Activation of the hypoxia-inducible factor pathway by roxadustat improves glucose metabolism in human primary myotubes from men.

AIMS/HYPOTHESIS: Hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes. METHODS: Primary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser. RESULTS: Exposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg-1 min-1) and type 2 diabetes (25.3 ± 4.4 pmol mg-1 min-1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g-1 h-1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g-1 h-1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345). CONCLUSIONS/INTERPRETATION: Roxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.

Investigating the therapeutic effects and mechanisms of Roxadustat on peritoneal fibrosis Based on the TGF-ß/Smad pathway.

Peritoneal fibrosis (PF) is particularly common in individuals undergoing peritoneal dialysis (PD). Fibrosis of the parenchymal tissue typically progresses slowly. Therefore, preventing and reducing the advancement of fibrosis is crucial for effective patient treatment. Roxadustat is a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), primarily used to treat and improve renal anemia. Recent studies have found that HIF-1α possesses antioxidant activity and exerts a certain protective effect in ischemic heart disease and spinal cord injury, while it can also delay the progression of pulmonary and renal fibrosis. This study establishes the mice model through intraperitoneal injection of 4.25 % peritoneal dialysate fluid (PDF) and explores the therapeutic effects of Roxadustat by inducing TGF-ß1-mediated epithelial-mesenchymal transition (EMT) in Met-5A cells. The aim is to investigate the protective role and mechanisms of Roxadustat against PD-related PF. We observed thicker peritoneal tissue and reduced permeability in animals with PD-related PF samples. This was accompanied by heightened inflammation, which Roxadustat alleviated by lowering the levels of inflammatory cytokines (IL-6, TNF-α). Furthermore, Roxadustat inhibited EMT in PF mice and TGF-ß1-induced Met-5A cells, as evidenced by decreased expression of fibrotic markers, such as fibronectin, collagen I, and α-SMA, alongside an elevation in the expression of the epithelial marker, E-cadherin. Roxadustat also significantly decreased the expression of TGF-ß1 and the phosphorylation of p-Smad2 and p-Smad3. In conclusion, Roxadustat ameliorates peritoneal fibrosis through the TGF-ß/Smad pathway.

Roxadustat reduces left ventricular mass index compared to rHuEPO in haemodialysis patients in a randomized controlled trial.

BACKGROUND: Left ventricular hypertrophy (LVH) is highly prevalent in haemodialysis (HD) patients and is associated with an increased risk of death. Roxadustat and recombinant human erythropoietin (rHuEPO, abbreviated as EPO) are the main treatment strategies for renal anaemia in HD patients, but it has not been clear whether there is a difference in their effect on LVH. METHODS: In this multi-centre, prospective, randomized trial of 12-month duration, study participants were randomized in a 1:1 ratio to the roxadustat group or the EPO group. The doses of both treatment regimens were adjusted so that the patients had a haemoglobin level of 10.0-12.0 g per dL. The primary study endpoint was the change from baseline to 12 months in the left ventricular mass index (LVMI, g/m2) measured by echocardiography. RESULTS: In total, 114 patients were enrolled. The mean age was 50 years, and the median dialysis duration was 33 months. Sixty-one patients were men, and 24 were diabetic. LVMI decreased from 116.18 ± 27.84 to 110.70 ± 25.74 g/m2 in the roxadustat group. However, it increased from 109.35 ± 23.41 to 114.99 ± 28.46 g/m2 in the EPO group, with a significant difference in the change in LVMI between the two groups [-5.48 (-11.60 to 0.65) vs. 5.65 (0.74 to 10.55), p < 0.05]. Changes in left ventricular mass, end-diastolic volume and 6-min walk test seemed superior in the roxadustat group. There were no significant differences in other cardiac geometry, biochemical parameters and major adverse cardiovascular events between the two groups. CONCLUSIONS: Compared to EPO, roxadustat is more helpful in the regression of LVH in HD patients.

A preliminary study of roxadustat in the treatment of aplastic anemia patients with inadequate erythroid responses.

