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.

项目概要/摘要线粒体功能障碍是正常衰老、肾脏疾病和线粒体 DNA 的标志（mtDNA）损伤和突变积累被认为是根本原因之一。一个清晰的对体细胞 mtDNA 突变在年龄相关线粒体功能障碍中的功能作用的理解已经然而，现代突变检测技术的准确性有限以及复杂性阻碍了这一研究分离特定细胞及其成分的实验方法。此外，许多研究已经通过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对有机体水平的机制做出假设。通过实施双工测序，超精确的测序方法旨在检测频率低至 1x10-7 的突变，我们已经能够表征 10 种年轻和成年组织的体细胞 mtDNA 突变的组织特异性模式年老的老鼠。在此过程中，我们发现了器官之间独特的衰老突变模式，其中肾脏皮质显示体细胞 mtDNA 突变频率最高。即使在肾脏内，我们也发现了区域差异将富含肾小管的肾皮质与分离的肾小球的突变率进行比较，从而揭示肾小球点突变频率明显较低，氧化线粒体DNA突变频率较低与整个皮质相比，mtDNA 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具有细胞特异性。基于前提肾脏中与年龄相关的体细胞 mtDNA 突变是由细胞特异性能力差异决定的为了应对突变积累，我们将利用先进的技术方法，包括 Duplex 测序，以实现两个目标。在目标 1 中，来自独特肾细胞群的线粒体将被准确地利用与线粒体杂交的 Cre-Lox 线粒体报告小鼠 (MITO-Tag) 进行分离和分析表达肾小球足细胞（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从单细胞类型群体中进行分析通过自然衰老积累体细胞突变。在目标 2 中，肾脏特异性线粒体功能障碍将是通过单肾切除术和引入高脂肪/高蔗糖饮食作为早产儿模型产生肾脏老化；这将使我们能够阐明参与肾细胞体细胞突变的分子机制。氧化应激下的线粒体 DNA 以及对旨在保护线粒体的干预措施的反应；具体来说， SS-31，一种具有潜在转化应用的再生肽。该项目将开发新颖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，并提供新的方法 DNA突变和衰老对慢性肾病等肾脏疾病的影响的观点以及老年人的急性肾损伤。