"Tissue transplantation is fantastic but it would be so much better if we could instead raise organs from a patient's own cells," says lead investigator Raphael Kopan, Ph.D., associate professor of medicine and of molecular biology and pharmacology at Washington University School of Medicine in St. Louis. "Before we can actually trick cells into doing what we want them to do we really need to understand every detail about how the organ is put together."
Using an antibody that specifically identifies the active form of Notch, Kopan's group observed that the protein is extremely active in the kidney at an earlier stage than previously thought. So they teamed up with kidney development expert Jeffrey H. Miner, Ph.D., associate professor of medicine and of cell biology and physiology, to investigate further. First, though, they had to resolve a methodological conundrum: How do you study the effect of Notch in the kidney if animals without Notch die before the kidney begins to form?
The answer came from an entirely different field: Alzheimer's disease. In 2001, Kopan's team discovered that a group of potential Alzheimer's drugs that inhibit a protein complex called gamma-secretase also interfere with Notch. For clinical purposes, the drugs have since been refined to minimize their potentially dangerous effects on Notch. But drugs that severely inhibit this protein are perfect for studying its activity in laboratory animals.
"We took advantage of developments in different fields to allow us to do this analysis," says Kopan. "Without collaborating and combining our knowledge, we would not have been able to conduct this study."
The team removed both kidneys from normal mice during early development and placed them in organ culture. They treated one kidney from each mouse with a gamma-secretase inhibitor and showed that this process prevented all Notch signaling. The second kidney from each animal was used for comparison.
After three days of treatment with the inhibitor there were fewer and less developed tubular structures in the treated compared to the untreated kidneys. These differences became more pronounced after five days of treatment: Tubes in untreated tissue branched an average of 10 times and the tips of these branches had consistent, small diameters; tubes in treated tissue only branched a maximum of eight times and their branches were more irregularly shaped.
For the most part, the treated cells successfully passed through the first stage of development, in which they evolved from embryonic, precursor cells into epithelial cells, which form the lining of the organ. But the most pronounced abnormalities occurred in the next stage of development, in which the cells become more specialized.
Urine is formed in the kidney's functional units, called nephrons. Within each nephron are several structures, including a long, winding tube called the proximal tubule and octopus-shaped cells called podocytes that wrap their "feet" around blood vessels. After two days of treatment with the gamma-secretase inhibitor, neither podocytes nor proximal tubule cells formed. Another nephron structure, the distal tubule, was not disturbed.
"The most exciting finding was that Notch signaling appears to tell some cells to become podocytes from a mass of non-specialized epithelial cells," Miner says. "This shows that Notch is involved at an earlier stage of podocyte development than any other factor that's been identified so far."
Even more surprising was that the tissue lost the ability to form podocytes after a certain amount of time. If Notch signaling resumed after two days, podocytes recovered. But if it did not resume until three days or more, the cells instead developed into those that comprise the proximal tubule.
"It's as if the cell can tell time," Kopan explains. "After three or four days without Notch signaling, it realizes it will never become a podocyte and decides to respond to the next signal it receives."
Next, the team hopes to further differentiate the role of Notch in formation of each component of the nephron, and to determine the specific genes responsible for this particular developmental pathway.
Cheng HT, Miner JH, Lin MH, Tansey MG, Roth K, Kopan R. g-Secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podoctye and proximal tubule formation in developing mouse kidney. Development, vol. 130, pp. 5031-5042, Oct. 15, 2003.
Funding from the National Institutes of Health, the Alzheimer's Association, the Zenith award, the American Heart Association and the March of Dimes supported this research.
The full-time and volunteer faculty of Washington University School of Medicine are the physicians and surgeons of Barnes-Jewish and St. Louis Children's hospitals. The School of Medicine is one of the leading medical research, teaching and patient-care institutions in the nation. Through its affiliations with Barnes-Jewish and St. Louis Children's hospitals, the School of Medicine is linked to BJC HealthCare.