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. 2014 Jul;34(7):1399-411.
doi: 10.1161/ATVBAHA.114.303508. Epub 2014 May 22.

S100/Calgranulin-mediated inflammation accelerates left ventricular hypertrophy and aortic valve sclerosis in chronic kidney disease in a receptor for advanced glycation end products-dependent manner

Ling Yan  1 , Liby Mathew  1 , Bijoy Chellan  1 , Brandon Gardner  1 , Judy Earley  1 , Tipu S Puri  1 , Marion A Hofmann Bowman  2
Affiliations

S100/Calgranulin-mediated inflammation accelerates left ventricular hypertrophy and aortic valve sclerosis in chronic kidney disease in a receptor for advanced glycation end products-dependent manner

Ling Yan et al. Arterioscler Thromb Vasc Biol. 2014 Jul.

Abstract

Objective: S100A12 and fibroblast growth factor 23 are biomarkers of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). We tested the hypothesis that human S100/calgranulin would accelerate cardiovascular disease in mice subjected to CKD.

Approach and results: A bacterial artificial chromosome of the human S100/calgranulin gene cluster containing the genes and regulatory elements for S100A8, S100A9, and S100A12 was expressed in C57BL/6J mouse (hBAC-S100) to generate a novel humanized mouse model. CKD was induced by ureteral ligation, and hBAC-S100 mice and wild-type mice were studied after 10 weeks of chronic uremia. hBAC-S100 mice with CKD showed increased fibroblast growth factor 23 in the hearts, left ventricular hypertrophy, diastolic dysfunction, focal cartilaginous metaplasia, and calcification of the mitral and aortic valve annulus together with aortic valve sclerosis. This phenotype was not observed in wild-type mice with CKD or in hBAC-S100 mice lacking the receptor for advanced glycation end products with CKD, suggesting that the inflammatory milieu mediated by S100/receptor for advanced glycation end products promotes pathological cardiac hypertrophy in CKD. In vitro, inflammatory stimuli including interleukin-6, tumor necrosis factor-α, lipopolysaccarides, or serum from hBAC-S100 mice upregulated fibroblast growth factor 23 mRNA and protein in primary murine neonatal and adult cardiac fibroblasts.

Conclusions: Myeloid-derived human S100/calgranulin is associated with the development of cardiac hypertrophy and ectopic cardiac calcification in a receptor for advanced glycation end products-dependent manner in a mouse model of CKD. We speculate that fibroblast growth factor 23 produced by cardiac fibroblasts in response to cytokines may act in a paracrine manner to accelerate left ventricular hypertrophy and diastolic dysfunction in hBAC-S100 mice with CKD.

Keywords: S100 proteins; glycosylation end products, advanced; hypertrophy, left ventricular; renal insufficiency, chronic.

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Conflict of interest statement

