The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-α.

Prolyl hydroxylase domain protein 2 (PHD2)-catalyzed modification of hypoxia-inducible factor (HIF)-α is a key event in oxygen sensing. We previously showed that the zinc finger of PHD2 binds to a Pro-Xaa-Leu-Glu (PXLE) motif. Here, we show that the zinc finger binds to this motif in the ribosomal chaperone nascent polypeptide complex-α (NACA). This recruits PHD2 to the translation machinery to cotranslationally modify HIF-α. Importantly, this cotranslational modification is enhanced by a translational pause sequence in HIF-α. Mice with a knock-in Naca gene mutation that abolishes the PXLE motif display erythrocytosis, a reflection of HIF pathway dysregulation. In addition, human erythrocytosis-associated mutations in the zinc finger of PHD2 ablate interaction with NACA. Tibetans, who have adapted to the hypoxia of high altitude, harbor a PHD2 variant that we previously showed displays a defect in zinc finger binding to p23, a PXLE-containing HSP90 cochaperone. We show here that Tibetan PHD2 maintains interaction with NACA, thereby showing differential interactions with PXLE-containing proteins and providing an explanation for why Tibetans are not predisposed to erythrocytosis.

Dimethyloxalylglycine Suppresses SREBP1c and Lipogenic Gene Expressions in Hepatocytes Independently of HIF1A.

Dimethyloxalylglycine (DMOG) is a representative inhibitor of the prolyl hydroxylase domain (PHD), which mediates the degradation of hypoxia-inducible factor-1-alpha (HIF1A). DMOG exerts its pharmacological effects via the canonical pathway that involves PHD inhibition; however, it remains unclear whether DMOG affects lipogenic gene expression in hepatocytes. We aimed to elucidate the effects of DMOG on sterol regulatory element-binding protein-1c (SREBP1c), a master regulator of fatty acid synthesis in hepatocytes. DMOG treatment inhibited SREBP1c mRNA and protein expression in HepG2 and AML12 hepatocytes and reduced the transcript levels of SREBP1c-regulated lipogenic genes. A luciferase reporter assay revealed that DMOG inhibited the transcriptional activity of SREBP1c. Moreover, DMOG suppressed SREBP1c expression in mice liver. Mechanistically, treatment with DMOG enhanced the expression of HIF1A and insulin-induced gene 2 (INSIG2), which inhibits the activation of SREBP1c. However, HIF1A or INSIG2 knockdown failed to reverse the inhibitory effect of DMOG on SREBP1c expression, suggesting a redundant role of HIF1A and INSIG2 in terms of repressing SREBP1c. DMOG did not function through the canonical pathway involving inhibition of SREBP1c by PHD, highlighting the presence of non-canonical pathways that mediate its anti-lipogenic effect.

Role of TRPA1 in Painful Cold Hypersensitivity.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that plays a pivotal role in pain generation after exposure to irritant chemicals and is involved in the sensation of a wide variety of pathological pain. TRPA1 was first reported to be sensitive to noxious cold, but its intrinsic cold sensitivity still remains under debate. To address this issue, we focused on cold hypersensitivity induced by oxaliplatin, a platinum-based chemotherapeutic drug, as a peculiar adverse symptom of acute peripheral neuropathy. We and other groups have shown that oxaliplatin enhances TRPA1 sensitivity to its chemical agonists and reactive oxygen species (ROS). Our in vitro and animal model studies revealed that oxaliplatin, or its metabolite oxalate, inhibits hydroxylation of a proline residue within the N-terminus of human TRPA1 (hTRPA1) via inhibition of prolyl hydroxylase domain-containing protein (PHD), which induces TRPA1 sensitization to ROS. Although hTRPA1 is insensitive to cold, PHD inhibition endows hTRPA1 with cold sensitivity through sensing the small amount of ROS produced after exposure to cold. Hence, we propose that PHD inhibition can unveil the cold sensitivity of hTRPA1 by converting ROS signaling into cold sensitivity. Furthermore, in this review, we summarize the role of TRPA1 in painful cold hypersensitivity during peripheral vascular impairment.

Structural basis for binding of the renal carcinoma target hypoxia-inducible factor 2α to prolyl hydroxylase domain 2.

