Arterial Klotho Expression and FGF23 Effects on Vascular Calcification and Function

<div><p>Recent studies support a role for FGF23 and its co-receptor Klotho in cardiovascular pathology, yet the underlying mechanisms remain largely elusive. Herein, we analyzed the expression of Klotho in mouse arteries and generated a novel mouse model harboring a vascular smooth muscle cell specific deletion of Klotho (<i>Sm22-KL^−/−</i>). Arterial Klotho expression was detected at very low levels with quantitative real-time PCR; Klotho protein levels were undetectable by immunohistochemistry and Western blot. There was no difference in arterial Klotho between <i>Sm22-KL^−/−</i> and wild-type mice, as well as no changes in serum markers of mineral metabolism. Intravenous delivery of FGF23 elicited a rise in renal (0.005; p<0.01) but not arterial Egr-1 expression, a marker of Klotho-dependent FGF23 signaling. Further, the impact of FGF23 on vascular calcification and endothelial response was evaluated in bovine vascular smooth muscle cells (bVSMC) and in a murine <i>ex vivo</i> model of endothelial function, respectively. FGF23 treatment (0.125–2 ng/mL) did not modify calcification in bVSMCs or dilatory, contractile and structural properties in mice arterial specimen <i>ex vivo</i>. Collectively, these results demonstrate that FGF23-Klotho signaling is absent in mouse arteries and that the vascular response was unaffected by FGF23 treatment. Thus, our data do not support Klotho-mediated FGF23 effects in the vasculature although confirmative studies in humans are warranted.</p> </div

Immunohistochemical staining and Western blotting for expression of Klotho in different arteries from <i>Sm22-KL^−/−</i> mice.

<p>(A) Klotho protein was undetectable in the vascular wall of different arteries (aorta, mesenteriac, femoral and lung arteries), as determined by immunohistochemical staining, when compared with a distinct positive staining for Klotho in kidney distal tubule. Upper panels show Klotho staining of aorta with two different antibodies (KM2076 and KM2119). Lower panel shows Klotho staining of mesenterial, femoral and lung arteries, using the same antibodies. All results are shown in 40x magnification. Scale bar, 10 µM. (<b>B</b>) Immunoblot analysis of pooled whole aortic lysates from three wild-type and three <i>Sm22-KL^−/−</i> mice revealed no Klotho protein (using two different antibodies; KM2076 and KM2119), when compared with the positive control of whole kidney extracts from wild-type mice, which showed a strong band for Klotho at 130 kDa.</p

No effect of FGF23 on βGP-induced mineral deposition by bVSMCs.

<p>(<b>A</b>) bVSMCs were cultured in 6-well plates in 10% FCS-DMEM till 90% confluent, and then incubated in medium containing 5 mM βGP and mock transfected media (control), or with medium containing βGP supplemented with FGF23 in concentrations ranging from 0.125–2 ng/mL. Representative phase contrast images of cells stained with alizarin red on days 10 are shown. The red stain indicates areas of mineralization. Scale bar = 500 µm. (<b>B</b>) Alizarin red dye elution was performed in order to quantify mineralization. Results represent data from day 10. Data are shown as mean ± SEM (n = 3 individual samples).</p

Long-term effects of FGF23 on the passive length-tension relationships.

<p>Long-term effects of FGF23 (6 ng/mL) or vehicle (DMSO 0.17% v/v) on the passive length-tension relationships. Bold solid black lines indicate responses in the presents of vehicle with thin dashed lines showing ± SEM range; bold gray lines indicate responses in the presence of FGF23 with thin dashed gray lines showing ± SEM range.</p

Long term effects of FGF23 on induced contractions or relaxations.

<p>Long term effects of FGF23 or vehicle (DMSO 0.17% v/v) on phenylephrine (A), thromboxane A2 (TXA₂) analog U46619 (B), acetylcholine (C) and sodium nitroprusside (D) induced contractions or relaxations, respectively. Data are shown as means ± SEM. Solid black lines indicate responses in the presence of vehicle and gray lines indicate responses in the presence of FGF23.</p

Serum biochemistries in <i>Sm22-KL</i>^−/− and wild-type mice.

<p>At 8 weeks of age <i>Sm22-KL^−/−</i> mice had normal serum biochemistries reflecting mineral metabolism as compared to wild-type controls. Results are displayed as median (range).</p

Klotho gene expression in mouse arteries.

<p>Gene expression of Klotho in kidney extracts, arteries and lung of wild-type mice, <i>Sm22-KL^−/−</i> mice, <i>β-KL^−/−</i> mice, and DCT209 cells. The Klotho transcript levels remained unchanged between wild-type and <i>Sm22-KL^{<b>−/−</b>}</i> mice in all arteries investigated and were 7000 times lower when compared to kidney transcript levels. <i>Sm22-KL^−/−</i> transcript levels remained at same levels or lower when compared with the <i>β-KL^−/−</i> mice and the DCT209 cell line.</p

Vascular Egr-1 response to FGF23 injection.

<p>Transcript levels of Egr-1 were analyzed in aorta and kidneys from wild-type mice injected with 0.9% NaCl (n = 3) or 0.15 mg/kg FGF23 (n = 4). In contrast to the kidney, no distinct rise in Egr-1 mRNA was seen in aorta 30 min after an FGF23 injection. Data are presented as fold change, with NaCl set to 1.</p

Targeted deletion of Klotho in <i>Sm22-KL^{<b>−/−</b>}</i> mice.

<p>Mice with loxP sites inserted into intron 1 and 2 of Klotho, were crossed with transgenic mice expressing Cre recombinase under the control of the mouse smooth muscle protein 22-alpha (Sm22) promoter, resulting in targeted deletion of the Klotho gene specifically in vascular smooth muscle cells by Cre recombination.</p