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Abstract: PO2472

ROCK-Binding ASD2-Domain of Shroom3 Has a Profibrotic Role

Session Information

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Lin, Qisheng, Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital, Shanghai, Shanghai, China
  • Banu, Khadija, Yale University School of Medicine, New Haven, Connecticut, United States
  • Reghuvaran, Anand, Yale University School of Medicine, New Haven, Connecticut, United States
  • Wei, Chengguo, Mount Sinai Health System, New York, New York, United States
  • He, John Cijiang, Mount Sinai Health System, New York, New York, United States
  • Murphy, Barbara T., Mount Sinai Health System, New York, New York, United States
  • Menon, Madhav C., Yale University School of Medicine, New Haven, Connecticut, United States
Background

We previously showed that a CKD-associated Shroom3 allele was a quantitative trait locus for SHROOM3 expression and promoted CKD and allograft nephropathy.

Methods

Here we investigated the mechanism of increased fibrosis downstream of Shroom3. We systematically deleted consensus domains in Shroom3 and evaluated profibrotic signaling in tubular cells (Fig-1).

Results

Overexpression of ASD2-domain deleted SHROOM3 mutant (ASD2-SH3 with deficient ROCK binding) consistently reduced TGF-B signaling responses in TGF-B reporter 293-Tcells and in Smad-reporter luciferase assays (vs ASD1-, PDZ-, Fyn-binding domain deletion mutants and intact SHROOM3; n=3 sets each). Based on these data we generated doxycycline inducible transgenic mice for SHROOM3 and ASD2-SH3 mutants. Two founder lines of each transgene crossed with CAGS-rTTA mice were selected based on transgene expression in kidney tissues upon DOX-feeding. Adult CAGS-rTTA:SHROOM3-Tg, ASD2-S3-Tg and control mice were dox fed, and UUO surgery performed (n=5 each). At 7 days, UUO kidneys from ASD2-SH3-Tg mice showed reduced Phospho-Smad3, and reduced profibrotic transcripts (Col1a1, Fn1) vs other groups. ASD2-SH3 UUO kidneys had significantly reduced interstitial fibrosis (masson trichrome and sirius red), and reduced COL1A1 immunofluorescence (fig 2).

Conclusion

Our sequential findings show that the profibrotic role of Shroom3 excess is mediated via its ASD2-domain.

Funding

  • NIDDK Support