The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.

Inhibition of hypoxia-inducible factor-prolyl hydroxylation protects from cyclophosphamide-induced bladder injury and urinary dysfunction.

Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Activation of the oxygen-regulated hypoxia-inducible factor (HIF) pathway has been shown to protect mucosal membranes by increasing the expression of cytoprotective genes and by suppressing inflammation. The activity of HIF is controlled by prolyl hydroxylase domain (PHD) dioxygenases, which have been exploited as therapeutic targets for the treatment of anemia of chronic kidney disease. Here, we established a mouse model of acute cyclophosphamide (CYP)-induced blood-urine barrier disruption associated with inflammation and severe urinary dysfunction to investigate the HIF-PHD axis in inflammatory bladder injury. We found that systemic administration of dimethyloxalylglycine or molidustat, two small-molecule inhibitors of HIF-prolyl hydroxylases, profoundly mitigated CYP-induced bladder injury and inflammation as assessed by morphological analysis of transmural edema and urothelial integrity and by measuring tissue cytokine expression. Void spot analysis to examine bladder function quantitatively demonstrated that HIF-prolyl hydroxylase inhibitor administration normalized micturition patterns and protected against CYP-induced alteration of urinary frequency and micturition patterns. Our study highlights the therapeutic potential of HIF-activating small-molecule compounds for the prevention or therapy of bladder injury and urinary dysfunction due to blood-urine barrier disruption.NEW & NOTEWORTHY Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Here, we demonstrate that pharmacological inhibition of hypoxia-inducible factor (HIF)-prolyl hydroxylation prevented bladder injury and protected from urinary dysfunction in a mouse model of cyclophosphamide-induced disruption of the blood-urine barrier. Our study highlights a potential role for HIF-activating small-molecule compounds in the prevention or therapy of bladder injury and urinary dysfunction and provides a rationale for future clinical studies.

Treatment with the Prolyl Hydroxylase Inhibitor JNJ Promotes Abdominal Aortic Aneurysm Progression in Diabetic Mice.

OBJECTIVE: Prolyl hydroxylase domain containing proteins (PHD) rigorously regulate intracellular hypoxia inducible factor-1 (HIF-1) protein expression and activity. Diabetes impairs PHD activity and attenuates abdominal aortic aneurysm (AAA) progression. The extent to which dysregulated PHD activity contributes to diabetes mediated AAA suppression remains undetermined. METHODS: AAAs were induced in diabetic and non-diabetic male C57BL/6J mice via intra-aortic elastase infusion. A PHD inhibitor (JNJ-42041935, aka "JNJ", 150 mmol/kg) or vehicle alone was administered daily starting one day prior to AAA induction for 14 days. Influences on AAA progression was assessed via ultrasonography and histopathology. Expression of aortic HIF-1α, three of its target genes and macrophage derived mediators were assayed via quantitative reverse transcription polymerase chain reaction. Aneurysmal sections from AAA patients with and without diabetes (two patients in each group) were immunostained for HIF-1α and vascular endothelial growth factor (VEGF)-A. RESULTS: Expression of HIF-1α target genes (erythropoietin, VEGF-A, and glucose transporter-1) was reduced by 45% - 95% in experimental diabetic aortas. Diameter enlargement was similarly limited, as were mural elastin degradation, leukocyte infiltration, and neo-angiogenesis (reduced capillary density and length) on histopathology. Pre-treatment with JNJ prior to AAA initiation augmented aortic HIF-1α target gene expression and aneurysm progression in diabetic mice, along with macrophage VEGF-A and matrix metalloproteinase 2 mRNA expression. No differences were noted in HIF-1α or VEGF-A expression on aortic immunohistochemical staining of human aortic tissue as a function of diabetes status. CONCLUSION: Small molecule PHD inhibitor treatment reduces or offsets impairment of experimental AAA progression in hyperglycemic mice, highlighting the potential contribution of dysregulated PHD activity to diabetes mediated aneurysm suppression.

Threshold of Serum Ferritin to Discriminate against Those at Greater Risk of Platelet Increase during Treatment with Hypoxia-Inducible Factor Prolyl Hydroxylase Domain Inhibitor.

