1Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; 2Max Planck Institute for Biology of Ageing Metabolomics Core Facility Köln, Deutschland; 3Universitätsklinikum Bonn Molekulare Kardiologie // Geb. 370 Bonn, Deutschland; 4University of Bonn Institute of Molecular Medicine and Experimental Immunology Bonn, Deutschland; 5Gemeinschaftspraxis Kardiologie Köln am Neumarkt Köln, Deutschland
Background: Aortic valve stenosis (AS) is the most common valve disease among adults. Chronic kidney disease (CKD) is closely associated with cardiovascular disease (CVD) including a higher incidence of AS and poor clinical outcomes. Major driving forces behind this association include calcification and fibrosis of the cardiovascular system which are induced and aggravated under uremic conditions. In this multifactorial context, the retention of uremic toxins such as indoxyl sulfate (IS) can be observed in advanced CKD, which is known to induce inflammatory and pro-calcific processes. However, the influence of IS on AS pathophysiology is only incompletely understood.
Methods and Results: Human valvular interstitial cells (VICs) from aortic valves were stimulated with phosphate (NaH2PO4) and IS in a concentration of 50 mmol/l to mimic uremic plasma. After 21 days of stimulation, calcified areas were stained using alizarin red solution (D). Photometric quantification of stained areas showed increased calcification of VICs stimulated with phosphate and IS compared to phosphate alone. Next-generation RNA sequencing identified pro-calcific genes, such as RUNX2 and inflammatory cytokines such as IL6 to be upregulated under the influence of IS (E). SLCO3A1 coding for OAT3A1 was shown to be the responsible gene for IS uptake and NKD2 was a molecular target in VIC calcification and IL6 upregulation upon IS exposure (F). A combined model of adenine-induced kidney injury and wire injury-induced AS was used to investigate the effects of uremia on AS in C57BL/6J mice (A). Valve function and morphology were assessed through echocardiography and histological analyses. In addition, wire injury in mice was performed during oral supplementation of IS. Echocardiography showed increased peak velocity and mean pressure gradient in uremic mice, indicating aggravated AS compared to control mice after wire injury (B). Explanted valves from uremic mice with AS exhibited a significant increase in macrophage infiltration, fibrotic areas and valvular NKD2 expression compared to controls (C). Valves of mice treated with IS showed more prominent fibrosis and macrophage infiltration as well as NKD2 expression compared to control mice.
Conclusion: Uremic conditions aggravate VIC calcification in vitro and AS development in mice and IS appears to participate in this process. NKD2 was identified as a downstream effector protein of IS involved in intracellular inflammation in VICs and murine AS. SLCO3A1 is responsible for IS uptake and downstream pro-inflammatory effects.