Sodium Butyrate Attenuates Diabetic Kidney Disease Partially via Histone Butyrylation Modification

Abstract

Inflammation and fibrosis are the important pathophysiologic processes in diabetic kidney disease (DKD), which is induced by epigenetics, especially histone posttranslational modification (HPTMs). Recent reports highlighted that butyrate, one of the short-chain fatty acids (SCFAs) primarily originated from the fermentation of dietary fiber in the gut, attenuates inflammation and fibrosis in the prevention and treatment of DKD; however, the molecular mechanisms are still unclear. Histone lysine butyrylation (Kbu), a novel histone modification marker induced by butyrate, has been found to be involved in the regulation of pathophysiological processes. To reveal the mechanisms of butyrate-induced histone (Kbu), in the prevention and treatment of DKD, both DKD models in vivo and in vitro were treated with sodium butyrate (NaB). Our results confirmed that exogenous NaB improved the disorder of glucose and lipid metabolism, prevented proteinuria and renal failure, and inhibited renal inflammation and fibrosis. Meanwhile, NaB also induced histone Kbu and H3K9 butyrylation (H3K9bu) in vivo and in vitro; however, inhibition of histone Kbu with the histone modification enzyme p300 inhibitor A485 reversed the anti-inflammatory and anti-fibrosis effects of NaB. In conclusion, our data reveal that NaB antagonizes renal inflammatory and fibrosis injury and attenuates DKD possibly via histone Kbu, suggesting that butyrate-induced histone Kbu or H3K9bu may be an important molecular mechanism in the pathogenesis and treatment of DKD.

Figure 1

NaB ameliorates glucose and lipid metabolism disorder in DKD mice. Mice were subjected to a high-fat diet (HFD) for 8 weeks, intraperitoneally (i.p.) injected with STZ, and then treated with sodium butyrate (NaB), for 12 weeks. Body weight (BW) (a) and random blood glucose (RBG) (b) were measured every 2 weeks; fasting blood glucose (FBG) (c), low-density lipoprotein-cholesterol (LDL-C) (d), total cholesterol (TC) (e), and total glyceride (TG) (f) values were measured at the 20th week of the experiment before sacrifice. Values are presented as the mean ± SD. ^∗P < 0.05,  ^∗∗0.001 < P < 0.01,  ^∗∗∗P < 0.001, and^∗∗∗∗P < 0.0001.

Figure 2

NaB alleviates inflammatory and fibrotic injury in DKD mice and high glucose-induced GMCs. Urine ACR (a), blood urea nitrogen (BUN) (b), blood crea (c), serum IL-6 (d), and serum MCP-1 (e) were assayed at the 20th week of the experiment; H&E and Masson staining of mice in each group (×400) and immunohistochemistry were used to detect the expression of Fn in mouse kidney of each group (×400) (f). The expression of mainly the contents of collagen type IV (COL IV) in kidneys of each group was detected by immunofluorescence (×400) (g); qRT-PCR of Fn, TGF-β, IL-6, and MCP-1 in kidney tissue after NaB treatment (h, i); Western blotting-based assays for the expression of TGF-β and MCP-1 in GMCs after NaB intervention (j); GMCs were stimulated with 30 mM high glucose in the presence of the indicated concentration of NaB for 24 h. MCP-1 and IL-6 in the cell culture supernatant were evaluated by the kit (k, l). Values are presented as the mean ± SD. ^∗P < 0.05,  ^∗∗0.001 < P < 0.01,  ^∗∗∗P < 0.001, and^∗∗∗∗P < 0.0001.

Figure 3

NaB induces histone Kbu in DKD kidney and renal mesangial cells. Immunohistochemistry (400x)-based assays for the expression of PanKbu and H3K9bu in DKD kidney tissue after NaB treatment (a). The expression of PanKbu in kidneys of each group was detected by immunofluorescence (×400) (b). The expression of PanKbu and H3K9bu after different concentrations of NaB intervened in GMCs (c). The expression of PanKbu and H3K9bu in GMCs of each group (d). The expression of H3K9bu in the nucleus in each group of GMCs (e). Values are presented as the mean ± SD. ^∗P < 0.05,  ^∗∗0.001 < P < 0.01,  ^∗∗∗P < 0.001, and^∗∗∗∗P < 0.0001.

Figure 4

A485 inhibits NaB-mediated histone Kbu and reverses anti-inflammatory and antifibrotic effects. Effect of the P300 inhibitor A485 on the expression of PanKbu and H3K9bu by Western blotting (a). The expression of H3K9bu in the nucleus of GMCs in each group by Western blotting (b). The expression of PanKbu in GMCs of each group was detected by immunofluorescence (×200) (c). A485 had no significant effect on NaB-induced PanKac modification of GMCs (d). The NaB inhibition of TGF-β and MCP-1 was reversed by A485 (e). qRT-PCR was performed to detect Fn and TGF-β mRNA levels (f). The expression of IL-6 in cells of each group was detected by immunofluorescence (×200) (g). Values are presented as the mean ± SD. ^∗P < 0.05,  ^∗∗0.001 < P < 0.01,  ^∗∗∗P < 0.001, and^∗∗∗∗P < 0.0001.