Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection
Authors: El Fekih Rania , Hurley James , Tadigotla Vasisht , Alghamdi Areej , Srivastava Anand , Coticchia Christine , Choi John , Allos Hazim , Yatim Karim , Alhaddad Juliano , Eskandari Siawosh , Chu Philip , Mihali Albana B. , Lape Isadora T. , Lima Filho Mauricio P. , Aoyama Bruno T. , Chandraker Anil , Safa Kassem , Markmann James F. , Riella Leonardo V. , Formica Richard N. , Skog Johan , Azzi Jamil R. 
Published: March 2021

Significance Statement
The traditional biomarkers currently used to monitor a kidney allograft for rejection are late markers of injury and they lack sensitivity and specificity. Allograft biopsies on the other hand, are invasive and costly. The authors describe the discovery and validation of two urinary exosomal mRNA multigene signatures for the diagnosis of acute T cell–mediated and antibody-mediated rejection and chronic, active antibody-mediated rejection in recipients of kidney transplant. Using a clinically validated platform for exosome isolation and analysis, they demonstrated the high stability of urinary exosomes and the reliability of this approach in monitoring patients for allograft rejection. One gene signature for all-cause rejection and another for discriminating T cell–mediated rejection from antibody-mediated rejection showed high predictive performances and offer clinicians the possibility of new tools for monitoring emergence of rejection in kidney allografts.

Background
Developing a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells’ proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection.

Methods
Using 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets.

Results
An exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature’s negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell–mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature’s negative predictive value was 90.6% and its positive predictive value was 77.8%.

Conclusions
Our findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.