fg-4592 and Disease-Models--Animal

Anemia, a common complication of chronic kidney disease (CKD), has previously been attributed primarily to decreased production of erythropoietin. More recently, it has become apparent that the etiology of anemia involves several other factors, most notably dysfunctional iron metabolism, mediated via increased hepcidin activity and reduced clearance. Current management of anemia in patients with advanced CKD is based on erythropoiesis-stimulating agents and iron supplementation, along with red blood cell transfusions when necessary; however, safety considerations associated with these therapies highlight the need to pursue alternative treatment options targeting other mechanisms such as hypoxia-inducible factors (HIFs) that act as central regulators of erythropoiesis by coordinating a series of graded hypoxic responses.. This review discusses the discovery of the HIF pathway and its regulation via HIF prolyl hydroxylase enzymes in the context of erythropoiesis and iron metabolism. The rationale for targeting this pathway and the clinical development of HIF prolyl hydroxylase inhibitors are reviewed, with a commentary on the potential implications of this class of agents in CKD anemia management. Key Messages: Pharmacologic activation of the HIF pathway results in a transient pseudo-hypoxic state that stimulates erythropoiesis in CKD patients with anemia. Results from clinical studies of a number of HIF prolyl hydroxylase inhibitors are increasingly available and provide support for the continued evaluation of the risk-benefit ratio of this novel therapeutic approach to the treatment of anemia in CKD.

Topics: Anemia; Animals; Barbiturates; Blood Pressure; Disease Models, Animal; Drug Design; Drug Interactions; Glycine; Hepcidins; Humans; Hypoxia-Inducible Factor 1; Inflammation; Isoquinolines; Kidney Failure, Chronic; Picolinic Acids; Protein Domains; Pyrazoles; Triazoles

Damage of Intestinal Stem Cells (ISCs) is the main cause of radiation induced-intestinal injury (RIII). Recently, hypoxia Inducible factor (HIF) was verified to be critical for promoting proliferation of ISCs, which suggested a protective role of HIF in the RIII. Thus, we investigated the effect of FG-4592, a novel up-regulator of HIF, on the protection of RIII. With/without FG-4592 treatment, the abdomen of mice was radiated, and intestinal injury was assessed. Especially, by intestinal organoid culture, the multiplication capacity and differentiation features of ISCs were detected. As a result, FG-4592, a novel up-regulator of HIF could remit RIII and promote regeneration and differentiation of ISCs after radiation, which were depended on HIF-2 rather than HIF-1.

Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Disease Models, Animal; Glycine; Hypoxia-Inducible Factor 1; Intestinal Mucosa; Intestines; Isoquinolines; Male; Mice; Mice, Inbred C57BL; Radiation Injuries; Stem Cells; Up-Regulation

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).. 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).. 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.. 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.

Topics: Animals; Cell Hypoxia; Cell Proliferation; Disease Models, Animal; Fatty Liver; Glycine; Hepatectomy; Hypoxia-Inducible Factor 1, alpha Subunit; Isoquinolines; Liver; Liver Regeneration; Male; Prolyl-Hydroxylase Inhibitors; Protein Stability; Proteolysis; Rats, Inbred Lew; Transcriptome

Acute kidney injury (AKI) is a known risk factor for the development of chronic kidney disease (CKD), with no satisfactory strategy to prevent the progression of AKI to CKD. Damage to the renal vascular system and subsequent hypoxia are common contributors to both AKI and CKD. Hypoxia-inducible factor (HIF) is reported to protect the kidney from acute ischemic damage and a novel HIF stabilizer, FG4592 (Roxadustat), has become available in the clinic as an anti-anemia drug. However, the role of FG4592 in the AKI-to-CKD transition remains elusive. In the present study, we investigated the role of FG4592 in the AKI-to-CKD transition induced by unilateral kidney ischemia-reperfusion (UIR). The results showed that FG4592, given to mice 3 days after UIR, markedly alleviated kidney fibrosis and enhanced renal vascular regeneration, possibly via activating the HIF-1α/vascular endothelial growth factor A (VEGFA)/VEGF receptor 1 (VEGFR1) signaling pathway and driving the expression of the endogenous antioxidant superoxide dismutase 2 (SOD2). In accordance with the improved renal vascular regeneration and redox balance, the metabolic disorders of the UIR mice kidneys were also attenuated by treatment with FG4592. However, the inflammatory response in the UIR kidneys was not affected significantly by FG4592. Importantly, in the kidneys of CKD patients, we also observed enhanced HIF-1α expression which was positively correlated with the renal levels of VEGFA and SOD2. Together, these findings demonstrated the therapeutic effect of the anti-anemia drug FG4592 in preventing the AKI-to-CKD transition related to ischemia and the redox imbalance.

