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Abstract: PO2512

Hyaluronan Synthase-2 Antisense (HAS2-AS1) Is a Novel Long Non-Coding RNA That Regulates Pro-Fibrotic Cell Responses in the Kidney

Session Information

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Grigorieva, Irina, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Woods, Emma L., Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Khalid, Usman, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Bowen, Timothy, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Steadman, Robert, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Meran, Soma, Cardiff University, Cardiff, South Glamorgan, United Kingdom
Background

Renal interstitial fibrosis drives CKD and increased synthesis of hyaluronan (HA) in the tubulointerstitium correlates with fibrosis and renal outcomes. Our work shows that of the three HA synthase enzymes, HAS2 expression is causally linked with fibrosis in vivo, a pro-fibrotic cell phenotype in vitro, and is regulated by a long non-coding RNA, HAS2-AS1. Here we investigated the mechanisms that regulate HAS2-AS1 expression and function and influence HA-dependent regulation of pro-fibrotic cell phenotype.

Methods

Primary human fibroblasts were used to test effects of siRNA-mediated HAS2-AS1 knockdown on TGFβ1-driven myofibroblast differentiation and HA levels by ELISA, RT-qPCR and immunofluorescence. ChIP-Seq determined binding of HYAL2 to HAS2-AS1 and HAS2 promoters. Alterations in HAS2-AS1 expression were assessed from acute to chronic kidney injury and in fibrosis prevention using kidneys from a rat-model of bilateral ischaemia-reperfusion-injury (IRI) or from rats that underwent ischaemic-preconditioning prior to IRI (prevention model).

Results

In fibroblasts, TGFβ1 increased HAS2-AS1 expression concomitantly with HAS2. HAS2-AS1 knockdown resulted in significant attenuation of HAS2 expression demonstrating that HAS2-AS1 is a positive regulator of HAS2. HAS2-AS1 knockdown led to a decrease in soluble and cell-surface HA, attenuated TGFβ1-driven expression of pro-fibrotic markers, and modified expression of the principal HA receptor, CD44 and its variant isoforms suggesting a link between HAS2-AS1 and CD44 alternative splicing relevant to progression of renal disease. In turn, we showed that HYAL2 (a novel regulator of DNA/RNA processing) regulates HAS2-AS1 expression. Anti-HYAL2 ChIP-Seq analysis identified enrichment of HAS2-AS1, but not HAS2 promoter sequence, and HYAL2 knockdown resulted in attenuation of TGFβ1-driven HAS2-AS1 and decrease in myofibroblast marker expression. Kidneys with progressive fibrosis had significantly increased HAS2-AS1 expression versus kidneys that were protected from fibrosis through IPC, suggesting an in vivo role for HAS2-AS1 in modulation of pro-fibrotic renal responses.

Conclusion

HAS2-AS1 is a novel lncRNA causally-linked with pro-fibrotic responses both in vitro and in vivo and a new potential therapeutic target for intervention in fibrosis.