Some aplastic anemia(AA) patients only have partial hematological responses after immunosuppressive therapy. Failure to achieve complete normalization of blood counts, particularly hemoglobin, will reduce their quality of life. This open-label pilot study was conducted to evaluate the efficacy and safety of roxadustat in this setting. A total of 14 patients with AA who had inadequate erythroid response after immunosuppressive therapy were included in the study. The primary efficacy endpoint was hemoglobin response at week 8 after roxadustat treatment. The median duration of roxadustat therapy was 14 (4-30) weeks, with 12 patients receiving roxadustat for ≥ 8 weeks. At week 8, nine patients (9/14, 64.3%) had their hemoglobin rising for at least 15 g/L, with two patients (2/14, 14.3%) achieving normal hemoglobin levels. By the last follow-up, hemoglobin responses were observed in 10 patients (10/14, 71.4%), with 4 patients(4/14, 28.6%) having normal hemoglobin levels. Roxadustat was tapered or discontinued in four responded patients; one relapsed after 12 weeks of tapering, and three maintained their response. Four patients (4/14, 28.6%) experienced mild adverse effects during therapy. Roxadustat is safe and well tolerated by patients with AA. Treatment with the hypoxia-inducible factor prolyl hydroxylase inhibitor improves hemoglobin levels in AA patients with inadequate erythroid responses.

Compassionate use of roxadustat for treatment of refractory renal anemia in an infant.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) have played an important role in the treatment of renal anemia in children, but cannot improve hemoglobin to target level in some cases. Roxadustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor, can stimulate endogenous erythropoietin production and regulate iron metabolism even in patients with kidney failure. However, roxadustat has not yet been approved for use in children. CASE-DIAGNOSIS/TREATMENT: We report a case of refractory renal anemia in an 80-day-old boy, who was hyporesponsive to ESAs even in combination with iron supplementation and transfusion. Compassionate use of roxadustat successfully corrected the intractable anemia. Hyperkalemia is a manageable adverse event of concern during follow-up. CONCLUSION: The successful experience in this case may inform the clinical utility of roxadustat for refractory renal anemia in children, which should be further confirmed by well-designed prospective clinical trials.

Roxadustat Versus Erythropoietin: The Comparison of Efficacy in Reversing Ventricular Remodeling in Dialysis Patients with Anaemia.

Background: Renal anaemia and left ventricular hypertrophy are the main complications of chronic kidney disease and are shared among dialysis patients. This retrospective study aimed to compare the efficacies of the hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat and recombinant human erythropoietin in reversing ventricular remodeling in dialysis patients with renal anaemia. Methods: A total of 204 participants underwent baseline examinations, including echocardiograms and laboratory tests, before being administered either treatment for at least 24 weeks from January 2018 to October 2021, after which follow-up examinations were conducted at 6 months. Propensity score matching based on key variables included age, gender, cardiovascular diseases, cardiovascular medications, dialysis course and the vascular access at baseline was performed to include populations with similar characteristics between groups. Results: In total, 136 patients were included with roxadustat or recombinant human erythropoietin. The left ventricular mass index after treatment with roxadustat and recombinant human erythropoietin both significantly decreased after 6 months, but there was no significant difference in the change in left ventricular mass index between the two groups. In addition, the left ventricular end-diastolic diameters and left ventricular wall thickness, systolic blood pressure, and diastolic blood pressure significantly decreased in the roxadustat group. Roxadustat and recombinant human erythropoietin also increased haemoglobin significantly, but there was no significant difference in the change in haemoglobin between the two groups. The results of multiple linear regression showed that the change in haemoglobin was independent factor affecting the improvement of left ventricular mass index. Conclusions: The increase of haemoglobin was associated with improving left ventricular hypertrophy in dialysis patients. However, the beneficial effects between roxadustat and recombinant human erythropoietin on left ventricular mass index did not show clear superiority or inferiority in six months.

