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.

Cataract, an opacification in the crystalline lens, is a leading cause of blindness. Deposition of hydroxyapatite occurs in a cataractous lens that could be the consequence of osteogenic differentiation of lens epithelial cells (LECs). Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the transcription of a wide range of cytoprotective genes. Nrf2 upregulation attenuates cataract formation. Here we aimed to investigate the effect of Nrf2 system upregulation in LECs calcification. We induced osteogenic differentiation of human LECs (HuLECs) with increased phosphate and calcium-containing osteogenic medium (OM). OM-induced calcium and osteocalcin deposition in HuLECs. We used heme to activate Nrf2, which strongly upregulated the expression of Nrf2 and heme oxygenase-1 (HO-1). Heme-mediated Nrf2 activation was dependent on the production of reactive oxygens species. Heme inhibited Ca deposition, and the OM-induced increase of osteogenic markers, RUNX2, alkaline phosphatase, and OCN. Anti-calcification effect of heme was lost when the transcriptional activity of Nrf2 or the enzyme activity of HO-1 was blocked with pharmacological inhibitors. Among products of HO-1 catalyzed heme degradation iron mimicked the anti-calcification effect of heme. We concluded that heme-induced upregulation of the Nrf2/HO-1 system inhibits HuLECs calcification through the liberation of heme iron.

Background: Information regarding the prevalence of PKU in the Middle East in comparison to other world regions is scarce, which might be explained by difficulties in the implementation of national newborn screening programs.Objective: This study seeks for the first time to genotype and biochemically characterize patients diagnosed with hyperphenylalaninemia (HPA) at the Pediatric Metabolic Genetics Clinic at the King Hussein Medical Center, Amman, Jordan.Methods: A total of 33 patients with HPA and 55 family members were investigated for pterins (neopterin and biopterin) and dihydropteridine reductase (DHPR) activity in dried blood spots. Patients with HPA were genotyped for phenylketonuria (PKU) and the genes involved in tetrahydrobiopterin (BH₄) metabolism.Results: In total 20 patients were diagnosed with PKU due to phenylalanine hydroxylase (PAH) deficiency, 2 with GTP cyclohydrolase I (GTPCH) deficiency, 6 with DHPR deficiency, and 3 with the 6‐pyruvoyl‐tetrahydropterin synthase (PTPS) deficiency. Diagnosis was not possible in 2 patients. This study documents a high percentage of BH₄ deficiencies within HPA patients. With one exception, all patients were homozygous for particular gene variants.Conclusions: This approach enables differentiation between PKU and BH₄ deficiencies and, thus, allows for critical selection of a specific treatment strategies.