Background: Chronic kidney disease (CKD) is closely associated with calcifying processes of the blood vessels and heart valves which contributes to the high cardiovascular morbidity and mortality in such patients. Calcific aortic valve stenosis (AS) is the most common valve disease among adults. In patients with CKD, incidence of AS is high and clinical outcome after surgical or interventional valve replacement worsened. Impaired renal function leads to a retention of uremic toxins, such as indoxyl sulfate (IS), which is known to mediate harmful effects on the heart and the vasculature. The influence of IS on AS pathophysiology is currently unknown.

Methods and results: Human aortic valvular interstitial cells (VICs) were cultured, and calcification was induced by use of pro-calcifying-medium (PCM), containing NaH₂PO₄ and L-ascorbic acid. In addition, 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 leading to a total of 4 different conditions. After 21 days of stimulation, calcified areas were stained using alizarin red solution (A). Photometric quantification of stained areas revealed an increased calcification of VICs stimulated with PCM and IS together compared to PCM alone (B). Next generation RNA sequencing of VICs stimulated for seven days showed a differential regulation of genes associated to osteoblastic differentiation as well as inflammatory cytokines (C). In a next step, uremia was induced in 15 C57BL6/J mice with adenine-rich diet. Control mice (n=15) received a standard chow (D). After 4 weeks of treatment, we performed a wire injury to induce aortic valve stenosis (n=9) or sham operation (n=6) (E). Echocardiographic evaluation of AS was performed every 2 weeks. After a total of 10 weeks, mice were sacrificed, and aortic valves explanted. Liquid chromatography-Mass spectrometric plasma analyses showed a significant increase in IS plasma-levels in uremic mice as compared to control animals (D). AS + Uremia mice showed a significant increase in echocardiographic parameters of AS (G).

Conclusion: Our findings demonstrate that in vitro conditions of uremia induce calcification of aortic VICs and that uremic toxin IS acts as a pro-calcific factor in this setting. CKD mice with higher plasma levels of IS show a higher degree of AS after wire injury measured by echocardiographic parameters. Further research is needed to identify the underlying molecular mechanisms.