Background: 
Cardiovascular disease (CVD) is a major cause of morbidity and mortality in patients with chronic kidney disease (CKD). This association is largely driven by calcifying processes of the cardiovascular system including the vasculature and the heart valves. Calcific aortic valve stenosis (AS) is the most common valve disease among adults. CKD is associated with increased incidence of AS and impaired outcomes after surgical or interventional valve replacement. Differentiation of valvular interstitial cells (VICs) to a pro-calcific phenotype is known to be a key feature in the pathogenesis of AS. Progressive CKD leads to retention of uremic toxins, such as indoxyl sulfate (IS), which is known to induce vascular calcification. The influence of IS on VICs is currently not known.

Methods and results: 
Human VIC cultures were established and calcification was induced by use of pro-calcifying-medium (PCM), containing NaH₂PO₄ and L-ascorbic acid. Additionally, IS dissolved in dimethyl sulfoxide (DMSO) or DMSO alone were added in a concentration of 50 mmol/l, which is comparable to IS plasma concentrations in patients with advanced CKD. A previously performed photometric viability assay using MTT as dye proved this concentration of IS to be non-toxic to VICs. After 7 days of incubation, VICs were fixed in formalin and calcification was evaluated through staining with alizarin red solution. Staining was quantified by extracting alizarin with 100 mmol/l cetylpyridinium chloride and photometric measurements at 540 nm. We observed a significantly higher degree of VIC calcification under the influence of IS in addition to PCM compared to PCM alone. In the control medium (CM) group no calcification could be detected. To identify molecular mechanisms behind VIC calcification, VICs were cultured for seven days and next generation RNA Sequencing (RNA Seq) and mass spectrometric proteomic analysis were performed. We observed a downregulated expression of the myofibroblastic markers calponin (CNN1) and transgelin (TAGLN) as well as protein levels. This effect appeared to be accelerated by IS. Further, we observed an upregulation of the osteoblastic markers runt-related transcription factor 2 (RUNX2) and bone morphogenic protein 2 (BMP2) indicating a phenotype switch of VICs to an osteoblastic phenotype.

Conclusion: 
Our findings show that in vitro conditions of uremia induce calcification of aortic VICs. In AS both a myofibroblastic and osteoblastic differentiation of VICs represent important pathophysiological mechanisms that lead to progressive fibrosis and calcification of the valve cusps. In our in vitro model, osteoblastic differentiation of VICs appears to be the main pathology. Further research is needed und identify the underlying molecular mechanisms.