Background: Cardiovascular disease is a major cause of death among patients with chronic kidney disease (CKD) and cardiovascular calcification is one of the driving forces behind this association. Calcific aortic valve stenosis (AS) is one of the most frequently observed valve diseases and CKD is associated with increased incidence of AS and poor prognosis. A crucial step in the development of AS is the calcification of valvular interstitial cells (VIC) of the valve leaflets. Uremia leads to retention of the uremic toxin indoxyl sulfate (IS), which is widely known for its cardiovascular toxicity and associated with endothelial dysfunction and vascular calcification in CKD. The influence of IS on VIC-calcification is currently unknown.

Methods and results: Human VIC cultures were established and incubated in control medium (CM) or pro-calcifying-medium (PCM), containing NaH₂PO₄ and L-ascorbic acid. Further, IS solved 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 VIC. After 7 days of incubation, VIC 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 CM group no calcification could be detected. To identify potential molecular mechanisms mediating IS-induced calcification, RNA was isolated from VIC cultured under the above-mentioned conditions to identify key genes involved in the process of valvular calcification. Next generation RNA-sequencing identified a large number of significantly regulated genes under the 4 experimental conditions. Among the most significantly regulated genes we identified naked cuticle homologue 2 (NKD2), a regulator of the WNT-signaling pathway, as significantly up-regulated under the influence of IS. This finding was confirmed using quantitative PCR in different biological specimens, in which the combination of PCM+IS showed a significant induction of NKD2 expression in contrast to PCM alone. To investigate the influence of NKD2 on VIC-calcification, a genetic knockdown was performed by incubating VIC with small-interference RNA (siRNA) for 24 hours. After 21 days of incubation under PCM and PCM+IS, alizarin-staining showed less calcified areas in NKD2-knockdown cells compared to scrambled siRNA as negative control.