Soluble Klotho Pretreatment Improves Endothelial Dysfunction Induced By FGF23 Halee Patel, Neerupma Silswal, and Michael Wacker UMKC School of Medicine, Kansas City, MO INTRODUCTION RESULTS CONCLUSION FGF23 at 9000 pg/ml increased endothelial ROS production and impaired vascular relaxation. FGF23 induced increase in ROS levels in endothelial cells were reduced by Klotho pretreatment. Klotho pretreatment of aortic rings restored the FGF23 mediated impairment of vascular relaxation. REFERENCES/ACKNOWLEDGEMENTS SIGNIFICANCE These findings strongly suggest that chronic elevations of serum FGF23 as occurs during chronic kidney disease are linked to increases in superoxide levels leading to impaired endothelial function. Impaired endothelial function and relaxation of the vasculature can lead to arterial stiffness and atherosclerosis. Superoxide scavengers such as Klotho may be a therapeutic strategy for patients with chronic kidney disease to help reduce the vascular impairment caused by elevated FGF23 levels. (B) (A) VehicleFGF23 Klotho + FGF23 Klotho reduces ROS levels Figure 2. (A) Endothelial cells were loaded with DHE and pretreated with vehicle, FGF23 (9000 pg/ml), or Klotho (100 ng/ml) plus FGF23 (9000 pg/ml). Images show increased fluorescence in the FGF23 treated cells and reduced fluorescence in the Klotho + FGF23 treated cells. (B) Mean fold changes in fluorescence compared to vehicle. Data are shown as mean + SEM. * Statistically significant difference from vehicle (P<0.05). ^ Statistically significant difference from FGF23 (P<0.05). Figure 1. Hypothesized mechanism for increased superoxide production leading to reduced NO bioavailability by FGF23. Reduced NO bioavailability impairs the ability of the vasculature to relax. One potential mechanism involves the production of superoxide via the activation of enzymes like NADPH oxidase. Klotho is a soluble protein that may reduce superoxide levels. Klotho pretreatment restores the impairment in endothelial mediated relaxation induced by FGF23 Figure 3. Aortic rings were pretreated with vehicle, FGF23 (9000 pg/ml), FGF23 with Klotho (100 ng/ml) and then precontracted with PGF 2α (10  M ) and relaxed with ACh. FGF23 induced impairment in relaxation while the rings pretreated with Klotho plus FGF23 showed normal relaxation. Data are shown as mean + SEM. * Statistically significant difference from vehicle (P<0.05). ^ Statistically significant difference from FGF23 (P<0.05). RESULTS BACKGROUND Endothelial cell OO OO eNOS OONO- Smooth muscle GTP cGMP relaxation FGF23 activate NADPH oxidase sGC NO FGFR Klotho chelate METHODS Animals: Primary endothelial cells were isolated from CD1 mice and cultured for 4-5 days DHE Staining for Reactive Oxygen Species: Endothelial cells were incubated with vehicle, FGF23 (9000 pg/mL), or FGF23 plus Klotho (100 ng/mL) for 20 minutes and then loaded with DHE (5 mM) for 20 min. DHE fluoresces at 615 nm when bound to ROS. Analysis: Cells were analyzed and quantified by epifluorescence microscopy using Slidebook imaging software. Either a one factor or two factor ANOVA followed by Bonferroni’s post hoc tests were used for statistical analysis using GraphPad Prism software. Significance was set at P<0.05. Aortic Rings: 2- 3 mm rings were extracted from adult mice and treated with either vehicle, FGF23, or FGF23 plus Klotho and then preconstricted with 10  M of PGF 2α followed by acetylcholine (Ach) to relax the vessel. Force changes were recorded using an ADinstruments PowerLab. Fibroblast growth factor 23 (FGF23) is a hormone secreted by osteocytes that serves as an important regulator of serum phosphate and vitamin D via direct actions on the kidney and parathyroid (Bergwitz et al. 2010). High circulating levels of FGF23 have been clinically associated with the development of cardiovascular disease (CVD), especially during chronic kidney disease (CKD) where serum FGF23 is substantially increased up to 100–1,000 fold (Arnlov et al. 2013). There are clinical associations between elevated FGF23 and atherosclerosis, impaired flow-mediated dilation, and impaired vasoreactivity and arterial stiffness (Yilmaz et al. 2010). FGF23 impairs endothelial relaxation by reducing NO bioavailability and increasing superoxide production (Silswal et al. 2014). Klotho is a transmembrane protein that has a role in aging (Ifanas’ev, I. 2010). Klotho can also exist in soluble form, which potentially binds with reactive oxygen species (ROS) in the endothelium (Rakugi et al. 2007). We hypothesize that Klotho pretreatment reduces the FGF23 induced increase in ROS levels and restores vascular relaxation. 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Patel) and the American Heart Association grants 11SDG (to M.J. Wacker). ^