Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease

Abstract

Chronic kidney disease (CKD) is linked to greater prevalence and rapid progression of calcific aortic valve disease (CAVD) characterized by valvular leaflet fibrosis and calcification. Fibroblast growth factor 23 (FGF23) level is elevated, and anti-aging protein Klotho is reduced in CKD patients. However, the roles of FGF23 and Klotho in the mechanism of aortic valve fibrosis and calcification remain unclear. We hypothesized that FGF23 mediates CKD-induced CAVD by enhancing aortic valve interstitial cell (AVIC) fibrosis and calcification, while soluble Klotho inhibits FGF23 effect. Methods and Results: In an old mouse model of CKD, kidney damages were accompanied by aortic valve thickening and calcification. FGF23 levels in plasma and aortic valve were increased, while Klotho levels were decreased. Recombinant FGF23 elevated the inflammatory, fibrogenic, and osteogenic activities in AVICs. Neutralizing antibody or shRNA targeting FGF23 suppressed the pathobiological activities in AVICs from valves affected by CAVD. FGF23 exerts its effects on AVICs via FGF receptor (FGFR)/Yes-associated protein (YAP) signaling, and inhibition of FGFR/YAP reduced FGF23's potency in AVICs. Recombinant Klotho downregulated the pathobiological activities in AVICs exposed to FGF23. Incubation of FGF23 with Klotho formed complexes and decreased FGF23's potency. Further, treatment of CKD mice with recombinant Klotho attenuated aortic valve lesions. Conclusion: This study demonstrates that CKD induces FGF23 accumulation, Klotho insufficiency and aortic valve lesions in old mice. FGF23 upregulates the inflammatory, fibrogenic and osteogenic activities in AVICs via the FGFR/YAP signaling pathway. Soluble Klotho suppresses FGF23 effect through molecular interaction and is capable of mitigating CKD-induced CAVD.

Keywords: Chronic kidney disease; Klotho; YAP; calcific aortic valve disease; fibroblast growth factor 23.

Figure 1

CKD increases FGF23 levels and decreases Klotho levels in the plasma and aortic valves of old mice. (A) Schematic diagram outlining the experimental protocols. Old mice (18-20 months) were on AIN-76A diet for 6 weeks and subjected to treatment with adenine and calcitriol from week 3 to week 5. (B) Representative images of von Kossa staining show valvular thickening and calcified nodule formation in the aortic valves of CKD mice. Scale bar = 100 μm. (C) CKD mice have higher FGF23 levels and lower Klotho levels in plasma in comparison to sham controls. (D) Representative images of immunofluorescence staining show increased FGF23 levels and reduced Klotho levels in aortic valves of CKD mice. Original magnification 40x. Scale bar = 100 µm. Quantitative data are expressed as mean ± SEM. n = 4 mice per group. *P < 0.05 versus sham controls.

Figure 2

FGF23 induces AVIC fibrosis and calcification. (A) Human AVICs from normal valves were treated with recombinant FGF23 in different concentrations for 72 hours. Representative immunoblots (left) and densitometric data (right) show that FGF23 upregulates the expression of inflammatory (ICAM-1 and VCAM-1), fibrogenic (collagen I and collagen IV) and osteogenic (RUNX2 and ALP) mediators in AVICs. (B) Representative images of Picrosirius Red (PSR) staining (upper) and Alizarin Red S staining (lower), along with corresponding spectrophotometric data, show that prolonged treatment with recombinant FGF23 (10 days or 14 days) induces collagen and calcium deposition in AVICs. Images were taken using a 10x objective. Scale bar = 100 µm. Quantitative data are expressed as mean ± SEM, n = 4 cell isolates from distinct donor valves in each group. *P < 0.05 versus untreated control.

