Targeting NINJ1 protein could make donor livers more resilient

Key study findings

• The protein NINJ1 is highly expressed in human liver tissue  
• It is directly linked to injury and dysfunction after transplant  
• Cells most susceptible to NINJ1 injury can be protected by drug therapy

Results suggest that NINJ1 is a potential target for therapy to ensure donor livers can withstand injury during transplant. 

During transplantation, blood flow to the donor liver must stop as it’s moved to the recipient. Once transplanted, oxygen-rich blood is restored but the act of doing so can cause what’s called ischemia-reperfusion injury. To protect transplanted livers from this tissue damage, researchers at The Hospital for Sick Children (SickKids) have discovered a way to protect dying cells from rupturing and affecting the surrounding area.  

Published in Science Advances, their study identifies a specific protein, ninjurin-1  (NINJ1), as a key driver of ischemia-reperfusion injury (IRI) resulting from that sudden return of blood. By blocking NINJ1, the research team reduced liver injury in pre-clinical models, opening a potential new strategy to improve transplant outcomes.  

The study brought together cell biology and paediatric liver transplantation expertise at SickKids with adult liver transplantation at Toronto General Hospital (TGH), part of University Health Network (UHN). 

"There are over 600 liver transplants in Canada each year and, while each one saves a life, that new organ can risk being injured in the process," says Dr. Blayne Amir Sayed, Surgical Lead of Liver Transplantation and Hepatobiliary Surgery at SickKids, Associate Scientist in Cell & Systems Biology and co-senior author of the study. "We sought a new path to intervene before an injury could happen in order to deliver a strong, healthy liver to each recipient."

Why dying cells can damage a transplanted organ 

During transplantation, donor livers temporarily lose normal blood flow. As circulation is restored, the return of oxygen can trigger a second wave of injury. IRI causes cells to die and can provoke intense inflammation, compromising the liver's function after surgery and risking long-term complications. 

NINJ1 controls an important final step in the process of IRI. When activated, its molecules assemble in the outer membrane of dying cells and cause it to break open, releasing inflammatory contents into the surrounding tissue. 

"NINJ1 acts very late in cell death," says Dr. Benjamin Steinberg, co-senior author, Staff Anesthesiologist and a member of the Liver Transplant Anesthesia team, and Scientist in Neurosciences & Mental Health at SickKids. "Whether a dying cell stays contained or bursts open is essential and can dramatically change how the surrounding tissue  responds to a new liver.” 

The researchers believe that, rather than blocking every pathway that can lead to cell death, they could intervene at this final step instead. That makes NINJ1 a promising target for future treatments. 

Mapping NINJ1 in the human liver 

Using single-cell sequencing and spatial transcriptomics to measure gene activity in individual cells, the researchers mapped NINJ1 across the human liver and found it was prominent in hepatocytes and Kupffer cells, two cell types that play important roles in liver function and immune responses. 

Could NINJ1 play a similarly impactful role in transplantation? The team analyzed liver samples from adults undergoing transplantation at TGH and found that increased NINJ1 activity shortly after blood flow returned was linked to a severe complication called early allograft dysfunction, whereby the liver is less effective in the days following surgery.  

"We identified the mechanism in the lab, understood where it occurs in the human liver, and then saw the same process associated with injury in transplanted organs," says Sayed.   

The researchers then tested whether NINJ1 was actually causing liver injury. In lab models of IRI, removing NINJ1 significantly reduced reduced acute liver damage. Removing NINJ1 specifically from hepatocytes or immune cells called macrophages protected the liver, showing that both cell types are involved in injury.  

Dr. Jan Mossemann, a postdoctoral fellow in the Sayed Lab and co-first author of the study, helped show that both hepatocytes and Kupffer cells suffer membrane rupture linked to NINJ1 in transplant-like conditions. Importantly, this process could be blocked with a drug. 

Improving donor organ health before transplantation 

One advantage of targeting NINJ1 is that a donor organ could be treated directly, outside the body, using machine perfusion technology. In the future, the researchers say such therapies could be added while blood flow returns to a donor liver before it reaches the recipient, making the organ more resilient and improve transplant safety.  

"Every donor organ is extraordinarily valuable," Steinberg says. "If we can make them more resilient to the unavoidable stresses of transplantation, we may be able to improve outcomes and expand the number of organs that can be used."

This October marks 40 years since the first liver transplant performed at SickKids. Today, it runs one of the busiest liver transplant programs in North America, performing about 40 transplants each year. Sayed says that researchers are looking to the biology of individuals cells to find newer ways to ensure success.  

"We have become remarkably good at replacing a failing liver," Sayed says. "Outcomes for young patients have improved dramatically in recent decades, but we still have work to do to ensure that a child’s liver transplant is for life. We must ask how to make every transplanted liver as healthy and resilient as possible."

** Top photo: Liver image showing immune cells (magenta), architecture (cyan), and cellularity (yellow). Using advanced imaging, the Sayed and Steinberg labs have been able to visualize cell death and tissue damage over space and time. (Credit: Dr. Ori Scott) 

This study was supported by Genetech and funded by the Canadian Institutes of Health Research (CIHR), University of Toronto, Translational Research Seed Grant through Ajmera Transplant Centre at UHN, American Society of Transplant Surgeons, and the Team Izzy Innovator Award through SickKids Foundation.