Noninvasive Diagnosis of Acute Antibody Mediated Rejection of Human Kidney Transplant Recipients by Measurement of Serum MicroRNAs

Abstract

Introduction: Micro(mi)RNAs are single stranded RNAs that have a profound impact on mRNA/protein expression and have been advanced as cancer biomarkers. Herein, we investigated the hypothesis that extracellular/circulating levels of miRNAs are diagnostic biomarkers of acute antibody mediated rejection (AMR) of human kidney allografts. Methods: We isolated total RNA from 36 individuals; 12 patients with AMR biopsies, 20 patients with normal biopsies and 4 healthy volunteers. Discovery: We did global expression profiling of serum miRNAs from 7 kidney transplant recipient (3 AMR and 4 Normal) and 4 healthy volunteers. Differentially expressed (P< 0.1) miRNAs were queried and a panel of miRNAs associated with endothelial and immune cell functions were identified. The miRNA with least variability within 4 healthy volunteers was used for normalizing other miRNAs to adjust for biological variability. Verification: The differential expression was verified by measuring miRNAs in the same serum samples by targeted miRNA specific realtime (RT) PCR assays. Validation: We did targeted miRNA specific RT-PCR assay of the miRNAs in the serum of 25 kidney transplant recipients; 9 AMR and 16 Normal. Sera were spiked with C. Elegans miR-54 synthetic RNA and were used for normalizing other miRNAs to adjust for technical variability. The fold differences between AMR and Normal were calculated. Results: Of the 377 mature miRNAs analyzed in the Discovery set, 38% were measureable (Ct < 35) in sera from AMR patients and 41% in sera of normal patients. Unsupervised hierarchical clustering of circulating miRNA levels correctly classified the two groups and circulating levels of 17 miRNAs were significantly different between AMR and Normal (Figure 1).[Figure 1]Six of the 17 distinguishing miRNAs were associated with endothelial and/or immune cells. We measured their levels in the sera from 25 kidney transplant recipients (AMR=9 and Normal=16). We validated that the circulating levels 5 of the 6 miRNAs are significantly higher in AMR than Normal (Figure 2).[Figure 2]Conclusion: Our data demonstrate that extracellular/circulating levels of miRNA expression are associated with AMR of human renal allografts. Our discovery and validation of circulating levels of miRNAs as biomarkers of AMR introduces a novel strategy to noninvasively diagnose AMR.