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. 2011;33(3):250-9.
doi: 10.1159/000324693. Epub 2011 Mar 2.

Vascular remodeling and arterial calcification are directly mediated by S100A12 (EN-RAGE) in chronic kidney disease

Joseph Gawdzik  1 , Liby Mathew, Gene Kim, Tipu S Puri, Marion A Hofmann Bowman
Affiliations

Vascular remodeling and arterial calcification are directly mediated by S100A12 (EN-RAGE) in chronic kidney disease

Joseph Gawdzik et al. Am J Nephrol. 2011.

Abstract

Background: The proinflammatory cytokine S100A12 (also known as EN-RAGE) is associated with cardiovascular morbidity and mortality in hemodialysis patients. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in nonatherosclerosis-prone C57BL/6J mice on normal rodent chow diet, but exposed to the metabolic changes of chronic kidney disease (CKD), would develop vascular disease resembling that observed in patients with CKD.

Methods: CKD was induced in S100A12 transgenic mice and wild-type littermate mice not expressing human S100A12 by surgical ligation of the ureters. The aorta was analyzed after 7 weeks of elevated BUN (blood urea nitrogen), and cultured aortic smooth muscle cells were studied.

Results: We found enhanced vascular medial calcification in S100A12tg mice subjected to CKD. Vascular calcification was mediated, at least in part, by activation of the receptor for S100A12, RAGE (receptor for advanced glycation endproducts), and by enhanced oxidative stress, since inhibition of NADPH-oxidase Nox1 and limited access of S100A12 to RAGE attenuated the calcification and gene expression of osteoblastic genes in cultured vascular smooth muscle cells.

Conclusion: S100A12 augments CKD-triggered osteogenesis in murine vasculature, reminiscent of features associated with enhanced vascular calcification in patients with chronic and end-stage kidney disease.

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Figures

Fig. 1
Fig. 1
Development of CKD after ureteral obstruction. a WT and S100A12tg (TG) mice had the right ureter obstructed (RUO) for 6 days followed by the release of obstruction (RRUO), 7 days of recovery and then removal of contralateral kidney function by left ureteral obstruction (LUO). BUN levels were measured post-LUO to assess function of the previously obstructed kidney. b Serum S100A12 was measured in WT and S100A12tg mice at sacrifice. ∗ p < 0.01.
Fig. 2
Fig. 2
Advanced medial calcification in the aorta of S100A12tg mice (TG) subjected to CKD (a, upper right panel) but not in WT-CKD mice (a, lower right panel). The distal thoracic aorta is shown in 5× magnification stained for alizarin red (AR), and 40× magnification insets show calcification upon alizarin red staining, and elastic fibers in black on Verhoeff-Van Giessen-stained serial sections. Scale bars = 10 μm. b Quantification of calcification nodules seen on alizarin red-stained sections. c Total calcium content was measured in the ascending aorta and arch and normalized to protein content.
Fig. 3
Fig. 3
Effect of S100A12 on oxidative stress. a 8-Isoprostane in the urine of WT and S100A12tg (TG) mice undergoing sham and CKD. b Semiquantitative expression of S100A12, RAGE and Nox1 protein of whole aortic lysate (∗ p < 0.05 compared to WT). c Representative histology from S100A12tg-CKD aorta showing grade 3 degradation of elastic fibers (fibers are black on Verhoeff-Van Giessen stain, left panel) with no corresponding calcification on alizarin red stain (middle panel), and hematoxylin and eosin stain (right panel). Original magnification is 10× with 40× insets. d Quantification of elastic fiber breakdown shows more elastolysis in S100A12tg aortas (∗∗ p < 0.001 compared to WT; p = 0.06 comparing S100A12tg-sham and -CKD).
Fig. 4
Fig. 4
In vitro calcification and osteoblastic gene expression in cultured VSMC (harvested from the aortas of WT and S100A12tg mice). a Total calcium content of VSMC cultured in DMEM and ossification medium containing H2O2 and high phosphate (H2O2/HP). b Quantitative reverse transcription-polymerase reaction for gene expression of bone morphogenic protein 2 (BMP2), bone-gla protein (BGLAP) and Runx2 in VSMC cultured in ossification medium. GAPDH expression was used as internal control (∗ p < 0.001). Effect of shRNA Nox1, shRNA control, soluble RAGE (sRAGE) and BSA on total calcium content (c) and mRNA expression (d) for BMP2, BGLAP and Runx2 (p < 0.001). VSMC transfected with shRNA plasmids (S100A12 and control) were selected by flow cytometry for GFP expression prior analysis.
Fig. 5
Fig. 5
Principle of S100A12-induced ROS in VSMC. S100A12 (red circle in cartoon, online version only) binds and activates RAGE to promote oxidative stress, including translocation of regulatory subunit Rac1 to associate with Nox. S100A12 binds to NADPH-oxidase Nox1 either directly or via binding proteins, as shown recently by our group [12], and the generated ROS promotes VSMC dysfunction, including osteoblastic differentiation. We speculate that S100A12 may act similarly in VSMC to its homolog S100A8/9 in phagocytes where it activates p67phox upon sensing bacteria or other danger molecules resulting in electron transfer from NADPH to molecular oxygen, and thereby generates a respiratory burst to destroy bacteria [30].
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