Detecting hidden threats: proteomic changes in urinary extracellular vesicles are associated with pediatric kidney allograft rejection

BACKGROUND: Early detection of kidney allograft injury is essential to preserve long-term graft function. Current diagnostics, particularly biopsy, are invasive and may be inconclusive. Extracellular-vesicles (EVs) carry molecular cargo that reflects renal cellular states. Although EV proteomics has shown promise for detecting acute rejection (AR) in adults, its value in pediatrics(PKT) is not well defined. METHODS: In this pilot study, urine samples from 29 PKT recipients and 3 healthy controls were analyzed, including 7 with biopsy-confirmed AR, 9 with chronic rejection (CR), and 13 with stable graft function (ST). Classification was based on urine findings, creatinine trends, and biopsy results. EVs were isolated from fresh-second-void urine by ultracentrifugation and analyzed using LC/MS. Longitudinal samples from three AR patients assessed temporal proteomic changes. RESULTS: Overall, 192 proteins were differentially abundant across groups (FDR p < 0.05). AR showed enrichment of immune-related pathways and increased free hemoglobin proteins compared with ST, while CR closely resembled ST. EV concentration and size distribution were similar across groups, indicating proteomic differences were independent of EV abundance. Longitudinal analyses revealed dynamic proteomic changes corresponding to rejection and recovery. CONCLUSIONS: EV proteomics is a promising approach for detecting and monitoring AR in PKT recipients and warrants larger multicenter studies. IMPACT: This pilot study demonstrates urinary extracellular vesicle (EV) proteomics as a promising non-invasive approach to detect and monitor acute rejection (AR) in pediatric kidney-transplant (PKT) recipients. EVs carry molecular cargo reflecting kidney cell states, providing a dynamic readout of graft health and injury. Early identification of rejection may reduce reliance on invasive biopsies, enable timely intervention, and support precision-based, personalized management. As the first study to characterize EV proteomic profiles in PKT, it lays important groundwork. Larger multicenter studies are warranted to validate these findings.