Objective: We determined whether urinary cell mRNAs predict response to anti-rejection therapy in kidney allograft recipients with Banff grade IA or higher ACR biopsies.

Methods: We developed real-time quantitative PCR assays and measured absolute levels of mRNAs in urinary cells from 4300 urine specimens from 485 kidney allograft recipients enrolled in the Clinical Trials of Transplantation 04 study. We measured 25 mRNAs encoding proteins pivotal to T cell activation, inhibition, co-stimulation, and regulation, and mRNAs for innate immunity, cytokines, and cytotoxic proteins, and 18S rRNA.

Results: Kruskal-Wallis tests of 18S rRNA-normalized and log-transformed urinary cell levels of mRNAs demonstrated that CD3, CD4, CD8, CD25, CD27, CD28, CD80, CD86, FOXP3, CTLA4, TGF-β1, interferon gamma, granzyme B, perforin, IP-10, or CXCR3 were significantly different among urine specimens matched to ACR biopsies (43 samples, 34 patients), urines matched to No Rejection biopsies (162, 126), and urines from Stable Patients (1522, 201). Among the mRNAs in urine collected at the time of an ACR biopsy and analyzed for predicting ACR reversal (defined as return of eGFR to within 15% of pre-treatment levels), mRNA for FOXP3 (area under the receiver operating characteristic curve (ROC AUC): 0.80, 95% CI, 0.64 to 0.96); IL-2 (0.74, 0.57 to 0.91); CD28 (0.72, 0.52 to 0.92); CD103 (0.73, 0.55 to 0.90); or CXCR3 (0.71, 0.51 to 0.91) predicted ACR reversal. Stepwise logistic regression was used to develop a parsimonious gene signature; a weighted sum of FOXP3, TGF-β1, and IL-6 mRNAs was found to be the best predictor of ACR reversal (0.89, 0.77 to 1.00, Fig.1A) and was superior to the predictive accuracy of FOXP3 mRNA alone (P<0.01, Fig.1B).

Conclusions: In addition to discovering novel diagnostic biomarkers of ACR, we have developed a noninvasive and mechanistically-informative three-gene signature for predicting reversal of ACR. FOXP3, a specification factor for T-regulatory cells, is the dominant component of this signature.

CITATION INFORMATION: Luan D., Schwartz J., Ding R., Muthukumar T., Dadhania D., Suthanthiran M. Identification of Noninvasive and Mechanistically-Informative Biomarkers Predictive of Reversal of Acute Cellular Rejection (ACR) in Kidney Allografts Am J Transplant. 2017;17 (suppl 3).

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