Chronic antibody-mediated rejection (AMR) is a major cause of renal allograft rejection. Yet despite its clinical importance an integrated understanding of how responses to antibody and complement mediated attack are regulated by the transplanted kidney has not been established. This gap in our knowledge is due at least in part to an incomplete understanding of responses made by the kidney that result in gene regulation promote or prevent injury. MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression post-transcriptionally. miRNAs play an important role in regulating renal injury. However, the study of miRNAs in rejection and renal injury has largely been based on analysis of total cellular miRNAs that are differentially expressed during disease. This approach is problematic because it does not provide information on the mRNAs targeted by these miRNAs. To address this issue, we asked whether it is possible to isolate miRNAs and the mRNAs they are targeting in the RNA-Induced Silencing Complex (RISC) by isolating RNAs cross-linked to the RNA Binding Protein (RBP) AGO2. Using this approach we defined the first miRNA-mRNA interaction map for transplant related renal injury. These proof-of-principle studies revealed that within the miRNA-mRNA targetome it is possible to defined miRNAs and the mRNAs they target that undergo unique changes in cells undergoing injury. Pathway enrichment analysis indicated that miRNAs present in the RISC complex target mRNAs encoding proteins in pathways that may contribute to injury. Based on these studies, we hypothesize that the miRNA-mRNA targetome can be used to identify gene pathways that contribute to AMR. To test this hypothesis, we will use a clinically relevant murine model to determine the miRNA-mRNA map for AMR and use information elucidated by the targetome to examine gene pathways under regulation by miRNAs. We examine the clinical relevance of our findings by examining whether similar changes occur in human kidney transplants. These studies will provide unique insight into process that drive pathology associated with AMR, information that could be used to distinguish AMR from other types of injury, and provides a novel resource to the transplantation and wider scientific community.

In this proposal we will examine the role of post-transcriptional regulation of gene expression by microRNAs in antibody-mediated rejection of renal transplants.