Kidney Week

Abstract: SA-PO912

Targeting Alternative Splicing of Fibronectin (Fn) to Reduce Extra Domain A (EDA)+ Fn Production and Inhibit Kidney Fibrosis

Session Information

• Pathology and Lab Medicine - 2
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Pathology and Lab Medicine

• 1800 Pathology and Lab Medicine

Authors

• Phanish, Mysore K., SW Thames Institute for Renal Research, Renal Unit, St helier Hospital, Epsom and St Helier |University hospitals NHS Trust, St Georges' University of London, London, United Kingdom

Mysore K. Phanish, MD, MBBS, PhD

• Heidebrecht, Felicia, SW Thames Institute for Renal Research, Renal Unit, St helier Hospital, Epsom and St Helier |University hospitals NHS Trust, St Georges' University of London, London, United Kingdom

Felicia Heidebrecht, PhD

• Virdee, Pritpal Singh, SW Thames Institute for Renal Research, Renal Unit, St helier Hospital, Epsom and St Helier |University hospitals NHS Trust, St Georges' University of London, London, United Kingdom

Pritpal Singh Virdee, MBChB

• Rigo, Frank, Ionis Pharmaceuticals Inc, Carlsbad, California, United States

Frank Rigo

• Dockrell, Mark Edward, SW Thames Institute for Renal Research, Renal Unit, St helier Hospital, Epsom and St Helier |University hospitals NHS Trust, St Georges' University of London, London, United Kingdom

Mark Edward Dockrell, MSc, PhD

Group or Team Name

• Research Team, SW Thames Institute for Renal Research.

Background

EDA+ isoform of Fn is overexpressed in fibrosis. We investigate the effect of blocking ‘splicing in’ of EDA exon with antisense oligonucleotides (ASO) on EDA+Fn production and TGFβ1-induced fibrotic events in human primary proximal tubule cells (PTEC) and murine model of aristolochic acid (AA)-induced tubular injury.

Methods

PTEC were treated with TGFβ1 for 48 h, transfected with RNase H-independent ASO designed to block EDA exon inclusion (ASO5, selected after screening targeting 20 ASOs). EDA+Fn RNA and protein expression were analysed along with the expression of pro-fibrotic TGFβ target genes. In vivo, we assessed the expression of EDA+ Fn in murine AA model (IP inj of AA, 3.5mg/kg, D1 & 5; kidney lysate analysed by PCR for target genes on D0, D12, D20 & D100. We assessed the effect of ASO5 (50mg/kg) in mouse models: Short model (D-1 ASO5, neg control, NC or PBS S/C inj followed by a single dose of IP AA, cull D3) and long model (ASO5 or NC-ASO or PBS D-1, D3, x2/wk for 3 wks, weekly until cull D96, IP AA D1 and D5).

Results

ASO5 was effective and selective in reducing EDA+Fn RNA and protein in human PTEC (n=6, p<0.0001). TGFβ1 induced endogenous TGFβ, αSMA, MMP2, MMP9 and Col I mRNA and reduced K-Cadherin expression. These changes were attenuated by ASO5 (n=3-9, p <0.0001). Further increases in αSMA, MMP2, MMP9 (N 3-6, p<0.001) observed 48h after removal of TGFβ was inhibited by ASO5 (P<0.001). In vivo, compared to D0, there was a significant increase in EDA+/EDA- RNA ratio (30-fold, N=6, P<0.0001) on D12 which dropped to 2-fold on D100 (P<0.01). Similar pattern of induction was observed for CTGF, TGFβ1 and LTBP1 mRNA. The effect of ASO5 (50mg/kg) in mouse models: Short model (D-1 ASO5,neg control, NC or PBS S/C inj followed by a single dose of IP AA, cull D3) and long model (ASO5 or NC-ASO or PBS D-1, D3, x2/wk for 3 wks, weekly until cull on D96, IP AA D1 and D5). ASO5 treatment significantly reduced the mRNA levels of EDA+Fn, TGFβ1 (short and long models) and LTBP1 (long model P<0.05). Immunostaining for EDA+ Fn was attenuated in animals treated with ASO5.

Conclusion

EDA+ Fn plays a key role in TGFβ driven pro-fibrotic responses in renal tubule cells and blocking its production with ASO (in vitro and vivo) offers a potential therapy to limit the progression of renal fibrosis.