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kidney transplant

Inside Pediatrics, Nephrology

Researchers Get One Step Closer to Non-invasive Test for Kidney Rejection

Doctor conducting kidney exam on child.

Monitoring kidney transplant rejection in children is akin to sticking your hand into five pots of water, four of which could burn you. The only surefire way to know if a child is rejecting the organ is with a biopsy. The procedure is invasive, requires anesthesia, carries risks of complications and is expensive. In other words, it’s very hot water. 

Yet every kidney transplant patient at Children’s of Alabama receives a routine biopsy six months after transplant. Only about one 1 in 5, however, actually show signs of rejection, meaning most of those biopsies were unnecessary. Now imagine there was a simple blood or urine test to tell which children were likely to reject the kidney and need a biopsy. That could mean going from 1 in 5 biopsies positive for rejection to 4 in 5 or 5 in 5, sparing hundreds of children from a painful procedure they don’t need.

Pediatric nephrologist Michael E. Seifert, MD, and his team have been working for years on developing such a test, using a large biorepository of patients’ blood, urine and kidney biopsy tissue collected throughout and after the transplant process.

Their work involves investigating gene expression in the tissue samples to find signals of rejection. But while they are good at identifying abnormalities from a piece of biopsy tissue, the process still has room for improvement. With the way tissue is processed, it’s difficult to determine if the abnormal signals are coming from cells that are relevant for rejection—such as immune cells—or from cells that don’t play a role in rejection.

Now, Seifert and his lab are using a novel technique called spatial transcriptomics, or spatial gene expression assays, which enable them to “see” the signals in the context of their natural habitat without destroying the underlying tissues.

“Spatial transcriptomics allows you to develop non-invasive biomarkers that are more reflective of the underlying biology of the disease you’re interested in, such as rejection,” Seifert said. And those more precise biomarkers could narrow down the number of patients who require biopsies. “This will help us understand the mechanisms of kidney transplant injury and rejection with much higher precision,” he said.

Before this technique, they used one of two methods to study gene expression in the tissue. One is to take the tissue, grind it up, see which genes are high and which are low, then develop a test based on the findings. The other is to separate a piece of biopsy tissue into its component cells and individually examine their gene expression. That’s more precise than the bulk gene expression or grinding method, Seifert said, but you lose any spatial context as to where in the tissue the cell came from.

One way to think about it is having all your furniture jammed into a pod in the front yard, taking a chair into the house, and hoping it’s the right piece for that spot by the window. But without the rest of the furniture in the room, it’s hard to know. With spatial transcriptomics, he said, you’re viewing the chair in context with the rest of the furniture.

“The spatial platform is a really incredible tool in that it allows you to be so precise in the areas of the kidney that you’re studying,” Seifert said. He can also isolate cells he’s interested in from the rest of the tissue without disturbing the tissue itself. “Being able to keep the tissue intact enables you to assign geographic locations for the different signals you’re getting when you test the tissue,” he said. 

“We’re just beginning to learn all the ways we can apply it to kidney transplant diseases.”

He and his team presented their first paper on their findings using the new platform at the American Transplant Congress in Boston in June.

Inside Pediatrics, Nephrology

Tracking Kidney Transplant Rejection in the Blood and Urine 

Michael Seifert, M.D., is a pediatric nephrologist at Children’s of Alabama and an associate professor in the Division of Nephrology in the University of Alabama at Birmingham Department of Pediatrics.

Michael Seifert, M.D., is a pediatric nephrologist at Children’s of Alabama and an associate professor in the Division of Nephrology in the University of Alabama at Birmingham Department of Pediatrics.

Children who receive kidney transplants at Children’s of Alabama undergo a routine biopsy six months after the procedure to look for signs of rejection. In about 20 percent of patients, those signs are there, even if their blood and urine tests look normal. “On the flip side,” said Children’s pediatric nephrologist Michael E. Seifert, M.D., “that means about 80 percent of our patients are getting biopsies that are normal.” 

The holy grail, then, would be a non-invasive biomarker test using blood or urine that can identify kidney transplant injury without needing a biopsy — which is exactly what Dr. Seifert and his lab are working on. Their research relies on a biorepository of patients’ blood, urine, and kidney biopsy tissue collected throughout and after the transplant process. 

One of the blood tests they’re working on uses technology originally developed to screen maternal blood for signs of fetal abnormalities. The test looks for cell-free DNA, or cfDNA, which comes from the fetus and differs from maternal DNA. Since transplanted kidneys also have DNA different from the patient’s own kidney, “you can look in the bloodstream for the proportion of the DNA coming from the transplant versus the normal background from the recipient’s non-transplant cells,” Dr. Seifert said. High levels of cfDNA is a sign of acute kidney injury that could be due to rejection.  

