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Autophagy plays a critical role in Klotho gene deficiency-induced arterial stiffening and hypertension

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Abstract

Klotho is an anti-aging gene that shortens the life span when disrupted and extends the lifespan when overexpressed. This study investigated whether autophagy plays a role in Klotho gene deficiency-induced arterial stiffening and hypertension. Klotho mutant heterozygous (KL+/−) mice and age- and sex-matched wild-type (WT) mice were used. Arteries were examined for autophagy using Western blot assays. Pulse wave velocity (PWV), a direct measure of arterial stiffness, and blood pressure (BP) increased significantly in KL (+/−) mice. The autophagy level, as measured by LC3-II expression and autophagy flux, increased in aortas of KL (+/−) mice, indicating that Klotho gene deficiency upregulated autophagy. Chloroquine diminished Klotho gene deficiency-induced increases in PWV and BP and eliminated the upregulation of autophagic flux in KL (+/−) mice. Klotho gene deficiency-induced arterial stiffness was accompanied by upregulation of MMP9, TGFβ-1, TGFβ-3, RUNX2, and ALP, but these changes were effectively mitigated by chloroquine. Chloroquine also halted an increase in scleraxis expression in aortas of Klotho (+/−) mice. In cultured mouse aortic smooth muscle cells, Klotho gene deficiency increased autophagy, leading to upregulation of scleraxis, a key transcription factor of collagen synthesis. Klotho gene deficiency failed to upregulate scleraxis expression when autophagy was inhibited, suggesting that autophagy is a critical mediator of Klotho gene deficiency-induced upregulation of scleraxis. Suppression of enhanced autophagy by chloroquine lessens Klotho gene deficiency-induced arterial stiffening and hypertension by stopping upregulation of MMP9 and scleraxis. The enhanced autophagic activity plays a crucial role in Klotho gene deficiency-induced arterial stiffening and hypertension.

Key messages

  • Klotho gene deficiency upregulates autophagy.

  • Upregulation of autophagy plays a role in the pathogenesis of arterial stiffening.

  • Autophagy regulates MMP9 activity and scleraxis expression.

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Abbreviations

ALP:

Alkaline phosphatase

BFA:

Bafilomycin A1

bHLH:

Basic helix-loop-helix

KL (−):

Klotho free medium

KL (+/−):

Klotho mutant heterozygous

LC3-II:

Microtubule-associated proteins 1A/1B light chain 3B

MMP:

Matrix metalloproteinases

MOVAS:

Mouse vascular aortic smooth muscle cell

P62/SQSTM1:

Sequestosome 1

PBS:

Phosphate-buffered saline

PWV:

Pulse wave velocity

Runx2:

Runt-related transcription factor 2

SCX:

Scleraxis

sKL:

Recombinant secreted Klotho

TGFβ:

Transforming growth factor beta

TIMP:

Tissue inhibitors of metalloproteinases

VSMC:

Vascular smooth muscle cells

WT:

Wild type

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Acknowledgments

We would like to thank Dr. Nathan Tipton for his assistance in editing the manuscript.

Support and funding

This work was supported by NIH R01 HL118558, AG049780, HL122166, HL116863, DK093403, AG062375, HL102074, and HL105302.

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Authors and Affiliations

  1. Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Kai Chen & Zhongjie Sun

  2. Department of Physiology, College of Medicine, The University of Tennessee Health Science Center, C302B Coleman Bldg., 956 Court Ave, Memphis, TN, 38163-2116, USA

    Kai Chen & Zhongjie Sun

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  1. Kai Chen
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Z.S. created the experimental design, provided management for the funding, and contributed to the writing and editing of the manuscript. K.C. executed the experiments, analyzed the data, and contributed to the writing of the manuscript.

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Correspondence to Zhongjie Sun.

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Chen, K., Sun, Z. Autophagy plays a critical role in Klotho gene deficiency-induced arterial stiffening and hypertension. J Mol Med 97, 1615–1625 (2019). https://doi.org/10.1007/s00109-019-01841-6

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