Epigenetic Influence of Diet on Bladder Physiology

项目摘要 下尿路功能障碍（LUTD）的高发病率和患病率， 非洲人群中许多膀胱疾病的因素（约66%，20%和6%的男性人群） 分别显示轻微、中度及严重症状），与环境及 生活方式因素，如饮食。我们以前在正常和患病膀胱中报道过， 饮食改变膀胱形态，特别是细胞增生和肥大，引起膀胱功能的变化。 功能和影响负责逼尿肌收缩的关键钙信号传导机制。 对LUTD和其他膀胱疾病的机制的理解仍然非常有限; 表观遗传学可能发挥了作用。因此，长期目标是确定饮食对人的表观遗传影响 膀胱生理学和病理生理学，同时也达到所需的培训和专业知识水平 成为一名独立的研究者。本申请的总体目标是（i） 表征饮食对特定组蛋白修饰和基因表达变化的影响， 膀胱和（ii）将组蛋白和基因表达中观察到的任何改变与表型和 膀胱的功能变化。核心假设是，表观遗传修饰有助于 不同饮食导致膀胱形态和功能改变的分子变化。 该项目的基本原理是，确定饮食对膀胱的表观遗传影响， 提供基础和支持性的初步数据，以便随后研究 饮食在正常膀胱和膀胱功能障碍。饮食，作为一个可以改变的因素， 健康影响，可以提供一个深入了解LUTD发生的机制。该项目还 让我有机会作为一名表观遗传学家独立发展核心假设将 通过追求三个具体目标进行测试：1）确定膀胱形态和功能的变化， 消费不同的饮食（高脂肪、高碳水化合物和高蛋白饮食）; 2）识别膀胱 使用RNAseq进行饮食处理后转录组的变化;以及3）鉴定饮食对 用于增殖状态（H3 S10 P，H3 S28 P），转录激活（H3 K9 Ac， H3 K27 Ac）和DNA复制（H4 K5 Ac，H4 K12 Ac）。 喂食在第一个目标下，将使用切除的整个大鼠膀胱组织来鉴定膀胱的变化。 体重、逼尿肌收缩力和膀胱组织学（苏木精和伊红染色， 免疫组织化学），以确定每种饮食导致的形态和功能变化。为 第二个目标是进行RNA测序，以确定转录组丰度的全球变化 在每个饮食组中。为了达到第三个目的，蛋白质分离技术（SDS-PAGE）和 Western免疫印迹将用于鉴定特定组蛋白靶的变化。拟议 研究是创新的，因为它专注于膀胱生理学研究的新方向， 病理生理学，调查饮食，所有患者中常见的可改变的公共卫生因素， 几种非传染性疾病病因的共同点，以及其对 影响基因转录和表达的表观遗传修饰可导致LUTD。的 拟议的研究是重要的，因为它预计将推进科学知识的 膀胱功能的机制，并提供一个初步的观点和数据，随后 将建立饮食在膀胱功能障碍中所起的表观遗传作用的研究。它还将提供数据 支持管理LUTD和其他非传染性疾病的新干预战略， 疾病

PROJECT SUMMARY The high incidence and prevalence of Lower Urinary Tract Dysfunction (LUTD), a common factor in many bladder disorders in African populations (about 66%, 20% and 6% of the male population show mild, moderate and severe symptoms respectively), has been related to environmental and lifestyle factors such as diet. We have previously reported in both normal and diseased bladders, that diet alters bladder morphology, specifically cell hyperplasia and hypertrophy, causing changes in function and influencing key calcium-signalling mechanisms responsible for detrusor contractility. Understanding of the mechanisms responsible for LUTD and other bladder disorders is still very limited; epigenetics may play a role. Thus, the long-term goal is to determine the epigenetic effects of diet on bladder physiology and pathophysiology, while also attaining the level of training and expertise required for me to become an independent researcher. The overall objectives in this application are to (i) characterize the influence of diet on specific histone modifications and gene expression changes in the bladder and (ii) relate any observed alterations in histones and gene expression to phenotypic and functional changes in the bladder. The central hypothesis is that epigenetic modifications contribute to the molecular changes underlying alterations in bladder morphology and function with different diets. The rationale for this project is that identification of the epigenetic effects of diet in the bladder will provide a foundation, and supportive preliminary data, for subsequent studies on the roles played by diet in the normal bladder and in bladder dysfunction. Diet, being a modifiable factor with great public health impact, could provide an insight into the mechanisms by which LUTD occur. This project also offers me the opportunity to establish my independence as an epigeneticist. The central hypothesis will be tested by pursuing three specific aims: 1) Identify changes in bladder morphology and function after consumption of different diets (high fat, high carbohydrate, and high protein diets); 2) Identify bladder transcriptome changes after dietary treatment using RNAseq; and 3) Identify effects of the diets on histone acyl marks for proliferative status (H3S10P, H3S28P), transcriptional activation (H3K9Ac, H3K27Ac) and DNA replication (H4K5Ac, H4K12Ac) in the bladder of rats from each dietary group after feeding. Under the first aim, excised whole rat bladder tissue will be used to identify changes in bladder weight, detrusor contractility, and bladder histology (haematoxylin and Eosin stain, and immunohistochemistry) to determine morphological and functional changes as a result of each diet. For the second aim, RNA sequencing will be done to identify global changes in transcriptome abundance in each dietary group. To achieve the third aim, protein separation techniques (SDS-PAGE) and western immunoblotting will be employed to identify changes in specific histone targets. The proposed research is innovative, because it focuses on a new direction for research into bladder physiology and pathophysiology, investigating diet, a common modifiable public health factor in all patients and a common denominator in the aetiologies of several non-communicable diseases, and how its effects on epigenetic modifications, influencing transcription and expression of genes could result in LUTD. The proposed research is significant because it is expected to advance scientific knowledge on the mechanisms of bladder function, and provide an initial standpoint and data upon which subsequent studies on the epigenetic roles played by diet in bladder dysfunction will build. It will also provide data to support new intervention strategies for the management of LUTD and other non-communicable dieases.