Transforming growth factor beta1, MicroRNAs and Diabetic Nephropathy

描述（由申请人提供）：糖尿病肾病（DN）是糖尿病的主要并发症之一，可导致终末期肾病。DN的主要特征包括由于细胞外基质（ECM）沉积导致的肾小球硬化和系膜细胞增宽增加。虽然一些生化途径和关键的促纤维化因子，如转化生长因子-β 1（TGF-β）和ECM蛋白胶原蛋白，已被牵连在DN的发病机制，微妙的分子和核机制调节他们还不清楚。我们最近发现了系膜细胞（MCs）中TGF-β关键基因靶点与称为micro-RNAs（miRs）的小非编码RNA家族成员之间的新联系。越来越多的证据表明miR在基因调控中发挥重要作用，因为它们可以通过与靶基因的mRNA结合来抑制靶基因的表达。尽管已经预测了miR的几个靶点，但仍需要做很多工作来确定其生物学和疾病相关性。miR在正常和疾病条件下的调节机制也不清楚。我们建议在DN的背景下探索这些方面。我们发现TGF-β下调参与胶原调节的关键抑制因子，并且这些因子也被肾脏和MC中表达的两种特异性miR靶向和下调。这些miR的表达在用TGF-β处理的MC中增加，并且在糖尿病小鼠的肾小球中也增加。此外，我们观察到这些miR可以触发增强胶原蛋白表达的调节和前馈机制。因此，我们提出了新的假设，即DN中TGF-β的增加导致关键miR及其靶基因的异常产生和作用，从而导致肾小球硬化增强。具体目标1将检查TGF-β上调MC中主miR及其下游效应miR的转录机制。Specific Aim 2将评估下游miR的特异性基因靶点以及它如何促进TGF-β介导的胶原调节。具体目标3将通过评估抗miR寡核苷酸在糖尿病小鼠中的治疗潜力以及通过检查miR敲除小鼠中DN的进展来测试体内功能相关性。我们的初步结果揭示了迄今为止尚未探索的TGF-β作用机制。这项最先进的研究可以通过破译肾脏中这些难以捉摸的小RNA的生物学功能，在肾脏研究领域开辟新天地并产生重大影响。它们还可以为糖尿病肾病的新疗法铺平道路。公共卫生相关性：糖尿病在美国非常普遍，是一个主要的医疗保健问题。这进一步放大了它与显著加速和衰弱的并发症如糖尿病肾病相关的事实。该项目旨在确定可能导致开发急需的新疗法以降低糖尿病肾病发病率和死亡率的新机制。

DESCRIPTION (provided by applicant): Diabetic nephropathy (DN) is one of the major complications of diabetes that can lead to end stage renal disease. Key features of DN include increased glomerulosclerosis and mesangial cell widening due to extracellular matrix (ECM) deposition. Although several biochemical pathways and key profibrotic factors, such as transforming growth factor-beta 1 (TGF-¿) and the ECM protein collagen, have been implicated in the pathogenesis of DN, the subtle molecular and nuclear mechanisms regulating them are unclear. We recently uncovered a novel connection between key gene targets of TGF-¿ in mesangial cells (MCs) and members of a family of small non-coding RNAs called micro-RNAs (miRs). Increasing evidence shows that miRs play important roles in gene regulation since they can suppress the expression of target genes by binding to their mRNAs. Although several targets of miRs have been predicted, much needs to be done to determine their biological and disease relevance. The mechanisms by which miRs are regulated under normal and disease conditions are also not clear. We propose to explore these aspects in the context of DN. We discovered that TGF-¿ downregulates key repressive factors involved in collagen regulation, and that these factors are also targeted and downregulated by two specific miRs expressed in the kidney and MCs. The expressions of these miRs are increased in MCs treated with TGF-¿ and also in the glomeruli of diabetic mice. Furthermore, we observed that these miRs can trigger regulatory and feed forward mechanisms that enhance the expression of collagen. We therefore put forward the novel hypothesis that increased TGF-¿ in DN leads to the aberrant production and actions of key miRs and their target genes which result in enhanced glomerulosclerosis. Specific Aim 1 will examine the transcriptional mechanisms by which TGF-¿ upregulates a master miR and its downstream effector miR in MCs. Specific Aim 2 will evaluate the specific gene target of the downstream miR and how it contributes to TGF-¿ mediated collagen regulation. Specific Aim 3 will test the in vivo functional relevance by evaluating the therapeutic potential of anti-miR oligo-nucleotides in diabetic mice, and by examining the progression of DN in a miR knockout mouse. Our preliminary results have uncovered novel hitherto unexplored mechanisms of action of TGF-¿. This state-of-the-art study could break new ground and have a major impact in the field of renal research by deciphering the biological functions of these elusive small RNAs in the kidney. They could also pave the way for novel new therapies for diabetic kidney disease. PUBLIC HEALTH RELEVANCE: Diabetes is highly prevalent in the USA and a major healthcare problem. This is further magnified by the fact that it is associated with significantly accelerated and debilitating complications such as diabetic nephropathy. This project proposes to identify novel new mechanisms involved that could lead to the development of sorely needed newer therapies to reduce the morbidity and mortality of diabetic nephropathy.