Role of NRF2 in oxidative stress pathophysiology of diabetes kidney disease

1型（T1D）和2型糖尿病（T2D）的患病率仍在增加，但直到最近人们才认为它们的病因完全不同：胰岛β细胞不能产生足够的胰岛素来补偿T2D的胰岛素抵抗，而胰岛β细胞的自身免疫破坏则是T1D。然而，发现GLIS3基因的多态性通过改变β细胞对压力的敏感性而易患T1D和T2D，强调了β细胞健康是一个共同点。该项目结合了DIL （Todd）在研究T1D的遗传和分子原因及其与T2D的遗传重叠方面的独特优势，以及NNRCO （Johnson）在研究胰腺β细胞存活的细胞生物学以及实施高通量筛选和最新成像技术方面的独特优势，以研究β细胞健康和糖尿病的遗传和机制基础。我们正在进行的分析表明，超过60个T1D风险区域中的bbb150基因在β细胞中表达，并且一些T1D候选变体与在人类胰岛中特异性活性的调控基序共定位，而不是在免疫细胞中。然而，类似于糖尿病前期胰腺所遇到的应激条件，可能会显著改变染色质状态和基因调控。拟议的项目有两个平行的目标：-研究几个新发现的T1D区域（其中一些与T2D风险位点重叠）中的基因如何通过β细胞脆弱性导致糖尿病。通过研究应激和糖尿病状态对人类供体胰岛染色质状态和基因调控的影响，发现β细胞健康所需的新基因/途径。目的1将使用基因敲低方法筛选在胰岛中特异性活性调节区域附近的蛋白质编码基因。我们将比较在化学（thapsigargin）、免疫（细胞因子处理、与细胞毒性T细胞共培养）和遗传（如GLIS3功能丧失）应激与非应激条件下对β细胞（内源性β - ah1细胞组装成球状/假胰岛和供体胰岛）的影响。在目标2中，我们将使用ATAC-seq和RNA-seq来定义应激和非应激供体胰岛之间染色质状态的变化，以鉴定在对照胰岛中可能看起来是静止的基因和调控区域。最近这些方法的小型化将使我们能够将一个供体的胰岛分成几个实验组。转录组学和蛋白质组学数据的整合将使数据的硅组装成为功能途径。根据组织的可用性，我们的目标是比较来自糖尿病和非糖尿病供体胰岛的结果。

The prevalences of type 1 (T1D) and type 2 diabetes (T2D) are still increasing, but their aetiologies were until recently thought to be entirely distinct: islet beta-cell failure to produce sufficient insulin to compensate for insulin resistance in T2D versus autoimmune destruction of pancreatic islet beta cells in T1D. However, the discovery that a polymorphism of the GLIS3 gene predisposes to both T1D and T2D, through altering beta-cell sensitivity to stress, has highlighted beta-cell health as a common denominator.This project combines the unique strengths of the DIL (Todd) - in studying the genetic and molecular causes of T1D and its genetic overlaps with T2D - and the NNRCO (Johnson) -in studying the cell biology of pancreatic beta-cell survival and in implementing high-throughput screens and the latest imaging technology - to investigate the genetic and mechanistic bases underlying beta-cell health and diabetes.Our ongoing analyses show that >150 genes in over 60 T1D risk regions are expressed in beta cells and that several T1D-candidate variants colocalise to regulatory motifs active specifically in human islets and not in immune cells. However, stress conditions, similar to those encountered in the pre-diabetic pancreas, are likely to alter chromatin states and gene regulation dramatically.The proposed project has two parallel aims:-Investigate how genes in several newly identified T1D regions (some of which overlap with T2D risk loci) cause diabetes through beta-cell fragility. 2-Discover new genes/pathways required for beta-cell health by investigating the effects of stress and diabetes status on chromatin states and gene regulation in human donor islets.Aim 1 will use gene knockdown approaches to screen through protein-coding genes proximal to regulatory regions active specifically in islets. We will compare effects in beta cells (EndoC- betaH1 cells assembled into spheroids/pseudoislets and donor islets) subjected to chemical (thapsigargin), immune (cytokine treatment, co-culture with cytotoxic T cells) and genetic (e.g. GLIS3 loss-of-function) stresses versus non-stressed conditions.In aim 2 we will define the changes in chromatin states between stressed and non-stressed donor pancreatic islets to identify genes and regulatory regions that may otherwise appear to be quiescent in control islets, using ATAC-seq and RNA-seq. The recent miniaturisation of these methods will allow us to split the islets from one donor into several experimental groups. Integration of transcriptomics and proteomics data will enable the in silico assembly of the data into functional pathways. Depending on tissue availability, we aim to compare findings with those from diabetic versus non-diabetic donor islets.