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Abstract: SA-PO569

Regulation of Fibronectin Splicing with Antisense Oligonucleotides in an Aristolochic Acid Model of AKI to CKD

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Virdee, Pritpal Singh, South West Thames Institute For Renal Research, London, United Kingdom
  • Dockrell, Mark E., South West Thames Institute for Renal Research, Surrey, United Kingdom
  • Phanish, Mysore K., Epsom and St helier University Hospitals NHS Trust, Carshalton, London, United Kingdom
Background

Extra Domain A Fibronectin (EDA+FN) is an active glycoprotein present at sites of fibrosis and is produced through post transcriptional alternative splicing of mRNA. Bonventre et al have previously described an early peak at 3 hours with significant fall in EDA+FN at 6 weeks post ischaemia reperfusion injury. We developed a murine model of aristolochic acid nephropathy (AAN) exhibiting acute to chronic progression and investigated the effects of RNAse H-Independent ASO on EDA+FN expression and subsequent impact on markers and mediators of tubulointerstitial fibrosis.

Methods

In a short model of AAN, CD1 mice aged 10 weeks received subcutaneous (SC) PBS (n=6), RNAse H-independent negative control (NC-ASO, n=6) or target antisense (T-ASO, n=6) at 50mg/kg at day -1 followed by one dose of intraperitoneal (IP) AA 3.5mg/kg on day 1 for each arm, mice culled at day 3. In the long model they were injected with IP AA 3.5mg/kg on days 1 and 5 followed by SC PBS (n=6) or 50mg/kg NC-ASO (n=12) or T-ASO (n=12) at day -1 and 3 and then weekly injections until day 96. Whole kidney tissue was extracted and RNA analysis undertaken using RT PCR.

Results

In the short model KIM1, CD68 and EDA + FN mRNA expression was significantly increased at 48 hr post AA compared to Day 0 (p<0.05). T-ASO reduced EDA+FN mRNA significantly compared to NC-ASO (p<0.0001) with no effect on CD68 or KIM1. TGFβ1 expression was downregulated by T-ASO (p<0.05). In PBS arm of the long model at day 96, compared to day 0 there was statistically significant increase in mRNA expression of EDA+FN, EDA-FN, TGFβ1, LTBP1, MMP2, MMP9, αSMA, KIM1 and Coll1a1. At day 96, T-ASO resulted in significant knock down of mRNA expression of EDA+FN, TGFβ1and LTBP1 compared against NC-ASO with no effect on expression of αSMA, Coll1a1, MMP2 or KIM1.

Conclusion

In this model of AAN we have demonstrated increased mRNA expression of EDA+FN in the early phase but also sustained at day 96. We have demonstrated the ability to knockdown EDA+FN mRNA at both time points and also noted knockdown of TGFβ1 and LTBP1 mRNA with our target antisense suggesting a functional interaction. Antisense did not have an effect on markers of AKI or other mediators of fibrosis. Further analysis of samples with immunohistochemistry will help determine whether T-ASO has an impact on attenuation of fibrosis in this model.