#5629 EFFECT OF HIGH PHOSPHORUS CONTENT/DIET ON MIR-145 LEVELS AND VASCULAR SMOOTH MUSCLE CELLS OSTEOGENIC DIFFERENTIATION

Abstract

Abstract Background and Aims Phosphorus (P) is found in most foods as a natural component, but nowadays, with the rise in the consumption of processed foods, P intake has increased in the developed world. Elevated serum P in patients with chronic kidney disease (CKD) contributes to development of vascular calcification. However, it is poorly understood the relationship between high P dietary intake and vascular calcification in healthy population with normal renal function, and the molecular pathways responsible for the transdifferentiation of vascular smooth muscle cells (VSMCs) that precedes it. Recent studies show that miR-145 is the most abundant microRNA (miR) in VSMCs, and its decrease is associated with loss of contractile phenotype -lower α-actin levels- and increased osteogenic differentiation. In addition, low serum levels of miR-145 has been associated with the presence of vascular calcification. Thus, the aim of the study was to evaluate the effect of high P dietary intake on aortic and serum miR-145 levels, phenotypic alterations of VSMCs and vascular calcification. Method The study was performed: a) in vivo in aortas and serums from rats with normal renal function fed normal P (NP, 0,6% P -n = 10-) and high P diets (HP, 0,9% P -n = 10-), and b) in vitro exposing the VSMCs to different concentrations of P (up to 3 mM), and overexpressing and silencing miR-145 to investigate its potential role in the VSMCs phenotypic differentiation. Results a) In vivo study: The HP group showed higher serum P (NP: 1.29 [1.15-1.35] vs HP:1.40 [1.33-1.48] mmol/L, p = 0.02) and PTH levels (NP: 235.58 ± 75.58 vs HP: 498.67 ± 142.07 pg/ml, p = 0.0001), without significant differences in serum Ca and FGF23 levels. There were not differences in aortic Ca content, although there was a significant reduction in α-actin expression in the HP group (NP: 2.49 [2.10-3.25] vs HP:1.23 [1.04-1.50] R.U., p = 0.002). Moreover, the aortic and serum levels of miR-145 were lower in the HP group: aortic miR-145 (NP: 1.26 [0.97-1.49] vs HP: 0.56 [0.38-0.69] R.U., p = 0.004) and serum miR-145 (NP: 0.84 [0.70-0.99] vs HP: 0.37 [0.31-0.52] R.U., p = 0.001). b) In vitro study: A7r5 cells exposure to high P reduced miR-145 (1mM P: 1.00 [0.88-1.28] vs 3mM P: 0.41 [0.32-0.52], p = 0.001), and α -actin (1mM P: 1.03 [0.96-1.15] vs 3mM P: 0.73 [0.61-0.74], p = 0.0008) levels increasing Ca content in a P concentration-dependent manner (1 mM P: 0.24 [0.00-1.17]; 1.5 mM P: 0.66 [0.47-2.61]; 2 mM P: 2.72 [1.06-4.01]; 2.5 mM P: 4.60 [3.02-4.91]; 3 mM P: 5.81 [5.58-6.34], p = 0.000). the silencing of miR-145 under standard P content (1mM), decreased α -actin (control: 1.02 [0.99-1.04] vs miR-145 antagomir: 0.75 [0.69-0.90] R.U., p = 0.0007), meanwhile its overexpression increased it (control: 1.02 [0.99-1.04] vs miR-145 mimic: 2.70 [2.68-3.07] R.U., p = 0.001). The α -actin levels and Ca content were not modified after silencing miR-145 under high P conditions C (3 mM), meanwhile miR-145 overexpression under high P conditions increased α -actin levels (control: 0.54 [0.41-0.61] vs miR-145 mimic: 1.82 [1.68-2.04] R.U., p = 0.0005), and partially prevented the increases in Ca content (control: 182.77 [176.78-194.84] vs miR-145 mimic: 117.16 [112.27-122.69] R.U., p = 0.0005). Conclusion The results indicate the high P exposure could lead to an impairment of vascular health by causing decrease in miR-145 levels with the consequent reduction in α -actin expression in VSMC cells.