Mitochondrial overload and proximal tubular cell atrophy

近端肾小管上皮细胞(PTC)是对能量要求很高的肾脏细胞。他们的能源需求得到了满足 主要来自线粒体脂肪酸的氧化。如果没有足够的ATP来满足高能量需求 产生后，肾小管上皮细胞发生凋亡和萎缩。线粒体脂肪酸脱轨 因此，代谢是肾小管细胞死亡的主要候选机制。它最近一直在 在2型糖尿病小鼠和人类中发现糖尿病肾脏表现出更多的脂肪酸 代谢和氧化，但这与三磷酸腺苷的产生不匹配。肾皮质蓄积 代谢产物氧化不完全，这是线粒体超载的典型表现。我们的中心假设是 这种不完全的脂肪酸氧化通过关键的途径引起近端小管的细胞凋亡 线粒体功能。 我们的一组令人信服的初步数据表明，细胞中线粒体超载会导致能量不足和 氧化剂生产。在我们的新小鼠模型中，不完全脂肪酸氧化和线粒体超载 会导致肾脏疾病。这是通过PTC特异性缺失肉碱乙酰转移酶(CrAT)实现的。这个 酶将多余的脂肪酸产物从线粒体中排出；因此，缺乏CrAT模型 线粒体超载。我们的目标是明确肾小管上皮细胞凋亡的先驱机制。 线粒体超载。三个相互关联但又相互独立的目标将使用最新情况来检验我们的假设 分子生物学和氧化还原生物学的艺术方法。在目标1中，我们将测试不完全脂肪的预测 酸性氧化导致线粒体能量不足，从而对肾小管细胞有害。目标2将测试 不完全脂肪酸氧化导致线粒体ROS产生过多和细胞凋亡的假说。 前两个目标将使用功能丧失方法(PTC特异的CrAT基因敲除小鼠单独或在 结合肥胖症和2型糖尿病)，从这些分离的原发PTC中进行机制研究 模型和高级生物物理测量(细胞外流量分析仪、电子自旋共振 光谱学)。在目标3中，我们将测试是否通过增加线粒体的外流来缓解线粒体超载 不完全氧化的产品提供了预防。我们将使用体外和体内的功能获得实验 (crat过度表达和重新表达)作为救援实验。该实验策略旨在 确定不完全线粒体脂肪酸氧化在肾小管损伤中的作用，破译潜在的 生化机制以及这些途径是否能为肾小管细胞的保存提供基础 在肥胖症和2型糖尿病的背景下，很久以前就出现了细胞凋亡。前提是这些 机制是肾小管细胞凋亡的先导，靶向线粒体脂肪酸超载可能是一种 突出新领域要预防，而不是治疗肾小管萎缩和慢性肾脏疾病。

Proximal tubular epithelial cells (PTC) are highly energy demanding kidney cells. Their energy need is covered mostly from mitochondrial fatty acid oxidation. If the high energy demand is not met with sufficient ATP production, tubular epithelial cells undergo apoptosis and atrophy. Derailments in mitochondrial fatty acid metabolism are therefore the main underlying candidate mechanisms in tubular cell death. It has recently been discovered in type 2 diabetic mice and humans that the diabetic kidney exhibits increased fatty acid metabolism and oxidation, but this is not matched with ATP production. The kidney cortex accumulates incompletely oxidized metabolic products which is typical of mitochondrial overload. Our central hypothesis is that this incomplete fatty acid oxidation causes proximal tubule apoptosis through pathways critical to mitochondrial function. Our compelling set of preliminary data show that mitochondrial overload in cells causes energy deficit and oxidant production. In our new mouse model, incomplete fatty acid oxidation and mitochondrial overload causes kidney disease. This was achieved by PTC-specific deletion of carnitine-acetyltransferase (CrAT). The enzyme shuttles excess fatty acid products out of the mitochondria; therefore, lack of CrAT models mitochondrial overload. Our goal is to define the mechanisms that are forerunners of tubular apoptosis due to mitochondrial overload. Three interconnected but independent aims will test our hypothesis using state-of-the- art approaches of both molecular and redox biology. In Aim 1, we will test the prediction that incomplete fatty acid oxidation causes mitochondrial energy deficit, which is then detrimental to tubular cells. Aim 2 will test the hypothesis that incomplete fatty acid oxidation leads to excess mitchondrial ROS production and apoptosis. The first two aims will use loss-of-function approaches (PTC-specific CrAT knockout mice alone or in combination with obesity and type 2 diabetes), mechanistic studies from primary PTCs isolated from these models and advanced biophysical measurements (extracellular flux analyzer, electron spin resonance spectroscopy). In Aim 3, we will test whether alleviating mitochondrial overload by enhancing the efflux of incompletely oxidized products offers prevention. We will use in vitro and in vivo gain-of-function experiments (CrAT overexpression and re-expression) as rescue experiments. The experimental strategy is designed to establish the role of incomplete mitochondrial fatty acid oxidation in tubular injury, decipher the underlying biochemical mechanisms and address whether such pathways can offer the basis for tubular cell preservation well before the appearance of apoptosis, in the context of obesity and type 2 diabetes. Provided that these mechanisms are forerunners of tubular cell apoptosis, targeting mitochondrial fatty acid overload can be a prominent new area to prevent, rather than treat tubular atrophy and chronic kidney disease.