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Abstract: FR-OR048

Whole Genome Bisulfate Sequencing and dCas9-Mediated Epigenome Editing Identifies a Key Role for Tumor Necrosis Factor Alpha in Progressive Diabetic Kidney Disease

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Park, Jihwan, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Guan, Yuting, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Seasock, Matthew J., University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Qiu, Chengxiang, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Gluck, Caroline A., Nemours/AI DuPont Hospital for Children, Wilmington, Delaware, United States
  • Palmer, Matthew, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Susztak, Katalin, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Background

Epigenetic changes have been proposed as the biological mechanism that encodes the long-lasting impact of metabolic changes on diabetic kidney disease (DKD) development. Understanding the contribution of methylation changes to DKD has been limited by a lack of comprehensive base resolution methylation data and specific methylome editing.

Methods

We have performed genome-wide based resolution methylation analysis of microdissected human control and DKD kidney tubule samples using whole genome bisulfate sequencing to identify methylation differences in human DKD kidneys. We used RNA sequencing to define transcriptional changes in the same samples. We validated results with larger array-based and animal model results.We applied dCas9-Tet1 system to define the causality of individual cytosine methylation changes and gene expression under specific sgRNAs.

Results

Methylation differences with genome-wide significance were detected in kidney tubule samples of patients with DKD. The changes were replicated by large cohort array-based methylation analysis. Differentially methylated regions (DMRs) were enriched on regulatory regions and associated with gene expression changes; however, it is likely that most changes were actually the result of cell type-specific changes rather than cell-specific changes. Pathway analysis indicated coordinated (methylation and gene expression) changes in metabolic and immune response pathways; specifically, changes in tumor necrosis factor alpha (TNFα) signaling. dCas9-Tet1 based lowering of cytosine methylation level of the TNFα DMR region resulted in an increase in TNF transcription indicating that methylation of this locus plays an important role in controlling gene expression. Increasing the TNF levels in diabetic animal models resulted in high grade albuminuria due to increased cytokine levels and cell death indicating TNFα is functionally important in DKD development.

Conclusion

Our results indicate wide spread methylation differences in DKD kidneys.We propose that epigenetic dysregulation of TNF locus likely contributes to nephropathy in patients with diabetes.