Topics: Acute Kidney Injury; Animals; Antioxidants; Disease Models, Animal; Fibrosis; Glycine; Isoquinolines; Kidney; Male; Mice, Inbred C57BL; Pharmaceutical Preparations; Regeneration; Renal Insufficiency, Chronic; Reperfusion Injury; Vascular Endothelial Growth Factor A

Objective: Roxadustat is a new medication for the treatment of renal anemia. EPO (erythropoietin)-the current treatment standard-has been reported to enhance platelet activation and production. However, to date, the effect of roxadustat on platelets is unclear. To address this deficiency, herein, we have evaluated the effect of roxadustat on platelet production and function.\ \ Approach and Results: We performed several mouse platelet functional assays in the presence/absence of in vitro and in vivo roxadustat treatment. Both healthy and 5/6 nephrectomized mice were utilized. The effect of roxadustat on platelet function of healthy volunteers and chronic kidney disease patients was also evaluated. For platelet production, megakaryocyte maturation and proplatelet formation were assayed in vitro. Peripheral platelet and bone marrow megakaryocyte counts were also determined. We found that roxadustat could not stimulate washed platelets directly, and platelet aggregation, spreading, clot retraction, and P-selectin/JON/A exposure were similar with or without in vitro or in vivo roxadustat treatment among both healthy and 5/6 nephrectomized mice. In vivo mouse thrombosis models were additionally performed, and no differences were detected between the vehicle and roxadustat treatment groups. EPO, which was considered a positive control in the present study, promoted platelet function and production as reported previously. Megakaryocyte maturation and proplatelet formation were also not significantly different between control mice and those treated with roxadustat. After receiving roxadustat for 14 days, no difference in the peripheral platelet count was observed in the mice.\ \ Conclusions: Administration of roxadustat has no significant impact on platelet production and function.

Topics: Animals; Blood Coagulation; Blood Platelets; Case-Control Studies; Disease Models, Animal; Erythropoietin; Glycine; Hematinics; Humans; Isoquinolines; Male; Mice, Inbred C57BL; Platelet Activation; Renal Insufficiency, Chronic; Thrombopoiesis; Thrombosis

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Ischemic preconditioning (IPC) is an effective procedure to protect against ischemia/reperfusion (I/R) injury. Hypoxia-inducible factor-1α (Hif-1α) is a key molecule in IPC, and roxadustat (RXD), a first-in-class prolyl hydroxylase domain-containing protein inhibitor, has been recently developed to treat anemia in patients with chronic kidney disease. Thus, we investigated whether RXD pretreatment protects against I/R injury.Methods and Results:RXD pretreatment markedly reduced the infarct size and suppressed plasma creatinine kinase activity in a murine I/R model. Analysis of oxygen metabolism showed that RXD could produce ischemic tolerance by shifting metabolism from aerobic to anaerobic respiration.. RXD pretreatment may be a novel strategy against I/R injury.