INTRODUCTION: Hypoxia-inducible factor prolyl hydroxylase domain inhibitors (HIF-PHI) are a new treatment for renal anemia. HIF-PHI is believed to increase iron usage to improve availability of iron for erythropoiesis. Therefore, there is concern that HIF-PHI might be prone to iron deficiency and that thrombosis might be induced by increased platelet and transferrin levels due to this iron deficiency. METHODS: Relationship of iron-related factors with platelet count (PLT) and total iron-binding capacity (TIBC; which reflects the transferrin level) were examined in 29 patients who were treated with darbepoetin alfa (DA) and then switched to roxadustat (Rox). To determine how changes in PLT and TIBC related to changes in iron-related factors, univariable and multivariable linear regression models were applied. To examine what iron-related factors on Day 0 influenced change in PLT, we used receiver operating characteristic (ROC) curves and logistic regression analysis for a rate of change in PLT ≤0% as the endpoint. Logistic regression analysis was performed with the reference group having serum ferritin (s-ft) or Transferrin saturation below the corresponding cutoff value (low vs. high). RESULTS: Multivariable analysis showed significant positive correlations between the rate of change in PLT and the change in s-ft and red blood cells (RBC) count {ß-coefficients; 0.40 [95% confidence interval (CI): 0.17-0.62], p = 0.001} (ß-coefficients; 30.45 [95% CI: 10.90-50.00], p = 0.004). The rate of change in TIBC was significantly positively correlated with only the change in RBC count. The ROC showed a significant cutoff value for s-ft of 77.2 ng/mL (sensitivity 63.6%, specificity 83.3%, area under the curve 0.76, 95% CI 0.55-0.96). Multivariable logistic regression also showed that only high s-ft was significantly elevated (9.46, 95% CI 1.42-63.30, p = 0.020). CONCLUSIONS: This study showed that changes in PLT were correlated with s-ft and amount of hematopoiesis. This suggests that an increase in PLT due to iron levels is less likely when s-ft is 77.2 ng/mL or higher at the time of switching from DA to Rox. In contrast, TIBC was only related to hematopoiesis in these patients. Control of s-ft before initiation of HIF-PHI treatment and gradual hematopoiesis might reduce the risk of thrombosis when switching from erythropoiesis-stimulating agents to HIF-PHI.

Selective HIF stabilization alleviates hepatocellular steatosis and ballooning in a rodent model of 70% liver resection.

BACKGROUND: Small-for-size syndrome (SFSS) looms over patients needing liver resection or living-donor transplantation. Hypoxia has been shown to be crucial for the successful outcome of liver resection in the very early postoperative phase. While poorly acceptable as such in real-world clinical practice, hypoxia responses can still be simulated by pharmacologically raising levels of its transducers, the hypoxia-inducible factors (HIFs). We aimed to assess the potential role of a selective inhibitor of HIF degradation in 70% hepatectomy (70%Hx). METHODS: In a pilot study, we tested the required dose of roxadustat to stabilize liver HIF1α. We then performed 70%Hx in 8-week-old male Lewis rats and administered 25 mg/kg of roxadustat (RXD25) at the end of the procedure. Regeneration was assessed: ki67 and 5-ethynyl-2'-deoxyuridine (EdU) immunofluorescent labeling, and histological parameters. We also assessed liver function via a blood panel and functional gadoxetate-enhanced magnetic resonance imaging (MRI), up to 47 h after the procedure. Metabolic results were analyzed by means of RNA sequencing (RNAseq). RESULTS: Roxadustat effectively increased early HIF1α transactivity. Liver function did not appear to be improved nor liver regeneration to be accelerated by the experimental compound. However, treated livers showed a mitigation in hepatocellular steatosis and ballooning, known markers of cellular stress after liver resection. RNAseq confirmed that roxadustat unexpectedly increases lipid breakdown and cellular respiration. CONCLUSIONS: Selective HIF stabilization did not result in an enhanced liver function after standard liver resection, but it induced interesting metabolic changes that are worth studying for their possible role in extended liver resections and fatty liver diseases.

Prolyl Hydroxylase Domain Inhibitor Protects against Metabolic Disorders and Associated Kidney Disease in Obese Type 2 Diabetic Mice.

