PROJECT SUMMARY The overarching objectives of this proposal are to investigate whether existing urinary mRNA and metabolite profiles, which are diagnostic and prognostic for early renal allograft injury in adults, can be validated in pediatric kidney transplant recipients. Children with end-stage renal disease (ESRD) have a life expectancy at least 20 years shorter than children without renal disease. During their lifetime, most children with ESRD will require several kidney transplants to prolong duration and quality of life. Each transplant incurs new surgical and immunological risk. Despite substantial gains in improving short-term allograft outcomes through improved immunosuppressive regimens, the half-life of a kidney transplant is still only 10-12 years. Thus, there is a critical need to prolong allograft survival. A major hindrance to advancing long-term outcomes is the inability to reliably detect early allograft injury before clinical manifestations arise. Early allograft injury results from acute cellular or antibody- mediated rejection or early emergence of viruses, primarily BK virus which leads to BK virus nephropathy. This project’s specific aims are: 1) to validate highly sensitive and specific adult urinary cell mRNA signatures to diagnose and predict acute cellular rejection (ACR) in pediatric recipients, 2) to evaluate the clinical efficacy of urine metabolite profiling to diagnose and prognosticate ACR in pediatric recipients and to validate the sensitivity and specificity of a combined adult kidney allograft urinary mRNA and metabolite signature for ACR in pediatric recipients and 3) to investigate if BKV-VP-1 mRNA levels in urinary cells are diagnostic of BK virus associated nephropathy, and if urinary cell levels of Plasminogen Activator Inhibitor-1 mRNA and serum creatinine, predict future graft failure. These aims will be achieved through the collaborative effort of key pediatric kidney transplant programs across the United States and Canada, supported by two outstanding labs at Weill Cornell Medicine and Stanford. This study’s results will advance the ability to identify and characterize early allograft injury in pediatric kidney allograft recipients through non-invasive immune surveillance. By doing so, we will create opportunities to better inform clinical decision-making, improving practice paradigms to promote positive long-term graft outcomes.