Background: Patients with chronic kidney disease (CKD) are highly susceptible to cardiovascular calcification. We hypothesize that sortilin is crucially involved in the development of cardiovascular disease (CVD) in patients with CKD by regulating calcification and fibrosis. However, inducers and the underlying molecular mechanism of sortilin induction have not been reported.

Methods and Results: Calcified lesions from CKD patients and CKD mice display high sortilin expression. Treatment of SMCs with serum from CKD patients undergoing hemodialysis promoted sortilin expression and vascular smooth muscle cell (SMC) calcification compared to control serum. To identify potential sortilin inducers, human hemodialysates were fractionated by hydrophobicity using liquid chromatography. Hydrophobic uremic toxins increased SMC sortilin protein expression 4.6±2.3 fold (p=0.001). Serum glucocorticoid levels are higher in patients with declined kidney function. The natural glucocorticoid cortisol and the synthetic glucocorticoid dexamethasone increased sortilin expression 2.7±1.1 fold (p=0.006) and 4.4±2.0 fold (p=0.001), respectively. Cortisol increased sortilin protein stability (p=0.005) but not mRNA stability (p=0.735). RNA-binding proteins govern the translation of mRNA into their corresponding protein products. In transcriptomic analyses of calcifying SMCs, the RNA-binding protein with multiple splicing (RBPMS) was significantly increased compared to control (1.9 fold, p=0.023). Silencing of RBPMS by trend promoted dexamethasone-induced sortilin protein expression (2.1±1.3 fold, p=0.12) and reduced glucocorticoid receptor (GR) protein expression (-88%, p=0.01). Inhibition of the GR by mifepristone inhibited sortilin expression on mRNA (-30%, p=0.001) and protein level (-51%, p=0.001). Inhibition of the GR coactivator histone acetyltransferase p300/CBP by the competitive inhibitor C646 reduced sortilin expression (-68%, p=0.022) but did not affect GR expression. Further, inhibition of p300/CBP inhibited SMC calcification assessed by tissue non-specific alkaline phosphatase activity (-66%, p=0.001) and matrix mineralization. Moreover, increased histone H3K4 methylation by inhibition of lysine-specific demethylase 1 promoted sortilin expression 4.1±1.8 fold (p=0.001), that could be prevented by mifepristone.
 
Conclusion: Our findings provide evidence that GR-signaling in SMCs plays an important role in both sortilin transcriptional regulation by histone H3K4 methylation and acetylation and sortilin translational regulation by the RNA-binding protein RBPMS.