Topics: Animals; Cell Respiration; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Glycine; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Isoquinolines; Male; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Signal Transduction

Acute lung injury (ALI) is one of the most severe outcomes of sepsis which still waiting for effective treatment method. Roxadustat (FG-4592) which is often used for treatment of anemia in patients with chronic kidney disease (CKD), its affection on LPS-induced ALI haven't been evaluated. MH-S and MLE-12 cell injury and ALI mouse model was induced LPS. Several assays were used to explore the role of FG-4592 in reducing the damage caused by LPS. FG-4592 treatment significantly upregulated HIF-1α and HO-1 and strikingly attenuated inflammation in vivo and in vitro. Furthermore, septic mice overexpressing HIF-1α had high level of survival rate and lower expression of inflammatory factors while down-regulation can enhance the damage of LPS. HIF-1α has a protective effect on acute lung injury in LPS induced septic mice. FG-4592 treatment remarkably ameliorated the LPS-induced lung injury through the stabilization of HIF-1α. Besides the role in treating CKD anemia, the clinical use of FG-4592 also might be extended to ALI.

Topics: Acute Lung Injury; Animals; Cells, Cultured; Disease Models, Animal; Glycine; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Isoquinolines; Male; Mice; Mice, Inbred BALB C; Sepsis

Diabetic foot ulcers are a major health-care burden worldwide. One primary cause of the delayed wound healing in diabetic patients is impaired function of the hypoxia-inducible factor-1α/vascular endothelial growth factor (HIF-1α/VEGF) axis, which results in compromised neovascularization in response to hypoxia. In the present study, we aimed to investigate the effect of roxadustat, a novel HIF prolyl-4-hydroxylase inhibitor, on angiogenesis and its therapeutic effect on cutaneous wound healing in diabetic rats. In vitro, we found that roxadustat could promote the angiogenic activity of human umbilical vein endothelial cells, accompanied by up-regulation of HIF-1α/VEGF/VEGFR2 signaling. Next, we demonstrated that Ki8751, a VEGFR2-specific inhibitor, could inhibit the increased angiogenic activity of human umbilical vein endothelial cells induced by roxadustat. In vivo, we performed a Matrigel plug assay and demonstrated that roxadustat induced vascularization of the Matrigel plugs, and this effect could be partially inhibited by Ki8751. Finally, we utilized a streptozotocin-induced diabetic rat model and found that roxadustat could accelerate cutaneous wound healing and promote angiogenesis in the wound sites. In conclusion, roxadustat promotes angiogenesis via activation of the HIF-1α/VEGF/VEGFR2 pathway and exhibits therapeutic effects on diabetic wound healing by increasing angiogenesis. Our findings suggest that roxadustat can be a promising strategy to promote diabetic cutaneous wound healing.

Topics: Angiogenesis Inducing Agents; Animals; Blotting, Western; Cells, Cultured; Diabetes Mellitus, Experimental; Disease Models, Animal; Glycine; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Isoquinolines; Neovascularization, Pathologic; Rats; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Wound Healing

Prolyl hydroxylase domain enzyme inhibitors (PHDIs) stabilize hypoxia-inducible factors (HIFs), and are protective in models of acute ischemic and inflammatory kidney disease. Whether PHDIs also confer protection in chronic inflammatory kidney disease models remains unknown. Here we investigated long-term effects of PHDI treatment in adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis. After three weeks, renal dysfunction and tubulointerstitial damage, including proximal and distal tubular injury, tubular dilation and renal crystal deposition were significantly attenuated in PHDI-treated (the isoquinoline derivative ICA and Roxadustat) compared to vehicle-treated mice with adenine-induced nephropathy. Crystal-induced renal fibrosis was only partially diminished by treatment with ICA. Renoprotective effects of ICA treatment could not be attributed to changes in adenine metabolism or urinary excretion of the metabolite 2,8-dihydroxyadenine. ICA treatment reduced inflammatory infiltrates of F4/80+ mononuclear phagocytes in the kidneys and supported a regulatory, anti-inflammatory immune response. Furthermore, interstitial deposition of complement C1q was decreased in ICA-treated mice fed an adenine-enriched diet. Tubular cell-specific HIF-1α and myeloid cell-specific HIF-1α and HIF-2α expression were not required for the renoprotective effects of ICA. In contrast, depletion of mononuclear phagocytes with clodronate largely abolished the nephroprotective effects of PHD inhibition. Thus, our findings indicate novel and potent systemic anti-inflammatory properties of PHDIs that confer preservation of kidney function and structure in chronic tubulointerstitial inflammation and might counteract kidney disease progression.

