Abstract: SA-OR25
Shroom3-Rock Interaction and Profibrotic Function: Resolving the Mechanism of a CKD Genome-Wide Association Study Risk Allele
Session Information
- CKD: New Insights into Mechanisms and Treatment Strategies
October 26, 2024 | Location: Room 24, Convention Center
Abstract Time: 05:30 PM - 05:40 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Reghuvaran, Anand, Yale University School of Medicine, New Haven, Connecticut, United States
- Kumar, Ashwani, Yale University School of Medicine, New Haven, Connecticut, United States
- Caldato Barsotti, Gabriel, Yale University School of Medicine, New Haven, Connecticut, United States
- Pell, John F., Yale University School of Medicine, New Haven, Connecticut, United States
- Tanvir, E M, Yale University School of Medicine, New Haven, Connecticut, United States
- He, John Cijiang, Icahn School of Medicine at Mount Sinai, 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 common intronic SNP (A-allele at rs17319721) is an enhancer for Shroom3 expression in donor kidneys promoting fibrosis. Given known therapeutic roles for Rho-kinase (ROCK) inhibitors in CKD, and the established interaction between Shroom3 and Rock via its ASD2 domain, we proposed that Shroom3-mediated ROCK activation played a crucial role in its profibrotic function in high expressors.
Methods
To test this, we developed transgenic mice and cell lines that inducibly overexpress wild-type- (WTS3) or ASD2-domain deletion- Shroom3 (ASD2ΔS3) and evaluated in fibrosis models.
Results
Prior scRNAseq data showed Shroom3 and Rock co-expression in injured PCT, distal nephron and fibroblasts during fibrosis [Fig A]. We used tubular cell lines (mIMCD3, HEK293T) to confirm absent ROCK binding (Co-IP)[B], reduced Rock activation (phospho-MYPT1) and reduced fibroblast proliferation (3T3) in ASD2ΔS3 cells vs WTS3 tubular cells [C-D]. . In vivo, we studied ureteric obstruction (UUO) and Aristolochic nephropathy (AAN) as fibrosis models ASD2ΔS3. In vivo, in AAN models, both global (CAGS) and Pan-tubular(PAX8)specific WTSh3 mice showed increased fibrosis (Trichrome [E-F] and Collagen I staining [G]), and ASD2Δ mice showed reduced Azotemia (N=5 mice each [H]). Fibroblast specific mice are pending. For therapeutics, using homology modelling we developed 7 boron-based agents to inhibit Shroom3-Rock interaction (BT1131-BT1137). BT1135-BT1137 showed significant inhibition pMYPT1 [I] and 3T3 proliferation. Subsequent dose-response efficacy and toxicity studies identify BT1137 as optimal candidate for in vivo work.
Conclusion
We show the critical role of Shroom3-Rock interaction in renal fibrosis and show druggability of this interaction with applicability to individuals with the high expressor Shroom3 risk-allele.
Funding
- NIDDK Support