Disclosure: All authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Transgenic expression of human S100/calgranulin in myeloid cells is associated with inflammation
A) Transgenic expression of a bacterial artificial chromosome containing the human S100/calgranulin genes (hBAC-S100) up regulates protein expression of S100A12 in myeloid blood cells (red-stained cells on immunofluorescent microscopy insert b) and not in WT peripheral blood cells (insert e). B) S100A12 is increased in serum of hBAC-S100 mice and not detectable in WT serum (* p<0.001). C) qRT-PCR for S100A12mRNA normalized to β-actin and expressed as fold change compared to the expression of a tissue-specific abundant gene (myosin heavy chain 7, Myh7 for heart tissue and isolated cardiac myocytes; connective tissue growth factor, CTGF for isolated cardiac fibroblasts; CD68 for peripheral blood leucocytes, spleen, and bone marrow). D/E) qRT-PCR from peripheral blood cDNA of 6-week-old WT and hBAC-S100 littermate mice with intact RAGE signaling (D) and from 6-week-old WT and hBAC-S100 littermate mice with deleted RAGE signaling (RAGE-KO) (E) for LPS-binding protein (LBP), myeloperoxidase (MPO), neutrophil lactoferrin (LTF), vascular cell adhesion molecule 1 (VCAM1), neutrophil proteinase 4 (PRTN3), human neutrophil alloantigen 2a (CD177), neutrophil gelatinase-associated lipocalin (LCN2), proteoglycan 2, bone marrow (PRG2), CCAAT/enhancer binding protein (C/EBP), epsilon (CEBPE), cathepsin G (CTSG), and chitinase 3-like 3 (CHI3l3). F/G) Serum IL-6 and IL-22 are increased in hBAC-S100 mice with intact RAGE, H) attenuated IL-6 and IL-22 in mice lacking RAGE (WT and hBAC-S100) despite expression of S100A12 in hBAC/S100-RAGE KO mice (*P<0.03).
Figure 2
Figure 2. Model of CKD in hBAC-S100 and WT littermate mice
A) Reversible surgical ligation of the right ureter followed by irreversible ligation of the left ureter. B) BUN was measured in the serum at various time points until sacrifice as an indicator for developing CKD after reversible right ureter obstruction (RUO) for 6 days, followed by release of RUO (RRUO), followed 7 days later by irreversible left ureter obstruction (LUO). * P=0.001 vs. sham). C/D) Representative H&E stained slides of the right kidney were scored for interstitial fibrosis with tubular atrophy, and for interstitial inflammation using scores of 0–3 for semi-quantitative assessment with 0 =<5%, 1= 6–25%, 2= 26–50%, and 3=>50% involvement. There was no difference in renal pathology between the four CKD groups. Scale bar 10 μm. E) Systolic and diastolic blood pressure were measured in the tail artery (* P=0.04 vs. respective sham group). F) Serum FGF23 and S100A12 (G) were measured by ELISA (* P=0.01 vs. respective sham group). All values are mean ±SEM, n>20 mice per group.
Figure 3
Figure 3. hBAC-S100 mice with CKD develop LVH and impaired diastolic function
A) M-mode Doppler for measurement of fractional shortening (FS, quantified in D) and Left Ventricular Posterior Wall Thickness (LVPW, quantified in E). B) Continuous wave Doppler over the mitral valve for early (E) and atrial (A) flow was measured to calculate E/A (quantified in F). C) Continuous wave Doppler over the aortic valve was measured to calculate mean and peak aortic valve velocity timed integral (AoVTI, quantified in G). H) Representative gross pathology sections from the cardiac mid-chamber (hematoxylin and eosin stain, 5x magnification, scale bare = 20 μm) demonstrate LVH, confirmed by (I) increased ratio of heart weight to body weight in hBAC-S100/CKD mice (* P= 0.01 compared to WT/CKD and S100/sham). All values are mean ±SEM, n>20 mice per group.
Figure 4
Figure 4. hBAC-S100 mice with CKD develop ectopic cardiac calcification
A) Serial stained sections of the mitral valve of hBAC-S100 (upper panel) and WT mice (lower panel) and B) of the aortic valve of hBAC-S100 (upper panel) and WT mice (lower panel). Serial sections were stained for hematoxylin and eosin (H&E), Alizarin Red S staining calcium deposits in red color as marked by arrow, and Masson’s Trichrome. P=pigment, Scale bar 10 μm. C) Cardiac calcification developed spontaneously in 10-month-old hBAC-S100 mice (without CKD and on regular rodent chow diet) as shown by red color on Alizarin Red S stained sections of the aortic valve and co-localizing to hypertrophic chondroblast-like cells (upper panel in C), and to calcification on the valve leaflet tip (lower panel in C). D) Red-colored calcification foci (shown by arrow) but not the black-colored pigment deposition (marked as P) on Alizarin Red S-stained serial sections (n= 50–70 per heart) were counted from the apex to the ascending aorta. E) Quantification of the maximal aortic valve thickness of the three leaflets measured as depicted in panel B. All values are mean ±SEM, n=10 mice per group.
Figure 5
Figure 5. hBAC-S100 mice with CKD develop left ventricular hypertrophy
A) Quantitative real time RT-PCR in murine heart for genes associated with hypertrophy and fibrosis. B) Immunoblotting of cardiac tissue lysates for FGF23 and β actin, and semiquantitave densitometry of bands. C–J) Serial sections of the aortic valve of hBAC-S100 (C–E) and WT control (F–H) stained with Alizarin Red (C, F) and hematoxylin and eosin (E, H) shows calcifying chondroblast-like cells (marked with an arrow) and expression of FGF23 in interstitial valve cells in hBAC-S100 mice (D) but not in WT mice (G). Non-immune IgG was used as negative control (insert in D & G, J), and newborn murine WT bone tissue was used as positive control (I). 400x magnification, scale bare = 10 μm, P=pigment. K. Quantitative real time RT-PCR in cultured primary murine cardiac fibroblasts (CF) and aortic smooth muscle cells (ASMC) for FGF23 mRNA after 6h stimulation with IL-6 (25 ng/ml), TNFα (25 ng/ml) LPS (25 ng/ml), recombinant S100A12 (2 ug/ml), serum from hBAC-S100 mice (3%) or serum from WT mice (3%). L. FGF23 protein measured by ELISA in cell culture supernatant from primary neonatal cardiac fibroblast from WT/RAGE+/+ and WT/RAGE−/− mice after 24 hours stimulated with cytokines or mouse serum (* P<0.01 compared to control)
Figure 6
Figure 6. hBAC-S100 mice with CKD and lacking RAGE are protected from cardiac hypertrophy and calcification
A) Systolic (SBP), diastolic (DBP) and mean (MAP) arterial blood pressure were measured in the tail artery. B) Serum FGF23 was measured by ELISA. C) Indexed heart weight to body weight measured at sacrifice. D) Quantitative real time RT-PCR in murine hearts for selected genes associated with hypertrophy and fibrosis. E) Immunoblotting of cardiac tissue lysates for FGF23 and β actin, including hBAC-S100/RAGE+/+/CKD as positive control (left lane), and from mice lacking RAGE (lane 2–7), and semiquantitative densitometry of bands (F). G) E/A ratio of continuous wave Doppler over the mitral valve for early (E) and atrial (A) flow is calculated as a measure of diastolic function. H) Quantification of red-colored calcification foci on Alizarin Red S-stained serial cardiac sections 200 μm apart was counted from the apex to the ascending aorta.
Figure 7
Figure 7. Proposed model of LVH in CKD
Increased serum concentration of S100/calgranulins in mice with CKD promotes systemic inflammation in a RAGE-dependent manner, and this is associated with cartilagenous metaplasia and calcification of the valve annulus. Importantly, systemic inflammation in vivo (and in cultured cardiac fibroblasts upon treatment with cytokines), up regulates endogenous FGF23 in cardiac fibroblasts, which may act in a paracrine manner to promote LVH and diastolic dysfunction. Elevated serum FGF23 has been previously associated with LVH and recombinant FGF23 was shown to directly cause hypertrophy of cardiac myocytes in vitro and in vivo (ref. 24).
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