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Kidney Transplant Rejection Biomarkers Sought in Newly Funded Urinary Omics Study


NEW YORK (GenomeWeb) – An international team led by investigators at the University of Pennsylvania is gearing up to do a large, multicenter project involving multi-omic profiling on urine samples to search for markers related to kidney transplant outcomes in children and adults.

Although the final tally of participating sites is still somewhat in flux, the researchers expect to enroll hundreds of children with kidney transplants for a newly-funded arm of the effort. They are also in the processes of following many more adult kidney transplant cases in the US and internationally.

The group plans to do prospective testing on urine samples from kidney transplant recipients, including targeted mRNA profiling and urinary metabolomics focused on 16,000 metabolites, in the hopes of finding metabolite and/or messenger RNA clues to identifying acute transplant rejection before symptoms of rejection appear. The team is also on the hunt for insights into rejection-related complications such as BK viremia or infections with other viruses following transplant-related immunosuppression.

If successful, the researchers hopes to find markers for staying ahead of rejection and secondary complications that can damage the transplanted organ and put recipients at risk by tapping a method that's less invasive — and widely implementable — than serum creatinine levels or organ graft biopsies levels currently used to detect early or impending rejection.

"Typically by the time people present with clinical symptoms of rejection, there's often irreversible graft damage by that point. It can also take about four years off the lifetime of the transplant graft," said study leader Brendan Keating, a genetics and transplantation researcher affiliated with the Children's Hospital of Philadelphia and the University of Pennsylvania.

Keating co-leads iGeneTrain, a consortium that is turning to omic approaches in its search for markers to track and understand everything from transplant rejection to secondary complications of organ transplantation. At the 2016 American Society of Human Genetics annual meeting, for example, Keating outlined efforts to understand ties between kidney or liver transplant outcomes using microRNA, metabolite, or single-cell RNA sequencing data.

Prior analyses hint that at least some mRNA levels are altered in urine samples from individuals who experience acute rejection after kidney transplantation. In particular, Keating pointed to a 2013 study in the New England Journal of Medicine by investigators from the Clinical Trials in Organ Transplantation project.

That study, which involved 485 kidney transplant recipients enrolled prospectively, revealed higher-than-usual levels of 18S ribosomal RNA gene transcripts and transcripts for CD3-epsilon and IP-10 (now called CXCL10) genes in the urine cell pellets of the 38 individuals who experienced biopsy-confirmed acute kidney transplant rejection.

Such findings may reflect immune functions involved in rejection, Keating explained, since CD-epsilon is a T-cell specific marker and IP-10 is involved in other T-cell mediated processes.

With that in mind, he and his colleagues plan to try to translate results from earlier research, while adding in a metabolite-profiling component, in a new longitudinal prospective study involving some pediatric kidney transplant recipients between the ages of two and 18-years-old.

The "Validating injury to the renal transplant using urinary signatures in children," or VIRTUUS, study is slated to include around 450 individuals with childhood kidney transplants and will involve investigators at up to a dozen sites in the US and Canada. Keating cautioned that the project may have to drop one or two study sites due to cuts in the budget of the National Institute of Child Health and Human Development (NICHD).

The investigators have been waiting on final approval of their R01 grant from NICHD to get the urinary omics study into full swing. Though the budget has shrunk by roughly 17 percent, the award is expected to start this month.

In the original proposal, the team requested roughly $4 million, though the final grant is expected to come in at least 17 percent lower. The project may lose two centers as a result, though the coordinators are looking at strategies for keeping as many centers and participants as possible.

With this general strategy, the team hopes to detect potential biomarkers for rejection and other relevant kidney transplant outcomes, and also obtain a better sense of the methods that are most effective for reliably detecting these markers, Keating explained.

"Because this is an opportunity to look at different methods together in [samples from] the same time points, we're also using an exosome method for looking at mRNA — and microRNA as well — and a third independent method," he said.

Using urine samples collected during the year after each individual's kidney transplant, the researchers intend to use quantitative real time-PCR to assess mRNA levels for genes such as CD3-epsilon, CXCL10, and 18S rRNA. The same samples will be subjected to metabolomic and, potentially, lipidomic analyses at Michael Snyder's Stanford University genetics lab, as well as validation testing with orthogonal mRNA profiling methods.

"We've spent the last four years optimizing both metabolomics and lipidomics," Snyder said, noting that current metabolic profiles may contain as many as 20,000 peaks or more.

"I think that opens the possibility of being able to see what's going on at a much better level than has been done before," he explained, noting that his team also plans to do some of the RNA profiling for the study.

Some of those analyses have been underway for a few months already, Keating said, noting that each urinary sample will be processed for several different types of analyses — from whole-urinary metabolites to experiments on urinary cell pellets.

In parallel, the investigators plan to collect saliva samples from the study participants for parallel, array-based genotyping, exome sequencing, and/or genome sequencing. That genetic data is expected to help in exploring patient genetic features associated with kidney transplant complications or rejection. If and when blood extra blood samples are available, they may also profile T-cell and other immune patterns.

On the adult transplant side, meanwhile, Keating and his team have enrolled roughly 170 adult participants at the University Pennsylvania so far and expect to ultimately get 350 kidney transplant recipients on board to participate at UPenn.

They are teaming up with international groups to add thousands more samples from adult kidney recipients to their analyses. For example, they plan to collaborate with investigators at King Faisal Specialist Hospital, which has a large pediatric transplant program and is ramping up to establish a clinical genomics lab, a precision medicine program, and a metabolomics and proteomics lab.

The American team is also working on the European Union-funded BIOMARGIN project — an effort by investigators at transplant centers across Europe to search for non-invasive biomarkers for kidney transplant injuries using multiple omics platforms.

"We are collecting data on RNA, so transcriptomics, but also miRomics, metabolomics, proteomics, and even peptidomics on all the same samples from the same patients," said Maarten Naesens, a nephrology researcher at the University of Leuven and member of the BIOMARGIN team.

For the first stage of BIOMARGIN, which began four years ago, the researchers carefully selected 120 well-characterized transplant cases, Naesens noted. Many more transplant individuals have been enrolled for subsequent stages of the project, including an ongoing prospective study involving around 500 patients.

Being able to compare candidate markers from BIOMARGIN with those identified in samples from the US is expected to be beneficial, he explained, since transplant rejection patterns can differ in the European and North American settings.

"It's extremely important to interact with teams like the team of Brendan Keating in the United States to have an independent validation possibility," Naesens said, noting that such proposed collaborations should help both teams validate candidate markers.

Keating and his colleagues plan to analyze metabolite patterns in samples from the adult transplant recipients for adult-specific markers, though the team also plans to do a pediatric-focused investigation and a joint analysis that includes metabolite profiles for both adult and pediatric cases.

Although the study is observational, it may lay the groundwork for markers that can be applied down the road to introduce interventions or enhanced monitoring for those who appear to be en route to transplant rejection. On the other hand, the availability of such markers might make it possible to ease off on immune suppression in individuals at lower rejection risk, which might protect against complications stemming from reduced immunity.

"If you had a highly robust — with high sensitivity and specificity — assay for prognostication in kidney, you could potentially use it to reduce immune suppression," Keating said, noting that "the side effects of immune suppression are pretty immense, especially in pediatric populations who are exposed to immune suppression for the rest of their lives."