#590 Inhibition of Histone deacetylase 6 restores kidney fibrosis in a mouse model of unilateral ureteral obstruction

Abstract Background and Aims Due to the critical role of histone acetylation in kidney development and pathology, numerous preclinical and clinical studies have explored the therapeutic potential of histone deacetylase (HDAC) inhibitors in managing kidney diseases. However, most available HDAC inhibitors are pan-HDAC inhibitors, which lack specificity and are associated with notable side effects, highlighting the need for selective HDAC inhibitors. Among the various HDACs, we are particularly interested in HDAC6. In our previous study, we synthesized selective HDAC6 inhibitors (HDAC6i) #60, featuring a quinazolinone-based structure and a difluoromethyl oxadiazole zinc-binding group, designed to reduce side effects. We conducted in vitro and in vivo experiments to investigate the role of HDAC6 in chronic kidney disease (CKD), and to analyze the impact of HDAC6 inhibition on renal fibrosis. Method We analyzed the gene expression of HDAC isoforms in the kidneys and used immunostaining with Collagen I (α)1-GFP+ transgenic mice to localize HDAC6 expression. Subsequently, we employed Slc34a1CreERT2/+; Rosa26fsEGFP-L10a/+ mice to study mRNA expression in slc34a1-expressing proximal tubular epithelial cells (PTECs) through translating ribosomal affinity purification (TRAP). Additionally, we used C57BL/6 mice to perform qPCR, western blotting and picrosirius red staining to assess fibrosis marker and acetylation levels. We used HDAC6i #60 to conduct both in vitro and in vivo tests. Results First, we performed unilateral ureteral obstruction (UUO) surgery to induce pericyte-myofibroblast transition and progressive renal fibrosis in mice. Protein levels of α-SMA and HDAC6 in the UUO kidney increased over time. Acetyl-H3 levels initially decreased but later normalized during the later stages, suggesting that HDAC6 suppresses acetyl-H3 during the early stages of fibrosis. Through immunostaining, we also observed an upregulation of HDAC6 expression following UUO, particularly in tubular epithelial cells. We further confirmed the localization of HDAC6 by TRAP of mRNA populations in Slc34a1-expressing PTECs. Next, we treated PTECs with the HDAC6i #60 and found that, although the protein levels of HDAC6 were not reduced by HDAC6i treatment, the acetylation levels of H3 and α-tubulin were increased. This indicates that HDAC6i effectively restores acetylation in tubular cells. Finally, after 10 days of oral gavage with HDAC6i #60 following UUO surgery, qPCR and western blot analysis revealed a significant decrease in Acta2 and Pdgfrβ expression, along with a downward trend in Col1a1, Col3a1, and Tgfβ1 levels. Quantification of picrosirius red staining further confirmed reduced fibrosis following HDAC6i treatment. Conclusion Our study demonstrates the critical role of HDAC6 in the early stages of UUO-induced renal fibrosis. The selective HDAC6i #60 effectively attenuates fibrosis, underscoring its potential as a therapeutic strategy for CKD.