Direct, acute effects of Klotho and FGF23 on vascular smooth muscle and endothelium - PubMed (original) (raw)

Direct, acute effects of Klotho and FGF23 on vascular smooth muscle and endothelium

Isabelle Six et al. PLoS One. 2014.

Abstract

Chronic kidney disease (CKD) is regarded as a state of Klotho deficiency and FGF23 excess. In patients with CKD a strong association has been found between increased serum FGF23 and mortality risk, possibly via enhanced atherosclerosis, vascular stiffness, and vascular calcification. The aim of this study was to examine the hypothesis that soluble Klotho and FGF23 exert direct, rapid effects on the vessel wall. We used three in vitro models: mouse aorta rings, human umbilical vein endothelial cells, and human vascular smooth muscle cells (HVSMC). Increasing medium concentrations of soluble Klotho and FGF23 both stimulated aorta contractions and increased ROS production in HVSMC. Klotho partially reverted FGF23 induced vasoconstriction, induced relaxation on phosphate preconstricted aorta and enhanced endothelial NO production in HUVEC. Thus Klotho increased both ROS production in HVSMC and NO production in endothelium. FGF23 induced contraction in phosphate preconstricted vessels and increased ROS production. Phosphate, Klotho and FGF23 together induced no change in vascular tone despite increased ROS production. Moreover, the three compounds combined inhibited relaxation despite increased NO production, probably owing to the concomitant increase in ROS production. In conclusion, although phosphate, soluble Klotho and FGF23 separately stimulate aorta contraction, Klotho mitigates the effects of phosphate and FGF23 on contractility via increased NO production, thereby protecting the vessel to some extent against potentially noxious effects of high phosphate or FGF23 concentrations. This novel observation is in line with the theory that Klotho deficiency is deleterious whereas Klotho sufficiency is protective against the negative effects of phosphate and FGF23 which are additive.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Direct effects of phosphate, Klotho and FGF23 on vascular reactivity and on H2O2 concentration.

A.Vascular reactivity on aortic rings ex vivo. Direct effects of phosphate (1.0–3.0 mM), Klotho (0–2.0 nM) and FGF23 (0–400 ng/ml) on isolated aortic rings in absence or in the presence of ROS inhibitor, dimethylthiourea (10 mM). Contraction values are expressed as percentage of the contraction obtained with 70 mM KCl. * p<0.001, ** p<0.0001 vs. mouse vessels alone. N, 4 per experiment. B. Direct effects of phosphate (2.0 mM), Klotho (1.6 nM) and FGF23 (10 ng/ml), alone or associated, on H2O2 concentration in human umbilical vein endothelial cells (HUVECs) and human vascular smooth muscle cells (HVSMCs). * p<0.0005 vs. HVSMCs control group, $ p<0.005 vs HVSMCs phosphate group, £ p<0.05 vs. HUVECs control group. N, 7 per experiment.

Figure 2

Figure 2. Vascular reactivity on aortic rings ex vivo.

Direct effects of phosphate (1.0–3.0 mM), Klotho (0–2.0 nM) and FGF23 (0–400 ng/ml) on isolated aortic rings in absence or in the presence of ERK inhibitor, U0126 (10 μM). Contraction values are expressed as percentage of the contraction obtained with 70 mM KCl. ** p<0.0001 vs. mouse vessels alone. N, 4 per experiment.

Figure 3

Figure 3. Effects of Klotho associated with FGF23 on vascular reactivity, NO production and eNOS and iNOS expressions.

A.Vascular reactivity on aortic rings ex vivo. Vascular effects of FGF23 (10, 100 ng/ml) in association with Klotho (0.8, 1.2, 1.6 nM). Contraction values are expressed as percentage of the contraction obtained with 70 mM KCl. N, 4 per experiment. B. Effects of FGF23 (10 ng/ml), Klotho (1.6 nM) or phosphate (2.0 mM), alone or associated, on NO production, (N, 6 per experiment) in HUVECs. * p<0.05, ** p<0.005 vs. control group, $ p<0.05 vs FGF23 group, £ p<0.005 vs phosphate group, § p<0.05 vs. Klotho group. C. Effects of FGF23 (10 ng/ml), Klotho (1.6 nM), or the two combined on eNOS expression (N, 4–6 per experiment), and iNOS de novo expression (N, 4–6 per experiment) in HUVECs. * p<0.05 vs. control group.

Figure 4

Figure 4. Effects of phosphate associated with Klotho on vascular reactivity and eNOS and iNOS expressions.

A. Effects of phosphate (2.0 mM) combined with Klotho (0.4, 0.8, 1.2, 1.6, 2.0 nM) on aortic rings ex vivo. Contraction values are expressed as percentage of the contraction obtained with 70 mM KCl. Relaxation was calculated taking into account the maximal contraction obtained with phosphate 2.0 mM alone. The possible involvement of NO was studied by incubating aortic rings with L-NNA for 30 min before exposure to phosphate and Klotho. N, 4 per experiment. B. Effects of phosphate (2.0 mM), Klotho (1.6 nM) or the two combined on eNOS expression (N, 4–6 per experiment) and iNOS de novo expression (N, 4–6 per experiment). * p<0.05 vs. control group.

Figure 5

Figure 5. Vascular effects of phosphate (2.0 mM) associated with FGF23 (10–400 ng/ml) (A) and contraction obtained with phenylephrine and relaxation obtained with acetylcholine after incubation of aortic rings in presence of phosphate (2.0 mM), Klotho (1.6 nM), FGF23 (10 ng/ml) or the three combined (B).

* p<0.005 vs. all other groups, $ p<0.05 or p<0.001 vs control group.

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Grants and funding

Hirokazu Okazaki and Priscilla Gross were supported by grants from Conseil Régional de Picardie, Amiens, France (Marno-MPCC for HO and Feder for PG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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