Reagents and antibodies

Sodium phosphate (Cat#342483), cobalt chloride (CoCl2) (Cat#769495), CaCl2 (Cat#G5670), and β-glycerophosphate (β-GP) (Cat#G9422) were from Sigma-Aldrich (MO, USA). Alizarin red S (Cat#G8550) was from Solarbio (Beijing, China). Dapagliflozin (Cat#HY-10450), EX527 (Cat#HY-15452), and Vitamin D3 (Cat#HY-15398) were from MedChemExpress (NJ, USA). Cetylpyridinium chloride was from Aladdin (Shanghai, China). Trypsin, penicillin, streptomycin, fetal bovine serum (FBS), and Dulbecco’s Modified Eagle’s Medium (DMEM) were from GIBCO. A glucose kit (Cat#A154-1-1), a calcium assay kit (Cat#C004-2-1), and an ALP assay kit (Cat#A059-2-1) were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A NAD+/NADH assay kit (Cat#S0175) was from Beyotime (Shanghai, China). A Von Kossa staining kit (Cat#G1034) from Servicebio (Wuhan, China). Anti-α-SMA (Cat#67735-1-Ig, 1:10000 for WB, 1:200 for IF), anti-RUNX2 (Cat#20700-1-AP, 1:500 for WB, 1:200 for IF), anti-BMP2 antibody (Cat#66383-1-Ig, 1:1000 for WB), anti-HIF-1α (Cat#66730-1-Ig, 1:2000 for WB, 1:200 for IF), and anti-GAPDH (Cat#10494-1-AP, 1:10000 for WB) were from Proteintech (Wuhan, China). Anti-SM22α (Cat# ab14106, 1:2000 for WB) was from Abcam. Anti-RUNX2 (Cat#12556, 1:1000 for WB), anti-SIRT1 (Cat#9474, 1:1000 for WB, 1:200 for IF), and anti-HIF-1α (Cat#36169, 1:1000 for WB) were from Cell Signaling Technology (MA, USA). Anti-acetyl-HIF-1α (Cat#HY0140, 1:2000 for WB) was from Immunoway.

Cell culture

The thoracic aorta of a 6-week-old C57BL/6 mouse was used to harvest primary VSMCs as in a prior report [5]. Briefly, these mice were euthanized using sodium pentobarbital (150 mg/kg, ip), after which the thoracic aorta was removed and separated from the adventitia and endothelium, with the remaining tissue being cut into 1 mm2 tissue blocks. These aortic tissue blocks were then cultured for 1–2 weeks in complete media supplemented with 20% FBS. After the VSMCs migrated from the explants, the tissue blocks were removed and the VSMCs were cultured with DMEM containing 10% FBS. All subsequent experiments were conducted with primary VSMCs between passages 5 and 8. Human aortic smooth muscle cells (HASMCs) were obtained from the American Type Culture Collection (ATCC, USA). DMEM supplemented with 10% FBS and penicillin/streptomycin was used to culture all cells in a 5% CO2 incubator at 37ºC. To induce VSMCs calcification, cells were cultured for 7 days in DMEM supplemented with 1% FBS and 3 mmol/L sodium phosphate (Pi) for 7 days, replacing the media every other day. To determine the role that DAPA plays in VSMCs calcification, these cells were treated with various DAPA concentrations (2.5µM, 5µM, 10µM).

Animal experiments

The Ethics Committee for Animal Experiments of the Second Hospital of Shanxi Medical University provided approval for all animal studies (Approval No. DW2023045), which were conducted as per the NIH Guide for the Care and Use of Laboratory Animals. For these assays, C57BL/6 mice (males, 6 weeks old) were obtained from the Laboratory Animal Centre of Shanxi Medical University. A 5/6 nephrectomy model of CKD-associated vascular calcification was established as reported previously [5]. Briefly, isoflurane (2%) was used to anesthetize these mice, followed by the removal of two-thirds of the left kidney, with the entirety of the right kidney then being removed after a one-week interval. These mice were separated into the following groups: a sham control group fed a normal diet, a DAPA group in which sham control mice fed a normal diet were administered 5 mg/kg DAPA per day via gastric perfusion, a CKD model group in which mice were fed a 1.8% high phosphorus diet, and a CKD + DAPA model group in which mice were fed a 1.8% high phosphorus diet and administered 5 mg/kg DAPA per day via gastric perfusion. Animal treatments were maintained for an 8-week period, followed by the euthanasia of these mice with sodium pentobarbital (150 mg/kg, ip). The aorta was then isolated and murine serum was harvested for further experimentation. A separate model of vascular calcification was established through vitamin D3 (VitD3) overload [4, 15]. At 8 weeks of age, mice were randomly divided into three groups (n = 10/group): control, VitD3, and VitD3 + DAPA groups. Mice in the appropriate groups were subcutaneously injected with VitD3 (5 × 105 IU/kg) on three consecutive days, with mice in the VitD3 + DAPA group additionally receiving 5 mg/kg DAPA daily.

Rat arterial ring organ culture

Sprague-Dawley rats (6 weeks old, males) were euthanized with sodium pentobarbital (150 mg/kg, i.p.), followed by the isolation of the thoracic aorta. The resected aortas were then cut to produce 2–3 mm arterial rings that were subsequently cultured for 7 days in DMEM supplemented with 10 mM β-GP, 3 mM CaCl2, and 10% FBS, exchanging media every other day. Calcification of the aortic rings was detected by Von Kossa staining.

