VIRTUUS Children's Study

Advances in immunosuppressive regimens have significantly improved short-term allograft
survival for kidney transplant recipients. Yet, long-term allograft survival remains static.
For children with end-stage renal disease (ESRD), improvements in long-term outcomes are
greatly needed. Children with ESRD require multiple transplants over a lifetime, incurring
repeated surgical and immunological risks with each newly transplanted organ. Allograft
injury occurs primarily due to acute cellular rejection (ACR) and/or antibody mediated
rejection (AMR) and viral infections, such as BK virus associated nephropathy (BKVN). A major
hindrance to promoting long-term allograft survival is the lack of non-invasive diagnostic
and prognostic biomarkers to reliably detect early injury in the allograft before clinical
manifestations arise.

The incidence of acute rejection (AR) in children in the first year post-transplant is
10-13%[1]. The current gold standard for diagnosing AR is core needle biopsy; however, biopsy
is highly invasive, incurs risk of bleeding and graft loss, is subject to sampling error and
lacks sensitivity and specificity for early injury. Since immune responses are dynamic over
time, single biopsies do not adequately capture anti-allograft immunity, but repeat biopsies
are impractical in children who often require sedation and hospitalization for biopsies.
Other markers, such as serum creatinine, have low sensitivity and specificity for early
kidney allograft damage.

The ability to identify sub-clinical kidney allograft injury using minimally invasive,
robust, biomarkers with high sensitivity and specificity in pediatric recipients would
represent a major advance in pediatric kidney transplant care. In the Clinical Trials in
Organ Transplantation (CTOT)-04 study, a NIH-sponsored, multicenter, prospective study of
adult kidney allograft recipients, members of the VIRTUUS team were able to diagnose and
predict ACR using urinary cell mRNA and metabolite profiles with high sensitivity and
specificity[2, 3]. In addition, the investigators validated a urinary cell mRNA signature
that distinguishes acute rejection (AR) from acute tubular injury (ATI) and ACR from AMR as
well as a urinary cell mRNA signature diagnostic and prognostic of BKVN[3-5]. The overarching
objective of this VIRTUUS proposal is to adapt existing validated adult noninvasive
diagnostic and prognostic biomarkers to characterize allograft status in pediatric recipients
of kidney allografts. Specifically, the investigators will investigate 1) whether the adult
urinary cell 3-gene signature is diagnostic and prognostic of ACR in pediatric recipients of
kidney allografts, 2) whether the combined metabolite and the urinary cell 3-gene signature
is diagnostic and prognostic of ACR in pediatric recipients of kidney allografts, 3) whether
levels of BKV VP-1 mRNA in urinary cells are diagnostic of BKVN, and 4) whether urinary cell
levels of plasminogen activator inhibitor -1 (PAI-1) mRNA and serum creatinine levels predict
allograft failure.

Investigators propose to validate early immunologic markers that have shown to be prognostic
and diagnostic in adult kidney transplant recipients in pediatric kidney transplant
recipients. Investigator findings will significantly advance the field of pediatric
transplantation by moving toward proactive, tailored immunosuppressive regimens that minimize
morbidity and optimize long-term allograft survival.

The Investigator's primary objective is to hypothesize that: (i) the adult urinary cell
3-gene signature will be diagnostic and prognostic of ACR in longitudinally collected urine
samples from children with kidney transplants; and (ii) combined metabolite and mRNA
biomarkers have greater ability to diagnose ACR than the mRNA or metabolite signature alone
and (iii) levels of BKV VP-1 mRNA are diagnostic of BK virus nephropathy (BKVN) and (iv)
urinary cell levels of plasminogen activator inhibitor-1 (PAI-1) mRNA and serum creatinine
levels predict allograft failure.

Investigators seek to:

1. Determine if the adult urinary cell 3-gene signature is diagnostic and prognostic of ACR
in pediatric kidney allograft recipients,

2. Evaluate whether a combined metabolite and the 3-gene urinary mRNA signature is
diagnostic and prognostic of ACR and

3. Test the hypothesis that BKV-VP-1 mRNA levels in urinary cells are diagnostic of BKVN,
and to test the hypothesis that a two variable prediction model composed of urinary cell
level of PAI-1 mRNA and serum creatinine levels, both measured at the time of BKVN
biopsy diagnosis, predict future graft failure

Secondary objectives include the following:

1. Create a repository of DNA samples from urine, saliva, discarded blood and tissue, and
deceased donor blood and tissue to use for future research studies that will examine
genome-wide associations with rejection and viral infections in pediatric kidney
transplant recipients and

2. Create a biobank of samples of left-over blood and deceased donor blood to later examine
associations between urine and blood proteomics and metabolomics.

Inclusion Criteria:

- Males or females between 2 to 18 years at the time of recruitment

- Receiving the first, or additional, incident kidney transplants.

- Have an existing/prevalent transplant with a scheduled kidney allograft biopsy.

- Parental/guardian permission (informed consent) and, if appropriate, child assent.

Exclusion Criteria:

• Patient's primary medical team feels the subject's participation is not safe.