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Indoxyl-sulfate activation of the AhR-NFκB pathway promotes IL-6 secretion and subsequent osteogenic differentiation in human valvular interstitial cells
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Edité par CCSD ; Elsevier/French Society of Cardiology -
International audience. IntroductionCalcific aortic valve disease (CAVD) results from fibro-calcific degeneration of the aortic valve leaflets, causing major cardiovascular complications. In chronic kidney disease (CKD) patients, this pathological remodelling is more frequent, appears earlier and progresses faster leading to a mortality rate three times higher than in the general population. The uremic toxin Indoxyl-sulfate (IS), a powerful predictor of cardiovascular mortality in CKD patients, is a strong promoter of ectopic calcification, which role in CAVD has never been studied.ObjectiveThis work aimed to evaluate whether IS influences primary human valvular interstitial cells (hVICs) mineralization in-vitro.MethodPrimary hVICs isolated from aortic tricuspid valves collected from CAVD patients were exposed to increasing concentrations of IS (IS normal [Isn]: 0.5 Mg/mL, IS uremic [Isu]: 37 Mg/mL or IS maximum [Ismax]: 233 Mg/mL) in the presence of an osteogenic medium (OM) containing 2.4 mmol/L calcium and 2.5 mmol/L phosphate. HVICs osteogenic transition was assessed by qRT-PCR following Bmp2 and Runx2 mRNA. Their mineralization was quantified by the o-cresolphthalein method. Inflammation was assessed following NFκB activation by western blot and IL-1β, IL-6 and TNF-α secretion by ELISA. A siRNA approach allowed determining the signaling pathway involved.ResultsIS increased OM-induced hVICs osteogenic transition and calcification concentration-dependently, an effect blocked after transfection with siRNA targeting IS receptor AhR. Exposure to [ISu] or [ISmax] promoted P65 phosphorylation, the blockade of which inhibited IS-induced mineralization, suggesting a key role for inflammation in this process. HVICs did not secrete TNF-α, produced few IL-1β but secreted high amounts of IL-6 in response to IS, which was blocked by silencing AhR or P65. The use of an antibody neutralizing IL-6 abolished IS-induced procalcific effects.ConclusionIS promotes hVICs mineralization through AhR-dependent activation of NFκB pathway and subsequent release of IL-6. The fact that IL-6 neutralization blocks IS procalcific effects is of particular interest since interventional studies evaluating the cardiovascular impact of anti-IL-6 antibodies in CKD are underway.
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