Metabolic regulation of diabetic nephropathy

描述(由申请人提供)： 糖尿病肾病是糖尿病的主要并发症，是世界范围内终末期肾病的主要原因。高血糖通过形成晚期糖基化终末产物(AGEs)、激活依赖于NOX4的活性氧自由基(ROS)生成和线粒体功能障碍等复杂途径导致器官损伤。我们实验室产生的初步和公开的数据表明，当培养的肾脏细胞暴露在高糖(HG)或AGEs以及1型糖尿病大鼠的肾皮质和肾小球时，NOX4在线粒体上的新定位和NAD(P)H依赖的ROS在这个间隔内的产生增加。组织学上，糖尿病肾病的特征是细胞外基质蛋白(纤维连接蛋白和胶原蛋白)过度积聚。基质蛋白的积累是蛋白质合成增加和蛋白质降解减少的结果。高糖通过激活mTOR信号通路诱导蛋白质合成，通过诱导纤溶酶原激活物抑制物-1(PAI-I)等基质降解酶抑制蛋白质降解。我们的初步发现和已发表的数据表明，依赖于HG和年龄的mTOR激活是氧化还原敏感的。最近的证据表明，转录因子缺氧诱导因子(HIF)-1α及其靶基因PAI-1在糖尿病动物模型肾小球中表达上调。事实上，我们的初步数据显示，HIF-1α在暴露于HG或AGEs的MC中上调，并介导FN的积累。重要的是，我们展示了mTOR氧化还原激活介导HIF-1α积聚的潜在机制。在糖尿病患者和糖尿病动物模型的细胞和组织中检测到线粒体功能障碍，伴随着ATP水平的降低和氧气消耗的减少。我们的初步数据表明，HG介导的细胞ATP水平的降低与NADPH氧化酶活性的增加有关。此外，我们发现NOX4在其C端序列中含有一个ATP结合盒，并且ATP抑制MC匀浆中依赖NADPH的超氧化物的产生。综上所述，这些结果表明存在一种新的机制，通过这种机制，HG中ATP的减少和能量稳态的变化来调节线粒体NOX4的活性。参与线粒体生物发生的线粒体基因表达减少与细胞代谢和氧耗的改变有关。支持这一点的是，我们发现线粒体生物发生的转录调节因子盈阳1(YY1)在暴露于HG的MC中下调，从而提供了一种改变能量稳态的潜在机制。综上所述，我们将在体外和体内的实验动物模型中，确定高糖和晚期糖基化终产物降低细胞内ATP水平和激活线粒体NOX4依赖的氧自由基，从而稳定低氧诱导因子-1α和细胞外基质在肾小球积聚的机制。尖端技术将被用来阐明这些机制，并将为糖尿病肾病的发病机制提供新的见解。识别对ATP敏感的NOX4的生物物理特性将允许特定的小分子靶向，而不会干扰线粒体的重要呼吸功能。这项工作可能具有重大的治疗意义。

DESCRIPTION (provided by applicant): Diabetic nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease worldwide. Hyperglycemia induces organ injury through complex pathways including the formation of advanced glycation end products (AGEs), activation of Nox4-dependent generation of reactive oxygen species (ROS), and mitochondrial dysfunction. Preliminary and published data generated in our laboratory demonstrate a novel localization of Nox4 to the mitochondria and enhanced NAD(P)H- dependent ROS generation within this compartment when cultured renal cells are exposed to high glucose (HG) or AGEs as well as in the renal cortex and glomeruli of rats with type 1 diabetes. Histologically, diabetic nephropathy is characterized as an excessive accumulation of extracellular matrix proteins (Fibronectin and Collagens). Accumulation of matrix proteins is a result of increased protein synthesis and decreased protein degradation. High glucose induces protein synthesis through activation of the mTOR signaling pathway and inhibits protein degradation by inducing inhibitors of matrix-degrading enzymes such as plasminogen activator inhibitor-1 (PAI-I). Our preliminary findings and published data demonstrate that HG- and AGE-dependent activation of mTOR is redox-sensitive. Recent evidence indicates the transcription factor, hypoxia inducible factor (HIF)-1 alpha, and its target gene PAI-1, are upregulated in glomeruli of diabetic animal models. Indeed, our preliminary data show that HIF-1alpha is up-regulated in MCs exposed to HG or AGEs and mediates FN accumulation. Importantly, we show a potential mechanism by which redox-activation of mTOR mediates HIF-1alpha accumulation. Mitochondrial dysfunction, with reduced ATP levels and reduced oxygen consumption, has been detected in cells and tissues of diabetic patients and animal models of diabetes. Our preliminary data indicate that HG-mediated reduction of cellular ATP levels is associated with increased NADPH oxidase activity. Moreover, we find that Nox4 harbors an ATP-binding cassette in its C-terminal sequence and that ATP inhibits NADPH-dependent superoxide generation in MC homogenates. Together, these results suggest the existence of a novel mechanism by which the decrease in ATP and changes in energy homeostasis in HG regulate mitochondrial Nox4 activity. Reduced expression of mitochondrial genes involved in mitochondrial biogenesis is associated with altered cellular metabolism and oxygen consumption. In support of this, we find Ying Yang1 (YY1), a transcriptional regulator of mitochondrial biogenesis is downregulated in MCs exposed to HG, providing a potential mechanism by which energy homeostasis is altered. Taken together, we will identify the mechanisms by which high glucose and advanced glycation end products reduce intracellular ATP levels and activate mitochondrial Nox4-dependent oxygen radicals with subsequent stabilization of hypoxia-inducible factor-1 alpha and extracellular matrix accumulation in renal glomeruli, in vitro and in experimental animal models in vivo. Cutting edge techniques will be applied to elucidate these mechanisms and will provide novel insights into the pathogenesis of diabetic nephropathy. Identifying the biophysical properties of Nox4 sensitive to ATP will allow specific small molecule targeting without interfering with vital respiratory functions of mitochondria. This work may have major therapeutic implications.