy Masyuk, both basic scientists with advanced academic degrees from the Ukraine, to his lab. Building upon the work of their predecessors, the Masyuk’s were instrumental in helping the LaRusso lab develop innovative biochemical techniques and culture systems to isolate and fractionate cholangiocytes into their different parts. The team defined many of the channel, transporter receptor and exchanger proteins that are involved in normal cholangiocyte function and pinpointed their location in the various cellular compartments.

Two research tracks converge

By 2000, the LaRusso team had established that cholangiocytes were important contributors to, and modulators of bile formation. The efforts meant that much more was known about how cholangiocytes transport fluid, electrolytes, and dissolved substances; and also about the complex hormonal and physiological factors that regulate bile formation. They were also developing an interest in the function of cilia, which extend from cholangiocytes in the bile stream.

“What heightened interest in cilia was nephrologists discovering that the genes that are mutated in polycystic kidney disease primarily reside in the cilium,” explains Dr. LaRusso.”

The need for collaboration now became clear — cysts develop from epithelial cells that line both the liver and the kidney, and cilia are somehow involved.

Homing in on the malfunction

In 2003, the two groups published a cell study that located fibrocystin on cilia (Human Molecular Genetics, 2003). Fibrocystin is a protein associated with ARPKD so the discovery linked cilia to kidney cysts. Follow up animal studies determined that fibrocystin is also expressed in cholangiocyte cilia and that a mutation in the ARPKD gene causes defects in cholangiocyte cilia resulting in liver cysts.

Simultaneously, Dr. Torres’s team reported a treatment that stops the development of cysts and prevents loss of kidney function in animals (Nature Medicine, 2003). The drug, known as OPC31260, inhibits the production of cyclic AMP and is the focus of a multicenter, international clinical trial with 1,400 participants in which the group is currently participating.

The LaRusso lab also became interested in cyclic AMP when they observed high levels of it in cholangiocytes isolated from an animal model of polycystic kidney and liver disease. Cyclic AMP is called a second messenger and plays an important role in how a cell sends the signals that regulate its processes. The LaRusso lab deduced that it was causing liver cysts to grow by contributing to excessive water transport and increased proliferation.

Targeting a potential therapy

In 2006, the LaRusso lab developed a cholangiocyte cell line from a PKD animal model allowing them to isolate and characterize cholangiocyte cilia and focus on cholangiocyte cilia function. By 2007, much more was known about the molecular mechanisms of bile secretion and the roles that cholangiocytes play in signaling, transport of water, ions and solutes; and defects that result in reduced bile flow (cholestasis). By this time, cholangiocytes were increasingly recognized as target cells of a variety of cholangiopathies.

In earlier studies, the LaRusso lab discovered that a receptor for a hormone called somatostatin was expressed on cholangiocytes. Noting that cyclic AMP levels decrease when they stimulated the receptor with somatostatin, the two research teams experimented with a drug similar to somatostatin, called octreotide, to try to decrease cyclic AMP cellular levels and reduce cyst formation.

When the experiment worked in cultured cells, they developed a way of growing bile ducts that form cysts in a 3D culture to facilitate further testing. After equally successful animal studies (Gastroenterology, 2007) octreotide, which is approved to treat other diseases and has proven safe and well tolerated, was ready for human testing

In January 2007, with Dr. Hogan as principal investigator, the clinical trial to evaluate the effect of octreotide on the size of the liver in patients with severe polycystic liver disease began. The 42 participants — all diagnosed with polycystic liver disease — receive monthly intramuscular injections of the drug and Mayo radiologist Bernard King, M.D., is refining MRI techniques for precise liver measurement. Similar studies are underway in the Netherlands and Italy and initial data is promising.

Signaling between cilia and exosomes

The two research teams saw another opportunity to collaborate when, in 2004, another group discovered that proteins associated with some kidney diseases were actively shed in the urine. Polycystin-1, polycystin-2, and fibrocystin are proteins associated with PKD. What intrigued the Mayo teams was the hypothesis that the functional site of these proteins is on the cilia, in which case they could be available in urine for non-invasive diagnostic PKD tests.

“It was a Friday night in 2006 when we decided to spin down some urine,” says Dr. Hogan. “Saturday morning we were very excited to see a huge amount of polycystins.”

Testing then intensified. Dr. Ward hurdled the first problem by devising a method to filter out a common contaminating protein. The PKD proteins shed in urine are contained within membrane particles that the group named exosome-like vesicles (ELVs), which had to be broken down before they could isolate and analyze the resulting proteins. The results were astonishing — of the 552 proteins that they identified, 232 of them were new to proteomic databases.

In addition, they showed that fibrocystin in human urine is cleaved, or split into simpler molecules, as it does in cell studies. That observation urged them to examine human and animal ARKPD kidney tissue under the electron microscope where they observed vesicles binding to cilia. Using the bile units developed by Dr. Masyuk, and exosomes that Dr. Ward labeled with gold, the team was able to show exosomes fusing with and then being shed from cilia. The studies provided the first in vivo evidence of a dynamic reaction between exosomes and cilia, which raises the possibility of a novel form of signaling between the two structures (Journal American Society Nephrology, Jan 2009)

The success of the cholangiocyte and PKD groups exemplifies Mayo’s hallmark of collaboration.

“Mayo really promotes internal cooperation and networking,” says Dr. LaRusso. “It’s the first time I’ve been able to directly link what I do in the lab to a clinical trial. We never could have done it without the PKD group. And it’s encouraging that we have a dozen other potential drugs to test based on other abnormalities we’ve identified.”

That’s good news for patients who come to Mayo Clinic desperate for immediate innovative treatments and for patients worldwide who may find the help they need from one of these discoveries.

— Yvonne Hubmayr, May 2009