O6-Benzylguanine Decreases Fibrotic Markers via Transforming Growth Factor (TGF)-β Canonical and Noncanonical Pathways, Leading to Decreased Epithelial-Mesenchymal Transition in a Model of Unilateral Ureteral Obstruction

Background: O6-benzylguanine (BG) is an O6-methylguanine-DNA-methyltransferase inhibitor and is used only in combination with methylating agents to treat chemoresistant tumors. In our previous studies, we observed after BG treatment decreased proliferation of vascular smooth muscle cells along with lower expression of α-SMA (α-smooth muscle actin). Therefore, we hypothesized that BG may also inhibit fibroblast proliferation, thus reducing fibrosis development. Methods: The rats were randomized into three groups: SHAM – the control group, with a standard surgical procedure performed; UUO – the group with Unilateral Ureteral Obstruction performed; and BG – the group with Unilateral Ureteral Obstruction, treated with BG at a dose of 30 mg/kg p.o. every 24 hours for 7 days. After 7 days, experiment was terminated and the kidneys were harvested. Proteins were isolated using RIPA buffer, separated by SDS-PAGE (12%) and analysed by western blot. Results: In the BG-treated group, we observed a significant decrease in the levels of: pJNK/JNK, pSMAD2, β-catenin, vimentin, α-SMA, E-cadherin and HMGB1. The results also showed decreased levels of signaling molecules such as pERK/ERK, pPI3K p85, pAKT, N-cadherin, NF-kB. No changes were observed in the levels of pMEK/MEK, pp38, MnSOD. Conclusion: Our results show for the first time that BG possesses antifibrotic properties. It interferes with TGF-β canonical (pSMAD pathway) and non-canonical pathways involved in fibrogenesis, including the JNK pathway, which regulates proinflammatory and growth factors and supports the TGF-β canonical pathway by phosphorylating SMAD2/3. BG also reduces β-catenin in the Wnt pathway, which modulates epithelial-mesenchymal transition and inhibits antifibrotic pSMAD7, leading to higher pSMAD2/3 activity. Additionally, BG affects the PI3K/AKT pathway, which influences extracellular matrix proteins and β-catenin activation. By inhibiting these pathways, BG reduces markers of epithelial-mesenchymal transition, such as vimentin, N-cadherin, and α-SMA, key components in fibrosis. This work was supported by: UK/3163/24, APVV 23-0399, VEGA 1/0513/24 Funding: Government Support – Non-U.S.