Effects of roxadustat on thyroid hormone levels and blood lipid metabolism in patients undergoing hemodialysis: a retrospective study.

Background: Roxadustat is commonly used to treat renal anemia. However, the potential effects of roxadustat on metabolism and organs other than the kidneys have recently attracted increased attention. Objective: This study aimed to examine the regulatory effects of roxadustat on thyroid hormones and blood lipid metabolism in patients with end-stage kidney disease (ESKD) undergoing hemodialysis. Methods: Eighty ESKD patients on hemodialysis and taking roxadustat were enrolled. Hemoglobin, thyroid hormones (TSH, FT3, FT4), and blood lipid profiles (TC, LDL-C, TG, HDL-C) were assessed before and after treatment. Changes in these parameters were compared, and relevant causative factors were analyzed. Results: Roxadustat significantly increased Hb, lowered TSH, FT4, TC, and LDL-C levels (all P<0.001). Patients were categorized into three groups based on post-treatment TSH inhibition percentage: Q1(≥70%), Q2(30%-70%), Q3(≤30%). Pre-treatment TSH decreased with reduced TSH inhibition (P<0.05). Post-treatment, TC, LDL-C, TSH, FT3, and FT4 increased with reduced TSH inhibition (all P<0.05).TC and LDL-C significantly decreased post-treatment in Q1 and Q2 (P<0.05). Correlation analysis showed a positive correlation between ΔTSH and pre-treatment TSH levels (r=0.732, P<0.001). The proportion of patients with ≥70% TSH inhibition increased with higher pre-treatment TSH levels (P for trend <0.05). ΔLDL-C and ΔTSH were positively correlated (r=0.278, P<0.05), with ΔTSH identified as an influencing factor in multiple linear regression (ß=0.133, 95% CI [0.042, 0.223], P<0.05). Conclusion: Roxadustat effectively improves anemia in ESKD patients while inhibiting TSH and FT4 secretion and reducing TC and LDL-C levels. Decreases in TSH levels correlate with baseline TSH levels, and lowered blood lipid levels are associated with decreased TSH levels.

Effects of the Omicron variant of coronavirus disease 2019 on anaemia treatment for patients undergoing peritoneal dialysis.

AIM: Patients undergoing dialysis are at high risk for coronavirus disease 2019 (COVID-19). Haemoglobin (Hb) levels may be changed in patients infected with the Omicron variant of COVID-19 who are undergoing peritoneal dialysis (PD). Therefore, we aimed to explore the effect of anti-anaemia treatment on such patients. METHODS: This study enrolled patients undergoing PD who were infected with the Omicron variant of COVID-19 at our centre between November 2022 and February 2023. We retrospectively analysed changes in Hb levels and explored the effectiveness of roxadustat and recombinant human erythropoietin (rhEPO) for patients infected with the Omicron variant of COVID-19. RESULTS: Among 125 enrolled patients, 83 (66.4%) were infected with the Omicron variant and 75 (90.4%) patients mainly experienced mild or moderate disease. During infection, C-reactive protein (CRP) levels were higher than those before infection (p < .001). Additionally, their CRP levels were negatively correlated with Hb levels (p = .002). However, Hb levels were decreased 1 month after infection (109.61 ± 10.64 g/L vs. 115.69 ± 12.04 g/L; p < .001). The roxadustat treatment group did not exhibit significantly decreased Hb levels 1 month after infection (114.57 ± 14.11 vs. 117.51 ± 10.74 g/L; p = .225). Conversely, the rhEPO treatment group experienced a mild decrease in Hb levels (108.69 ± 11.20 vs. 115.03 ± 12.23 g/L; p = .001). Ferritin levels increased in both groups during infection (p = .495). Two months after infection, ferritin levels (median, 205.0 ng/mL) were significantly decreased compared with during infection (median, 377.4 ng/mL) (p < .001) in the roxadustat treatment group. CONCLUSION: Roxadustat was effective than rhEPO for treating anaemia in patients undergoing PD who were infected with the Omicron variant of COVID-19.

