Induction of cardiac FGF23/FGFR4 expression is associated with left ventricular hypertrophy in patients with chronic kidney disease

Abstract

Background: In chronic kidney disease (CKD), serum concentrations of fibroblast growth factor 23 (FGF23) increase progressively as glomerular filtration rate declines, while renal expression of the FGF23 coreceptor Klotho decreases. Elevated circulating FGF23 levels are strongly associated with mortality and with left ventricular hypertrophy (LVH), which is a major cause of cardiovascular death in CKD patients. The cardiac FGF23/FGF receptor (FGFR) system and its role in the development of LVH in humans have not been addressed previously.

Methods: We conducted a retrospective case-control study in 24 deceased patients with childhood-onset end-stage renal disease (dialysis: n = 17; transplanted: n = 7), and 24 age- and sex-matched control subjects. Myocardial autopsy samples of the left ventricle were evaluated for expression of endogenous FGF23, FGFR isoforms, Klotho, calcineurin and nuclear factor of activated T-cells (NFAT) by immunohistochemistry, immunofluorescence microscopy, qRT-PCR and western blotting.

Results: The majority of patients presented with LVH (67%). Human cardiomyocytes express full-length FGF23, and cardiac FGF23 is excessively high in patients with CKD. Enhanced myocardial expression of FGF23 in concert with Klotho deficiency strongly correlates with the presence of LVH. Cardiac FGF23 levels associate with time-averaged serum phosphate levels, up-regulation of FGFR4 and activation of the calcineurin-NFAT signaling pathway, an established mediator of cardiac remodelling and LVH. These changes are detected in patients on dialysis but not in those with a functioning kidney transplant.

Conclusions: Our results indicate a strong association between LVH and enhanced expression levels of FGF23, FGFR4 and calcineurin, activation of NFAT and reduced levels of soluble Klotho in the myocardium of patients with CKD. These alterations are not observed in kidney transplant patients.

Keywords: Klotho; chronic kidney disease; fibroblast growth factor 23; fibroblast growth factor receptor 4; left ventricular hypertrophy.

FIGURE 1:

Patients with CKD develop LVH. (A) The heart weight/body weight ratio of each patient in relation to its respective control is significantly higher. (B) Representative sections from the left ventricle stained with HE or WGA are shown (both magnification, ×20; scale bar, 100 μm). (C) Quantification of cardiomyocyte cross-sectional area of patients in relation to respective controls (n = 100 cells per section) demonstrate increased surface area of individual cardiomyocytes. (D) Correlation of cardiomyocyte cross-sectional area with duration of ESRD. (E) Quantification of BNP mRNA expression in the myocardium of patients in relation to matched controls. Values are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 2:

Cardiac FGF23 is expressed in human left ventricular myocardium, and correlates with LVH in CKD patients. (A) Representative immunoblots for cardiac FGF23 protein and GAPDH (loading control) in myocardial tissue of healthy subjects. Plasma and recombinant human FGF23 (rhFGF23) reveals full-length O-glycosylated species, whereas cleaved FGF23 products are not detected. (B) Immunofluorescence co-staining for FGF23 and sarcomeric α-actinin demonstrating predominantly expression of FGF23 in human cardiomyocytes (magnification, ×63; scale bar, 50 µm). (C) Cardiac FGF23 mRNA expression in myocardium of patients in relation to matched controls is elevated, and associates with LVH. (D) Representative immunohistochemistries of human myocardial sections stained for FGF23 (brown) with hematoxylin counterstain (blue) (magnification, ×63; scale bar, 50 µm). (E) Quantification of FGF23% area in human myocardial tissue is enhanced in the LVH⁺ group. (F) Correlation of cardiac FGF23 with cardiomyocyte cross-sectional area, and (G) with the expression of BNP, indicating that local FGF23 expression is related to cardiac hypertrophy. Values are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 3:

