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.

项目摘要/摘要线粒体功能障碍是规范性衰老和肾脏疾病和线粒体DNA的标志（mtDNA）已提出损伤和突变积累是一个根本原因。清晰了解体细胞mtDNA突变在与年龄相关的线粒体功能障碍中的功能作用已有然而，由于现代突变检测技术和复杂性的有限准确性而阻碍了隔离特定细胞及其成分的实验方法。此外，许多研究有低估了通过广泛使用单器官的组织特异性分析的组织特异性分析的重要性研究有机体机制的假设。通过实施双链测序，精确的测序方法旨在检测频率低至1x10-7的突变，我们一直是能够表征来自年轻和的10个组织中体细胞mtDNA突变的组织特异性模式老鼠。这样，我们确定了器官之间的独特衰老突变模式，肾皮质显示体细胞mtDNA突变的最高频率。即使在肾脏中，我们也发现了区域差异比较富含小管的肾皮质中的突变率与孤立的肾肾小球，从而表明肾小球具有明显较低的点突变频率，氧化mtDNA突变的频率较低与整个皮质相比，MTDNA基因中突变的差异积累。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。基于前提肾脏中与年龄相关的体细胞mtDNA突变由细胞特异性差异确定为了应对突变积累，我们将采用高级技术方法，包括双工测序，解决两个目标。在AIM 1中，来自独特的肾细胞种群的线粒体将是准确的通过利用CRE-Lox线粒体记者小鼠（mito-tag）进行分离和分析表达肾小球足细胞（Podocin）或小管上皮（KSP）CRE的小鼠。突变负担，线粒体的能量和线粒体将在单个细胞类型种群中分析体细胞突变通过自然衰老积累。在AIM 2中，肾脏特异性线粒体功能障碍将是通过单否切除术并引入高脂肪/高蔗糖饮食作为早产的模型肾脏衰老；这将使我们能够阐明肾脏诱变所涉及的分子机制 mtDNA在氧化应激下，并响应旨在保护线粒体的干预措施；具体来说， SS-31，一种具有潜在翻译应用的复兴肽。该项目将开发出新颖的工具阐明细胞类型和与年龄相关的体细胞mtDNA突变在肾脏中的作用，并提供了新的关于DNA突变和衰老对肾脏疾病（例如慢性肾脏疾病）的贡献的观点和老年人的急性肾脏受伤。