OAT3A1/NKD2 mediates increased calcification of aortic valvular interstitial cells under in vitro conditions of uremia

Abstract

Background Chronic kidney disease is closely associated with cardiovascular disease which contributes to a high mortality and morbidity in these patients. Calcific aortic valve stenosis is the most common valve disease among adults. CKD leads to a higher incidence of AS and is associated with impaired clinical outcomes after valve replacement. CKD leads to retention of uremic toxins, such as indoxyl sulfate (IS), which is known to induce inflammatory and pro-calcific processes. The influence of IS on AS pathophysiology is currently not known. Methods and results Human aortic valvular interstitial cells (VICs) were stimulated by 4 different conditions: Control medium (CM), CM and IS in a concentration of 50 mmol/l, pro-calcifying medium (PCM) which was produced by adding NaH2PO4 and L-ascorbic acid to CM and PCM+IS. After 7 days of incubation, VICs were fixed in formalin and calcification was evaluated through staining with alizarin red solution (B). Staining was quantified by photometric measurements at 540 nm. We observed a significantly higher degree of VIC calcification under the influence PCM+IS compared to PCM alone (C). VICs were stimulated for 7 days and next-generation RNA Sequencing (RNA Seq) was performed. Analysis of RNA Seq dataset identified naked cuticle homologue 2 (NKD2) to be strongly regulated by IS. This effect was confirmed by qPCR using VICs from a total of three different human donors and immunoblot analysis (D, E). We observed a more pronounced upregulation of NKD2 gene expression under PCM+IS vs. PCM alone (p<0.0001) (D). To investigate the influence of NKD2 on VIC calcification genetic knockdown of NKD2 was performed by transfecting VICs with NKD2 siRNA compared and scrambled siRNA as negative control (nc siRNA) (F). After 21 days of stimulation and subsequent alizarin staining, we observed a significant decrease in VIC calcification under NKD2 knockdown compared to negative control under PCM+IS conditions (G, H). To identify upstream mechanisms that lead to NKD2 upregulation by IS we investigated the influence of organic anion transporters (OAT) which are responsible for cellular uptake of IS. Probenecid, known as a potent inhibitor of OATs showed a dose dependent suppression of NKD2 upregulation under PCM+IS (I). Systematic analysis of RNA Seq data regarding SLCO genes coding for OAT and OAT-peptides revealed that only SLCO3A1, coding for OAT3A1 is highly abundant in VICs (J). SLCO3A1 knockdown showed a significant decrease of NKD2 expression after stimulation with PCM+IS for 7 days compared to negative control (K). Conclusion Our findings show that in vitro conditions of uremia increase calcification of aortic VICs. NKD2 expression is regulated by PCM and IS, and PCM+IS treated cells show the strongest upregulation of NKD2. Genetic knockdown of NKD2 attenuates VIC calcification suggesting a role of NKD2 in this process. Further, our findings suggest that OAT3A1 is involved in IS mediated regulation of NKD2 expression. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Medical faculty of the University of Bonn, German Cardiac Society (DGK), German Research Foundation (DFG), TRR259