FGFR1 is not regulated in human myocardial tissue of CKD patients independent of LVH. (A) Quantitative RT-PCR analysis, calculated according to the 2^−ΔCt method with normalization to β-actin (ACTB), reveals predominant mRNA expression of FGFR1 and FGFR4, but not KLOTHO (n.d., not detectable) in the left ventricle of human myocardial tissue of healthy subjects. (B) Immunoblots of lysates from human myocardial autopsy samples confirm protein expression of FGFR1 and FGFR4, and demonstrates clearly the presence of Klotho protein, with kidney protein lysates as positive control. (C) Quantitative RT-PCR analysis of FGFR1 mRNA expression in heart tissue of our patient cohort is unaffected in the presence of CKD or LVH. (D) Representative immunohistochemistries of human myocardial sections and aorta as positive control stained for FGFR1 (brown) with hematoxylin counterstain (blue) (magnification, ×63; scale bar, 50 µm). (E) Quantification of FGFR1% area of human myocardial tissue demonstrates no changes between patients and controls. Values are presented as mean ± SEM.

FIGURE 4:

LVH correlates with declining Klotho, and induction of cardiac FGFR4 expression in CKD patients. (A) Detection of Klotho (brown) with hematoxylin counterstain (blue) in cardiac tissue sections of patients and controls by immunohistochemistry, with human kidney sections as positive control (magnification, ×63; scale bar, 50 µm). (B) Quantification of Klotho % area demonstrates an association of Klotho deficiency with LVH in CKD. (C) Fold increase of Klotho % area correlates negatively with duration of ESRD, and (D) with cardiomyocytes cross-sectional area in CKD patients. (E) Representative immunohistochemistries of human myocardial sections stained for FGFR4 (brown) with hematoxylin counterstain (blue), and human liver tissue as positive control (magnification, ×63; scale bar, 50 µm). (F) FGFR4 mRNA expression is induced in the myocardium of CKD patients in relation to matched controls, and is higher in individuals with LVH. (G) Cardiac FGFR4 correlates positively with cardiomyocyte cross-sectional area in CKD. Values are presented as mean ± SEM; *P < 0.05, ***P < 0.001, ****P < 0.0001.

FIGURE 5:

The calcineurin–NFAT-associated signalling pathway and pro-hypertrophic gene programs are induced in LVH⁺ CKD patients. (A) mRNA expression of the regulatory subunit of calcineurin (CNB) is up-regulated in myocardial autopsy samples of LVH⁺ CKD patients. (B) Likewise, the mRNA of NFAT is higher in LVH⁺ group. (C) Representative immunohistochemistries of human myocardial sections stained for NFAT (brown) with hematoxylin counterstain (blue) (magnification, ×63; scale bar, 50 µm). Note nuclear localization of NFAT protein in LVH⁺ but not in LVH⁻ CKD patients. (D) Indicating cardiac remodelling, the ratio of ACTA to ACTC mRNA is significantly higher in the LVH⁺ group. Values are presented as mean ± SEM; *P < 0.05, **P < 0.01.

FIGURE 6:

Cardiac FGF23 mRNA expression correlates with time-averaged serum phosphate, and FGFR4 expression in patients with CKD, and is low after kidney transplantation. (A) Cardiac FGF23 mRNA correlates positively with time-averaged serum phosphate levels, and (B) FGFR4 expression, and (C) cardiac FGF23 correlates negatively with eGFR in the whole-patient cohort. (D) Cardiac FGF23 mRNA expression is high in dialysis but not in transplant patients. (E) Representative immunohistochemistries of human myocardial sections stained for FGF23 (brown) with hematoxylin counterstain (blue) (magnification, ×63; scale bar, 50 µm), and quantification of FGF23% area demonstrates higher FGF23 protein in dialysis but not in transplant patients. (F) Cardiac FGFR4 mRNA expression is elevated significantly in dialysis but not in transplant patients. (G) Representative immunohistochemistries of human myocardial sections stained for Klotho (brown) with hematoxylin counterstain (blue) (magnification, ×63; scale bar, 50 µm). Klotho protein is significantly lower in dialysis patients, but not in KTx patients. (H) In myocardial autopsy samples, mRNA expression of calcineurin subunits B (CNB) is up-regulated in dialysis patients compared with respective controls, and slightly down-regulates after KTx. (I) The expression of the transcription factor NFAT is up-regulated significantly in patients on dialysis, and reduced in KTx patients. (J) Myocardial hypertrophy is induced in dialysis patients as demonstrated by marked up-regulated BNP mRNA expression, whereas no significant difference is observed in KTx group. Values are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.