Receptor for advanced glycation end products: a key molecule in the genesis of chronic kidney disease vascular calcification and a potential modulator of sodium phosphate co-transporter PIT-1 expression

Abstract

Background Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, frequent vascular calcification (VC) and accumulation of uraemic toxins. Advanced glycation end products and S100 proteins interact with the receptor for advanced glycation end products (RAGE). In the present work, we aimed to investigate the role(s) of RAGE in the CKD–VC process. Methods Apoe−/− or Apoe−/−Ager (RAGE)−/− male mice were assigned to CKD or sham-operated groups. A high-phosphate diet was given to a subgroup of Apoe−/−and Apoe−/−Ager−/− CKD mice. Primary cultures of Ager+/+ and Ager−/− vascular smooth muscle cells (VSMCs) were established and stimulated with either vehicle, inorganic phosphate (Pi) or RAGE ligands (S100A12; 20 µM). Results After 12 weeks of CKD we observed a significant increase in RAGE ligand (AGE and S100 proteins) concentrations in the serum of CKD Apoe−/− mice. Ager messenger RNA (mRNA) levels were 4-fold higher in CKD vessels of Apoe−/− mice. CKD Apoe−/− but not CKD Apoe−/− or Ager−/− mice displayed a marked increase in the VC surface area. Similar trends were found in the high-phosphate diet condition. mRNA levels of Runx2 significantly increased in the Apoe−/− CKD group. In vitro, stimulation of Ager+/+VSMCs with Pi or S100A12 induced mineralization and osteoblast transformation, and this was inhibited by phosphonoformic acid (Pi co-transporters inhibitor) and Ager deletion. In vivo and in vitro RAGE was necessary for regulation of the expression of Pit-1, at least in part through production of reactive oxygen species. Conclusion RAGE, through the modulation of Pit-1 expression, is a key molecule in the genesis of VC.

Uremic Toxins Affecting Cardiovascular Calcification: A Systematic Review

“…Similarly, addition of vascular cell adhesion molecule-1 (VCAM-1) or the pro-inflammatory cytokine interleukin-8 (IL-8) to human VSMC in pro-calcifying medium increased calcification as shown by alizarin red staining and increased ALP activity (for VCAM-1) or calcium content (for IL-8) [30,48]. The RAGE ligand S100A12 induced calcification in mouse VSMC as revealed by alizarin red staining, which was linked to an increased generation of reactive oxygen species (ROS) as well as an increased mRNA expression of the phosphate co-transporter PIT-1 and the osteoblast markers BMP2 and CBFA1/RUNX2 [36]. Overexpression of the peptide salusin-β, shown to be increased in patients undergoing hemodialysis [47], increased calcification of human VSMC and rat aortic rings, not only in calcification media, but also in media without calcification stimulation, as demonstrated by calcium content, alizarin red, or von Kossa staining as well as ALP activity.…”

“…For the substances in Tables 1-3, following links with inflammation were identified: Phosphate (Table 1): Treatment of mouse VSMC with high phosphate medium induced ROS production and ROS-mediated expression of S100 proteins as pro-inflammatory RAGE ligands along with osteogenic marker expression and calcification. The phosphate co-transporter PIT-1, shown to be crucial for phosphate-induced calcification, was upregulated by high phosphate medium through involvement of the RAGE receptor, which could be blocked by anti-oxidant treatment [36].…”

“…In line with this, incubation of pluripotent undifferentiated MSC with TNF-α as well as with the pro-inflammatory cytokine interleukin-1β (IL-1β) enhanced osteoblastic transdifferentiation and calcification [41], linking pro-inflammatory mediators to vascular calcification over osteogenic transdifferentiation. Additionally, the pro-inflammatory RAGE ligand S100A12 induced expression of osteoblast markers BMP2 and CBFA1/RUNX2 in mouse VSMC along with increased calcification, with ROS-induced PIT-1 expression revealed to be important in S100A12-induced calcification [36]. For the pro-inflammatory cytokine interleukin-8 (IL-8), increased calcification extent in human VSMC in pro-calcifying conditions was mechanistically linked to a downregulation of the calcification inhibitor osteopontin [30].…”

“…Low Molecular Weight Substances, Increased in Blood in CKD (Table 1) A high ambient phosphate concentration triggers calcification in a variety of situations, as shown in vitro in human VSMC [29,30,[32][33][34], rat and mouse VSMC [35,36], rat aortic rings [33], as well as in aortas of rats subjected to 5/6 nephrectomy [35]. Apo −/− mice with 5/6 nephrectomy displayed aortic valve calcification 12 weeks after surgery both when fed a high phosphate diet as well as a control diet, though with increased calcium deposits detected upon high phosphate diet [36]. Additionally, elevated calcium levels triggered calcification in human VSMC in vitro as well as ex vivo studies with rat aortic rings [38,40].…”

“…Phosphate (Table 1): Along with vascular calcification, a high concentration of phosphate induced a significant increase in CBFA1/RUNX2, BMP2 and osteopontin expression in mouse VSMC [36,37], with an important role for the RAGE receptor and oxidative stress [36]. This latter finding demonstrates a link between inflammation and VSMC osteogenic transdifferentiation induced by high phosphate, with (RAGE receptor-mediated) oxidative stress contributing to increased expression of the phosphate co-transporter PIT-1 as well as RUNX2 upon high-phosphate treatment of VSMC, and with pharmacological inhibition of the phosphate co-transporters PIT-1 and PIT-2 interfering with high phosphate-induced calcification of VSMC [36]. Similarly, high phosphate levels significantly increased MSX2, SOX9, CBFA1/RUNX2, BMP2, and osteopontin in human aortic SMC along with vascular calcification and increased expression of PIT-1 [32,33].…”