Aims: Chronic kidney disease (CKD) is frequently associated with other chronic diseases including anemia. Daprodustat (DPD) is a prolyl hydroxylase inhibitor, a member of a family of those new generation drugs that increase erythropoiesis via activation of the hypoxia-inducible factor 1 (HIF-1) pathway. Previous studies showed that HIF-1 activation is ultimately linked to acceleration of vascular calcification. We aimed to investigate the effect of DPD on high phosphate-induced calcification.Methods and Results: We investigated the effect of DPD on calcification in primary human aortic vascular smooth muscle cells (VSMCs), in mouse aorta rings, and an adenine and high phosphate-induced CKD murine model. DPD stabilized HIF-1α and HIF-2α and activated the HIF-1 pathway in VSMCs. Treatment with DPD increased phosphate-induced calcification in cultured VSMCs and murine aorta rings. Oral administration of DPD to adenine and high phosphate-induced CKD mice corrected anemia but increased aortic calcification as assessed by osteosense staining. The inhibition of the transcriptional activity of HIF-1 by chetomin or silencing of HIF-1α attenuated the effect of DPD on VSMC calcification.Conclusion: Clinical studies with a long follow-up period are needed to evaluate the possible risk of sustained activation of HIF-1 by DPD in accelerating medial calcification in CKD patients with hyperphosphatemia.

Calcific aortic valve stenosis (CAVS) is a heart disease characterized by the progressive fibro-calcific remodeling of the aortic valves, an actively regulated process with the involvement of the reactive oxygen species-mediated differentiation of valvular interstitial cells (VICs) into osteoblast-like cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of a variety of antioxidant genes, and plays a protective role in valve calcification. Heme oxygenase-1 (HO-1), an Nrf2-target gene, is upregulated in human calcified aortic valves. Therefore, we investigated the effect of Nrf2/HO-1 axis in VIC calcification. We induced osteogenic differentiation of human VICs with elevated phosphate and calcium-containing osteogenic medium (OM) in the presence of heme. Heme inhibited Ca deposition and OM-induced increase in alkaline phosphatase and osteocalcin (OCN) expression. Heme induced Nrf2 and HO-1 expression in VICs. Heme lost its anti-calcification potential when we blocked transcriptional activity Nrf2 or enzyme activity of HO-1. The heme catabolism products bilirubin, carbon monoxide, and iron, and also ferritin inhibited OM-induced Ca deposition and OCN expression in VICs. This study suggests that heme-mediated activation of the Nrf2/HO-1 pathway inhibits the calcification of VICs. The anti-calcification effect of heme is attributed to the end products of HO-1-catalyzed heme degradation and ferritin.

Vascular calcification (VC) is associated with a number of cardiovascular diseases, as well as chronic kidney disease. The role of smooth muscle cells (SMC) has already been widely explored in VC, as has the role of intracellular Ca2+ in regulating SMC function. Increased intracellular calcium concentration ([Ca2+]i) in vascular SMC has been proposed to stimulate VC. However, the contribution of the non-selective Piezo1 mechanosensitive cation channels to the elevation of [Ca2+]i, and consequently to the process of VC has never been examined. In this work the essential contribution of Piezo1 channels to arterial medial calcification is demonstrated. The presence of Piezo1 was proved on human aortic smooth muscle samples using immunohistochemistry. Quantitative PCR and Western blot analysis confirmed the expression of the channel on the human aortic smooth muscle cell line (HAoSMC). Functional measurements were done on HAoSMC under control and calcifying condition. Calcification was induced by supplementing the growth medium with inorganic phosphate (1.5 mmol/L, pH 7.4) and calcium (CaCl2, 0.6 mmol/L) for 7 days. Measurement of [Ca2+]i using fluorescent Fura-2 dye upon stimulation of Piezo1 channels (either by hypoosmolarity, or Yoda1) demonstrated significantly higher calcium transients in calcified as compared to control HAoSMCs. The expression of mechanosensitive Piezo1 channel is augmented in calcified arterial SMCs leading to a higher calcium influx upon stimulation. Activation of the channel by Yoda1 (10 μmol/L) enhanced calcification of HAoSMCs, while Dooku1, which antagonizes the effect of Yoda1, reduced this amplification. Application of Dooku1 alone inhibited the calcification. Knockdown of Piezo1 by siRNA suppressed the calcification evoked by Yoda1 under calcifying conditions. Our results demonstrate the pivotal role of Piezo1 channels in arterial medial calcification.

IntroductionValve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered.Methods and resultUsing in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1α and HIF-2α) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1α, HIF-2α, Glut-1), and calcification in VICs. Down-regulation of HIF-1α and HIF-2α inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics [desferrioxamine, CoCl2, Daprodustat (DPD)] promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted aortic VC in the CKD mice model.DiscussionHIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1α and HIF-2α, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate aortic VC.

Objective: Valve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients on hemodialysis. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered. Approach and Results: Using in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1α and HIF-2α) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1α, HIF-2α, Glut-1), and calcification in VICs. Down-regulation of HIF-1α and HIF-2α inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics (desferrioxamine, CoCl2, Daprodustat (DPD)) promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted VC in the CKD mice model. Conclusion: HIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1α and HIF-2α, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate VC. Translational perspective: One in four hemodialysis-dependent CKD patients on DPD treatment experience a major cardiovascular event during a 2.5-year follow-up period. This work provides a possible explanation for this phenomenon and should initiate further studies to address whether DPD-mediated acceleration of valve calcification triggers the unbeneficial effect of DPD.