Developing kidneys from scratch

To Alex Hughes, Assistant Professor in Bioengineering within Penn Engineering and in Cell and Developmental Biology within Penn Medicine, the kidney is a work of art. “I find the development of the kidney to be a really beautiful process,” says Hughes.

Most people only ever see the organ in cross-section, through textbooks or by dissecting animal kidneys in high school biology class: a bean-shaped slice with lots of tiny tubes. “I think that really undersells how amazing the structure is,” says Hughes, who points out that kidneys grow in utero like forests of pipes, branching exponentially.

The Rhythm of the Kidneys

Densely packed with tubules clustered in units known as nephrons, kidneys cleanse the blood, maintaining the body’s fluid and electrolyte balance, while also regulating blood pressure. The organ played a crucial role in vertebrates emerging from the ocean: as one paper puts it, kidneys preserve the primordial ocean in all of us.

Unfortunately, kidneys struggle in the modern world. Excessively salty food, being overweight, not exercising enough, drinking too much and smoking can all raise blood pressure, which damages the kidney’s tiny blood vessels, as does diabetes.

In some cases, damage to the kidney’s nephrons can be slowed with lifestyle changes, but, unlike the liver, bones and skin, which can regrow damaged tissue, kidneys have a limited capacity to regenerate. At present, without a transplant, the nephrons we have at birth must last a lifetime.

The Burden of Chronic Kidney Disease

Today, one in ten people worldwide — more than 850 million in all, including more than one in seven Americans — suffers from chronic kidney disease, or CKD. The condition is hard to initially detect. According to the Centers for Disease Control, as many as 90% of Americans with CKD are completely unaware of the condition. The disease is also progressive and incurable. By 2040, CKD is expected to be the fifth-leading cause of years of life lost globally.

Eventually, CKD leads to kidney failure, at which point there are only two treatments: dialysis — which costs tens of thousands of dollars per year, frequently causes pain and requires patients to spend hours each week hooked up to machines that filter the blood — or kidney transplantation. The waiting list to receive a new kidney in the United States is roughly 100,000 people and three to five years long.

Even if everyone born today adopted healthier lifestyles, millions would still suffer from the disease. The most common prenatal developmental abnormalities involve the kidneys and urinary tract, impacting 2% of all births, or nearly three million babies each year. “There is a huge clinical burden of kidney disease,” says Hughes. “And there are relatively few engineers trying to come up with new solutions.”

To that end, the Hughes Lab focuses on elucidating the mechanisms behind kidney development and using those insights to create kidney tissue from scratch, which could reduce the need for both dialysis and transplantation. “I think there’s just enormous opportunity to think about synthetically reconstituting kidney tissues for regenerative medicine,” says Hughes.

Building without a Blueprint

To grow artificial kidneys, researchers like Hughes first need to understand how nature builds the organ. This is harder than it sounds. Everyone’s heart and circulatory system look more or less the same, but no two pairs of kidneys are exactly alike.

Kidneys form as their tubules branch, a variable process that leads to some people’s kidneys having nine times as many nephrons as others — and potentially many more times the filtration power and lifespan. “There’s a lot of variability in how many nephrons we have,” Hughes points out, referring to the kidney’s tiny, functional unit. “If you have fewer nephrons, does that mean that you have a higher chance of chronic kidney disease? The research seems to support this.”

The mechanisms that govern that branching process and nephron formation have long been poorly understood. “It’s like a city’s water distribution network,” says Hughes, “but it’s being built by these cells that somehow collectively know what to build and where their neighbors are and what junctions to make, all without a blueprint.”

In a recent paper in Nature Materials, Hughes and his lab discovered a potential governor of kidney growth: tiny mechanical stress waves, which occur when the kidney’s densely packed tubules bump into one another. “Imagine being in an elevator and the elevator’s packed with people already,” says Hughes. “If you keep adding people, it will create this mechanical stress— you’d literally be pushing people away with your elbows.”

Hughes and his collaborators — including co-first authors Louis Prahl, a postdoctoral scholar in the Hughes Lab, Jiageng Liu, a Bioengineering doctoral student, and John Viola, a Bioengineering Ph.D. graduate  — carefully analyzed microscopic images of developing animal kidneys at different times to determine their geometry and pressed on the organs with tiny tools to measure their rigidity. The more tightly packed the tubules, which increases over time, the stiffer the tissue. As tubule branching continues, they found that each additional branch caused a pulse of mechanical stress, which the team believes may constitute one of the signals for nephron formation.

