Role of NRF2 in oxidative stress pathophysiology of diabetes kidney disease

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.