Discovery and Validation of a
Urinary Exosome mRNA Signature
for the Diagnosis of Human Kidney Transplant Rejection

Key Takeaways

• Urinary exosomes originate from cells in the kidney and contain information from the parent cell; they are stable for up to two weeks at 4°C
• Urinary exosome mRNA profiles are shown to be strongly associated with biopsy-proven rejection
• The profiles can help discriminate between T cell–mediated rejection (TCMR) and antibody-mediated rejection (ABMR)
• This noninvasive test can improve the diagnosis of kidney transplant rejection and lead to better patient outcomes

Summary Statement

Examination of the urinary exosome mRNA profiles of patients undergoing biopsy for suspicion of rejection has identified a gene signature strongly associated with rejection. These profiles can be further subdivided by T cell–mediated rejection (TCMR) or antibody-mediated rejection (ABMR). Urinary exosome mRNA has a higher positive and negative predictive value than other rejection indicators, such as eGFR or donor-specific antibodies.

Background

Exosomes are small, 50–200 nm vesicles released by all cells. They mediate cell-to-cell communication by fusing with the target cell membrane and releasing contents, including messenger RNA (mRNA) from the parent cell, into the cell cytoplasm. The protein and mRNA content of exosomes reflects both the activation and disease state of the parent cell. The isolation and analysis of urinary exosome mRNA have been well validated for other tests, such as prostate cancer screening.

At the time of renal biopsy, 220 urine samples were collected from 175 patients who gave informed consent. Samples were divided into two groups based on the biopsy diagnosis of rejection or no-rejection (borderline diagnoses were initially excluded), and the rejection samples were subdivided into TCMR or ABMR. The results were compared with other established indicators of transplant rejection, such as a fall in estimated glomerular filtration rate (eGFR) or a rise in donor-specific HLA antibody (DSA) as detected by LABScreen™ Single Antigen assay.

The exosomes were shown to be stable in urine at 4°C for at least two weeks and longer at –80°C. The isolated mRNA was initially tested against a large panel of genes associated with inflammatory diseases. This was subsequently refined until a 15-gene signature panel was identified that could discriminate between any-cause rejection and no rejection. A second, overlapping five-panel signature was identified to help discriminate TCMR from ABMR.

There were 59 samples with biopsy-proven rejection and 133 samples without rejection. The AUC for all-cause rejection, as determined by the exosome panel, was 0.93 (95% CI, 0.87 to 0.98). This has a much higher predictive ability than eGFR (AUC 0.57) or DSA (AUC for ABMR 0.7, or 0.64 for any cause rejection).

The gene panel to distinguish TCMR from ABMR had an 87.5% sensitivity and 82.9% specificity, and the AUC was 0.87 (95% CI 0.76 to 0.97). Overall, the gene panel had a negative predictive value for any-cause rejection of 93.3% and a positive predictive value of 86.2%.

Of the samples initially excluded due to a borderline diagnosis on biopsy, only 25% with a negative exosome profile had a subsequent rise in eGFR, compared with 67% of patients with a positive profile, suggesting exosome analysis can assist in these borderline cases.

Summary

Urinary exosomes can be collected, stored, and easily transported at 4°C to provide a simple and effective method of post-transplant monitoring. The selected mRNA profiles have high negative and positive predictive abilities for both any-cause rejection and TCMR.

Conclusion

The analysis of the mRNA content of urinary exosomes is a safe, noninvasive method of post-transplant monitoring with a strong association with biopsy-proven rejection.

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