Deacetyl asperulosidic acid methyl ester attenuates renal fibrosis via Hsp90aa1-mediated TGFβR1 degradation

Background Renal fibrosis represents a common and critical pathological process underlying chronic kidney disease (CKD), invariably leading to progressive renal dysfunction and ultimately end-stage renal disease. Purpose This investigation sought to determine anti-fibrotic efficacy of Deacetyl Asperulosidic Acid Methyl Ester (DA), in ameliorating renal fibrosis, focusing on its interaction with heat shock protein 90 (Hsp90aa1) and its downstream effects on TGF-β/Smad signaling. Study design The anti-fibrotic efficacy of DA was evaluated in vivo using two murine models of CKD (unilateral ureteral obstruction and adenine-induced nephropathy) and in vitro using TGF-β1-stimulated NRK-49F fibroblasts and TCMK-1 tubular cells. Molecular targets were identified and validated through quantitative proteomics, Specific Pupylation as Identity Reporter (SPIDER) assay, and biophysical interaction analyses. Methods Renal function and histological changes were assessed by biochemical analysis, H&E, and Masson's trichrome staining. The expression of fibrotic markers and signaling pathway proteins was determined using IHC, Western blot, and qRT-PCR. The interaction between DA and Hsp90aa1 was verified by Cellular Thermal Shift Assay (CETSA), Drug Affinity Responsive Target Stability (DARTS), and Co-immunoprecipitation (Co-IP). Results Our data demonstrated that DA administration substantially ameliorated renal dysfunction and attenuated fibrotic progression in both animal and cellular models. Quantitative proteomic profiling results revealed a prominent suppression of extracellular matrix components and TGF-β type I receptor (TGFβRI) following DA treatment. Furthermore, biotin-labeled DA binding combined with SPIDER experiments identified Hsp90aa1 as a direct target of DA. Mechanistically, the binding of DA to Hsp90aa1 disrupts the interaction between Hsp90aa1 and TGFβR1, thereby destabilizing TGFβR1 and promoting its degradation. This regulation effectively impedes the transduction of the downstream TGF-β/Smad signaling cascade, ultimately mitigating renal fibrosis. Conclusion Our findings demonstrate that DA is a potent anti-fibrotic agent that targets Hsp90aa1 to modulate TGFBR1 stability and inhibit the TGF-β/Smad pathway. Download: Download high-res image (359KB) Download: Download full-size image