Vitamin D receptor deficiency increases systolic blood pressure by upregulating the renin-angiotensin system and autophagy

Abstract

The vitamin D receptor (VDR) may regulate blood pressure via multiple pathways. The present study investigated the underlying mechanism by which VDR deficiency increases blood pressure. A total of 16 8-week-old male littermate mice were randomly divided into the VDR knockout and wild-type groups (VDR^-/- and VDR^+/+ , respectively). Blood pressure was measured using a four-channel PowerLab data acquisition and ADI software analysis system. After euthanasia, vascular smooth muscle cells (VSMCs) were isolated from the VDR^-/- and VDR^+/+ mice. Oxidative stress, renin-angiotensin system (RAS) activation and autophagy markers were measured in the isolated VSMCs using reverse transcription-quantitative PCR (RT-qPCR), western blotting and transmission electron microscopy (TEM) assays. Mean systolic pressure was significantly higher in the VDR^-/- mice compared with the VDR^+/+ mice. RT-qPCR and western blotting analyses indicated that RAS markers (angiotensin II and II type 1 receptor) were significantly upregulated, oxidative stress was increased (evidenced by reduced superoxide dismutase and peroxiredoxin-4) and autophagy was activated (upregulation of autophagy related protein 7, Beclin 1 and microtubule-associated proteins 1A/1B light chain 3A) in the VDR^-/- VSMCs compared with the VDR^+/+ VSMCs. TEM demonstrated that there were more autophagy bodies in the VDR^-/- VSMCs compared with the VDR^+/+ VSMCs. In conclusion, VDR deficiency was associated with high blood pressure. The mechanism underlying the increase in blood pressure caused by VDR deficiency may involve activation of the RAS, as well as increased oxidative stress and autophagy of VSMCs.

Keywords: autophagy; hypertension; oxidative stress; vascular smooth muscle cell; vitamin D receptor.

Figure 1

Systolic blood pressure of the VDR^-/- and VDR^+/+ mice. Average systolic blood pressure was calculated from 10 measurements using the NIBP non-invasive blood pressure system in the KO and WT mice. BP, blood pressure; KO, knockout/VDR^-/-; WT, wild-type/VDR^+/+; VDR, vitamin D receptor.

Figure 2

Ang II and AT1R expression levels in VSMCs of VDRKO mice. (A) Protein expression levels of AT1R in the VSMCs isolated from VDRKO and VDRWT mice were detected using western blotting analysis and quantified. (B) mRNA levels of Ang II and AT1R in the VSMCs isolated from KO and WT mice were analyzed using reverse transcription-quantitative PCR. The data are presented as an average from three independent assays. WT levels were set at 1.0 for data normalization. KO, knockout/VDR^-/-; WT, wild-type/VDR^+/+; Ang, angiotensin; AT1R, Ang II type 1 receptor; VSMCs, vascular smooth muscle cells; VDR, vitamin D receptor.

Figure 3

Expression levels of SOD and Prdx4 in VSMCs of VDR^-/- mice. (A) mRNA levels of SOD in the VSMCs isolated from the VDRKO and VDRWT mice were analyzed using reverse transcription-quantitative PCR. The average intensity of the corresponding mRNA levels of the genes was calculated from three independent assays. (B) Protein expression of Prdx4 in the samples at the same conditions as A was detected using western blotting analysis. The average data are from three independent assays. WT levels were set at 1.0 for data normalization. SOD, superoxide dismutase; Prdx4, peroxiredoxin4; KO, knockout/VDR^-/-; WT, wild-type/VDR^+/+; VSMCs, vascular smooth muscle cells; VDR, vitamin D receptor.

Figure 4

Protein expression of ATG7, Beclin1, LC3A and p62 in VSMCs. Protein expression of ATG7, Beclin1, LC3A and p62 in VSMCs isolated from VDRKO and VDRWT mice was detected using western blotting analysis. (A) Representative images of the corresponding western blot images. The average intensities of (B) ATG7, (C) p62, (D) Beclin1 and (E) LC3A are summarized in the corresponding bar graphs from three independent assays. WT levels were set at 1.0 for data normalization. KO, knockout/VDR^-/-; WT, wild-type/VDR^+/+; VSMCs, vascular smooth muscle cells; ATG7, autophagy-related protein 7; p62, nucleoporin p62; LC3A, microtubule-associated proteins 1A/1B light chain 3A; VDR, vitamin D receptor.

Figure 5

Ultrastructural alterations in VSMCs of VDR^-/- mice. Changes in VSMC ultrastructure in the (A) VDRWT and (B) VDRKO mice were examined using TEM (scale bar, 500 nm). Autophagy bodies are indicated by the red arrows. PM, plasma membrane; VDR, vitamin D receptor; VSMCs, vascular smooth muscle cells; KO, knockout/VDR^-/-; WT, wild-type/VDR^+/+.