Figure 3

FGF23 is involved in mediating the pathobiological activities in AVICs of CAVD valves. (A) Immunoblots and densitometric data show that calcified aortic valves from patients with CAVD have higher levels of FGF23 and lower levels of Klotho in comparison to normal aortic valves. Values are means ± SEM. n = 4 aortic valve tissues from distinct donor in each group; *P < 0.05 versus normal aortic valves. N = normal. D = diseased. (B) Immunoblots and densitometric data show that human AVICs from diseased aortic valves have increased levels of FGF23 and decreased levels of Klotho. Values are means ± SEM. n = 4 cell isolates from distinct donor valves in each group. *P < 0.05 versus cells from normal valves. (C and D) Human AVICs from diseased valves were treated with lentivirus expressing FGF23 shRNA (100 nmol/L) or FGF23-neutralizing antibody (5 µg/mL) for 72 hours. Representative immunoblots and densitometric data show that AVICs treated with FGF23 shRNA or FGF23-neutralizing antibody have reduced levels of inflammatory, fibrogenic and osteogenic mediators. Values are mean ± SEM, n = 3 cell isolates from distinct donor valves in each group. *P < 0.05 versus control. Ctrl = control. N-AB = neutralizing antibody.

Figure 4

FGF23 upregulates AVIC pathobiological activities via activation of the FGFR/YAP signaling pathway. (A) Human AVICs from normal valves were pre-treated with global FGFR inhibitor PRN1371 (1.0 µM) for 2 hours, followed by treatment with FGF23 (40 ng/mL) for 72 hours. Representative immunoblots and densitometric data show that FGF23 induces the production of inflammatory, fibrogenic and osteogenic mediators in AVICs via FGFR. (B) Total RNA-sequencing was applied to analyze gene expression in AVICs incubated in the presence or absence of recombinant FGF23. The heat map illustrates diferent levels of gene expression, and the KEGG data show upregulation of the Hippo signaling pathway in FGF23-treated cells (significant enrichment at P < 0.05, Fisher's exact test). (C) Cells were treated with recombinant FGF23 for different time. Representative immunoblots and densitometric data show that FGF23 induces YAP phosphorylation. (D) AVICs were treated with PRN1371 prior to FGF23 stimulation for 2 hours. Representative immunoblots and densitometric data show that global FGFR inhibitor suppresses FGF23-induced YAP phosphorylation. (F) Cells were treated with verteporfin (200 nM) for 2 hours prior to FGF23 stimulation. Representative immunoblots and densitometric data show that inhibition of YAP reduces inflammatory, fibrogenic, and osteogenic responses following FGF23 stimulation. Quantitative data are expressed as mean ± SEM. n = 3 or 4 cell isolates from distinct donor valves in each group. *P < 0.05 versus control and ^#P < 0.05 versus FGF23 alone.

Figure 5

Klotho interacts with FGF23 to suppress its effects in AVICs. (A) Human AVICs from normal valves were treated with varied concentrations of recombinant Klotho prior to the exposure to recombinant FGF23 for 72 hours. Representative immunoblots and densitometric data show that Klotho downregulates the effect of FGF23 in inducing inflammatory, fibrogenic and osteogenic mediators. (B) Representative images of Picrosirius Red (PSR) staining, Alizarin Red S staining and corresponding spectrophotometric data normalized by cell density show that recombinant Klotho suppresses collagen and calcium deposition in cells following prolonged exposure to FGF23. Original magnification 10x. Scale bar = 100 µm. (C) Representative immunoblots and densitometric data show that heat-denatured Klotho has no effect on FGF23-induced expression of inflammatory, fibrogenic and osteogenic mediators. (D) Recombinant FGF23 was incubated with recombinant Klotho for 24 hours. Representative immunoblot probed with anti-Klotho or anti-FGF23 identified corresponding free protein and the formation of complexes between these two proteins. (E) Pre-mixed Klotho-FGF23 has minimal effects on the expression of inflammatory, fibrogenic and osteogenic mediators in AVICs. Quantitative data are expressed as mean ± SEM. n = 3 or 4 cell isolates from distinct donor valves in each group. *P < 0.05 versus control and ^#P < 0.05 versus FGF23 alone.

Figure 6

Recombinant Klotho attenuates aortic valve thickening and calcification in mice subjected to CKD protocol. Old mice subjected to CKD protocol were continuously treated with recombinant Klotho (20 µg/kg/day) in the period of week 3 to 6 using a subcutaneous osmotic pump. Representative images show that treatment with recombinant Klotho reduced aortic valve thickening and calcification. Scale bar = 100 μm. Values are mean ± SEM. n = 4 mice per group. *P < 0.05 versus sham and ^#P < 0.05 versus CKD.