High salt intake is the leading dietary risk factor for hypertension. Although clinical evidence suggests that high salt intake is associated with non-alcoholic fatty liver disease (NAFLD) that is an independent risk factor for hypertension, it remains elusive whether salt-induced hepatic damage leads to the development of hypertension. SIRT3 is the main protector in liver metabolism, and knockout of SIRT3 in mice results in fatty liver and metabolic syndrome. Therefore, we asked whether SIRT3 in liver participated in salt-induced hypertension and investigated the underlying mechanism.
Design and method:
6-week male wild-type or SIRT3-knockout (SIRT3−/−) mice were fed with 0.4% normal salt diet (NSD) or 8% high salt diet (HSD) for 16 weeks. Liver weight, body weight and blood pressure were measured. H&E, Oil-O red, and Sirius Red staining were performed on liver frozen sections to detect steatosis, lipid accumulation and fibrosis, respectively. Hepatic inflammation was reflexed by immunofluorescent staining of Cd11b, Mpo and F4/80, the markers of neutrophils, macrophages and Kupffer cells, respectively. Adeno-associated virus 8 (AAV8) with a Thyroxine-Binding Globulin (TBG) promoter was used to overexpress SIRT3 in liver. Chromatin immunoprecipitation sequencing (ChIP-seq) with an antibody against acetylated histone 3 lysine 27 (H3K27ac), an activated promoter marker, was used to explore the possible mechanism underlying the effect of SIRT3 on salt-induced hepatic inflammation.
HSD induced a significant increase in systolic blood pressure, a higher liver weight to body weight ratio, and stimulated lipid accumulation, steatosis, and fibrosis in the liver, accompanied by a lowered expression of SIRT3 in liver. Knockout of SIRT3 further exacerbated HSD-induced hepatic steatosis and inflammation, resulting in a more severe hypertension. Restoration of hepatic SIRT3 expression in SIRT3−/− mice by injection of AAV8-TBG-SIRT3 lowered all these pathological changes induced by HSD. ChIP-seq results suggest that knockout of SIRT3 mainly affected the H3K27ac level of promoters of genes related to AMP-activated protein kinase (AMPK) and insulin signaling pathways. Activation of AMPK by metformin significantly blocked salt-induced hepatic inflammation and hypertension, accompanied with an elevated SIRT3 expression in liver.
The present study proves that the reduced SIRT3 expression in liver plays a critical role in salt-induced hypertension by inducing hepatic inflammation. Restoration of hepatic SIRT3 expression or treatment with metformin helps to stave off hepatic inflammation that underlies salt-induced hypertension in clinical practice.