Background: Chronic kidney disease (CKD) is a highly prevalent condition with  cardiovascular disease (CVD) being a major cause of death among these patients. Calcific aortic valve stenosis (AS) is one of the most frequently observed valve diseases and CKD is known to be associated with aortic valve calcification, faster progression of valve stenosis and thus worse outcome for patients. Calcification of valvular interstitial cells (VIC) of the valve leaflets is a crucial step in the pathogenesis of valvular calcification. The uremic toxin indoxyl sulfate (IS) is an important mediator of CVD in CKD and in particular known to induce endothelial dysfunction. The influence of IS on VIC-calcification is currently unknown.

Methods and results: Human VIC culture was 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 μmol/l, which is comparable to IS plasma concentrations in CKD patients. 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 100mmol/l cetylpyridinium chloride and photometric measurements at 540 nm. After 7 days of incubation, we observed a significantly higher degree of VIC calcification under the influence of IS in addition to PCM compared to PCM alone (2.15±0.42 vs. 0.91± 0.11, p<0.0001). 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 showed a large number of significantly regulated genes under the 4 different conditions. Among those genes, we observed a significant upregulation of pro-calcific ABCG2 as well as a decreased expression of RGS5, which is known to be associated with cardiovascular disease. We suspect those genes, among others, to be involved in the pro-calcifying effect of IS. 

Conclusion: Our findings show for the first time that in vitro conditions of CKD induce the calcification of aortic VIC and a pro-calcific gene expression pattern. Further exploration of the underlying mechanisms could identify novel pathways mediating CKD-induced aortic valve calcification.