Topics: Adenine; Animals; Anti-Inflammatory Agents; Basic Helix-Loop-Helix Transcription Factors; Clodronic Acid; Complement C1q; Disease Models, Animal; Glycine; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Isoquinolines; Kidney Tubules; Male; Mice; Mice, Transgenic; Nephritis, Interstitial; Phagocytes; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Protective Agents; Renal Insufficiency, Chronic

We determined which metabolic pathways are activated by hypoxia-inducible factor 1-mediated (HIF-1-mediated) protection against oxygen-induced retinopathy (OIR) in newborn mice, the experimental correlate to retinopathy of prematurity, a leading cause of infant blindness. HIF-1 coordinates the change from oxidative to glycolytic metabolism and mediates flux through serine and 1-carbon metabolism (1CM) in hypoxic and cancer cells. We used untargeted metabolite profiling in vivo to demonstrate that hypoxia mimesis activates serine/1CM. Both [13C6] glucose labeling of metabolites in ex vivo retinal explants as well as in vivo [13C3] serine labeling of metabolites followed in liver lysates strongly suggest that retinal serine is primarily derived from hepatic glycolytic carbon and not from retinal glycolytic carbon in newborn pups. In HIF-1α2lox/2lox albumin-Cre-knockout mice, reduced or near-0 levels of serine/glycine further demonstrate the hepatic origin of retinal serine. Furthermore, inhibition of 1CM by methotrexate blocked HIF-mediated protection against OIR. This demonstrated that 1CM participates in protection induced by HIF-1 stabilization. The urea cycle also dominated pathway enrichment analyses of plasma samples. The dependence of retinal serine on hepatic HIF-1 and the upregulation of the urea cycle emphasize the importance of the liver to remote protection of the retina.

Topics: Animals; Carbon; Disease Models, Animal; Glycine; Glycolysis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Isoquinolines; Liver; Metabolic Networks and Pathways; Methotrexate; Mice; Mice, Knockout; Oxygen; Protein Stability; Retina; Retinopathy of Prematurity; Serine; Tissue Culture Techniques; Up-Regulation

Defects in the mitochondrial respiratory chain (RC) underlie a spectrum of human conditions, ranging from devastating inborn errors of metabolism to aging. We performed a genome-wide Cas9-mediated screen to identify factors that are protective during RC inhibition. Our results highlight the hypoxia response, an endogenous program evolved to adapt to limited oxygen availability. Genetic or small-molecule activation of the hypoxia response is protective against mitochondrial toxicity in cultured cells and zebrafish models. Chronic hypoxia leads to a marked improvement in survival, body weight, body temperature, behavior, neuropathology, and disease biomarkers in a genetic mouse model of Leigh syndrome, the most common pediatric manifestation of mitochondrial disease. Further preclinical studies are required to assess whether hypoxic exposure can be developed into a safe and effective treatment for human diseases associated with mitochondrial dysfunction.

Topics: Anaerobiosis; Animals; Antimycin A; Bacterial Proteins; Biomarkers; Body Temperature; Body Weight; CRISPR-Associated Protein 9; Disease Models, Animal; Electron Transport; Electron Transport Complex I; Endonucleases; Energy Metabolism; Gene Knockout Techniques; Genome-Wide Association Study; Glycine; Humans; Hypoxia-Inducible Factor 1; Isoquinolines; K562 Cells; Leigh Disease; Mice; Mice, Knockout; Mitochondria; Oxygen; Respiration; Suppression, Genetic; Von Hippel-Lindau Tumor Suppressor Protein; Zebrafish

Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions.

Topics: Animals; Biomarkers; Cell Transdifferentiation; Cells, Cultured; Disease Models, Animal; Glycine; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Isoquinolines; Male; Muscle, Smooth, Vascular; Phosphates; Rats; Rats, Wistar; Renal Insufficiency, Chronic; Signal Transduction; Vascular Calcification; Vascular Stiffness