Histone Demethylase Inhibitor, GSK-J4, Suppresses Tubulointerstitial Fibrosis in CKD

Background: Epigenetic modifications including histone modifications in the kidney have recently been reported to contribute to the progression of renal fibrosis. We have previously reported that one of histone modification inhibitors suppressed renal fibrosis by decreasing open chromatin region. Another histone modifier inhibitor, GSK-J4, is known to inhibit KDM6b (lysine demethylase 6b) which demethylates H3K27me3, resulting in the increase in H3K27me3, a repressive mark of histone modification. We investigated how GSK-J4 affected renal fibrosis. Methods: We performed in vitro experiments using normal rat kidney interstitial fibroblast cells (NRK49F) stimulated by transforming growth factor-beta (TGF-β) with or without exposure to GSK-J4. To identify the downstream target genes of GSK-J4, we performed comprehensive RNA-seq under normal condition and TGF-β stimulation with and without GSK-J4. In addition, we also performed RNA-seq using siRNA of KDM6b to identify the downstream targets of KDM6b because GSK-J4 inhibited KDM6b. Results: We confirmed that fibrosis-promoting genes including Ccn2 (cellular communication network factor2), also known as Ctgf (connective tissue growth factor), were inhibited by GSK-J4 in NRK49F under TGF-β stimulation. As a result of RNA-seq, we identified 113 downstream target genes of GSK-J4 that were upregulated by TGF-β and downregulated by GSK-J4. After siRNA knockdown of KDM6b with TGF-β stimulation, we identified 26 genes whose expression was upregulated by TGF-β but downregulated by knockdown of KDM6b. We compared the downstream genes of GSK-J4 to those with siRNA of KDM6b, and we identified seven genes commonly. Among them, Acta2 (alpha SMA; alpha smooth muscle actin), a fibrotic marker whose expression increases as renal fibrosis progresses, was suppressed by GSK-J4 treatment, which prevented KDM6b from demethylating H3K27me3 and increases H3K27me3, resulting in the reduction of renal fibrosis. Conclusion: GSK-J4 inhibited renal fibrosis by modulating H3K27me3 via KDM6b on fibrosis-related genes including Acta2. It is important to elucidate the epigenetic molecular mechanisms that accompany the progression of renal fibrosis, as this will lead to the discovery of new therapeutic targets in CKD. Funding: Government Support – Non-U.S.