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An unusual laboratory sits within Pitt’s medical research complex. It contains thousands of tanks of swirling, bubbling water. The tanks hold thousands of commonplace aquarium fish. But these minnows aren’t so ordinary—they’re helping scientists tackle some of biology’s toughest mysteries.

Water World

 


Cara J. Hayden


Researchers (clockwise from left) Michael Tsang, Neil Hukriede, Nathan Bahary, and Xiangyun Wei. (Steven Adams photo/Pittsburgh Tribune-Review)

Ages ago, an Israelite-priest spread the glistening kidneys of a young bull on a stone altar, preparing to offer them to God. The ancient Hebrews believed that kidneys were a precious organ. Through those curved orbs of flesh—set deep in the body where only God could see them—the temperament and character of a person could be judged.

And so, abiding by sacred rituals, the priest set the bull’s remains aflame. Smoke swirled to the heavens, disappearing into the sky.

Thousands of years later, the kidneys are known to have a more practical function. Most of us recognize that kidneys create urine, not character; but they are, indeed, integral to life. The kidneys filter our blood, removing biological waste and excess water. They stimulate red blood cell production, help to regulate blood pressure, and balance levels of fluids and minerals in the body.

Yet, even though science has revealed a lot about the twin organs near the small of our backs, University of Pittsburgh professor Neil Hukriede will tell you we’re not too far beyond the days when people thought that kidneys had spiritual properties. Many of the inner workings and quirks of the organ remain a mystery, and Hukriede is looking for clues in an unusual place.

Every day in a modern laboratory above Oakland’s Fifth Avenue, Hukriede pokes and prods at the kidney’s secrets. The primary question he ponders is: How do the kidneys grow? Understanding how cells “bud” into a kidney—or don’t—may lead to new treatments for kidney disease. Right now, for patients, there are only two survival options when both kidneys fail—mechanical dialysis to filter the blood or kidney transplantation. Hukriede and his research team want to expand those options, and they’re getting help from an unlikely source.

In an aquatic laboratory in Pitt’s Biomedical Science Tower 3, thousands of zebrafish dart about their tanks, an unexpected sight amid the robotic tools and supermicroscopes elsewhere in this leading research center. Hukriede—an assistant professor of microbiology and molecular genetics in the School of Medicine—helped to design the zebrafish facility, which uses the same technologies as corporate shrimp farms. It has a capacity of 11,000 tanks and a half-million fish. The tanks are stacked high and deep on industrial shelves, giving the lab the look of a library. It smells like a pet store instead of old-book must. There are water tanks instead of novels. But people here browse the stacks, too.This is where Hukriede studies the kidneys using zebrafish—simple aquarium dwellers that are helping scientists worldwide to unravel complex riddles of biology.

On a recent spring-semester morning, Lisa Antoszewski, a postdoctoral fellow on Hukriede’s team, glances over the tanks in one aisle. She checks labels that indicate the fishes’ dates of birth, genders, genetic defects, and spawning dates. In each tank, there are about 20 zebrafish—slim members of the minnow family, each about the size of an AA battery. They scoot through the water while Antoszewski decides which tank to choose. All the zebrafish have stripes, as one might guess from their name. They have kidneys, too. Any vertebrate—or animal with a backbone—has organs that are similar to those in humans. The ancient Hebrew priests knew this well from their animal sacrifices.

Eventually, Antoszewski pulls a tank off the shelf, disconnecting it from the matrix that filters and regulates the lab’s tanks. She carries the tub of fish—all plump females swollen with eggs—to the breeding area, careful not to spill any water. The tank’s label indicates the fish are kidney-defect carriers. Several years before, Hukriede and his team exposed some zebrafish to chemicals that caused genetic defects in their DNA. They kept the fish that showed symptoms of kidney problems, like swelling around the heart caused by excess water not released through urine. The team has continued breeding the fish as a way to investigate what isn’t working in the kidneys. They use that information to decipher how normal kidneys grow and function.

In the lab, Antoszewski heads back into the stacks, walking quickly in her purple lab clogs, to select a tank of males tagged as defect carriers. For her experiment, all of the fish need to have healthy kidneys, but they also need to have genes for kidney defects hidden in their DNA. When she returns, she scoops the male and female zebrafish into a special breeding tub. A spawning frenzy begins. By the next morning, hundreds of needlepoint-size balls have dropped through a filter at the tub’s bottom. They’re fertilized eggs, which will mature into embryos. According to Gregor Mendel’s long-taught principles of hereditary probability, a quarter of the embryos won’t grow proper kidneys because of the defective genes they inherited from their parents’ DNA.

The next morning, Antoszewski gently pours the hundreds of eggs, along with some water, into a Petri dish. The eggs are transparent. Using a microscope, she will be able to watch the embryos develop. Unlike mammalian babies that grow inside their mothers, or baby birds inside opaque eggshells, or other fish inside colored eggs, the zebrafish develop in clear bubbles, allowing rare glimpses into how bodies—and organs—grow.

As a bonus, zebrafish growth occurs rapidly. Within one day, a single cell emerges as an embryo with blood vessels, a heart, and a brain. Within three days, the embryo has a complete digestive system. And, as vertebrates, zebrafish share genetic similarities with humans. Their genes have counterparts in humans, so the fish offer an especially useful model for studying the molecular basis of human biological development.

