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，是全球终末期肾病的主要原因。高血压通过复杂的途径诱导器官损伤，包括形成晚期糖基化终产物（AGEs）、激活Nox 4依赖性活性氧（ROS）的产生和线粒体功能障碍。我们实验室产生的初步和公开的数据表明，当培养的肾细胞暴露于高葡萄糖（HG）或AGEs以及1型糖尿病大鼠的肾皮质和肾小球时，Nox 4在线粒体中的新定位和增强的NAD（P）H依赖性ROS产生。在组织学上，糖尿病肾病的特征在于细胞外基质蛋白（纤连蛋白和胶原蛋白）的过度积聚。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。高葡萄糖通过激活mTOR信号通路诱导蛋白质合成，并通过诱导基质降解酶抑制剂（如纤溶酶原激活物抑制剂-1（PAI-1））抑制蛋白质降解。我们的初步研究结果和已发表的数据表明，HG和AGE依赖的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）-1 α，及其靶基因派-1，在糖尿病动物模型的肾小球上调。事实上，我们的初步数据显示，HIF-1 α在暴露于HG或AGEs的MCs中上调，并介导FN积累。重要的是，我们显示了mTOR的氧化还原激活介导HIF-1 α积累的潜在机制。在糖尿病患者和糖尿病动物模型的细胞和组织中已经检测到线粒体功能障碍，伴随ATP水平降低和氧消耗减少。我们的初步数据表明，汞介导的细胞ATP水平的降低与NADPH氧化酶活性增加有关。此外，我们发现，Nox 4在其C-末端序列中含有ATP结合盒，ATP抑制MC匀浆中NADPH依赖的超氧化物生成。总之，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调节线粒体Nox 4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消耗改变有关。为了支持这一点，我们发现Ying Yang 1（YY 1），线粒体生物合成的转录调节因子在暴露于HG的MCs中下调，提供了一种潜在的机制，通过这种机制，能量稳态被改变。总之，我们将确定高葡萄糖和晚期糖基化终产物降低细胞内ATP水平和激活线粒体Nox 4依赖性氧自由基的机制，随后稳定缺氧诱导因子-1 α和细胞外基质积累在肾小球，在体外和在体内实验动物模型。尖端技术将被应用于阐明这些机制，并将提供新的见解糖尿病肾病的发病机制。确定对ATP敏感的Nox 4的生物物理特性将允许特异性小分子靶向而不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.