PROJECT SUMMARY Progressive renal decline is the central manifestation of diabetic nephropathy (DN) that leads to end-stage renal disease (ESRD). In participants of the Joslin Kidney Study (JKS), a longitudinal investigation of the natural history of DN in Type 1 diabetes (T1D), we recently demonstrated that microRNAs (miRNAs) involved in transforming growth factor (TGF)-β1 mediated renal fibrogenesis are deregulated early in patients who are at risk for progression to ESRD. Importantly, this deregulation occurs prior to the initiation of renal function decline, suggesting that these miRNAs have potential utility as novel biomarkers of the risk of progression to ESRD in DN. We hypothesize that additional miRNAs exist that play important roles in renal function decline in patients who are at risk of progression to ESRD but have not yet been studied. The identification of these miRNAs will lead to the discovery of novel factors involved in the pathogenesis of renal function decline. The goals of the proposed research project is to further leverage biobanked specimens from the JKS, along with innovative next-generation sequencing technology, to i) determine the expression profile of the full complement of miRNAs that are differentially expressed early in T1D patients who are at risk for progression to ESRD and prior to the initiation of renal function decline and ii) to begin to investigate the mechanisms by which these miRNAs contribute to disease pathogenesis. These goals will be accomplished through three Specific Aims. 1) To establish a comprehensive set of candidate miRNAs for early renal function decline. We will i) apply next-generation sequencing technology (miRNA-Seq) to assess miRNA levels in baseline plasma specimens from 80 non-progressors and 80 rapid progressors from the JKS and ii) define a set of candidate miRNAs for early renal function decline in T1D using robust statistical methodologies. 2) To validate the candidate miRNAs and examine their expression in additional T1D patients with early renal function decline. We will i) perform cross-platform validation (qPCR) of the candidate miRNAs identified in Aim 1 in all 80 non-progressors and 80 rapid progressors and ii) validate these miRNAs in 159 additional non-progressors and rapid progressors from the JKS. 3) To identify the miRNA:target interactions that are altered in early renal function decline. We will i) identify the biologically significant targets of the top-ranked differentially expressed candidate miRNAs identified in Aim 2 and ii) experimentally validate and functionally assess these miRNA:target interactions. Our approach is highly innovative and involves a multidisciplinary team of investigators with expertise in all aspects of the proposed research. Our implementation of this study will define currently unknown factors in DN and may lead to the identification of novel therapeutic targets to prevent progression of renal decline in T1D.