Discovery of Donor-Derived Exosomal DNA as an Exploratory Biomarker of Kidney Graft Rejection: A Cross-Sectional Study

Circulating donor DNA has emerged as a valuable tool for clinical decision-making in kidney transplantation. While most studies focus on cell-free DNA, the role of donor DNA associated with extracellular vesicles (EVs) remains unexplored. To address this, we analyzed donor-derived exosomal DNA (dd-exoDNA) in 100 kidney transplant recipients (KTR) undergoing surveillance or indicated biopsies. Serum exosomes were isolated using precipitation-based technology, and dd-exoDNA was analyzed via digital PCR targeting donor/recipient HLA-DRB1 mismatches. Dd-exoDNA levels were higher in rejection versus non-rejection (2.66 [0.56–7.10] ×10 −3 vs. 0.69 [0.28–1.71] ×10 −3 , p = 0.004) and were associated with Banff score items: glomerulitis ≥1 ( p = 0.037), peritubular capillaritis ≥1 ( p = 0.040), and tubulitis ≥2 ( p = 0.043). In multivariate analysis, dd-exoDNA remained independently associated with rejection, although with wide confidence intervals (OR [95%CI] 3.68 [1.32–10.26], P = 0.013). Exploratory threshold analyses suggested moderate discriminative performance. These findings indicate that donor DNA associated with circulating EVs may offer complementary information to existing biomarkers, warranting validation in external cohorts and comparison with established assays.