Enhancer variants in Shroom3 associate with renal fibrosis (TIF), but with reduced albuminuria. Detailed mechanisms for these pleiotropic effects are unclear. Here, we focus on identifying the specific profibrotic Shroom3 motif and separating this from its anti-proteinuric function. Given the role for Rho-kinases (Rock) in TIF, and the interaction of Rock with Shroom3 ASD2-domain, we hypothesized that Shroom3-mediated Rock-activation is crucial for profibrotic function. To test this, we develop transgenic tools that overexpress wild-type- (WT-Sh3) or ASD2-domain deletion- Shroom3 (ASD2 -Sh3). During TIF, Shroom3 and Rock co-expression occur in injured tubular cells and fibroblasts. In tubular- & fibroblast- lin... More
Enhancer variants in Shroom3 associate with renal fibrosis (TIF), but with reduced albuminuria. Detailed mechanisms for these pleiotropic effects are unclear. Here, we focus on identifying the specific profibrotic Shroom3 motif and separating this from its anti-proteinuric function. Given the role for Rho-kinases (Rock) in TIF, and the interaction of Rock with Shroom3 ASD2-domain, we hypothesized that Shroom3-mediated Rock-activation is crucial for profibrotic function. To test this, we develop transgenic tools that overexpress wild-type- (WT-Sh3) or ASD2-domain deletion- Shroom3 (ASD2 -Sh3). During TIF, Shroom3 and Rock co-expression occur in injured tubular cells and fibroblasts. In tubular- & fibroblast- lines, ASD2 -Sh3 overexpression reduce Rock activation, and pro-fibrotic/pro-inflammatory transcripts downstream of TGF 1/Wnt/Ctnnb1-signaling vs WT-Sh3. In vivo, inducible global-, or tubular-specific-, but not fibroblast-specific-, ASD2 -Sh3 overexpression mitigate TIF, vs WT-Sh3 overexpression. Importantly, ASD2 -Sh3 mice do not develop albuminuria, while overexpression of a distinct Fyn-binding deficient mutant Shroom3 (FBDM-Sh3) induces albuminuria. We then develop small molecule inhibitors of Shroom3-Rock interaction (P2Is) and confirm Rock inhibition with these agents in WT-Sh3 cell lines. Our lead P2I from these studies, BT1137, mitigates Rock-activation, profibrotic signaling and TIF in WT-Sh3 mice. Hence, we delineate the profibrotic Shroom3 motif and develop therapeutics for kidney disease from Shroom3 excess.Shroom3 genetic variants increase Shroom3 levels and promote kidney fibrosis but reduce proteinuria, complicating Shroom3 targeting for precision medicine. Here, the authors show increased fibrosis mediated by Shroom3 Rock signaling and blocking this interaction genetically or with new compounds reduces tubular Rock activation and fibrosis.