Each tubule, Hughes’ group concluded, essentially competes for space with its neighbors. In other words, there’s no master plan the kidneys follow, helping to explain why the number of nephrons in mature kidneys differs from person to person. The finding suggests that kidney development is something like an improvised dance, with each tubule reacting to the touch of its neighbors.

In videos created by the Hughes Lab to visualize the process, adjacent nephrons form one after the other, as if they were following a beat. “It’s still a hypothesis,” adds Hughes, “but we think that the stem cells that are around these tubules are effectively listening for these mechanical stress waves to guide their decision making about when to form a nephron or when not to.” If researchers can simulate that rhythm, they might be able to guide the development of artificial kidneys, which would represent a tremendous leap forward in treating CKD.

The Right Ingredients

At the moment, artificial kidney tissue — in the form of clusters of cells known as organoids — is far from clinical usefulness. Whereas normal kidneys involve an ordered collection of different cell types, organoids typically wind up as chaotic masses of cells in the wrong places.  “You can create the right cell types,” says Hughes, “but their spatial organization is incorrect for the most part.”

Kidneys’ spatial organization is crucial — a water filtration plant can’t work if the pipes don’t line up. Unfortunately, the tubules in organoids typically display insufficient branching and fail to drain into a single exit point. In other words, they can’t fulfill the kidney’s most crucial functions: filtering waste from the blood and ensuring that waste exits the body. “There needs to be a lot of engineering innovation in how we guide those tissues to be more lifelike,” says Hughes.

Part of the problem is that kidney organoids require at least three different types of stem cells: one for the tubules, one for the nephrons and one for support structures like blood vessels. Unlike, say, gut organoids, which model intestinal tissue and can be grown from a single type of stem cell, kidney organoids are inherently more complicated.

In a second recent paper, in Cell Systems, the Hughes Lab proposed a novel solution: create tiny communities of the various cell types, patterned in a mosaic. By adjusting the ratios of each stem cell type, the researchers were able to influence the composition of the organoid.

Hughes and his coauthors — first author Catherine Porter and Samuel Grindel, both Bioengineering doctoral students, and Grace Qian, a 2023 Bioengineering graduate and current doctoral student at the University of California Berkeley and University of California San Francisco — developed custom microwells, in which they grew a variety of different combinations of kidney stem cells, almost like bakers trying out different recipes.

As the ratios changed, the researchers noticed a “peak” in tubule formation, suggesting an optimal composition for growing kidney tissue, which they termed the “goldilocks” ratio. “If we change the ratio, we see quite different compositions of the organoid,” says Hughes. “So you can treat these as designer organoids where you have control over the outcome.”

From Lab to Clinic

Ultimately, Hughes hopes to combine these dual insights — into the mechanical stress waves that influence kidney development, and the ratios that shape organoid formation — into clinical applications. “You can imagine as these organoids are differentiating,” he says, “you could simulate that rhythmic process and see if suddenly you can kick off a larger-scale outcome.”

The urgency of developing alternatives to transplantation and dialysis is hard to overstate. At present rates, there will never be enough kidneys for transplantation. “I think that’s a big gap that engineers can hope to fill,” says Hughes. In his office, he keeps his great-grandfather’s pocket watch, a reminder of how function and form go hand in hand when it comes to designing intricate mechanical objects. The watch still runs.

These studies were conducted at the University of Pennsylvania School of Engineering and Applied Science and supported by the National Science Foundation (DMR-2309043 and CAREER Awards 2339278 and 2047271) and National Institutes of Health National Institute of General Medical Sciences (R35GM133380), National Institute of Diabetes and Digestive and Kidney Diseases (R01DK132296), and Eunice Kennedy Shriver National Institute of Child Health and Human Development (K25HD097288 and R21HD112663).

Additional co-authors of the papers referred to include Aria Zheyuan Huang, Trevor J. Chan, Gabriela Hayward-Lara and Catherine M. Porter of Penn Engineering, and Chenjun Shi and Jitao Zhang of Wayne State University.