Urine exosomes are small vesicles shed into the urine by exocytosis from all renal epithelial cell types under normal physiologic states and disease states, and contain proteins that can mirror disease specific proteomics perturbations in kidney injury. Methodologies for analyses of the exosomal fraction of the urinary proteome require additional development and the benefit of urinary exosomal versus unfractionated proteomic analysis for biomarker discovery is as yet unknown. The purpose of this study was to determine specific proteomic components and their differences between the soluble (Uw) and exosomal (Ue) fractions of urine and the differential impact on both Uw and Ue in biopsy confirmed acute renal allograft rejection (AR). Methods: Mid-stream, clean catch urine samples were collected from 30 demographically matched kidney transplant recipients with and without AR (nAR). A centrifugal filtration approach was adapted for improved isolation of urine exosomes. The proteomic components of Uw and Ue were analyzed by mass spectroscopy using iTRAQ and data analysis utilized SPSS for significance of differential abundance of proteins in Uw and Ue after transplantation with and without AR. Results: We identified 1018 proteins in Uw and Ue of transplantation as measured by iTRAQ and 349 proteins in Ue. 279 overlapped between the two urinary compartments and 70 of the Ue proteins were completely unique to the exosomal compartment. Eleven proteins, functionally involved in an inflammatory and stress response, were highly abundant in urine samples from patients with biopsy confirmed acute rejection, of which 3 were exclusive to the exosomal fraction. The AR specific proteins showed greater abundance in the Ue fraction [Figure 1], suggesting that these disease specific biomarkers are enriched in the Ue compartment due to exocytosis of renal tubular epithelial cells during the tubulitis injury of transplant rejection. Conclusions: We used a strategically rapid method for isolating urinary exosomes and demonstrate quantitative enrichment and perturbation of Ue proteins, specific to inflammatory and stress responses, in rejection. Further study of these specific Ue specific protein alterations is underway in the rejecting allograft to provide improved mechanistic insights into tissue injury in transplant rejection.

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