Targeting USP4 to inhibit TGF-β signaling: the antifibrotic potential of isovitexin in renal interstitial fibrosis.

Renal interstitial fibrosis (RIF), driven by persistent TGF-β signaling, is a hallmark of progressive kidney disease. Because deubiquitinases (DUBs) like USP4 stabilize TGF-β type I receptor (TβRI) to prolong signaling, targeting USP4 represents a promising antifibrotic strategy. To evaluate whether isovitexin (IVX), a plant-derived flavonoid, inhibits USP4 to destabilize TβRI, attenuate TGF-β/Smad signaling and EMT, and confer antifibrotic benefits in vitro and in vivo. We used a unilateral ureteral obstruction (UUO) mouse model and TGF-β1-stimulated renal tubular epithelial cells (HK-2, NRK-52E). Assays included biophysical target engagement (MST, CETSA, DARTS); biochemical analyses (ubiquitination, cycloheximide-chase, proteasome activity, co-immunoprecipitation); genetic perturbations (USP4 overexpression/knockdown, mutagenesis); histology (H&E, Masson's trichrome, immunohistochemistry); and molecular techniques (Western blotting, RT-qPCR, luciferase reporter assays). IVX alleviated UUO-induced renal injury, reduced ECM deposition, and normalized EMT markers (E-cadherin upregulated, α-SMA/vimentin downregulated), with efficacy comparable to losartan. IVX reduced TβRI and phosphorylated Smad3 while sparing TβRII. In tubular cells, IVX increased K48-linked ubiquitination of TβRI and accelerated proteasomal degradation, thereby suppressing Smad-dependent transcription. Binding assays confirmed direct IVX-USP4 engagement (Kd = 7.59 μM), inhibition of USP4 activity, disruption of the USP4-TβRI complex, and suppression of TGF-β1-induced USP4 expression. Overexpression of catalytically active USP4 reversed the effects of IVX. IVX functions as a natural USP4 inhibitor that modulates TGF-β signaling by selectively destabilizing TβRI, supporting a DUB-centered antifibrotic strategy and positioning IVX as a promising therapeutic lead for RIF treatment.