Western blot analysis

VSMCs were lysed on ice with RIPA buffer (Bosterbio) containing protease and phosphatase inhibitors for 15 min, while aortic tissue samples were added to lysis buffer and then lysed using a tissue homogenizer. Lysates were then centrifuged (12,000 xg, 20 min, 4 °C), and supernatant protein content was analyzed via BCA assay (Bosterbio). After combining proteins with loading buffer (Bosterbio), they were boiled for 5 min at 100 °C, separated via SDS-PAGE, and transferred onto PVDF membranes (Millipore). These blots were then probed with primary antibodies and HRP-conjugated secondary antibodies (Bosterbio, 1:10000), after which Image Lab (Bio-Rad) was used for ECL chemiluminescent signal detection, and ImageJ was used for densitometric quantification.

Glucose, Calcium, and ALP activity assay

Glucose levels, calcium content, and ALP activity were all analyzed with kits from commercial sources. Briefly, appropriate buffers were used to homogenize VSMCs and aortic tissue samples, followed by centrifugation and collection of the corresponding supernatants. Based on the provided instructions, the extracts were colorimetric analyzed at optical densities of 505 nm, 610 nm and 520 nm, and then glucose content, calcium content and ALP activity were calculated. Finally, the protein content of the extract was measured with a BCA assay and used to normalize those results.

NAD+/NADH ratio assay

The total amount of NAD+ and NADH were extracted from aortic or VSMCs samples as directed using appropriate extraction reagents, and then the extracts were subjected to colorimetric analysis at an optical density of 450 nm, with the total concentration was calculated. Next, a part of the extract was heated in 60℃ for 30 min to promote the degradation of NAD+, and the NADH content was obtained. Finally, the amount of NAD+ was obtained by subtracting the amount of NADH from the total amount of NAD+ and NADH. Based on the results, the NDA+/NADH ratio could then be calculated.

Alizarin Red S staining

VSMCs staining was performed by fixing these cells for 30 min using 4% paraformaldehyde, followed by adding Alizarin Red S stain (1%, pH 4.2) at room temperature for 3–5 min. Calcified cells appeared red in color following three washes with ddH2O. And then samples were dissolved with 500µL cetylpyridinium chloride solution (10%) and compared by colorimetric analysis of the eluated alizarin red at an optical density (OD) of 526 nm. When performing whole aorta staining, excessive connective tissue surrounding the aorta was separated, the aorta was fixed using 4% paraformaldehyde, followed by dehydration overnight in 95% alcohol. Aortas were then stained overnight in 0.003% Alizarin Red S stain with 1% KOH. Then, 2% KOH was used to wash away the staining solution, with the calcified aortic areas appearing purplish-red in color. Paraffin-embedded thoracic aorta sections were prepared by fixing the aorta with 4% paraformaldehyde prior to paraffin embedding and cutting into 5 μm posterior sections. These sections underwent standard deparaffinization and were stained for 2 min with 2% Alizarin Red S stain.

Von Kossa staining

Thoracic aortas were routinely deparaffinized, rehydrated, stained using silver nitrate, and illuminated for 4 h using an ultraviolet lamp. Sections were then rinsed with running water, stained in turn with hematoxylin and eosin, and imaged, with calcified areas appearing dark brown in color.

Immunofluorescence (IF) staining

VSMCs were initially fixed using 4% paraformaldehyde for 30 min, followed by permeabilization with 1% Triton-X. Samples were then blocked for 1 h with 5% BSA, rinsed thrice, and incubated overnight at 4 °C with primary antibodies. Cells were then incubated for 1 h at room temperature with a secondary antibody (Servicebio) in the dak. DAPI (Servicebio) staining solution was used to treat cells for 5 min to stain nuclei in the dark. Prepared 5-µm sections were routinely deparaffinized, rehydrated, and placed in an EDTA solution (Servicebio) for antigen repair in the microwave. After soaking sections for 1 h at room temperature using 5% BSA following 0.1% Triton X-100 treatment, sections were incubated with primary antibody at 4℃ overnight. Next day, sections were stained with secondary antibodies (Servicebio) at room temperature for 1 h in the dark. After staining with DAPI staining solution, cells were imaged via fluorescence microscopy.

Quantitative real-time PCR (qPCR)

Trizol (Takara) was used to isolate RNA from VSMCs and aortic tissue samples as directed, after which the PrimeScript RT Reagent Kit (Takara) was employed for reverse transcription and qPCR. The 2ΔΔCt method was employed to analyze relative gene expression, with GAPDH as a normalization control. All primers were from Sangon Biotech (Shanghai, China), and are compiled in Supplementary Table S1.

Blood pressure analysis

A non-invasive tail-cuff method (Visitech Systems BP-2000, NC, USA) was used for measurements of murine blood pressure. Briefly, following instrument calibration, animals were secured, with blood pressure measurements being taken following murine acclimatization to the test environment.

Cellular transfection

A SIRT1 siRNA (Cat# s223591) and SGLT2 siRNA (Cat# 289404) were obtained from Thermo Fisher Scientific (MA, USA), while an SGLT2 plasmid (Cat#HG19530-G) was from SinoBiological (Beijing, China). VSMCs were seeded in 6-well plates, and were transfected with these constructs using Lipofectamine 3000 (Thermo, MA, USA) as directed when cells were 60% confluent, utilizing serum-free medium (OPTI-MEM, GIBCO). At 6 h post-transfection, media was replaced. Western blotting was used to detect the efficiency of protein overexpression or knockdown.

Statistical analysis

All analyses were repeated a minimum of three times. All data were analyzed with GraphPad Prism 9.0 (CA, USA), are reported as means ± SEM, and were compared with Student’s t-tests or one-way ANOVAs. P < 0.05 was used to define statistical significance.