BACKGROUND: Prolyl hydroxylase domain (PHD) inhibitors, which stimulate erythropoietin production through the activation of hypoxia-inducible factor (HIF), are novel therapeutic agents used for treating renal anemia. Several PHD inhibitors, including enarodustat, are currently undergoing phase 2 or phase 3 clinical trials. Because HIF regulates a broad spectrum of genes, PHD inhibitors are expected to have other effects in addition to erythropoiesis, such as protection against metabolic disorders. However, whether such beneficial effects would extend to metabolic disorder-related kidney disease is largely unknown. METHODS: We administered enarodustat or vehicle without enarodustat in feed to diabetic black and tan brachyury (BTBR) ob/ob mice from 4 to 22 weeks of age. To elucidate molecular changes induced by enarodustat, we performed transcriptome analysis of isolated glomeruli and in vitro experiments using murine mesangial cells. RESULTS: Compared with BTBR ob/ob mice that received only vehicle, BTBR ob/ob mice treated with enarodustat displayed lower body weight, reduced blood glucose levels with improved insulin sensitivity, lower total cholesterol levels, higher adiponectin levels, and less adipose tissue, as well as a tendency for lower macrophage infiltration. Enarodustat-treated mice also exhibited reduced albuminuria and amelioration of glomerular epithelial and endothelial damage. Transcriptome analysis of isolated glomeruli revealed reduced expression of C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) in enarodustat-treated mice compared with the vehicle-only group, accompanied by reduced glomerular macrophage infiltration. In vitro experiments demonstrated that both local HIF-1 activation and restoration of adiponectin by enarodustat contributed to CCL2/MCP-1 reduction in mesangial cells. CONCLUSIONS: These results indicate that the PHD inhibitor enarodustat has potential renoprotective effects in addition to its potential to protect against metabolic disorders.

Effects of orally active hypoxia inducible factor alpha prolyl hydroxylase inhibitor, FG4592 on renal fibrogenic potential in mouse unilateral ureteral obstruction model.

Orally active hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors that stabilize HIF protein and stimulate the production of erythropoietin have been approved to treat renal anemia. Our previous report suggested that HIF-1α dependent fibrogenic mechanisms are operating at the early onset of renal fibrosis and its contribution declines with the progression in mouse unilateral ureteral obstruction (UUO) model. The aim of the study is to evaluate the renal fibrogenic potential of FG4592, a recently approved orally active HIF prolyl hydroxylase inhibitor in mouse UUO model. Male C57BL/6J mice orally given FG-4592 (12.5 mg/kg/day and 50 mg/kg/day) were subjected to UUO. Neither dose of FG-4592 affected renal fibrosis or macrophage infiltration. FG-4592 had no effects on increased mRNA of collagen I, collagen III or transforming growth factor-ß1. At 3 days after UUO, higher dose of FG-4592 potentiated the increased mRNA expression of profibrogenic molecules, plasminogen activator inhibitor 1 (Pai-1) and connective tissue growth factor (Ctgf) but such potentiation disappeared at 7 days after UUO. It is suggested that FG-4592 used in the present study had little effects on renal fibrosis even though high dose of FG-4592 used in the present study transiently potentiated gene expression of Pai-1 and Ctgf in the UUO kidney.

Endothelial HIF-2α contributes to severe pulmonary hypertension due to endothelial-to-mesenchymal transition.

Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1EC-/-), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1EC-/- mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.