Impact of C-reactive protein on the effect of Roxadustat for the treatment of anemia in chronic kidney disease: a systematic review of randomized controlled trials.

BACKGROUND: Chronic inflammation, reflected by an increased blood C-reactive protein (CRP) level, is common in patients with chronic kidney disease (CKD) and is involved in the development of renal anemia. This systematic review aims to investigate the impacts of CRP on the efficacy of hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) in the treatment of renal anemia in patients with CKD. METHODS: We conducted a comprehensive search of electronic databases including Pubmed, Web of Science, Embase, Cochrane Library, CNKI, Wanfang, and the International Clinical Trials Registry Platform (ICTRP), from their inception to May 19, 2022. We systematically reviewed evidence from randomized controlled trials using HIF-PHIs for renal anemia treatment. The mean difference (MD) in changes in hemoglobin concentration (∆Hb) before and after treatment served as the meta-analysis outcome, utilizing a random-effects model. We compared groups with CRP levels greater than or equal to the upper limit of normal (ULN) and less than the ULN. Additionally, further analysis was conducted in the CRP ≥ ULN group comparing HIF-PHIs and erythropoiesis-stimulating agents (ESA). RESULTS: A total of 7 studies from 6 publications were included in the analysis. In the comparison between the CRP ≥ ULN group and the CRP < ULN group, 524 patients from 4 studies were incorporated into the analysis. All patients received roxadustat as the primary intervention. The pooled results revealed no significant difference in ΔHb between patients with CRP ≥ ULN and CRP < ULN at baseline (Mean Difference: 0.00, 95% Confidence Interval: -0.32 to 0.33, P = 0.99). Moreover, within the CRP ≥ ULN group, three studies involving 1399 patients compared the efficacy of roxadustat and erythropoiesis-stimulating agents (ESAs). The results indicated no significant difference in ΔHb between patients treated with ESAs and HIF-PHIs (Mean Difference: 0.24, 95% Confidence Interval: -0.08 to 0.56, P = 0.14). In terms of medication dosage, an increase in ESA dose over time was observed across various studies, particularly evident in the CRP ≥ ULN group, while the dose of roxadustat remains constant over time and is not influenced by the baseline levels of CRP. CONCLUSIONS: Our systematic review demonstrates that roxadustat exhibits similar efficacy across different CRP levels. Moreover, within the CRP ≥ ULN group, roxadustat can maintain efficacy comparable to ESA without the necessity for dose escalation. TRIAL REGISTRATION: CRD42023396704.

Isoquinoline-based intrinsic fluorescence assessment of erythropoiesis-stimulating agent, Roxadustat (FG-4592), in tablets: applications to content uniformity and human plasma evaluation.

In the present study, a first validated and green spectrofluorimetric approach for its assessment and evaluation in different matrices was investigated. After using an excitation wavelength of 345 nm, Roxadustat (ROX) demonstrates a highly native fluorescence at an emission of 410 nm. The influences of experimental factors such as pH, diluting solvents, and different organized media were tested, and the most appropriate solvent choice was ethanol. It was confirmed that there was a linear relationship between the concentration of ROX and the relative fluorescence intensity in the range 60.0-1000.0 ng ml-1, with the limit of detection and limit of quantitation, respectively, being 17.0 and 53.0 ng ml-1. The mean recoveries % [±standard deviation (SD), n = 5] for pharmaceutical preparations were 100.11% ± 2.24%, whereas for plasma samples, they were 100.08 ± 1.08% (±SD, n = 5). The results obtained after the application of four greenness criteria, Analytical Eco-Scale metric, NEMI, GAPI, and AGREE metric, confirmed its eco-friendliness. In addition, the whiteness meter (RGB12) confirmed its level of sustainability. The International Council for Harmonisation (ICH) criteria were used to verify the developed method through the study in both spiked plasma samples and content uniformity evaluation. An appropriate standard for various applications in industry and quality control laboratories was developed.

Roxadustat ameliorates vascular calcification in CKD rats by regulating HIF-2α/HIF-1α.

