Beyond the role of the mineralocorticoid receptor in aortic stenosis pathogenesis: a novel interplay between MR, NGAL signaling, and the innate immunity-TLR4 pathway

Introduction Aortic stenosis (AS) is the most common valvular heart disease, particularly in the elderly, characterized by the loss of aortic valve (AV) homeostasis leading to valve thickening, calcification, and restricted blood flow[1]. Aldosterone (aldo), a key hormone in the regulation of sodium and water balance, has been implicated in various pathological processes beyond its traditional role in blood pressure maintenance. In the AV, aldo contributes to valve calcification, inflammation, and fibrosis during AS by activating mineralocorticoid receptor (MR) signaling [2]. Similarly, Neutrophil Gelatinase-Associated Lipocalin (NGAL) is implicated in AS, where it regulates tissue remodeling and inflammation[3]. Elevated NGAL levels correlate with worse outcomes, suggesting its role in valve degeneration[4]. In fact, NGAL can act downstream of MR signaling in kidney and cardiovascular diseases, though the precise mechanisms of their collaboration in the AV remain unclear. Objective The aim of this study was to uncover the signaling pathways responsible for mediating the effects of aldo-induced MR signaling and its downstream effectors on the differentiation, inflammation, and calcification of AV and its interstitial cells (VIC). Methods Healthy or stenotic wild-type (WT) or NGAL gene knockout (KO-NGAL) male rats, aged 12 weeks, were used for in vitro and ex vivo experiments, following NIH (no. 85-23) and EU animal care guidelines. VIC, isolated from the AV with collagenase, were exposed to aldo (10-8M) or recombinant (r)- NGAL (500 ng/mL) and preincubated with MR (finerenone 10-6M) or TLR4 (resatorvid 1 μM) antagonists, for 24h or 6 days. Western blot (WB), ELISA, immunohistochemistry and qPCR were used to assess inflammatory, fibrotic, and osteogenic markers in rat VIC and AV. Results Our study demonstrates that NGAL acts as a critical downstream effector of aldo-induced MR activation, playing a pivotal role in driving VIC differentiation, extracellular matrix (ECM) remodeling, osteogenesis, and inflammation as well as in vivo AV calcification. In response to MR activation, NGAL is not only produced intracellularly but also can be secreted. Extracellular rNGAL mimicked the effects observed in VIC following MR-NGAL pathway activation. The results also showed for the first time in the AV that the aldo-MR signaling led to a significant upregulation of the innate immunity TLR4-MYD88 pathway in an NGAL-dependent manner inducing VIC differentiation, ECM remodeling, osteogenesis and inflammation. Together, these findings highlight a novel signaling axis where the MR upregulates NGAL, which subsequently triggers the TLR4 pathway, leading to VIC and AV differentiation, osteogenesis, and inflammation. Conclusion This work provides new insights into the molecular mechanisms and the complex interplay between MR, NGAL, and TLR4 signaling pathways driving aortic valve stenosis, which could offer new therapeutic targets for disease management.Table-summary Graphical abstract