Increasing evidence strongly supports a role for growth factors and oxidized lipids in the pathogenesis of Diabetic Nephropathy (DN), largely due to their pro-fibrotic and growth promoting effects. However the molecular events involved are not well understood. In the previous funding period, we identified novel signaling mechanisms by which key factors associated with DN, namely transforming growth factor-beta1 (TGF-b), Angiotensin II and high glucose (HG), could cross talk with the lipoxygenase (LO) pathway of arachidonate metabolism and its oxidized lipid products to augment hypertrophy and extracellular matrix (ECM) gene expression in glomerular mesangial cells (MC). But the precise nuclear epigenetic mechanisms involved are not clear and will therefore be evaluated in this renewal. Gene transcription can be regulated by epigenetic mechanisms in the chromatin that include posttranslational modifications (PTMs) of nucleosomal histones. Histone PTMs, such as Histone H3 -lysine methylation (H3Kme), can modulate chromatin states to dictate the 'active'or 'inactive'states of gene promoters. Our preliminary results demonstrate that key epigenetic histone H3Kme dependent mechanisms are involved in the expression of fibrotic genes in MC in response to TGF-b and diabetic conditions. We now propose to extend these new observations to evaluate the MC epigenomic states and their functional relevance to DN. We will use state-of the-art approaches to determine epigenetic mechanisms by which TGF-b, HG and LO products induce transcriptional regulation and sustained expression of fibrotic genes in renal MC. We will also examine whether agents that block these diabetogenic stimuli can reverse the epigenetic events in vitro and in vivo in mouse models. The Central Hypothesis is that, under diabetic conditions, increased activity and expression of TGF-b and 12/15-LO and their cross-talk can lead to enhanced expression of extracellular matrix genes in MC via novel epigenetic mechanisms.
Specific Aim 1 is to perform candidate and genome-wide profiling of key H3Kme marks in MC treated with and without TGF-b, and then determine potential associations between the identified methylated candidates and gene expression levels.
Specific Aim 2 is to determine whether the effects of TGF-b and HG can be mediated by 12/15-LO.
Specific Aim 3 is to examine the in vivo relevance of TGF-b and 12/15-LO induced chromatin histone PTMs in mouse models of DN. The results of these conceptually and technologically innovative studies can greatly increase our understanding of the epigenetic mechanisms involved in the persistent microvascular complications of diabetes such as DN, with major therapeutic implications.
Diabetic Nephropathy is a major complication associated with diabetes that can lead to significantly increased rates of end stage renal disease and painful dialysis. Despite the availability of several therapies, the rates of diabetic nephropathy are soaring, leading to a drain on our healthcare resources. Since transforming growth factor-beta1 and 12/15-lipoxygenase have been implicated in these pathologies, we propose to identify new mechanisms by which these agents can induce the expression of pathologic genes in glomerular mesangial cells and promote renal dysfunction and diabetic nephropathy. We will use state-of-the-art technologies to achieve our Specific Aims and thereby advance our long-term goal to identify novel mechanisms and drug targets for diabetic nephropathy.
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