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Kidney Week

Abstract: SA-PO337

PRRX1 Is a Master Regulator of Renal Fibroblast Cell Fate and Regulates Myofibroblast Activation in Response to TGF-β via Alteration of the Chromatin Landscape

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 600 Development, Stem Cells, and Regenerative Medicine

Authors

  • Bonifacio, Ericka Janine, University College Dublin Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
  • Krupa, Ivan, University College Dublin Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
  • Carruthers, Niall Barry, University College Dublin Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
  • Scaife, Caitriona, University College Dublin Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
  • Davis, Jessie, University College Dublin Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
  • Crean, John, University College Dublin Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
Background

Emerging evidence suggests that changes in the chromatin landscape in response to TGFβ likely contributes to the pathogenesis of diabetic kidney disease (DKD). Using iPSC derived kidney organoids, we have recently shown that TGF-β signalling to chromatin unleashes a programme of gene expression intiating fibroblast to myofibroblast activation. We hypothesise that SMAD3 and the Polycomb Repressive Complex 2 core component, EZH2, co-occupy regulatory regions within chromatin to deposit histone post-translational modifications (PTMs), thereby facilitating a chromatin landscape which drives fibroblast activation in DKD. Our data suggests that PRRX1 acts as a master transcription factor in regulating fibroblast to myofibroblast differentiation through association with SMAD3/EZH2. To characterise this proposed epigenetic mechanism, proteomic and transcriptomic analysis was performed on iPSC-derived kidney organoids treated with TGFβ1.

Methods

Histone extraction was performed on undifferentiated iPSCs, iPSC-derived nephron progenitor cells, iPSC-derived kidney organoids, and kidney organoids treated with recombinant TGFβ1. Histones were then subjected to LC-MS/MS for analysis of global histone modifications. Single cell RNA-sequencing (scRNA-seq) was performed on iPSC derived kidney organoids treated with EZH2 inhibitor, GSK343, for 1h prior to TGFβ1 treatment for 48 hours.

Results

Mass Spectrometry data revealed differential abundance of trimethylated lysine residues on Histone 3 (H3K27me3) between TGFβ treated and control, indicating dynamic changes in transcriptionally permissive states. scRNA-seq analysis of kidney organoids treated with TGFβ1 revealed cluster specific changes in PRRX1 expression in response to TGFβ1, specifically in stromal cells and activated myofibroblast clusters. These changes in PRRX1 in response to TGFβ1 were consistently observed in other fibroblast cell lines.

Conclusion

TGF-β is a key driver of chromatin dynamics as evidenced by H3K27me3 and likely contributes to gene repression through interaction between SMAD3 and EZH2. We propose that subsequent silencing of PRRX1 expression mediates fibroblast to myofibroblast activation and represent a tractable therapeutic target for the amelioration of fibrosis in DKD.