Vascular calcification (VC) is a common complication of chronic kidney disease (CKD). VC is a gene-regulated process similar to osteogenic differentiation. There are still no convincing schemes to prevent and reduce the development of VC. It has been reported that hypoxia-inducing factor 1α (HIF-1α) and endothelin-1(ET-1) are related to VC. In this study, we found that the expression of ET-1 and HIF-1α was enhanced after VC, the interaction between HIF-1α and ET-1 was confirmed by CO-IP and luciferase experiments. We found that ET-1 was an upregulated differential gene of calcified vascular smooth muscle cells (VSMCs) through gene sequencing. However, hypoxia-inducing factor 2α (HIF-2α) and HIF-1α have antagonistic effects on each other. HIF-1α is a pro-inflammatory cytokine, and HIF-2α can improve inflammation and fibrosis. Roxadustat, as a selective PHD3 inhibitor, preferentially activates HIF-2α. It is still unclear whether roxadustat improves VC in CKD by regulating the expression of HIF-2α/HIF-1α. Alizarin red staining and western blot as well as immunohistochemical results showed that roxadustat could significantly reduce the degree of vascular and VSMCs calcification in CKD rats. Serum HIF-1α and ET-1 were significantly decreased after roxadustat treatment. In addition, western blot results showed that roxadustat could decrease the expression of HIF-1α and ET-1 in vascular tissues and calcified VSMC, but HIF-2α expression significantly increased. Interestingly, our study confirmed that activation of HIF-1α or inhibition of HIF-2α reversed the ameliorating effect of roxadustat on VC, proving that the effect mediated by roxadustat is HIF-2α/HIF-1α dependent. We have demonstrated for the first time that roxadustat improves VC in CKD rats by regulating HIF-2α/HIF-1α, thus providing a new idea for the application of roxadustat in VC of CKD.

Roxadustat protect mice from DSS-induced colitis in vivo by up-regulation of TLR4.

BACKGROUND: The incidence of inflammatory bowel disease (IBD) is growing in the population. At present, the etiology of inflammatory bowel disease remains unclear, and there is no effective and low-toxic therapeutic drug. The role of the PHD-HIF pathway in relieving DSS-induced colitis is gradually being explored. METHODS: Wild-type C57BL/6 mice were used as a model of DSS-induced colitis to explore the important role of Roxadustat in alleviating DSS-induced colitis. High-throughput RNA-Seq and qRT-PCR methods were used to screen and verify the key differential genes in the colon of mice between normal saline (NS) and Roxadustat groups. RESULTS: Roxadustat could alleviate DSS-induced colitis. Compared with the mice in the NS group, TLR4 were significantly up-regulated in the Roxadustat group. TLR4 KO mice were used to verify the role of TLR4 in the alleviation of DSS-induced colitis by Roxadustat. CONCLUSION: Roxadustat has a repairing effect on DSS-induced colitis, and may alleviate DSS-induced colitis by targeting the TLR4 pathway and promote intestinal stem cell proliferation.

Influencing factors of clinical efficacy of roxadustat among hemodialysis patients.

OBJECTIVE: To explore independent influencing factors for clinical efficacy of roxadustat in hemodialysis patients. METHODS: Hemodialysis patients treated with roxadustat were enrolled. The plasma trough concentrations of roxadustat were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A multiple logistic regression model was established to determine the factors that affect clinical efficacy of roxadustat in patients undergoing hemodialysis. RESULTS: A total of 67 hemodialysis patients were enrolled in the study. The results showed that age, blood trough concentration of roxadustat, and baseline hemoglobin (Hb) level were independent factors of clinical efficacy of roxadustat (OR = 1.06, p = .025 for age; OR = 1.001, p = .037 for plasma concentration; and OR = 0.941, p = .003 for baseline Hb), with an AUC score of 0.859. CONCLUSIONS: Age, blood trough concentration of roxadustat, and baseline Hb level were independent influencing factors of the response to roxadustat in hemodialysis patients.

