Abstract: TH-PO001

DNA Methylation Changes of Aging-Related Genes in Renal Proximal Tubular Epithelial Cells Are Regulated by Complement: A Whole-Epigenome Analysis

Session Information

Category: Acute Kidney Injury

  • 001 AKI: Basic

Authors

  • Castellano, Giuseppe, University of Bari, BARI, Italy
  • Franzin, Rossana, University of Bari, BARI, Italy
  • Sallustio, Fabio, University of Bari, BARI, Italy
  • Stasi, Alessandra, University of Bari, BARI, Italy
  • Curci, Claudia, University of Bari, BARI, Italy
  • Divella, Chiara, University of Bari, BARI, Italy
  • Grandaliano, Giuseppe, Nefrologia/Dialisi/Trapianto, Foggia, Italy
  • Gesualdo, Loreto, University of Bari, BARI, Italy
Background

Complement, in particular C5a, is the major player of Ischemia Reperfusion injury. Renal Proximal Tubular Epithelial Cells (RPTEC) express the C5aR, however the underlying mechanisms of C5a-C5aR interaction remain poorly understood. We aimed to investigate the impact of C5a exposition on DNA methylation profile in RPTEC.

Methods

Genome-wide array-based DNA methylation levels were measured in several RPTEC lots stimulated with C5a for 24h by the Illumina 450 BeadChips.The analysis was performed on site and region level considering the difference in mean methylation levels both in gene centric regions (promoter, 5'UTR, first exon, gene body, and 3'UTR) and in CpG islands regions (CpG islands and CpG islands shore and shelves). Gene Ontology tools were used to determine genes interaction. qPCR, WB, SA-β Gal staining and MTT were performed for validation analysis.

Results

Compared to basal, 144 sites were hypomethylated and 24 sites were hypermethylated by C5a. Several sites differentially methylated were located in CpG island regions and promoters of genes involved in DNA damage checkpoints in response to genotoxic events, in cell cycle regulation, apoptosis, Hedgehog, Wnt and p53 pathways. The most representative protein classes were: nucleic acid binding proteins (DNA topoisomerase), enzyme modulators, transcription factors and cytoskeletal protein (classification analysis: 21,9%, 9,40%, 7,80% and 7,70% respectively). qPCR analysis confirmed that the hypermethylated genes were downregulated and hypomethylated genes were upregulated. Interestingly, among these genes we found several effectors involved in acquirement of a SAPS (Senescent-Associated Secretory Phenothype). In accordance, 3h of exposure of RPTEC to C5a induced cellular senescence as observed by up-regulating SA-β Gal and by cell proliferation reduction (p<0.05). Moreover, senescent RPTEC showed a significant increase in p53 and p21 protein level, as sign of cell cycle arrest (p<0.05).

Conclusion

These results suggest a role of complement in inducing tubular senescence affecting epigenetic programs, in particular the DNA methylation profile. Targeting epigenetic mechanisms may represent a strategy to protect tubular cells from aging.