About 1 in 5 cases of the kidney-destroying condition nephrotic syndrome don’t respond to steroid treatment. They are a leading cause of end-stage kidney failure in children and young adults, who are quickly forced to go on dialysis or wait for a kidney transplant.
Thanks in large part to the lab of Friedhelm Hildebrandt, MD, chief of the Division of Nephrology at Boston Children’s Hospital, more is becoming known about this severe condition. Mutations in more than 30 genes have been implicated, all causing dysfunction of glomeruli, the kidney’s filtering units, specifically in cells known as podocytes. Test panels are now clinically available. Yet, in 70 percent of patients, the causative gene is still unknown.
A new study by Hildebrandt and colleagues in this week’s Nature Genetics pinpoints three new, completely unexpected genes, revealing the power of whole-genome sequencing and potentially opening a new treatment route for at least some steroid-resistant cases.
With exome sequencing, you can find a cause without having to speculate where to look.
The three genes encode a group of proteins that make up the nuclear pore complex (NPC), a structure found in every cell in the body. The NPC’s job is to help shuttle large molecules into and out of the cell nucleus. It’s such an essential part of cell operations that normally, a mutation in any of its components would be lethal — a baby wouldn’t survive beyond the embryonic stage.
“No one would have ever thought nuclear pore proteins would be a place to look in chronic kidney disease,” says Hildebrandt.
Yet, when Hildebrandt and research fellow Daniela Braun, MD, did whole-exome sequencing in 160 families with steroid-resistant nephrotic syndrome, they found 10 children with mutations in nuclear pore protein 93 (NUP93) or one of two companion NPC proteins. All had developed nephrotic syndrome between 1 and 6 years of age.
Why was their disease not lethal, given the fundamental functions of these proteins? The mutations in the genes were functionally very mild, says Hildebrandt.
“These patients can still make a little bit of the nuclear pore protein that has some residual function,” he says. “So they ‘only’ get nephrotic syndrome.”
A means to an end
The NPCs in children with NUP93 mutations were compromised to varying degrees, as shown by the broken-up nuclear membranes (in red) in columns 2 and 4-6 of this image. (The far-left column shows a normal cell nucleus with an intact membrane.)
“These fundamental proteins sometimes have multiple unrelated functions and do things completely novel and unexpected,” he explains. “Subtle mutations in these proteins can lead us directly to what goes wrong in these cells.”
Looking more closely at what goes wrong, Hildebrandt, Braun and colleagues found that NUP93 and its companion proteins were unable to bind to certain signaling proteins called SMADs. Normally, this binding enables SMADs to go into the nucleus, where they turn on genes that prevent fibrosis, or tissue scarring — thereby protecting against a key pathological process in nephrotic syndrome.
“Some of patients’ mutations abrogate this binding between SMAD and NUP93, so SMADs don’t go into the nucleus anymore,” says Hildebrandt.
In the image below, the far left column shows SMAD protein neatly tucked into the nucleus; in the other five columns, where NUP93 is mutated, a lot of SMAD is hanging around in the cytoplasm:
Delving further, the researchers identified two other molecules that also act on SMADs that could be plausible therapeutic targets: TGF-beta, which is known to cause fibrosis and organ damage in other diseases, and BMP7, which keeps TGF-beta signaling in check.
To efficiently screen for potential drugs that inhibit TGF-beta, or boost BMP7’s activity, Hildebrandt and colleagues have created and begun using a novel “podocyte migration assay.” Basically, researchers take videos of podocytes moving under a microscope: the speed and amount of movement is an indicator of their well-being before and after drugs are added to the system.
“Our results emphasize the power of whole-exome sequencing in identifying disease genes that would never have been predicted from cell biological data,” says Hildebrandt. “With exome sequencing, you can find a cause without having to speculate where to look.”