Aldosterone enhances high phosphate-induced vascular calcification through inhibition of AMPK-mediated autophagy

Abstract

It remains unclear whether the necessity of calcified mellitus induced by high inorganic phosphate (Pi) is required and the roles of autophagy plays in aldosterone (Aldo)-enhanced vascular calcification (VC) and vascular smooth muscle cell (VSMC) osteogenic differentiation. In the present study, we found that Aldo enhanced VC both in vivo and in vitro only in the presence of high Pi, alongside with increased expression of VSMC osteogenic proteins (BMP2, Runx2 and OCN) and decreased expression of VSMC contractile proteins (α-SMA, SM22α and smoothelin). However, these effects were blocked by mineralocorticoid receptor inhibitor, spironolactone. In addition, the stimulatory effects of Aldo on VSMC calcification were further accelerated by the autophagy inhibitor, 3-MA, and were counteracted by the autophagy inducer, rapamycin. Moreover, inhibiting adenosine monophosphate-activated protein kinase (AMPK) by Compound C attenuated Aldo/MR-enhanced VC. These results suggested that Aldo facilitates high Pi-induced VSMC osteogenic phenotypic switch and calcification through MR-mediated signalling pathways that involve AMPK-dependent autophagy, which provided new insights into Aldo excess-associated VC in various settings.

Keywords: aldosterone; autophagy; phenotypic switch; vascular calcification; vascular smooth muscle cell.

FIGURE 1

Aldosterone (Aldo) promoted vascular calcification only in the presence of high phosphate (Pi) in vivo and in vitro. A, Von Kossa staining of mouse thoracic aortas (scale bar: 100 μm). B, Calcium content of the thoracic aortas was measured and normalized to the protein levels. C, Quantitative analysis of alkaline phosphatase (ALP) activity in the aortas normalized to the protein content. D, Alizarin red S staining including whole well and microscopic (scale bar, 50 μm) images in rat VSMCs treated with Aldo at different doses plus high Pi or normal Pi stimulation for 7 d. E and F, Calcium content of VSMCs after Aldo induction at different doses (1, 10, 100 and 1000 nmol/L) and timepoints (3, 5, 7 and 9 d) with or without high Pi mellitus (2.6 mmol/L NaH₂PO₄). n = 10 per group for in vivo and n = 5 per group for in vitro experiments. *P < .05 vs control group; ^& P < .05 vs high‐phosphate diet (HPD) group; ^# P < .05 vs Pi group

FIGURE 2

Aldosterone (Aldo) accelerated high phosphate (Pi)–induced vascular smooth muscle cell (VSMC) calcification and phenotypic switch in a mineralocorticoid receptor‐dependent manner. A, Alizarin red S staining of VSMCs. B, Quantification of calcium deposition of VSMCs normalized to the protein content. C and D, Representative Western blot bands and semiquantitative analysis of runt‐related transcription factor 2 (Runx2), bone morphogenetic protein‐2 (BMP2) and osteocalcin (OCN) in VSMCs; E and F, Representative Western blot bands and semiquantitative analysis of smoothelin, alpha smooth muscle actin (α‐SMA) and smooth muscle 22 alpha (SM22α) in VSMCs after induction with different stimuli for 7 d, and 100 nmol/L Aldo and 20 μmol/L spironolactone (Sipro) were used. n = 5 per group. *P < .05 vs control group; ^# P < .05 vs Pi group; ^& P < .05 vs Aldo + Pi group

FIGURE 3

Aldosterone (Aldo) inhibited vascular smooth muscle cell (VSMC) autophagosomes formation. A and B, Representative Western blot bands and semiquantitative analysis of P62, Beclin‐1 and LC3 after Aldo stimulation at different doses for 7 d in the high phosphate (Pi) condition; C‐F, Representative Western blot bands and semiquantitative analysis of LC3 in untreated and Aldo‐treated cells. Note that chloroquine (CQ) (20 µmol/L) or bafilomycin A1 (Baf) (0.01 µmol/L) was added 3 h before Aldo. G and H, Representative confocal images (scale bar, 20 μm) and quantitative analysis of mRFP‐GFP‐LC3 puncta in VSMCs. Bar graph showed the mean number of autophagosomes (yellow dots) and autolysosomes (red dots) per cell. n = 5 per group. *P < .05 vs control group; ^# P < .05 vs Pi group; ^% P < .05 vs CQ or Baf group; ^& P < .05 vs Pi + CQ or Pi + Baf group

FIGURE 4

Aldosterone (Aldo) promoted calcification and osteogenic differentiation of vascular smooth muscle cells (VSMCs) via inhibition of autophagosomes. A, Alizarin red S staining. B, Quantitative analysis of the calcium content of VSMCs after induction with different stimuli for 7 d, normalized to the protein levels, and 100 nmol/L Aldo, 10 μmol/L 3‐methyladenine (3‐MA) and 10 μmol/L rapamycin (Rapa) were used. C‐E, Representative Western blot bands and semiquantitative analysis of LC3, smoothelin, α‐SMA, BMP2 and Runx2. n = 5 per group. *P < .05 vs control group; ^# P < .05 vs Pi group; ^& P < .05 vs Aldo + Pi group

FIGURE 5

Adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation mediated aldosterone (Aldo)‐mineralocorticoid receptor‐dependent inhibition of vascular smooth muscle cells (VSMC) autophagy. A and B, Representative Western blot bands and semiquantitative analysis of LC3, Beclin‐1, phospho‐AMPKα (P‐AMPKα) and total AMPKα (T‐AMPKα) in VSMCs. C, Alizarin red S staining of microscopic (scale bar, 50 μm) images in mice VSMCs. D, Quantitative analysis of the calcium content of VSMCs after induction with different stimuli for 7 d, normalized to the protein levels, and 100 nmol/L Aldo, 1 µmol/L Compound C and 20 μmol/L spironolactone (Sipro) were used. n = 5 per group. *P < .05 vs control group; ^# P < .05 vs Pi group; ^& P < .05 vs Aldo + Pi group; ^% P < .05 vs Aldo + Pi + Spiro group

FIGURE 6

Schematic representation of the molecular mechanism for aldosterone (Aldo)‐enhanced vascular smooth muscle cell (VSMC) calcification. In the presence of high phosphate (Pi), Aldo binds to mineralocorticoid receptor (MR) in cytoplasm and inhibits adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation, which then retards formation of VSMC autophagosome, in turn promotes phenotypic switch of VSMCs from the contractile to the osteogenic form, leading to enhanced VSMC calcification