Loss of smooth muscle cell hypoxia inducible factor-1α underlies increased vascular contractility in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an often fatal disease with limited treatment options. Whereas current data support the notion that, in pulmonary artery endothelial cells (PAECs), expression of transcription factor hypoxia inducible factor-1α (HIF-1α) is increased, the role of HIF-1α in pulmonary artery smooth muscle cells (PASMCs) remains controversial. This study investigates the hypothesis that, in PASMCs from patients with PAH, decreases in HIF-1α expression and activity underlie augmented pulmonary vascular contractility. PASMCs and tissues were isolated from nonhypertensive control patients and patients with PAH. Compared with controls, HIF-1α and Kv1.5 protein expression were decreased in PAH smooth muscle cells (primary culture). Myosin light chain (MLC) phosphorylation and MLC kinase (MLCK) activity-major determinants of vascular tone-were increased in patients with PAH. Cofactors involved in prolyl hydroxylase domain activity were increased in PAH smooth muscle cells. Functionally, PASMC contractility was inversely correlated with HIF-1α activity. In PASMCs derived from patients with PAH, HIF-1α expression is decreased, and MLCK activity, MLC phosphorylation, and cell contraction are increased. We conclude that compromised PASMC HIF-1α expression may contribute to the increased tone that characterizes pulmonary hypertension.-Barnes, E. A., Chen, C.-H., Sedan, O., Cornfield, D. N. Loss of smooth muscle cell hypoxia inducible factor-1α underlies increased vascular contractility in pulmonary hypertension.

Discovery of novel 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid derivatives as HIF prolyl hydroxylase inhibitors for treatment of renal anemia.

Prolyl hydroxylase domain-containing protein (PHD) inhibitors are useful as orally administered agents for the treatment of renal anemia. Based on the common structures of known PHD inhibitors, we found novel PHD inhibitor 1 with a 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid motif. The PHD2-inhibitory activity, lipophilicity (CLogP), and PK profiles (hepatocyte metabolism, protein binding, and/or elimination half-life) of this inhibitor were used as the evaluation index to optimize the structure and eventually discovered clinical candidate 42 as the suitable compound. Compound 42 was demonstrated to promote the production of erythropoietin (EPO) following oral administration in mice and rats. The predicted half-life of this compound in humans was 1.3-5.6 h, therefore, this drug may be expected to administer once daily with few adverse effects caused by excessive EPO production.

Therapeutic targeting of the HIF oxygen-sensing pathway: Lessons learned from clinical studies.

The oxygen-sensitive hypoxia-inducible factor (HIF) pathway plays a central role in the control of erythropoiesis and iron metabolism. The discovery of prolyl hydroxylase domain (PHD) proteins as key regulators of HIF activity has led to the development of inhibitory compounds that are now in phase 3 clinical development for the treatment of renal anemia, a condition that is commonly found in patients with advanced chronic kidney disease. This review provides a concise overview of clinical effects associated with pharmacologic PHD inhibition and was written in memory of Professor Lorenz Poellinger.

Regulation of ATP13A2 via PHD2-HIF1α Signaling Is Critical for Cellular Iron Homeostasis: Implications for Parkinson's Disease.

We previously reported that pharmacological inhibition of a class of enzymes known as prolyl hydroxylase domain proteins (PHDs) has neuroprotective effects in various in vitro and in vivo models of Parkinson's disease (PD). We hypothesized that this was due to inhibition of the PHD2 isoform, preventing it from hydroxylating the transcription factor hypoxia inducible factor 1 α (HIF1α), targeting it for eventual proteasomal degradation. HIF1α itself induces the transcription of various cellular stress genes, including several involved in iron metabolism. Although all three isoforms of PHD are expressed within vulnerable dopaminergic (DAergic) substantia nigra pars compacta neurons, only select downregulation of the PHD2 isoform was found to protect against in vivo neurodegenerative effects associated with the mitochondrial neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. These findings were corroborated in induced pluripotent stem cell-derived neurons, providing validation in a pertinent human cell model. PHD2 inhibition was found to result in increased expression of ATP13A2, mutation of which is responsible for a rare juvenile form of PD known as Kufor-Rakeb syndrome. Knockdown of ATP13A2 expression within human DAergic cells was found to abrogate restoration of cellular iron homeostasis and neuronal cell viability elicited by inhibition of PHD2 under conditions of mitochondrial stress, likely via effects on lysosomal iron storage. These data suggest that regulation of ATP13A2 by the PHD2-HIF1α signaling pathway affects cellular iron homeostasis and DAergic neuronal survival. This constitutes a heretofore unrecognized process associated with loss of ATP13A2 function that could have wide-ranging implications for it as a therapeutic target for PD and other related conditions. SIGNIFICANCE STATEMENT: Reductions in PHD2 activity within dopaminergic neurons in vivo and in cultured human induced pluripotent stem cell-derived neurons protects against mitochondrial stress-induced neurotoxicity. Protective effects are dependent on downstream HIF-1α expression. Knockdown of ATP13A2, a gene linked to a rare juvenile form of Parkinson's disease and recently identified as a novel HIF1α target, was found to abrogate maintenance of cellular iron homeostasis and neuronal viability elicited by PHD2 inhibition in vivo and in cultured dopaminergic cells under conditions of mitochondrial stress. Mechanistically, this was due to ATP13A2's role in maintaining lysosomal iron stores. This constitutes a novel mechanism by which alterations in ATP13A2 activity may be driving PD-related neuropathology.

