Increase in Cardiac Expression of Fibroblast Growth Factor 23 (FGF23) in Two Mouse Models of Chronic Kidney DiseaseDorothy Daniel1, Emily M. Adamic2, Jason R. Stubbs3, Chad D. Touchberry2, and Michael J. Wacker1

1Univ of Missouri-Kansas City School of Medicine, Kansas City, MO; 2Health and Sport Sciences, Univ. of Memphis, Memphis, TN; 3Kidney Inst, Univ of Kansas Medical Center, Kansas City, KS.

INTRODUCTION

METHODS

• Male Col4a3 null and wild-type (WT) mice were sacrificed at 10 weeks.

• For the adenine-diet model, male CD1 mice were fed 0.2% adenine daily for 12 weeks and asubset of these mice were exercised on a treadmill with a regimen of 60 minutes, 4days/week, at 65% eVO2.

• Whole hearts were removed and real time RT-PCR was used to measure expression of FGF23using β-actin as the housekeeping gene. Analysis was performed using the 2^ddct method.

• Statistical significance for data were performed using one-way ANOVA with post-hoc Tukey’sanalysis for multiple comparisons (Fig. 2-4), an unpaired t-test for one comparison (Fig. 6), or2-sided t-tests between individual groups at each time point (Fig. 5).

RESULTS

• Basal cardiac FGF23 expression was relatively low in control diet and Col4a3 WT mice, with anaverage delta CT of 17.3 and 15.9, respectively, in relation to β-actin expression.

• Cardiac FGF23 expression was increased 3.0 fold in adenine-fed mice and 3.2 fold in Col4a3null mice compared to control or WT mice, respectively.

• FGF23 expression was increased in the hearts of CKD mice indicating that FGF23 may haveparacrine effects on cardiac remodeling during CKD and could prove to be an importanttherapeutic target.

• Exercise did not improve kidney function in adenine-fed mice and did not abate the increasedcardiac FGF23 expression. With these factors still elevated, this particular exercise regimenmay not be beneficial for CKD outcomes even with the improved fitness adaptations.

CREDITS/DISCLOSURE/REFERENCES• This work was supported by funding from the American Heart Association 115065330016 (MW), NIH I-POI-AG039355-OIAI (MW), UMKC Sarah

Morrison Research Award (DD), and the University of Memphis Faculty Research Grant (CT)

1. Faul C, Amaral AP, Oskouei B, Hu MC, Sloan A, Isakova T, Gutiérrez OM, Aguillon-Prada R, Lincoln J, Hare JM, Mundel P, Morales A, Scialla J, Fischer M, Soliman EZ, Chen J, Go AS, Rosas SE,Nessel L, Townsend RR, Feldman HI, St John Sutton M, Ojo A, Gadegbeku C, Di Marco GS, Reuter S, Kentrup D, Tiemann K, Brand M, Hill JA, Moe OW, Kuro-O M, Kusek JW, Keane MG, WolfM. FGF23 induces left ventricular hypertrophy. J Clin Invest. 121: 4393-408, 2011

2. Grabner A, Amaral AP, Schramm K, Singh S, Sloan A, Yanucil C, Li J, Shehadeh LA, Hare JM, David V, Martin A, Fornoni A, Di Marco GS, Kentrup D, Reuter S, Mayer AB, Pavenstädt H, StypmannJ, Kuhn C, Hille S, Frey N, Leifheit-Nestler M, Richter B, Haffner D, Abraham R, Bange J, Sperl B, Ullrich A, Brand M, Wolf M, Faul C. Activation of cardiac fibroblast growth factor receptor 4causes left ventricular hypertrophy. Cell Metab. 22: 1020-32, 2015

3. Kao YH, Chen YC, Lin YK, Shiu RJ, Chao TF, Chen SA, Chen YJ. FGF-23 dysregulates calcium homeostasis and electrophysiologic properties in HL-1 atrial cells. Eur J Clin Invest 44: 795-801, 20144. Leifheit-Nestler M, Siemer R, Flasbart K, Richter B, Kirchhoff F, Ziegler W, Klintshar M, Becker J, Erbersdobler A, Aufricht C, Seeman T, Dagmar-Christiane F, Faul C, Haffner D. Induction of

cardiac FGF23/FGFR4 expression is associated with left Stubbs JR, Wacker MJ. FGF23 is a novel regulator of intracellular calcium and cardiac contractility in addition to cardiac hypertrophy.Am J Physiol Endocrinol Metab 304: E863-E873, 2013. ventricular hypertrophy in patients with chronic kidney disease. Nephrol Dial Transplant; 0:1-12, 2015.

