The RANKL receptor LGR4 contributes to PTH-induced vascular calcification.

Abstract

BACKGROUND: In Chronic Kidney Disease serum phosphorus elevations stimulate PTH production causing severe alterations in bone-vasculature axis. PTH is the main regulator of Receptor activator of NFKB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, essential for bone maintenance, which also plays an important role in vascular smooth muscle cells (VSMCs) calcification. The discovery of a new RANKL receptor, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), important for osteoblast differentiation but with unknown role in VC, led us to examine LGR4 contribution in high P/high PTH-driven vascular calcification (VC). METHODS: In vivo studies were conducted in subtotally nephrectomized rats fed normal or high phosphorus diet, with and without parathyroidectomy. Parathyroidectomized rat were supplemented with PTH 1-34 to achieve physiological serum PTH levels). In vitro studies were performed in rat aortic VSMCs cultured in control medium, calcifying medium (CM) or CM plus 10-7 vs 10-9M PTH. RESULTS: Rats fed high phosphorus, significantly increased aortic calcium content. Similarly, calcium deposition was higher in VSMCs exposed to CM. Both conditions were associated with increased RANKL and LGR4 and decreased OPG aorta expression, and exacerbated by high PTH. Silencing of LGR4 or PTH1R attenuated the high PTH-driven increases in calcium deposition. Furthermore, PTH1R silencing and pharmacological inhibition of protein kinase (PK) A, but not of PKC, prevented the increases in RANKL and LGR4 and decreased OPG. Treatment with PKA agonist corroborated that LGR4 regulation is a PTH/PKA-driven process. CONCLUSIONS: High PTH increases LGR4 and RANKL and decreases OPG expression in aorta, thereby favoring vascular calcification. The hormone's direct pro-calcifying actions involve PTH1R binding and PKA activation.