Mitochondrial overload and proximal tubular cell atrophy

近端肾小管上皮细胞（PTC）是高能量需求的肾细胞。他们的能源需求 主要来自线粒体脂肪酸氧化。如果没有足够的ATP来满足高能量需求 随着肾小管上皮细胞的产生，肾小管上皮细胞发生凋亡和萎缩。线粒体脂肪酸 因此，代谢是肾小管细胞死亡的主要潜在候选机制。最近有人 在2型糖尿病小鼠和人类中发现， 代谢和氧化，但这是不匹配的ATP生产。肾皮质积聚 不完全氧化的代谢产物，这是典型的线粒体过载。我们的核心假设是 这种不完全的脂肪酸氧化会通过关键途径引起近端小管凋亡， 线粒体功能 我们令人信服的一组初步数据表明，细胞中的线粒体过载会导致能量不足， 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过载 导致肾脏疾病。这是通过PTC特异性缺失肉毒碱乙酰转移酶（CrAT）实现的。的 酶穿梭多余的脂肪酸产品的线粒体;因此，缺乏CrAT模型 线粒体超载我们的目标是确定肾小管细胞凋亡的机制，这是由于 线粒体超载三个相互关联但独立的目标将使用状态来测试我们的假设- 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酸的预测， 酸氧化导致线粒体能量不足，这对肾小管细胞是有害的。目标2将测试 假设不完全的脂肪酸氧化导致过量的线粒体ROS产生和细胞凋亡。 前两个目标将使用功能丧失的方法（单独或联合应用PTC特异性CrAT敲除小鼠）。 与肥胖和2型糖尿病的组合），从这些分离的原发性PTC的机制研究 模型和先进的生物物理测量（细胞外通量分析仪，电子自旋共振 光谱学）。在目标3中，我们将测试是否通过增强线粒体的外排来减轻线粒体过载。 不完全氧化的产物提供了预防。我们将使用体外和体内功能获得实验 (CrAT过表达和再表达）作为拯救实验。实验策略旨在 确定线粒体脂肪酸不完全氧化在肾小管损伤中的作用， 生化机制，并解决这些途径是否可以提供肾小管细胞保存的基础 在肥胖和2型糖尿病的背景下，早在细胞凋亡出现之前。条件是这些 机制是肾小管细胞凋亡的先导，靶向线粒体脂肪酸超载可以是一种治疗方法。 突出的新领域，以预防，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.