Dietary phosphorus acutely impairs endothelial function

Abstract

Excessive dietary phosphorus may increase cardiovascular risk in healthy individuals as well as in patients with chronic kidney disease, but the mechanisms underlying this risk are not completely understood. To determine whether postprandial hyperphosphatemia may promote endothelial dysfunction, we investigated the acute effect of phosphorus loading on endothelial function in vitro and in vivo. Exposing bovine aortic endothelial cells to a phosphorus load increased production of reactive oxygen species, which depended on phosphorus influx via sodium-dependent phosphate transporters, and decreased nitric oxide production via inhibitory phosphorylation of endothelial nitric oxide synthase. Phosphorus loading inhibited endothelium-dependent vasodilation of rat aortic rings. In 11 healthy men, we alternately served meals containing 400 mg or 1200 mg of phosphorus in a double-blind crossover study and measured flow-mediated dilation of the brachial artery before and 2 h after the meals. The high dietary phosphorus load increased serum phosphorus at 2 h and significantly decreased flow-mediated dilation. Flow-mediated dilation correlated inversely with serum phosphorus. Taken together, these findings suggest that endothelial dysfunction mediated by acute postprandial hyperphosphatemia may contribute to the relationship between serum phosphorus level and the risk for cardiovascular morbidity and mortality.

Figure 1.

Effects of high P loading on ROS production. (A) P loading increased O^2- generation in a dose-dependent manner, and the effects of various oxidase inhibitors on the O^2- generation were tested by NBT assay. Data are expressed as mean ± SEM (n = 8 to 20). ^aP = 0.004 for column 3, <0.0001 for column 6, 0.005 for column 7, <0.0001 for column 8 versus column 1, ^bP = 0.019 for column 5 versus column 3. (B) Effect of ebselen or PFA on the P loading-mediated ROS production determined by time-lapse confocal microscopy analysis with APF. Data points shown are mean ± SEM for control (0.9 mM P, n = 3, closed circle), 2.8 mM P (n = 12, open circle), 2.8 mM P with 200 μM PFA (n = 6, square), or 2.8 mM P with 10 μM ebselen (n = 9, triangle). ^#P < 0.05 versus 2.8 mM P, and *P < 0.05 versus control. (C) Expression of type III sodium-dependent phosphate transporter in BAECs. RT-PCR analysis was performed with specific primer sets for four different types of sodium-dependent phosphate transporter (Type IIa, Type IIb, Type III [PiT-1 and PiT-2]) as described in Supplemental Appendix.

Figure 2.

Effects of high P loading on aortic vasodilation. (A) Dose-response curves of vasodilation induced by acetylcholine in rat aortic rings pretreated with Krebs-Henseleit bicarbonate (KHB) buffer containing 1.2 mM P (n = 3, closed circle) or 2.4 mM P (n = 2, open circle). Data are expressed as mean ± SEM. There was significant difference in intercepts between the curves of 1.2 mM P and 2.4 mM P (P = 0.0067). (B) Dose-response curves of vasodilation induced by sodium nitroprusside (SNP) in rat aortic rings stripped of the endothelium, preincubated with KHB buffer containing 1.2 mM P (n = 4, closed circle) or 2.4 mM P (n = 4, open circle). Data are expressed as mean ± SEM. (C) Effect of ebselen on the inhibition of vasodilation by high P medium in rat aortic rings. Rat aortic rings were preincubated with KHB buffer containing with 1.2 mM P (n = 4), 2.4 mM P (n = 3), or 2.4 mM P with 10 μM ebselen (n = 3). Data are expressed as mean ± SEM, *P < 0.05 versus column 1.

Figure 3.

Effect of high P loading on NO production. (A) Effect of bradykinin-induced NO production in BAECs measured by time-lapse confocal microscopy with DAF-2DA. The cells were pretreated with control (0.9 mM P, n = 8, closed circle), 1.8 mM P (n = 7, open circle), or 1.8 mM P with 10 μM ebselen (n = 6, closed triangle). *P < 0.05 versus control. (B) Effect of high P loading on intracellular Ca²⁺ increase induced by ATP in BAECs. BAECs were preincubated with control (0.9 mM P, solid line) or 2.8 mM P (dotted line) for 1 h before intracellular Ca²⁺ measurement. Intracellular Ca²⁺ increase induced by 0.5 μM and 2 μM ATP, and 10 μM calcium ionophore was sequentially estimated by time-lapse confocal microscopy analysis with Fluo-4 and Fura-Red. Change of intracellular Ca²⁺ concentration is expressed as change of fluorescence ratio (Fluo-4/Fura-Red). The curves are representative of three independent experiments. (C) Effects of high P loading on phosphorylation of eNOS at Thr⁴⁹⁷. Western blot analysis of phosphorylated eNOS at Thr⁴⁹⁷, total eNOS, and caveolin. BAECs were treated with control (0.9 mM P), 3 mM P, 3 mM P with 10 μM Gö6976, 3 mM P with 1 mM Tempol, or 3 mM Na₂SO₄ for 60 min. We subjected isolated caveolar membrane fractions from the cells to western blot analysis with anti-phospho-eNOS (Thr⁴⁹⁵) monoclonal antibody (mAb), anti-eNOS mAb, or anti-caveolin polyclonal antibody (pAb). Representative blots from four separate experiments and densitometric analysis data are shown. *P < 0.05 versus control, **P < 0.01 versus 3 mM P.

Figure 4.

Activation of conventional PKC by high P loading in BAECs. (A) Effect of high P loading on the PKC activity of BAECs. We treated BAECs with control (0.9 mM P), 1.5 mM P, 3 mM P, or 3 mM P with 200 μM PFA for 15 min, then pretreated whole cell lysates, and subjected lysates to measurement of conventional PKC activity using the TruLight PKCα Assay kit. *P < 0.05 versus column 1, ^†P < 0.05 versus 3 mM P (column 3). (B) Effect of high P loading on the subcellular localization of PKCα in BAECs. We treated BAECs with the indicated concentration of P or glucose in Medium 199 medium without serum for 1 h, and then fractionated the cells into cytosol and caveolar membrane fractions. We separated 20 μg of protein of each fraction by SDS-PAGE and performed western blot analysis with anti-PKCα mAb and anti-caveolin pAb. The data are representative from two separate experiments.

Figure 5.

Univariate association between serum P or serum glucose and %FMD. Univariate association between serum P and %FMD in a P400 meal (A) and in a P1200 meal (B). Symbols denote the same group in each graph (preprandial, closed circle; postprandial, open circle). Spearman's correlation coefficient (r_s) and its P value for r = 0 are represented in each association.

Figure 6.

Possible pathway of phosphate-mediated endothelial dysfunction. High P loading increases ROS production through PKC and NAD(P)H oxidase in endothelial cells. In addition, high P loading also decreases NO production through phosphorylation of eNOS. Increased ROS production and decreased NO production may cause endothelial dysfunction and CVD.