#571 Vitamin D receptor from VSMCs regulates vascular calcification during CKD: a potential role for miR-145a
Authors: Caus Maite , Alonso-Montes Cristina , Fernandez-Martin José Luis , Martí-Antonio Manuel , Sanjuan Auria Eritja , Pérez-Gómez Aurora , Tocados Juan Miguel Díaz , Bozic Milica , Valdivielso José Manuel 
Published: May 2024

Background and Aims

Vascular calcification (VC) is a highly prevalent complication of chronic kidney disease (CKD) and is associated with the higher morbidity-mortality of patients with CKD. Vitamin D receptor (VDR) has been proposed to play a role in the osteoblastic differentiation of vascular smooth muscle cells (VSMCs), but the involvement of vitamin D (1, 25D) in VC associated to CKD is controversial. Accumulating evidence points to microRNAs as important players in the process of VC and recent studies show that miR-145a expression decreases in VSMCs during VC. In addition, VDR has been considered as a potent regulator of these small molecules, while miR-145a was identified as a target for 1, 25D. Our aim was to determine the role of local vitamin D signalling in VSMCs during CKD-induced VC and the involvement of miR-145a in this process.

Method

We used epigastric arteries from CKD-affected patients and from individuals with normal renal function. In vivo, we employed an experimental model of CKD-induced VC in mice with conditional deletion of VDR in VSMC (Myh11-CreERT2+ VDRlox/lox), while in vitro, we used mouse VSMC with (VDRwt) or without VDR (VDRko) incubated in calcification media. Transfection experiments with mmu-miR-145a-5p mimic or mmu-miR-145a-5p inhibitor were carried out in mouse VDRwt VSMCs.

Results

CKD-affected patients showed an increase in VC, alongside an increased arterial expression of VDR compared with controls with normal renal function. Conditional gene silencing of VDR in arterial VSMCs led to a significant decrease of VC in the mouse model of CKD, despite similar levels of renal impairment and serum calcium and phosphate levels. This was accompanied by lower arterial expression of osteopontin (OPN) and lamin A, and a higher expression of sclerostin (SOST). Runx2 was increased in arteries of both CKD groups independently of the presence of VDR in VSMCs. Furthermore, CKD-affected mice showed a reduction of miR-145a expression in calcified arteries, which was significantly recovered in animals with deletion of VDR in VSMCs. In vitro, the absence of VDR prevented VC, inhibited the increase of OPN, and re-established the expression of miR-145a. Forced expression of miR-145a in vitro in VDRwt VSMCs blunted VC and decreased OPN levels. Mir-145a levels markedly decreased both in human and mouse VDRwt VSMCs incubated in calcification media and 1, 25D. Overexpression of miR-145a suppressed the levels of OPN induced by 1, 25D, while the inhibition of mir-145a led to a significant increase in OPN levels, even higher than in cells treated solely with 1, 25D. A predicted miR-145a-binding site was detected at the 3’UTR of OPN mRNA transcript, through the extended seed region of the miRNA, pointing to OPN as a possible target of mir-145a during VC in CKD. There were no significant differences in Runx2 levels in 1, 25D-treated cells after transfection with mir-145a mimic or inhibitor.

Conclusion

VDR is induced in calcified VSMCs during CKD where it can modulate the expression of promoters and inhibitors of VSMC transdifferentiation, as well as small noncoding RNA molecules such as mir-145a that modulate VC. The regulation of these pathways by VDR seems to be essential for the settings of CKD-induced VC, as deletion of VDR from VSMCs prevented it.