Cardiovascular and renal safety outcomes of hypoxia-inducible factor prolyl-hydroxylase inhibitor roxadustat for anemia patients with chronic kidney disease: a systematic review and meta-analysis.

This systematic review and meta-analysis were conducted to evaluate the cardiac and kidney-related adverse effects of roxadustat for the treatment of anemia in CKD patients. 18 trials with a total of 8806 participants were identified for analysis. We employed a fixed-effects model for analysis. The pooled result revealed no significant difference in the risk of occurrence of cardiac disorders when comparing CKD patients receiving roxadustat with the placebo (RR = 1.049; CI [0.918 to 1.200]) or ESA (RR = 1.066; CI [0.919 to 1.235]), in both dialysis-dependent (DD) (RR = 1.094; CI [0.925 to 1.293]) or non-dialysis-dependent (NDD) (RR = 1.036; CI [0.916 to 1.171]) CKD patients. No significant difference was observed in the risk of kidney-related adverse events when comparing roxadustat with the placebo (RR = 1.088; CI [0.980 to 1.209]) or ESA (RR = 0.968; CI [0.831 to 1.152]), in DD (RR = 2.649; CI [0.201 to 34.981]) or NDD (RR = 1.053; CI [0.965 to 1.149]) CKD patients. A high risk of hyperkalemia was observed in the roxadustat group in DD (RR = 0.939; CI [0.898 to 0.981]). Incidence of hypertension was higher in the roxadustat for NDD patients (RR = 1.198; CI [1.042 to 1.377]), or compared to the placebo (RR = 1.374; CI [1.153 to 1.638]). In summary, the risk of cardiac or kidney-related events observed in the roxadustat was not significantly increase whether in DD or NDD patients. However, attention must be paid to the occurrence of hyperkalemia for DD patients and hypertension in NDD patients using roxadustat.

Mitophagy mediated by HIF-1α/FUNDC1 signaling in tubular cells protects against renal ischemia/reperfusion injury.

Acute kidney injury (AKI) is associated with a high mortality rate. Pathologically, renal ischemia/reperfusion injury (RIRI) is one of the primary causes of AKI, and hypoxia-inducible factor (HIF)-1α may play a defensive role in RIRI. This study assessed the role of hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy in protection against RIRI in vitro and in vivo. The human tubular cell line HK-2 was used to assess hypoxia/reoxygenation (H/R)-induced mitophagy through different in vitro assays, including western blotting, immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reactive oxygen species (ROS) measurement. Additionally, a rat RIRI model was established for evaluation by renal histopathology, renal Doppler ultrasound, and transmission electron microscopy to confirm the in vitro data. The selective HIF-1α inhibitor LW6 reduced H/R-induced mitophagy but increased H/R-induced apoptosis and ROS production. Moreover, H/R treatment enhanced expression of the FUN14 domain-containing 1 (FUNDC1) protein. Additionally, FUNDC1 overexpression reversed the effects of LW6 on the altered expression of light chain 3 (LC3) BII and voltage-dependent anion channels as well as blocked the effects of HIF-1α inhibition in cells. Pretreatment of the rat RIRI model with roxadustat, a novel oral HIF-1α inhibitor, led to decreased renal injury and apoptosis in vivo. In conclusion, the HIF-1α/FUNDC1 signaling pathway mediates H/R-promoted renal tubular cell mitophagy, whereas inhibition of this signaling pathway protects cells from mitophagy, thus aggravating apoptosis, and ROS production. Accordingly, roxadustat may protect against RIRI-related AKI.

Roxadustat (FG-4592) protects against ischaemia-induced acute kidney injury via improving CD73 and decreasing AIM2 inflammasome activation.

