Kidney transplant rejection leads to short and long-term adverse clinical outcomes. Serum creatinine is a suboptimal late biomarker for rejection. Exosomes are nanometer-sized vesicles released by cells to mediate cell-to-cell communication by delivering genetic materials including mRNAs and microRNAs. In the transplanted kidney, exosomes originate from different cells including the immune cells during rejection. These cells release exosomes into the urine carrying parent cells' surface proteins and nucleic acids. The nucleic acid profile may carry disease signatures becoming potential biomarkers of disease activity and therapeutic targets.

We show that urinary exosomes, and their contents including RNA, are very stable and can be stored in the urine for 2 weeks at 4[deg]C, reinforcing exosomes as a potential source to identify disease signatures.

We isolated urinary exosomes, extracted mRNA, and performed differential gene expression analysis in 28 urine samples collected from 23 patients who underwent a kidney transplant biopsy for evaluation of acute rejection. The top performing gene expression signatures were validated in 38 samples collected from 32 patients who underwent a kidney transplant biopsy for suspicion of acute rejection. In addition, we isolated urinary exosomal protein profiles from 18 of the samples.

13/26 urine samples were from patients with any-cause acute rejection in the training set, and 23/38 urine samples in the validation set. Serum creatinine in patients with acute rejection in the training set was 2.4 [2.1 – 3.0] mg/dl and 2.0 [1.6-2.3] mg/dl in the validation set. Differential expression in the training set revealed 23 individual markers of rejection (P-value all < 0.05). A 3-gene exosomal mRNA signature of CXCL9 and CXCL10 normalized to IL17RA discriminated patients with any-cause acute rejection with c-statistics of 0.84 (0.68 – 1.00) and 0.74 (0.58 – 0.91) in the training and validation sets, respectively. The combined exosomal protein signature of CXCL9 and CXCL10 yielded a c-statistic of 0.83 (0.62 – 1.00).

Combination of exosomal CXCL9 and CXCL10 RNA normalized to IL17RA was able to predict any-cause acute rejection. Urinary exosomal gene expression may provide an accurate account of disease diagnosis.

CITATION INFORMATION: Assaker J., Srivastava A., Hurley J., Al Doujailan B., Eskandari S., Coticchia C., Mohamed M., Bentink S., Tadigotla V., Riella L., Chandraker A., Skog J., Azzi J. Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection Am J Transplant. 2017;17 (suppl 3).

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