Abstract TAC101: High Phosphate Induces Ligand-Independent Mineralocorticoid and Toll-Like Receptor 4 Signaling in Aortic Valve Interstitial Cells, Driving Key Mechanisms of Aortic Stenosis

Introduction: Aortic stenosis (AS) is the most prevalent valvulopathy in elderly adults, with increased incidence in chronic kidney disease (CKD). Elevated serum phosphate, due to FGF23-Klotho axis dysregulation and impaired excretion, has been identified as a potential pathogenic link between both conditions, along with disturbances in the renin-angiotensin system. Aortic valve interstitial cells (VIC) play a central role in AS pathogenesis; high inorganic phosphate (HP) promotes their osteogenic trans-differentiation via the type III phosphate transporter (Pit-1), triggering mitochondrial dysfunction, NADPH oxidase activation, and ROS generation. Pathways such as PI3K/Akt, MAPK, and TGF-β/Smad have been implicated in HP-driven osteogenesis and fibrosis. Objective: To elucidate the pathways activated in the aortic valve (AV) by the HP levels occurring during CKD and its potential role in activating mineralocorticoid receptor (MR) signaling. Methods: VIC were isolated from the AV of wild type or transgenic rats overexpressing the human (h)-MR. Cells were treated with HP levels (3.2 mM CaCl 2 and 2.6 mM NaH 2 PO 4 ) alone or in combination with aldosterone 10 -8 M in the presence of MR antagonist (MRA) finerenone (10 -6 M), or TLR4 inhibitor (TAK-242 10 µM). Control or CKD male rats, generated by 5/6 nephrectomy and having HP diet, were used for ex vivo experiments. WB, ELISA, IHQ and qPCR were used. Results: HP levels trigger ligand-independent transcription and signaling of the MR leading to VIC activation and fibrosis. Transgenic VIC overexpressing h-MR confirmed that elevated MR levels promote VIC activation and fibrotic responses. In parallel, HP increases Toll-like receptor 4 (TLR4) expression and signaling leading to the induction of fibrosis, osteogenesis and inflammation. These effects were intensified by co-treatment of VIC with aldosterone and HP because of the TLR4 signaling potentiation. In vivo , rats suffering from CKD showed significant extracellular matrix (ECM) alterations that were largely reversed by MRA. Exposure of VIC to serum from CKD rats disrupted the expression of key ECM-related and pro-fibrotic genes, including FGF-23 and biglycan, in an MR-dependent manner. Conclusions: In vitro studies revealed for the first time that HP levels trigger ligand-independent activation of the MR signaling, likely through the increase of oxidative stress and ROS generation. This links phosphate overload to MR-driven fibrosis and ECM remodeling in VIC.