Prolyl hydroxylase domain inhibitors as a novel therapeutic approach against anemia in chronic kidney disease.

Anemia is a common complication of chronic kidney disease and is mainly caused by the inability of injured kidneys to produce adequate amounts of erythropoietin. Studies elucidating the regulation of erythropoietin production led to the identification of hypoxia-inducible factor (HIF), which activates the transcription of genes that mediate adaptive responses to hypoxia. HIF is a heterodimer that consists of an α and ß subunit. While HIF-ß is constitutively expressed, HIF-α is subjected to ubiquitination and proteasomal degradation under normoxic conditions. This process is mediated by prolyl hydroxylase domain proteins, the inhibition of which results in an increased expression of hypoxia-induced genes, including erythropoietin. These findings led to the development of prolyl hydroxylase domain inhibitors as novel therapeutic agents against anemia in chronic kidney disease. Prolyl hydroxylase domain inhibition improves iron metabolism, which also contributes to erythropoiesis. To date, at least 6 small-molecule inhibitors of the prolyl hydroxylase domain have been tested in humans, and clinical trials have shown that they are effective without causing serious adverse events. However, there is a theoretical concern that the systemic activation of HIF could also induce deleterious effects such as tumorigenesis and severe pulmonary hypertension, which demands careful assessments in future clinical studies.

miR-21 contributes to renal protection by targeting prolyl hydroxylase domain protein 2 in delayed ischaemic preconditioning.

AIM: Upregulation of miR-21 in renal ischaemic preconditioning (IPC) was associated with increased hypoxia inducible factor (HIF)-1α expression. Hypoxic induction of HIF-1α is mediated by inhibition of prolyl hydroxylase domain protein 2 (PHD2) .We hypothesized that miR-21 regulated HIF-1α by targeting PHD2 in the renal IPC. METHODS: Luciferase reporter assay examined if miR-21 target the 3'-untranslated region of PHD2. In vitro, human proximal tubular cell line (HK-2) was incubated in hypoxia or hypoxia/ reoxygenation condition. Kidneys of Mice were respectively subjected to ischaemia/reperfusion injury (IRI) and IPC. Locked nucleic acid (LNA) modified anti-miR-21 was used to knockdown miR-21. Serum creatinine and histological changes estimated the renal injury. Levels of HIF-1α, PHD2, VEGF and miR-21 were examined by western blot or real-time PCR. RESULT: miR-21 targeting of PHD2 was confirmed by 3'-untranslated region reporter assay. miR-21 was significantly upregulated by hypoxia/reoxygenation in HK-2 cell, while PHD2 protein level decreased significantly. LNA anti-miR-21 significantly repressed miR-21 levels and increased the abundance of PHD2. In vivo, IPC upregulated miR-21 expression 24 h after the second ischaemia, while PHD2 expression decreased significantly with upregulation of HIF-1α protein and VEGF mRNA. MiR-21 induced by delayed IPC was effectively inhibited by the LNA anti-miR-21. With downregulation of miR-21, the protection of delayed IPC was attenuated and PHD2 protein was increased. Furthermore, upregulation of HIF-1α and VEGF were abolished after the LNA anti-miR-21 treatment. CONCLUSION: miR-21 could protect kidney against IRI via HIF-1α by inhibiting its target PHD2.The study suggested a new relationship between miR-21 and HIF-1α.

A mechanistic link between renal ischemia and fibrosis.

