PHILADELPHIA, July 30:  A human kidney is built once, before birth, and the rules guiding its construction have been largely inferred from animal models. Researchers have now read those rules directly in human tissue, showing that developing cells are steered by soluble signals released by their neighbors - and identifying insulin-like growth factor 2  as a key signal that sustains the kidney’s stem-like cells. The study was published today in Nature Genetics.

The study was conducted by the Penn-CHOP Kidney Innovation Center, a research collaboration between the Perelman School of Medicine at the University of Pennsylvania (Penn Medicine) and Children’s Hospital of Philadelphia focused on transforming patient care through early detection, prevention, and treatment of kidney disease - working together with bioengineers from Penn’s School of Engineering and Applied Science and the Institute for Regenerative Medicine.

How well a kidney is built before birth matters for the rest of a person’s life. Healthy people are born with anywhere from 200,000 to 2 million nephrons - the microscopic filtering units that clear waste from the blood — and no more are made after birth. A low nephron endowment carries a higher lifetime risk of high blood pressure and chronic kidney disease, and severe disruptions of kidney formation are the leading cause of chronic kidney disease in children.

The collaboration’s three-way structure was central to the result. The Kidney Innovation Center contributed the single-cell and spatial genomics used to build the map, along with the pediatric and fetal tissue that made it possible. The bioengineering group contributed the laboratory systems - cultured embryonic kidneys and stem-cell-derived organoids - needed to test whether a signal spotted in the map actually drives development. A map alone could not have shown that IGF2 does the work; an experimental system alone would not have known to test it.

How cells coordinate to build a kidney

To capture development as it occurs in intact tissue, the team combined single-cell RNA sequencing with two spatial transcriptomics platforms, analyzing more than 700,000 cells from human fetal kidneys between 12.5 and 20.5 weeks of gestation. Conventional single-cell sequencing requires dissolving tissue into separate cells, which discards exactly what development depends on: where each cell sits and which signals reach it. The spatial approach preserves that context, letting the researchers place every cell, and every stage of its differentiation, back onto the map of the developing organ.

“A kidney is not built by cells working alone,” said Katalin Susztak, MD, PhD, the Willard and Rhoda Ware Professor of Diabetes and Metabolic Diseases IV and co-director of the Penn-CHOP Kidney Innovation Center, who co-led the study. “Every cell has to know where it is, what its neighbors are releasing, and what it is supposed to become — and it has to get this right roughly a million times over. We could already see which cells were present. What we could not see were the signals passing between them.”

Because the researchers could follow differentiation across physical space rather than in the abstract, they were able to pinpoint the anatomical locations where developing cells commit to one path or another, and to revise the accepted sequence of those decisions in the human kidney.

A growth factor that keeps stem cells going

To find the signals driving these decisions, the researchers ran a genome-wide, spatially aware analysis of cell-to-cell communication, correlating the local presence of each soluble signal with the paths cells actually took. The analysis pointed to IGF2 as strongly associated with renewal of nephron progenitor cells, the stem-like population that gives rise to most of the nephron and is exhausted by late gestation. Two further signals, GDNF and RSPO3, were also linked to progenitor renewal.

The team then tested IGF2 directly. In cultured mouse embryonic kidneys, adding IGF2 increased branching of the ureteric tree and enlarged the caps of progenitor cells sitting at each branch tip. Blocking the receptor did the opposite, shrinking and disorganizing those caps and, at higher doses, eliminating the progenitor population. The same pattern held in human kidney organoids grown from stem cells: blocking the receptor reduced progenitor cells and tubular structures, while adding IGF2 expanded both.

The researchers also introduced a way of describing developing tissue that does not depend on classical anatomy. Rather than defining a cellular neighborhood by which cell types happen to sit nearby, they defined it by the full set of soluble signals a cell is bathed in. These signal-based neighborhoods tracked real developmental biology and, in several cases, cut across boundaries anatomists have traditionally drawn.

“Validating IGF2 as a stem cell renewal factor was important because it shows our approach works, but it also has implications for organoid technology and how the developmental environment can affect kidney health later in life,” said Jonathan Levinsohn, MD, PhD, a pediatric nephrologist at Children’s Hospital of Philadelphia and co-first author of the study. “Laboratory-grown kidney tissue is a rapidly developing field. Understanding how to improve growth is a concrete step towards generating organoids that function and better resemble the human kidney.”

Human genetics points the same way: variants near IGF2 and its receptor were recently associated with differences in the adult human kidney size in a large population study, and the new work offers a mechanism for that association.

“The kidney is a physical structure, and physical context is not incidental to how it forms — it is instructive,” said Alex J. Hughes, PhD, of Penn’s School of Engineering and Applied Science and the Institute for Regenerative Medicine, who co-led the study with Susztak. “Being able to read the signaling environment a cell actually experiences, rather than inferring it from the cell types nearby, changes what we can build.”

Cells can change their minds

One of the study’s central findings is that cell fate in the developing kidney is not sealed at the moment of commitment. Cells that had already started down the path toward becoming proximal tubule cells were observed converting into other cell types of the renal corpuscle, the kidney’s filtering structure.

That flexibility may resolve a longstanding puzzle. The chemical gradients thought to pattern the kidney are minute, yet able to produce a million correctly proportioned nephrons. The findings suggest these early gradients might not need to be perfect. Instead patterning appears to work in two passes: early signals lay down a permissive blueprint, while later signals fine-tune individual cells to match their final position.

“We have long assumed that once a kidney cell commits to a fate, it stays there,” Susztak said. “Our data show that development is more forgiving than that. The tissue lays down a rough plan and then corrects itself. I suspect this is how the kidney manages to build a million nephrons and get nearly all of them right.”

The framework is not specific to the kidney, and the same approach could be applied to any developing organ whose cells must coordinate across space.

“If we want to grow kidney tissue that behaves like the real thing, we have to know what the cells are saying to one another,” Susztak said. “This work gives us a first draft of that vocabulary. The same principles should apply to how tissues repair themselves after injury, and to diseases in which cells lose their identity.”

Additional collaborating institutions included Sheba Medical Center and Tel Aviv University in Israel, Meyer Children’s Hospital IRCCS and the University of Florence in Italy, Vanderbilt University Medical Center, and Northwestern University.

The study was funded in part by the U.S.-Israel Binational Science Foundation (2021164), the American Society of Nephrology Ben J. Lipps Research Fellowship, the DFG German Research Foundation (DU 2449/1-1), and Open Philanthropy (10080664 and 10095457).