In exactly 24 hours, Antoszewski examines the embryos through her microscope. Already, she can see their curved backbones. She notices that some of the embryos don’t have any podocytes, which are balls of specialized kidney cells that filter excess water and minerals from the blood to be processed into urine. In these fish, there are no balls of cells.Interesting, Antoszewski thinks. The team can use this malformation to help pinpoint the gene or genes responsible for the defect. She snaps digital photographs with her high-tech microscope. She can’t wait to show Hukriede.

Zebrafish originated in the Ganges River in India. In the late 1960s, George Streisinger, a biologist and amateur aquarist, began experimenting with the fish at the University of Oregon. Because zebrafish have backbones, he knew they shared anatomical features with humans. They reproduced regularly, laid clear eggs, and were easy to care for. When Streisinger died of cardiac arrest while scuba diving in 1984, his colleagues continued to use his fish in their research, and they published several papers.

Then, an eminent German biologist, Christiane Nusslein-Volhard, read those papers from Oregon. For years, she had used fruit flys to study genetics, but she immediately recognized the potential of zebrafish to advance the work of scientists who research how human bodies grow. She began encouraging colleagues worldwide to use zebrafish as an animal model, a message that carried even more weight after she won the 1995 Nobel Prize in Physiology or Medicine. One of her notable accomplishments was developing mutant animal models and then tracing the errors in their DNA, the same process that Hukriede and Antoszewski are applying to those zebrafish without podocytes.

In the early 1990s, Igor Dawid—a biologist from the National Institutes of Health (NIH) and Hukriede’s future mentor—attended an international zebrafish conference where Nobel Laureate Nusslein-Volhard spoke. Dawid was intrigued by the animal model’s possibilities and later shared his interest with Arthur S. Levine, who, at the time, was a scientific director at the NIH. Levine backed the idea and helped to open an NIH zebrafish facility, one of the first in the nation. Since 1998, Levine has been Pitt’s senior vice chancellor for health sciences and dean of the School of Medicine. He was the primary motivator in establishing Pitt’s zebrafish facility.

Hukriede, who earned his doctorate from the University of Rochester, joined Dawid’s NIH lab as a postdoctoral fellow in 1997. Dawid recalls that Hukriede “gave the lab spirit” and was always “throwing everybody else into excitement” with his questions. During Hukriede’s tenure as a postdoc, he mapped part of the zebrafish’s DNA, which Dawid describes as “a very valuable contribution to the zebrafish community as a whole.” Other scientists have used the DNA maps in their own research.

Hukriede also identified a protein that controls kidney development in the very beginning stages of embryo growth. And he screened zebrafish to search for genetic defects that might affect kidney development, which has formed the basis of the podocyte work that he and Antoszewski are conducting today.

In 2002, Hukriede was one of several top zebrafish researchers who were recruited by Levine to help build the Pitt aquatic laboratory and associated research program. Although all of these School of Medicine scientists/professors use zebrafish eggs in their research, they all study different parts of physiology: Nathan Bahary looks at gastrointestinal formation in relation to treatments for cancer and inflammatory bowel syndrome; Xiangyun Wei examines retinas and the possibility of engineering artificial retinas; and Michael Tsang investigates congenital skeletal defects. Meanwhile, Hukriede probes the origins of the kidney, an organ he considers beautiful even if it doesn’t hold souls. Since their arrival, they’ve drawn others from across the University into zebrafish research, too; and they launched the Fish and Frog Club, which meets monthly to share research findings and advice.

A few days after Antoszewski’s experiment with the kidney-defective fish, she pops into Hukriede’s office for their weekly meeting. Welcoming people and making them feel comfortable is characteristic of Hukriede. Colleagues describe him as approachable and friendly. He’s a similar “kidney” to those uncles or brothers who are always willing to talk about life while driving a few golf balls. (A secondary definition of the word kidney, “to describe a person’s temperament,” is a vestige of the ancient belief that kidneys housed personalities.)

After some small talk, Antoszewski tells her mentor about her experiment: “They don’t have any podocytes.”

“Really?” he responds, surprised. Most genetic defects don’t eliminate entire cell types.

On his computer, Hukriede clicks on his research team’s online lab journal. He opens the photos that Antoszewski took with her microscope. Together, they compare the fish with podocytes to those without. Many questions arise: Which gene caused the kidney defect? Did the podocytes not form at all, or did they start to grow and then die? How might this relate to human kidney formation?

Hukriede could ask long series of questions about almost anything. When he took lawn mowers apart as a kid, he constantly asked himself why this part went here or there, trying to figure out how it all worked. In college biology classes at Minnesota State University Moorhead, he enjoyed learning how human organs functioned. In his mind, they were like minimachines. Later on, at the University of Rochester, he realized that kidney research was a niche where he could pursue tough questions and potentially help a lot of people.

In his meeting with Antoszewski, Hukriede has many more questions about the podocyte experiment, but since it was an initial test, the two can’t be certain that defective DNA eliminated those embryos’ podocytes.

“Well,” Hukriede finally says, “do it again.” Like many scientists, he doesn’t believe in eureka moments or miracle discoveries. He believes in hard work and sporadic luck. That means repeating experiments and constantly pondering.

Since that meeting, Hukriede and Antoszewski have been making progress. They’ve determined which chromosome carries the defective gene, and they’re searching to find the culprit. Then, they will be able to figure out which proteins are involved, and how those proteins build podocytes in zebrafish embryos.

They’ll be closer to answering the all-important question: How do kidneys grow? Better yet, all kinds of answers will continue flowing from Pitt’s aquatic lab, thanks to the zebrafish whose swirling waters brim with hope.

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