Pathogenesis of Diabetic Nephropathy

描述(由申请人提供)：本提案的总体目标是描述近端肾小管特异性酶，即肌醇加氧酶(MIOX)在糖尿病肾病(DN)肾小管间质病理生物学中的作用。糖尿病肾病的特征是肾细胞中各种代谢/细胞信号通路的扰动，导致活性氧的产生。后者已成为糖尿病肾病发病机制的核心。代谢/信号事件主要是在肾小球细胞中描述的，与肾小管间质细胞相关的信息有限。葡萄糖反应MIOX通过葡萄糖-木糖(Gx)途径将肌醇分解为D-葡萄糖醛酸(Gx)，其代谢产物进入戊糖途径。MIOX启动的Gx途径导致氧化还原失衡，NADPH/NADP+和NAD+/NADH比值在4个步骤出现扰动[图1]，类似于多元醇途径；提示其激活将诱导氧化和低氧应激，最终导致糖尿病肾病患者ECM蛋白合成增加和肾小管间质损伤[图2]。扰乱NAD+/NADH比例也会导致NAD+的耗尽，结果是依赖NAD的脱乙酰基酶Sirutins的活性受到影响。Sirutins的靶标包括FOXO家族转录因子和转录辅助激活因子PGC-1，它们调节线粒体的生物发生和各种抗氧化基因。在这些扰动下，肾小管上皮细胞可能会经历能量应激和细胞凋亡。我们发表的(JBC 2011，AJP 2010)和初步数据表明，ROS在Gx途径中产生，Gx途径反过来增加MIOX的转录，从而建立ROS的循环生成。数据还表明Gx途径存在于肾脏中[图4]，糖尿病肾病患者的肾小管间室确实存在氧化应激[图3]。此外，MIOX在高糖中的过度表达导致ECM蛋白的合成增加[图10]。在此背景下，为实现该提案的目标，现提出以下3个具体目标。目的探讨高糖环境下肾小管上皮细胞MIOX体外过表达导致氧化应激加重、线粒体功能障碍和细胞外基质合成的机制。将评估NADPH：NADP+和NAD+：NADH比率、GSH、NOX4、PKC、TGF-、SIRT、转录因子、线粒体动力学以及促和抗凋亡基因的状况。将使用特定的抑制剂/激活剂来测试MIOX效应的特异性。目的研究MIOX诱导的Gx途径、氧化还原失衡及导致SIRTs和线粒体功能障碍、细胞凋亡和肾小管间质纤维化的下游信号转导事件。将使用患有STZ诱导的糖尿病的CD1小鼠和过度表达MIOX的小鼠与秋田小鼠杂交。目的III确定MIOX基因缺失是否能改善STZ诱导的杂合子(+/-)糖尿病小鼠的肾功能障碍和进展为肾小管间质损害，以及突变的Null(-/-)小鼠与秋田小鼠杂交的情况。预计Gx-SIRT途径的特征将有助于设计新的治疗策略来减缓糖尿病肾病的肾小管间质损伤。

DESCRIPTION (provided by applicant): Overall objective of the proposal is to delineate the role of a proximal tubular specific enzyme, i.e., myo- inositol oxygenase (MIOX), in tubulo-interstitial pathobiology in the context of diabetic nephropathy (DN). The DN is characterized by perturbation in various metabolic/cellular signaling pathways in renal cells resulting in the generation of reactive oxygen species (ROS). The latter have emerged as central to the pathogenesis of DN. The metabolic/signaling events have been delineated largely in renal glomerular cells, and information relating to tubulointerstitial cells is limited. Glucose responsie MIOX catabolizes myo-inositol to D-glucuronate via Glucuronate-Xylulose (GX) pathway, as described in "eye lens", and its metabolites enter pentose pathway. The GX pathway initiated by MIOX leads to "redox imbalance" with perturbed NADPH:NADP+ and NAD+:NADH ratios at 4 steps [Fig. 1], akin to polyol pathway; suggesting that its activation would induce oxidant and hypoxic stress culminating into an increased synthesis of ECM proteins and tubulo-interstitial injury in DN [Fig. 2]. Perturbed NAD+/NADH ratio would also cause depletion of NAD+, as a result the activity of NAD-dependent deacetylases, Sirutins, are compromised. Targets of Sirutins include FOXO family transcription factors and transcriptional coactivator PGC-1¿, which modulate mitochondrial biogenesis and various antioxidant genes. With these perturbations the tubular cells are likely to undergo energy stress and apoptosis. Our published (JBC 2011, AJP 2010) and preliminary data suggest that ROS are generated in the GX pathway, which in turn increases the transcription of MIOX, thus setting up cyclic generation of ROS. Data also suggest that GX pathway exist in the kidney [Fig. 4], and oxidant stress does occur in the tubular compartment in patients with DN [Fig. 3]. Also, MIOX over-expression in high glucose leads to accentuated synthesis of ECM proteins [Fig. 10]. With this background and to achieve objectives of the proposal the following 3 specific aims are proposed. AIM I is to delineate mechanisms by which MIOX overexpression in vitro in tubular cells leads to accentuation of the oxidant stress, mitochondrial dysfunctions and ECM synthesis in the presence of high glucose. Status of NADPH:NADP+ & NAD+:NADH ratios, GSH, NOX4, PKC, TGF-, SIRTs, transcription factors, mitochondrial dynamics, and pro- and anti-apoptotic genes will be assessed. Specific inhibitors/activators will be used to test the specificity of MIOX effects. AIM II is to characterie in vivo MIOX-induced GX pathway, redox imbalance and downstream signaling events leading to dysfunctions of SIRTs and mitochondria, apoptosis and tubulo- interstitial fibrosis. CD1 mice with STZ-induced diabetes and mice over-expressing MIOX cross bred with Akita mice will be used. AIM III is to determine if MIOX gene deletion leads to amelioration in renal dysfunctions and progression to tubulo-interstitial injury in STZ-induced diabetes in heterozygote (+/-) mice and when the mutant Null (-/-) mice are cross bred with Akita mice. It is anticipated that the characterization of GX-SIRT pathway would aid in devising novel therapeutic strategies to decelerate tubulo-interstitial injury in DN.