Studies in adults validate this as a good method to detect rejection. “But the problem is, those are done in adults whose native kidneys would be roughly similar in size to the transplant they’re carrying,” Dr. Seifert said. Children often have a much larger, adult-sized transplant compared to the size of their native kidneys, so the cutoff levels used for diagnosing rejection in adults may not work in children. “We’re trying to make these existing diagnostic tests more pediatric specific,” he said. 

The story is different for urinary biomarkers. It’s been known for years that the kidney releases certain proteins when it’s injured. Tests to detect those proteins, however, can take days. But a new device called SimplePlex, currently available only in the research setting, can measure several of these proteins at one time in a single sample in less than an hour.   

“We’re looking at ways to get this technology closer to the clinic, so you’re not just profiling kidney transplant patients’ risk for injury based on their standard blood tests like creatinine, but you’re also adding these additional biomarkers that can tell you more information about what’s happening inside the organ,” Dr. Seifert said.  

Being able to determine via blood and urine tests who needs a biopsy and who doesn’t would have a huge impact for pediatrics, particularly at Children’s, where these biopsies are done on a universal basis, he said. Ideally, the tests could also provide information on the underlying cause of the rejection. For instance, the cfDNA test is good at picking up antibody-mediated rejection, but not as good at recognizing cell-mediated rejection. The urine biomarkers are good at identifying both rejection types, but not at identifying rejection related to viral infections. 

“Once we understand more about the clinical scenarios in which the biomarkers perform well,” said Dr. Seifert, “we’ll be able to design interventional trials to treat the patient based on the biomarker changes rather than just the standard clinical tests such as creatinine.”  

Nephrology

Continual Performance Improvement in Pediatric Kidney Transplantation

The renal transplant team at Children’s of Alabama has performed more than 500 kidney transplants since 1968. To this day, continuous improvement remains at the heart of everything it does.

The hospital is part of the Improving Renal Outcomes Collaborative (IROC), a learning health system of 32 pediatric kidney transplant centers in the U.S. that share data and best practices in an effort to improve transplant outcomes.

“We know we can achieve better things more efficiently together than if we’re working individually,” said Children’s pediatric nephrologist Michael E. Seifert, M.D. 

Improvement Projects

One recent project involved improving blood pressure assessments. “We know that if we control blood pressure we get better patient outcomes and the transplant does better,” Seifert said. Yet an analysis of 17 IROC transplant centers found that blood pressure was being measured appropriately based on current guidelines at just 12% of transplant clinic visits.

Each IROC center had the freedom to design custom tools to fit its needs for improving blood pressure measurement. The answer at Children’s was paper-based tracking logs and regular meetings to review progress. The team also educated all staff on the importance of measuring blood pressure and how to measure it according to the most recent guidelines. “It was pretty simple things that, when applied systematically and consistently, led to prolonged improvement,” Seifert said. Today, at least 85% of clinic visits include an appropriately measured blood pressure and the team is working to improve that number.

The next project is to improve adherence to immunosuppressive drugs, a major risk factor for rejection and loss of the kidney transplant. The team is developing a questionnaire for parents and patients so they can identify the barriers to adherence and develop targeted interventions. “We have to get away from an accusatory approach to a partnership and ask, ‘How can we work with you to make it easier to take your medications?’” Seifert said.

Children’s is also a national leader in studying surveillance biopsies to help reduce acute rejection rates. Most pediatric transplant centers do not perform early surveillance biopsies at pre-specified time points because of their invasive nature, but Seifert and his team demonstrated that surveillance biopsies in the first six months after transplant can detect subclinical inflammation, which is associated with a nearly threefold increased risk of acute rejection and allograft failure. Treating patients who demonstrated such inflammation, they recently reported, significantly reduces that risk. Importantly, they also demonstrated that the  surveillance biopsy procedure was safe for pediatric patients, with extremely low rates of mild adverse events.

Bringing the Bench and the Clinic Closer

The transplant team also runs a robust translational research program, with half of transplant patients enrolled in at least one research study. One is a biorepository study in which patients’ blood, urine and kidney biopsy tissue is collected throughout and after the transplant process. “Then we can develop biomarkers of kidney transplant diseases that impact the survival of the transplant,” Seifert said.

The second study will identify determinants of cardiovascular health in pediatric and young adult kidney transplant recipients who have a high burden of cardiovascular risk. “Transplantation improves but doesn’t eliminate this risk,” Seifert said. “This study is

designed to understand certain unique cardiovascular risk factors, such as the impact of

early life stress, on cardiovascular and renal outcomes.”

The Transplant Experts

Learn more about kidney transplantation at Children’s of Alabama.