BACKGROUND: Hypoxia-mediated inflammation plays a crucial role in renal ischaemia-reperfusion (IR)-induced acute kidney injury (AKI) and may influence renal graft survival, with no available pharmacological treatments. Here we investigate the protective effects and mechanism of roxadustat (FG-4592), a hypoxia-inducible factor stabilizer, against renal IR injury. METHODS: The protein expression levels of CD73 and AIM2 inflammasome complex were examined in kidney biopsy specimens of AKI and post-renal transplantation (PRT) patients. The effects of FG-4592 on CD73 and absent in melanoma 2 (AIM2) inflammasome components were examined in IR mice (right nephrectomy, followed by 30 min of unilateral renal ischaemia and reperfusion for 24 h), and some of the model mice received intraperitoneal administrations of adenosine 5'-(α,ß-methylene)diphosphate sodium salt, which is an inhibitor of CD73. The function of FG-4592 was also investigated in vitro with HK-2 cells. RESULTS: In the AKI and PRT patients, the protein expression of AIM2 complex [AIM2-apoptosis-associated speck-like protein (ASC)-cleaved caspase-1) increased and the activation of CD73 signalling pathway was detected as well. The pretreatment of FG-4592 improved the creatinine elevation and renal tubular injuries induced by ischaemia. What's more, the administration of FG-4592 significantly enhanced CD73 synthesis in mouse kidney but suppressed the activation of the AIM2 inflammasome [decreased AIM2, ASC, caspase-1, interleukin (IL)-1ß and IL-18 levels]. Notably, the renoprotection of FG-4592 and the inhibition of AIM2 were abolished by the CD73 inhibitor. CONCLUSION: FG-4592-conveyed protection against AKI might be mediated by the induction of CD73 and the suppression of the AIM2 inflammasome, which may provide a novel therapeutic method for the treatment of AKI.

Roxadustat alleviates nitroglycerin-induced migraine in mice by regulating HIF-1α/NF-κB/inflammation pathway.

Sensitization of central pain and inflammatory pathways play essential roles in migraine, a primary neurobiological headache disorder. Since hypoxia-inducible factor-1α (HIF-1α) is implicated in neuroprotection and inflammation inhibition, herein we investigated the role of HIF-1α in migraine. A chronic migraine model was established in mice by repeated injection of nitroglycerin (10 mg/kg, i.p.) every other day for 5 total injections. In the prevention and acute experiments, roxadustat, a HIF-1α stabilizer, was orally administered starting before or after nitroglycerin injection, respectively. Pressure application measurement, and tail flick and light-aversive behaviour tests were performed to determine the pressure pain threshold, thermal nociceptive sensitivity and migraine-related light sensitivity. At the end of experiments, mouse serum samples and brain tissues were collected for analyses. We showed that roxadustat administration significantly attenuated nitroglycerin-induced basal hypersensitivity and acute hyperalgesia by improving central sensitization. Roxadustat administration also decreased inflammatory cytokine levels in serum and trigeminal nucleus caudalis (TNC) through NF-κB pathway. Consistent with the in vivo results showing that roxadustat inhibited microglia activation, roxadustat (2, 10, and 20 µM) dose-dependently reduced ROS generation and inflammation in LPS-stimulated BV-2 cells, a mouse microglia cell line, by inhibiting HIF-1α/NF-κB pathway. Taken together, this study demonstrates that roxadustat administration ameliorates migraine-like behaviours and inhibits central pain sensitization in nitroglycerin-injected mice, which is mainly mediated by HIF-1α/NF-κB/inflammation pathway, suggesting the potential of HIF-1α activators as therapeutics for migraine.

Roxadustat induces hepatotoxicity in zebrafish embryos via inhibiting Notch signaling.

Roxadustat is a novel and effective small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PHI). However, little research has been done on its toxicity to vertebrate embryonic development. In this study, we used zebrafish to assess the effects of roxadustat on early embryonic development. Exposure to 14, 28, and 56 µM roxadustat resulted in abnormal embryonic development in zebrafish embryos, such as shortened body length and early liver developmental deficiency. Roxadustat exposure resulted in liver metabolic imbalance and abnormal liver tissue structure in adult zebrafish. In addition, roxadustat could up-regulate oxidative stress, and astaxanthin (AS) could partially rescue liver developmental defects by down-regulation of oxidative stress. After exposure to roxadustat, the Notch signaling is down-regulated, and the use of an activator of Notch signaling can partially rescue hepatotoxicity. Therefore, our research indicates that roxadustat may induce zebrafish hepatotoxicity by down-regulating Notch signaling. This study provides a reference for the clinical use of roxadustat.

