Development of a Flexible Multiplex Urine RNA Assay for Detection and Differentiation of Kidney Allograft Injury

ABSTRACT Non-invasive approaches to detect kidney graft injury and distinguish donor-specific immune-mediated injury from other causes of inflammation are needed to guide recipient therapy while avoiding the morbidities of transplant biopsies. We used a multi-plex platform to interrogate RNA isolated from kidney transplant recipient urine to investigate gene expression patterns distinguishing grafts with no injury vs. ongoing injury and further differentiate acute T cell-mediated rejection (TCMR) from BK virus nephropathy (BKVN). As a training set we quantified expression of 796 immune function genes from 25 control recipients with stable graft function, 17 with biopsy-proven acute TCMR, and 13 with biopsy-proven BKVN. We identified a 20-gene signature that differentiated intragraft injury from grafts with stable function (area under the curve (AUC), 0.991) and a distinct 40-gene signature distinguishing acute TCMR from BKVN (AUC = 1.00). Validation in separate 118 urine RNA samples obtained at time of surveillance or for-cause biopsies from Clinical Trials in Organ Transplantation (CTOT)-08 and CTOT-19 studies showed AUC of 0.77 for the 20-gene injury signature and AUC of 0.79 for the 40-gene signature. Our results highlight the utility of this flexible, non-invasive biomarker platform for rapid detection and differentiation of immune processes causing ongoing kidney graft injury. Competing Interest Statement The authors have declared no competing interest. Funding Statement This study was funded by the National Institutes of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers UO1 AI84150, UO1 AI84146, and U01 AI063594 and by the Clinical Trials in Organ Transplantation Consortium NanoString Core (U01 AI063594). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRBs of Cleveland Clinic, University of Alabama Birmingham, University of Maryland School of Medicine, Northwestern University, Emory University, Icahn School of Medicine at Mt. Sinai gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes ↵* Drs. Mannon and Fairchild are co-senior authors of this report DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding authors upon reasonable request. Abbreviations - ABMR - antibody-mediated rejection - AUC - area under the curve - BKVN - BK virus nephropathy - CTOT - Clinical Trials in Organ Transplantation - DEG - differentially expressed genes - FSGS - focal segmented glomerular sclerosis - GEP - gene expression profiles - KTR - kidney transplant recipients - TCMR - T cell mediated rejection