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Mitochondrial DNA mutations in the renal cortex to elucidate cell-specific mechanisms of mitochondrial dysfunction in tubules and glomeruli

肾皮质线粒体 DNA 突变阐明肾小管和肾小球线粒体功能障碍的细胞特异性机制

基本信息

批准号：
10581517
负责人：
Monica Yicette Sanchez-Contreras
金额：
$ 19.44万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-01 至 2024-02-29
项目状态：
已结题

项目摘要

Project Summary/Abstract Mitochondrial dysfunction is a hallmark of normative aging and of kidney disease and mitochondrial DNA (mtDNA) damage and mutation accumulation has been proposed as one underlying cause. A clear understanding of the functional role of somatic mtDNA mutation in age-related mitochondrial dysfunction has been impeded, however, by the limited accuracy of modern mutation detection techniques and the complexities of experimental approaches to isolate specific cells and their components. Furthermore, many studies have underestimated the importance of tissue-specific analysis of mtDNA mutation by broadly applying single organ studies to make assumptions of organismal-level mechanisms. By implementing Duplex Sequencing, an ultra- accurate sequencing method designed to detect mutations with a frequency as low as 1x10-7, we have been able to characterize the tissue-specific patterns of somatic mtDNA mutation across 10 tissues from young and aged mice. In doing so, we identified unique aging mutation patterns between organs, with kidney cortex showing the highest frequency of somatic mtDNA mutations. Even within the kidney we found regional differences by comparing mutation rates in the tubule-rich kidney cortex to isolated renal glomeruli, thus revealing that the glomerulus has a significantly lower point mutation frequency, a lower frequency of oxidative mtDNA mutations and differential accumulation of mutations in mtDNA genes, as compared to the whole cortex. These results demonstrate that mtDNA somatic mutation accumulation is cell-specific within the kidney. Based on the premise that age-associated somatic mtDNA mutation in the kidney is determined by cell-specific differences in the ability to respond to mutation accumulation, we will utilize advanced technological approaches, including Duplex Sequencing, to address two Aims. In Aim 1, mitochondria from unique renal cell populations will be accurately isolated and analyzed by taking advantage of a Cre-Lox mitochondrial reporter mouse (MITO-Tag) crossed with mice expressing either a glomerular podocyte (podocin) or tubule epithelia (KSP) Cre. Mutation burden, mitochondrial energetics and mitophagy will be analyzed from single cell-type populations in the context of somatic mutation accumulation through natural aging. In Aim 2, kidney-specific mitochondrial dysfunction will be generated through uni-nephrectomy and by introducing a high fat/high sucrose diet as a model of premature kidney aging; this will allow us to elucidate the molecular mechanisms involved in somatic mutagenesis of renal mtDNA under oxidative stress and in response to interventions aimed at protecting the mitochondria; specifically, SS-31, a rejuvenating peptide with potential translational applications. This project will develop novel tools to clarify the role of cell-type and age-associated somatic mtDNA mutation in the kidney and provide a new perspective on the contribution of DNA mutation and aging to kidney diseases such as chronic kidney disease and acute kidney injury in the elderly.

项目总结/摘要线粒体功能障碍是正常衰老和肾脏疾病及线粒体DNA的标志线粒体DNA损伤和突变积累被认为是一个潜在的原因。一个明确了解体细胞mtDNA突变在年龄相关线粒体功能障碍中的功能作用，然而，由于现代突变检测技术的准确性有限，分离特定细胞及其成分的实验方法。此外，许多研究广泛应用单个器官，低估了mtDNA突变的组织特异性分析的重要性研究假设的有机体水平的机制。通过实施双重测序，精确的测序方法，旨在检测频率低至1x 10 -7的突变，我们已经能够描述10种组织中体细胞mtDNA突变的组织特异性模式，老老鼠在这样做的过程中，我们确定了器官之间独特的衰老突变模式，肾皮质显示体细胞mtDNA突变频率最高。即使在肾脏内部，我们也发现了区域差异，比较富含肾小管的肾皮质和分离的肾小球中的突变率，从而揭示了肾小球的点突变频率明显较低，线粒体DNA氧化突变频率较低，与整个皮层相比，线粒体DNA基因突变的差异积累。这些结果证明mtDNA体细胞突变积累在肾脏内是细胞特异性的。依据的前提肾脏中与年龄相关的体细胞mtDNA突变是由细胞特异性的能力差异决定的，为了应对突变的积累，我们将利用先进的技术方法，包括Duplex 排序，以解决两个目标。在目标1中，来自独特肾细胞群体的线粒体将被精确地分离并利用Cre-Lox线粒体报告小鼠（MITO-Tag）与表达肾小球足细胞（podocin）或肾小管上皮细胞（KSP）Cre的小鼠。突变负担，线粒体能量学和线粒体自噬将在以下背景下从单细胞型群体中进行分析：体细胞突变通过自然老化积累。在目标2中，肾脏特异性线粒体功能障碍将是通过单侧肾切除术和引入高脂肪/高糖饮食作为早产儿模型产生这将使我们能够阐明参与肾脏衰老的体细胞诱变的分子机制，线粒体DNA在氧化应激下和对旨在保护线粒体的干预措施的反应;具体而言， SS-31，一种具有潜在翻译应用的再生肽。该项目将开发新的工具，阐明细胞类型和年龄相关的体细胞mtDNA突变在肾脏中的作用，并提供新的关于DNA突变和衰老对慢性肾病等肾脏疾病的贡献的观点和老年人急性肾损伤。