Post-transcriptional regulation of gene expression by microRNAs in antibody-mediated rejection.

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: Specific Aims. Chronic antibody-mediated rejection (AMR) is the major cause of late chronic renal allograft failure. 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 how responses made by the kidney that result in changes 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 AMR 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 about which mRNAs these miRNAs target. To address this issue, we isolated 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. 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 model to determine the miRNA-mRNA interaction map or targetome for AMR and use the targeting information obtained to examine the importance of gene pathways under regulation by miRNAs in AMR. A key issue for these studies is how to interpret the relevance of changes in expression due to antibody mediated rejection (AMR). In this R56 we therefore propose to use digital special expression profiling to globally identify areas in renal tissue that exhibit changes in gene expression and refine this analysis into distinct cell types and assess clinical relevance. Specific Aim 1: Developing a digital special profiling approach to assess global changes in miRNA and mRNA expression in renal allografts. In heterogeneous tissue such as the kidney it is critical to determine cell types exhibit alterations in miRNA and mRNA expression as a result of AMR. Moreover, it is critical to define the extent to which changes occur in order to prioritize the importance in various cell types. To address this issue, we propose to use digital special expression profiling using NanoString’s GeoMx Digital Spatial Profiler (DSP) in proof-of- principle studies to use profiling to globally identify areas in mouse renal tissue undergoing AMR that exhibit changes in gene expression and refine this analysis into distinct cell types. We will focus on miR-21, which undergoes at least a ten-fold change in expression during AMR and targets known cell death pathway mRNAs. Using DSP coupled with histology, we will examine which regions of the kidney exhibit similar relative changes in miR-21and expression of its targets. Once regions showing similar differential expression are identified, we can conduct additional studies using labeled cell-type specific antibodies and RNA probes to identify cell types exhibiting changes to further refine our studies. Specific Aim 2: In this aim we will directly address clinical relevance by examining the extent to which changes in our murine model are observed in biopsy material from transplant patients. We will use an approach like that described above to determine if similar changes are observed in human kidneys undergoing AMR. Narrative Chronic antibody-mediated rejection (AMR) is the major cause of late chronic renal allograft failure. Yet in spite of 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 how responses made by the kidney that result in changes 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 AMR 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 about which mRNAs these miRNAs target. To address this issue, we isolated 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. 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 model to determine the miRNA-mRNA interaction map or targetome for AMR and use the targeting information obtained to examine the importance of gene pathways under regulation by miRNAs in AMR. A key issue for these studies is how to interpret the relevance of changes in expression due to antibody mediated rejection (AMR). In this R56 we therefore propose to use digital special expression profiling to globally identify areas in renal tissue that exhibit changes in gene expression and refine this analysis into distinct cell types. We will use this information to examine the extent to which these changes are observed in biopsy material from transplant patients that has been collected as part of the CTOT network.