Renal fibrosis is characterized by tubular cell atrophy and accumulation of extracellular matrix. Fibroblast activation becomes evident in areas surrounding atrophic tubules, with rarefaction of peritubular capillaries. Tubulointerstitial hypoxia is the final common pathway in progressive kidney disease. Hypoxia suppresses tubular epithelial growth and leads to failure of remodeling by facilitating dedifferentiation and apoptosis. Profibrotic factors such as transforming growth factor-ß (TGF-ß) mediate fibroblast activation, and recruited leukocytes, which appear in hypoxic areas, contribute to fibrosis. While resident renal cells adapt to the hypoxic environment via upregulation of relevant genes by hypoxia-inducible factor (HIF) family members, hypoxic adaptation via HIF may not be sufficient in chronic kidney disease (CKD) due to multiple factors. Thus, restoration of HIF-mediated responses may contribute to amelioration of CKD pathology. Studies to date have reported that HIF activation reduces inflammation and oxidative stress and ameliorates injury by decreasing tubular cell apoptosis and restoring peritubular capillary network. Prolyl hydroxylase domain (PHD) inhibitors that specifically activate HIF are currently evaluated for the treatment of renal anemia and may be effective for the treatment of CKD.

Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development.

In the adult, new vessels and red blood cells form in response to hypoxia. Here, the oxygen-sensing system (PHD-HIF) has recently been put into focus, since the prolyl-hydroxylase domain proteins (PHD) and hypoxia-inducible factors (HIF) are considered as potential therapeutic targets to treat ischemia, cancers or age-related macula degeneration. While the oxygen-sensing system (PHD-HIF) has been studied intensively in this respect, only little is known from developing vertebrate embryos since mutations within this pathway led to an early decease of embryos due to placental defects. During vertebrate embryogenesis, a progenitor cell called hemangioblast is assumed to give rise to blood cells and blood vessels in a process called hematopoiesis and vasculogenesis, respectively. Xenopus provides an ideal experimental system to address these processes in vivo, as its development does not depend on a functional placenta and thus allows analyzing the role of oxygen directly. To this end, we adopted a computer-controlled four-channel system, which allowed us to culture Xenopus embryos under defined oxygen concentrations. Our data show that the development of vascular structures and blood cells is strongly impaired under hypoxia, while general development is less compromised. Interestingly, suppression of Phd2 function using specific antisense morpholinos or a chemical inhibitor resulted in mostly overlapping vascular defects; nevertheless, blood cell was formed almost normally. Our results provide the first evidence that oxygen via Phd2 has a decisive influence on the formation of the vascular network during vertebrate embryogenesis. These findings may be considered in certain potential treatment concepts.

Disturbed hypoxic responses as a pathogenic mechanism of diabetic foot ulcers.

Diabetic foot ulceration (DFU) is a chronic complication of diabetes that is characterized by impaired wound healing in the lower extremities. DFU remains a major clinical challenge because of poor understanding of its pathogenic mechanisms. Impaired wound healing in diabetes is characterized by decreased angiogenesis, reduced bone marrow-derived endothelial progenitor cell (EPC) recruitment, and decreased fibroblast and keratinocyte proliferation and migration. Recently, increasing evidence has suggested that increased hypoxic conditions and impaired cellular responses to hypoxia are essential pathogenic factors of delayed wound healing in DFU. Hypoxia-inducible factor-1 (HIF-1, a heterodimer of HIF-1α and HIF-1ß) is a master regulator of oxygen homeostasis that mediates the adaptive cellular responses to hypoxia by regulating the expression of genes involved in angiogenesis, metabolic changes, proliferation, migration, and cell survival. However, HIF-1 signalling is inhibited in diabetes as a result of hyperglycaemia-induced HIF-1α destabilization and functional repression. Increasing HIF-1α expression and activity using various approaches promotes angiogenesis, EPC recruitment, and granulation, thereby improving wound healing in experimental diabetes. The mechanisms underlying HIF-1α regulation in diabetes and the therapeutic strategies targeting HIF-1 signalling for the treatment of diabetic wounds are discussed in this review. Further investigations of the pathways involved in HIF-1α regulation in diabetes are required to advance our understanding of the mechanisms underlying impaired wound healing in diabetes and to provide a foundation for developing novel therapeutic approaches to treat DFU.