Epigenetic Influence of Diet on Bladder Physiology

项目总结 下尿路功能障碍(LUTD)的高发和盛行率是一种常见的 非洲人口中许多膀胱疾病的因素(约占男性人口的66%、20%和6% 分别表现为轻度、中度和重度症状)，与环境和 生活方式因素，如饮食。我们之前在正常和患病的膀胱中都报告过， 饮食改变了膀胱的形态，特别是细胞的增殖和肥大，导致了 作用和影响逼尿肌收缩的关键钙信号机制。 对LUTD和其他膀胱疾病的机制的了解仍然非常有限； 表观遗传学可能起到了一定作用。因此，长期目标是确定饮食对健康的表观遗传效应。 膀胱生理学和病理生理学，同时达到所需的培训和专业知识水平 让我成为一名独立研究人员。本申请的总体目标是(I) 描述饮食对特定的组蛋白修饰和基因表达变化的影响 和(Ii)组蛋白和基因表达的任何观察到的变化与表型和 膀胱的功能改变。中心假设是表观遗传修饰有助于 不同饮食对膀胱形态和功能影响的分子变化。 这个项目的基本原理是确定饮食在膀胱意志中的表观遗传效应 为后续研究提供基础和支持性的初步数据 饮食在正常膀胱和膀胱功能障碍中。饮食，作为一个可修改的因素，与伟大的公众 对健康的影响，可以提供对LUTD发生机制的洞察。这个项目还 让我有机会确立我作为一名表观遗传学家的独立性。中心假说将 通过追求三个具体目标进行测试：1)确定术后膀胱形态和功能的变化 食用不同的饮食(高脂肪、高碳水化合物和高蛋白饮食)；2)识别膀胱 使用RNAseq进行饮食治疗后转录组的变化；以及3)确定饮食对 组蛋白酰基标记：增殖状态(H3S10P、H3S28P)、转录激活(H3K9Ac、 H3K27Ac)和DNA复制(H4K5Ac，H4K12Ac 喂食。在第一个目标下，切除的整个大鼠膀胱组织将被用来识别膀胱的变化 体重、逼尿肌收缩能力和膀胱组织学(苏木精-伊红染色，和 免疫组织化学)，以确定每种饮食引起的形态和功能变化。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 在每组饮食中。为了实现第三个目标，蛋白质分离技术(十二烷基硫酸钠-PAGE)和 Western免疫印迹将被用来识别特定组蛋白靶标的变化。建议数 研究具有创新性，因为它聚焦于膀胱生理学研究的新方向，以及 病理生理学，调查饮食，所有患者中的一个常见的可改变的公共健康因素，以及 几种非传染性疾病病因学上的共同点，及其对 表观遗传修饰，影响基因的转录和表达可导致LUTD。这个 拟议中的研究具有重要意义，因为它有望促进关于 膀胱功能的机制，并提供了初步的观点和数据，以供后续 关于饮食在膀胱功能障碍中的表观遗传学作用的研究将会建立起来。它还将提供数据 支持LUTD和其他非传染性疾病管理的新干预战略 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.