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Following kidney injury, pericytes differentiate into alpha-smooth muscle actin (α-SMA) positive myofibroblasts, the main collagen-producing cells in kidney fibrosis. The expression of Acta2 (which encodes α-SMA) repressors, such as Rasal1, Pura, Tead1, Ybx1, Ybx2, and others, is decreased in myofibroblasts. Short for suppressor of variegation 3(9) homologue 2 (SUV39H2), is a lysine methyltransferase capable of inducing trimethylation of histone 3 Lysine 9 (H3K9me3), resulting in transcriptional repression of gene. Therefore, we hypothesized that the Acta2 repressors on pericytes will have their gene expression reduced due to H3K9me3 induced by SUV39H2, resulting in increased α-SMA expression. We aim to observe whether chaetocin, an inhibitor of H3K9me3, can reverse this process and improve the degree of kidney fibrosis.
This study employed animal chronic kidney disease (CKD) model using C57BL/6 (B6) mice that were fed with a diet containing 0.2% adenine for eight weeks to investigate whether chaetocin can inhibit renal fibrosis and improve kidney function. The experiment involved three groups: the normal diet group, the adenine diet with vehicle group, and the adenine diet with chaetocin group. Renal function (BUN, Creatinine), hematocrit and body weight were checked regularly. Real-time quantitative polymerase chain reaction (qPCR) was used to analyze the gene expression related to fibrosis, histone methyltransferase and Acta2 repressors. Western blot analysis was employed to measure the levels of H3K9me3 and the expression of relevant protein. Finally, picrosirius red staining was used to assess the differences in the degree of fibrosis among the three groups.
In the model with 8-week 0.2% adenine diet, adenine diet significantly increased the BUN and creatinine compared to normal diet, while causing a significant decrease in HCT. It also led to a gradual decrease in body weight. In the qPCR experiments, fibrosis-related genes and Suv39h2 showed increased expression in the mice fed with adenine diet. When mice were injected intraperitoneally with chaetocin, the increase of fibrotic gene expression were reversed. Among the Acta2 repressors, Ybx2 showed a significant decrease in mice with adenine diet, while the chaetocin group demonstrated an increasing trend compared with vehicle group. In the Western blot experiments, collagen α1 type I precursor (pro-COL1A1) significantly increased in mice with adenine diet, while chaetocin showed a trend of reducing its expression. α-SMA increased significantly in mice with adenine diet, while the chaetocin group exhibited a trend of decreased expression. YBX2 showed a significant decrease in mice with adenine diet, while chaetocin group had a trend of increasing its expression. In the picrosirius red staining experiment, mice with adenine diet exhibited an increased degree of fibrosis, whereas chaetocin slowed down the formation of fibrosis.
Adenine diet resulted in an increase in fibrosis, reduced body weight and anemia in B6 mice, mimicking human CKD condition. Chaetocin group demonstrated an improvement in renal function, kidney fibrosis as well as the expression of Acta2 repressors. Therefore, the underlying mechanisms of chaetocin to inhibit renal fibrosis is a direction worth studying in the future.