5. Stubbs, JR, He N, Idiculla A, Gillihan R, Liu S, David V, Hong Y, Quarles LD. Longitudinal evaluation of FGF23 changes and mineral metabolism abnormalities in chronic kidney disease. J BoneMiner Res. 27: 38-46. 2012

6. Touchberry CD, Green TM, Tchikrizov V, Mannix, JE, Mao TF, Carney BW, Girgis M, Vincent RJ, Wetmore LA, Buddhadeb D, Bonewald LF,

Figure 4. Cardiac FGF23 expression in control, adenine-fed and adenine-fed + exercise mice. FGF23 expression was elevated in adenine-fed (n=6) and adenine-fed + exercised mice (n=5) compared to control mice (P<0.05). FGF23 expression was not significantly different between adenine-fed and adenine-fed, exercised mice (P>0.05).

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Figure 3. Induction of CKD in adenine-fed mice. Serum BUN (A), creatinine (B), phosphate (C) and calcium (D)were increased at week 12 in both AD (n=15) and AD+Ex (n=10) mice compared to control (n= 15; P<0.05). Therewere no statistical differences between serum values measured in AD mice compared to AD+Ex mice. Serumvalues were obtained using a Vetscan chemistry analyzer.

Figure 2. Changes in fitness in exercised adenine-fed mice. Run time (A), run speed (B) and run distance (C) weredecreased in adenine-fed (AD) mice (n=15) compared to control (n=10; t P>0.05). These values were increased inexercised adenine-fed (AD+Ex) mice (n=10) compared to AD alone (* P<0.05) or control mice (# P<0.05). Thesedata indicate that the adenine-fed mice were less fit than control mice and that the 12-week exercise regimeninduced significant fitness adaptation even with the progression of CKD caused by the adenine diet.

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Figure 6. Cardiac FGF23 expression in Col4a3 null and WT mice. FGF23 expression was elevated Col4a3 null mice compared to WT mice (n=5; P<0.05).

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Figure 5. Progression of CKD in Col4a3 null mice. Serum BUN (A), creatinine (B), phosphorus (C) and calcium(D) were increased in KO mice compared to WT mice by 12 weeks of age (n ≥5; * P<0.05). Modified fromStubbs et al. J Bone Miner Res 2012. Serum BUN was measured using the Quantichrome Urea Assay kit.Serum Creatinine was measured using a Vitros 250 analyzer. Serum phosphorus was measured by thephosphomolybdate-ascorbic acid method. Serum calcium was measured using the Liquicolor kit. * P<0.05compared to WT mice at the same age.

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• FGF23 is a hormone released primarily from osteoblasts and osteocytes in response to elevatedserum phosphate which acts to decrease phosphate reabsorption and promote phosphateexcretion.

• FGF23 serum levels rise with the progression of chronic kidney disease (CKD) and is associatedwith cardiovascular disease and mortality and can induce hypertrophy (Faul et al. 2011,Touchberry et al. 2013, Grabner et al. 2015).

• Our lab has previously shown that FGF23 exposure also induced an increase in cardiomyocyteintracellular Ca2+ levels and increased cardiac contractility (Touchberry et al. 2013).

• Recently, expression of FGF23 has been shown in cardiac tissue and cardiomyocytes fromdeceased adolescent CKD patients receiving renal replacement therapy in the form of dialysis orrenal transplant (Leifheit-Nestler et al. 2015). The aim of our study was to utilize adult mousemodels of untreated CKD to further examine changes in cardiac expression of FGF23 during thisdisease.

• While bone release of FGF23 increases during CKD, it has not been fully elucidated if FGF23expression in the heart can contribute to cardiac remodeling through paracrine mechanisms.

• We hypothesized that cardiac FGF23 expression would be elevated in two mouse models ofCKD: Col4a3 null (Alport syndrome) and adenine-fed (chronic tubulointerstitial nephritis). Wefurther hypothesized that exercise would reduce FGF23 levels in adenine-fed mice compared tosedentary adenine-fed mice.

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Figure 1. We hypothesize that stimulation of cardiac FGFR4 by FGF23 may lead to increased intracellular calcium(Touchberry et al. 2013), stimulation of SERCA or phospholambin to increase SR calcium stores (Kao et al. 2014), andactivation of NFAT hypertrophic signaling pathways (Faul et al. 2012, Grabner et al. 2015).

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CONCLUSION

Fitness adaptations in Adenine-Fed + Exercise Mice

Biomarkers of CKD in Adenine-Fed and Adenine-Fed + Exercise Mice

Cardiac FGF23 Expression is Elevated in Adenine-Fed and Adenine-Fed + Exercised Mice

Biomarkers of CKD in the Col4a3 null mouse model of CKD

Ca2+

Cardiac FGF23 Expression is Elevated in Col4a3 null Mice