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

• 批准号: 10190112
• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10190112
• 关键词: Acute; Acute Renal Failure with Renal Papillary Necrosis; Address; Age; Aging; Biological Assay; Cells; Characteristics; Chronic; Chronic Disease; Chronic Kidney Failure; Complex; Cre-LoxP; DNA Damage; DNA Maintenance; DNA Sequence Alteration; Detection; Diabetes Mellitus; Diet; Disease; Distributional Activity; Early Diagnosis; Elderly; Electron Transport; Epithelial; Fatty acid glycerol esters; Filtration; Frequencies; Functional disorder; Future; Generations; Genes; Genome; Heart; Homeostasis; Individual; Inherited; Injury; Injury to Kidney; Intervention; Kidney; Kidney Diseases; Link; Measures; Methods; Mitochondria; Mitochondrial DNA; Modeling; Modernization; Molecular; Mus; Mutagenesis; Mutation; Mutation Detection; NPHS2 protein; Natural regeneration; Nephrectomy; Nuclear; Organ; Organelles; Oxidative Stress; Patients; Pattern; Peptides; Pharmacology; Phenotype; Point Mutation; Population; Predisposition; Proliferating; Protein Subunits; Quality of life; Reactive Oxygen Species; Renal tubule structure; Replication Error; Reporter; Respiration; Ribosomal RNA; Role; Somatic Mutation; Specialized Epithelial Cell; Stress; Structure; Sucrose; Surveys; Techniques; Therapeutic; Tissues; Transfer RNA; Translating; age related; aged; base; cell type; design; detection method; genotoxicity; heteroplasmy; human old age (65+); improved; insight; kidney cell; kidney cortex; kidney dysfunction; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial genome; mouse model; next generation sequencing; novel; oxidative damage; podocyte; premature; prevent; regional difference; response; response to injury; single cell analysis; slit diaphragm; sugar; tissue regeneration; tool

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.

项目摘要/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一个潜在原因。一目了然 对体细胞线粒体DNA突变在年龄相关性线粒体功能障碍中的功能作用的理解 然而，现代突变检测技术的有限准确性和复杂性阻碍了这一进程 分离特定细胞及其成分的实验方法。此外，许多研究都有 低估了广泛应用单个器官进行线粒体DNA突变组织特异性分析的重要性 对生物体水平的机制进行假设的研究。通过实施双工测序，一种超 一种精确的测序方法，旨在检测频率低至1x10-7的突变，我们已经 能够表征来自年轻人和青少年的10个组织的体细胞线粒体DNA突变的组织特异性模式 衰老的小鼠。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 体细胞线粒体DNA突变频率最高。即使在肾脏内，我们也发现了区域差异 比较富含小管的肾皮质和孤立的肾小球的突变率，从而揭示 肾小球的点突变频率明显较低，氧化mtDNA突变的频率较低 与整个皮质相比，mtDNA基因突变的差异积累。这些结果 证明线粒体DNA体细胞突变在肾脏中的积累是细胞特有的。基于这样的前提 肾脏中与年龄相关的体细胞线粒体DNA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，包括双工 排序，以解决两个目标。在目标1中，来自独特的肾脏细胞群体的线粒体将被准确地 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， 线粒体能量学和有丝分裂将在以下背景下从单细胞类型群体中进行分析 通过自然衰老积累的体细胞突变。在目标2中，肾脏特有的线粒体功能障碍将是 通过单肾切除和引入高脂肪/高蔗糖饮食作为早产儿模型而产生 肾脏老化；这将使我们能够阐明肾脏体细胞突变的分子机制 线粒体DNA在氧化应激下以及对旨在保护线粒体的干预措施的反应；具体地说， SS-31，一种具有潜在翻译应用的年轻肽。该项目将开发新的工具来 阐明细胞类型和年龄相关的体细胞线粒体DNA突变在肾脏中的作用并提供新的 DNA突变和衰老在慢性肾脏病等肾脏疾病中的作用 和老年人的急性肾损伤。