SIRT6 protects vascular smooth muscle cells from osteogenic transdifferentiation via Runx2 in chronic kidney disease

Vascular calcification (VC) is regarded as an important pathological change lacking effective treatment and associated with high mortality. Sirtuin 6 (SIRT6) is a member of the Sirtuin family, a class III histone deacetylase and a key epigenetic regulator. SIRT6 has a protective role in patients with chronic kidney disease (CKD). However, the exact role and molecular mechanism of SIRT6 in VC in patients with CKD remain unclear. Here, we demonstrated that SIRT6 was markedly downregulated in peripheral blood mononuclear cells (PBMCs) and in the radial artery tissue of patients with CKD with VC. SIRT6-transgenic (SIRT6-Tg) mice showed alleviated VC, while vascular smooth muscle cell–specific (VSMC-specific) SIRT6 knocked-down mice showed severe VC in CKD. SIRT6 suppressed the osteogenic transdifferentiation of VSMCs via regulation of runt-related transcription factor 2 (Runx2). Coimmunoprecipitation (co-IP) and immunoprecipitation (IP) assays confirmed that SIRT6 bound to Runx2. Moreover, Runx2 was deacetylated by SIRT6 and further promoted nuclear export via exportin 1 (XPO1), which in turn caused degradation of Runx2 through the ubiquitin-proteasome system. These results demonstrated that SIRT6 prevented VC by suppressing the osteogenic transdifferentiation of VSMCs, and as such targeting SIRT6 may be an appealing therapeutic target for VC in CKD.

Data are expressed as mean ± SD., calculated using one-way ANOVA followed by Dunnett's test. *P < 0.05.

Supplemental Figure 4
WT mice were treated by AAV-sh-control and AAV-sh-SIRT6 for 4 weeks. Then the mice were sacrificed and collected aortas and kidneys. The SIRT6 expression among these groups were detected by Western blot to confirm the efficiency of AAV-sh-SIRT6 in aorta and kidney.

Supplemental Figure 5
A. WT mice were treated by AAV-sh-control and AAV-sh-SIRT6 for 4 weeks. Then these mice were treated with adenine and phosphorus diet (AP) for 12 weeks, or performed 5/6 nephrectomy and fed with high phosphorus for another 8 weeks. Then the mice were sacrificed and collected aortas. Alizarin red S images showing calcification in aortas among these groups. The calcified parts of aorta were shown in deep purple. Scale bars, 10 mm. B, C.
Calcium content (B) and ALP (C) were quantified in aortas tissue among these groups. D, E.
Analysis of osteogenic and contractile property factor expression of aortas tissue among these groups by Western blot. Data are expressed as mean ± SD., calculated using one-way ANOVA followed by Dunnett's test. *P < 0.05.

Supplemental Figure 6
The primary cells isolated from mouse aorta was identified by VSMCs marker (smooth muscle myosin heavy chain and SM-22α) in IF. Scale bar, 50μm Supplemental Fig. 7 A, B. WT and SIRT6-Tg VSMC were pre-transfected with siSIRT6 or si-negative control (siNC) and then exposed to Pi for 7 days; The VSMC was incubated with Pi together with DMSO or OSS-128167 for 7 days. The SIRT6 expression were analyzed by qPCR (A), and the cells extracts were immunoblotted for the indicated proteins (B). C-E. WT and SIRT6-Tg VSMC were incubated with Pi together with DMSO or OSS-128167 for 7 days. The VSMC was stained for mineralization by Alizarin red S (C), and the quantitative analysis of calcium content and ALP (D-E) were detected respectively. Statistical significance was assessed using one-way ANOVA followed by Dunnett's test and is presented as follows: *P < 0.05. All values are means ± S.D.

Supplemental Figure 8
A. Analysis of osteogenic and contractile property factor expression in WT and SIRT6-Tg VSMCs after Pi treatment by qPCR. B-E. The VSMC was pre-transfected with siSIRT6 or siNC together with Pi for 7 days, and the downstream osteogenic markers (OPN, OCN) and the contractile property marker (α-SMA, SM-22α) were analyzed by qPCR (B, C); VSMC was incubated with DMSO or OSS-128167 together with Pi for 7days and the same markers like above were analyzed by qPCR (D, E). Statistical significance was assessed using one-way ANOVA followed by Dunnett's test and is presented as follows: *P < 0.05. All values are means ± S.D.

Supplemental Figure 9
A. VSMCs were incubated with DMSO or OSS-128167 (0.1mM), a SIRT6 specific inhibitor, together with Pi (3.0Mm) for 7days and the osteogenic and contractile property markers were analyzed by Western blot. B-C. SIRT6-Tg VSMCs were pre-transfected with Runx2 plasmid or vector plasmid, and then exposed to Pi (3.0mM) for 7 days. Calcium content (B) and ALP (C) were quantified in the VSMCs. D. The VSMC was incubated with Pi for 7 days, then the mRNA level of Runx2 was detected through qPCR. E. SIRT6-Tg VSMC was transfected with shRNA targeting XPO1, XPO4, XPO7, or their vector negative control shRNA. The relative expression of XPO1, XPO4, XPO7 were analyzed by qPCR. Statistical significance was assessed using one-way ANOVA followed by Dunnett's test (B-C)and two-tailed t-test for two groups (D-E) and is presented as follows: *P < 0.05. All values are means ± S.D.

Supplemental Figure 10
WT VSMCs were treated with Pi for 7 days and incubated with the protein translation inhibitor CHX (0.2mM) for the indicated times before harvest, followed by immunoblotting with the anti-SIRT6 antibody and anti-Tublin anti-body. The curve shows the stability of SIRT6 protein (The SIRT6 protein of Pi group were normalized by no Pi group ).

Supplemental Figure 11
A. Anti-Runx2 IP followed by WB with anti-Runx2 or anti-Smurf1 antibody in WT VSMCs after treatment with Pi for 7days. Anti-rabbit IgG IP was used as a negative control. B.
Anti-Runx2 IP in WT and SIRT6-Tg VSMCs after treatment with Pi for 7days. WB was carried out with anti-Runx2 and anti-Smurf1 antibody. Anti-rabbit IgG IP was used as a negative control.

Supplemental Figure 12
The aortas slides were incubated with anti-rabbit-FITC antibody for 1h at room temperature.Nuclei were counterstained with DAPI. Prolong Gold antifade reagent was used to decrease fluorescence quenching of the slides. The elastic fibers are fluorescing. The picture shown as a negative control for IF staining. Scale bar, 50μm