Roxadustat Versus Epoetin Alfa for Treating Anemia in Patients with Chronic Kidney Disease on Dialysis: Results from the Randomized Phase 3 ROCKIES Study.

BACKGROUND: Concerns regarding cardiovascular safety with current treatments for anemia in patients with dialysis-dependent (DD)-CKD have encouraged the development of alternatives. Roxadustat, an oral hypoxia-inducible factor prolyl hydroxylase inhibitor, stimulates erythropoiesis by increasing endogenous erythropoietin and iron availability. METHODS: In this open-label phase 3 study, patients with DD-CKD and anemia were randomized 1:1 to oral roxadustat three times weekly or parenteral epoetin alfa per local clinic practice. Initial roxadustat dose depended on erythropoiesis-stimulating agent dose at screening for patients already on them and was weight-based for those not on them. The primary efficacy end point was mean hemoglobin change from baseline averaged over weeks 28‒52 for roxadustat versus epoetin alfa, regardless of rescue therapy use, tested for noninferiority (margin, -0.75 g/dl). Adverse events (AEs) were assessed. RESULTS: Among 2133 patients randomized (n=1068 roxadustat, n=1065 epoetin alfa), mean age was 54.0 years, and 89.1% and 10.8% were on hemodialysis and peritoneal dialysis, respectively. Mean (95% confidence interval) hemoglobin change from baseline was 0.77 (0.69 to 0.85) g/dl with roxadustat and 0.68 (0.60 to 0.76) g/dl with epoetin alfa, demonstrating noninferiority (least squares mean difference [95% CI], 0.09 [0.01 to 0.18]; P<0.001). The proportion of patients experiencing ≥1 AE and ≥1 serious AE was 85.0% and 57.6% with roxadustat and 84.5% and 57.5% with epoetin alfa, respectively. CONCLUSIONS: Roxadustat effectively increased hemoglobin in patients with DD-CKD, with an AE profile comparable to epoetin alfa. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Safety and Efficacy Study of Roxadustat to Treat Anemia in Patients With Chronic Kidney Disease, on Dialysis. CLINICALTRIALS: gov Identifier: NCT02174731.

Thyroid function analysis after roxadustat or erythropoietin treatment in patients with renal anemia: a cohort study.

PURPOSE: This cohort study was designed to explore whether roxadustat or erythropoietin could affect thyroid function in patients with renal anemia. METHODS: The study involved 110 patients with renal anemia. Thyroid profile and baseline investigations were carried out for each patient. The patients were divided into two groups: 60 patients taking erythropoietin served as the control group (rHuEPO group) and 50 patients using roxadustat served as the experimental group (roxadustat group). RESULTS: The results indicated that there were no significant differences in serum total thyroxine (TT4), total triiodothyronine (TT3), free triiodothyronine (FT3), free thyroxine (FT4) or thyroid stimulating hormone (TSH) between the two groups at baseline. After treatment, TSH, FT3, and FT4 were significantly lower in the roxadustat group than in the rHuEPO group (p < 0.05). After adjusting for age, sex, dialysis modality, thyroid nodules and causes of kidney disease, Cox regression showed that roxadustat was an independent influencing factor on thyroid dysfunction (HR 3.37; 95% CI 1.94-5.87; p < 0.001). After 12 months of follow-up, the incidence of thyroid dysfunction was higher in the roxadustat group than in the rHuEPO group (log-rank p < 0.001). CONCLUSION: Roxadustat may lead to a higher risk of thyroid dysfunction, including low TSH, FT3 and FT4, than rHuEPO in patients with renal anemia.