Erythropoietin mRNA Expression Research Articles

Siwu tablet attenuates high fructose-induced glomerular podocyte senescence in rats through increasing Nup155 to promote INO80 mRNA nuclear export

Ethnopharmacological relevanceSiwu tablet (SWT), derived from a traditional Chinese medicinal formula named Siwu decoction, is widely used for blood deficiency syndrome. Siwu decoction and its derived formulas have been proven to improve renal anemia and prevent senescence. Whether SWT prevents glomerular podocyte senescence and the underlying molecular mechanism remains unknow. Aim of the studyTo elucidate the protective effect and possible mechanism of SWT on glomerular podocyte senescence. Material and methodsLiquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to characterize components of SWT. Male Sprague-Dawley rats were given 10% fructose drinking water for 16 weeks. SWT (810 and 1620 mg/kg) was administered orally for the last 8 weeks. The assays of senescence-associated beta-galactosidase (SA-β-gal) staining, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot as well as enzyme linked immunosorbent assay were performed to evaluate rat glomerular podocyte senescence. The mRNA and protein levels of nucleoporin 155 (Nup155) and inositol requiring mutant 80 (INO80) in rat glomeruli were detected by qRT-PCR, Western blot and immunofluorescence. Foot processes and nuclear pore complexes (NPCs) of rat glomerular podocytes were visualized by transmission electron microscopy. ResultsOne hundred and fifty-nine components were preliminarily identified in SWT. The results of animal experiments showed that SWT decreased the activity of SA-β-gal, protein levels of p16, p21, p53 and phosphorylated histone H2AX (γ-H2AX), and mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in glomeruli of high fructose-fed rats. As expected, SWT increased renal cortex erythropoietin mRNA expression and serum erythropoietin concentration in this animal model. SWT reduced urine albumin-to-creatinine ratio and serum levels of uric acid, creatinine and blood urea nitrogen, and recovered glomerular structure injury in high fructose-fed rats. It up-regulated mRNA and protein levels of Nup155 and the number of podocyte NPCs, and subsequently reinforced mRNA nuclear export and protein expression of INO80 in rat glomeruli under high fructose stimulation. ConclusionsSWT ameliorates glomerular podocyte senescence in high fructose-fed rats possibly by increasing Nup155 to promote INO80 mRNA nuclear export.

Combined effects of Rhodiola rosea and caffeine supplementation on aerobic endurance and muscle explosiveness: a synergistic approach.

This study examined the synergistic effects of combining Rhodiola rosea (RHO) and caffeine (CAF) supplementation on muscle endurance and explosiveness in SD rats and human subjects, encompassing individuals without prior exercise training experience and seasoned aerobic athletes. Male SD rats and healthy human volunteers were randomly divided into four groups: CAF, RHO, CAF + RHO, and a control group (CTR). Nutritional supplements were administered throughout the training period, and pre-and post-measurement data were collected. In both the rat model and human subjects, the RHO+CAF group demonstrated significantly greater effects compared to the use of RHO or CAF supplements individually. Rats in the RHO+CAF group demonstrated extended running and swimming times and an increase in erythropoietin (EPO) mRNA expression in comparison to the CTR. Blood parameters, such as serum EPO levels, were enhanced in the CAF + RHO group, while blood urea nitrogen (BUN) and lactate (LA) levels significantly decreased in both the RHO and CAF + RHO groups. Hepatic and muscle glycogen contents were also higher in these groups. The gene expression analysis in rats demonstrated an elevation in the mRNA levels of glucose transporter-4 (GLUT-4), peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), Monocarboxylate transporter 1 (MCT-1), and Heme Oxygenase-1 (HO-1) in both the RHO and RHO+CAF groups. For individuals without prior aerobic training experience, the RHO+CAF group showed significant improvements compared to the CTR group in maximal oxygen consumption (VO2max), 5 km run, countermovement jump (CMJ), standing long jump, and 30 m sprint. For individuals with years of aerobic training experience, the RHO+CAF group exhibited enhanced performance in the 5 km run, CMJ, and standing long jump compared to the CTR group. In conclusion, the continuous 30 days supplementation of RHO, combined with a single dose of CAF, demonstrated superior effects on muscle endurance and explosiveness in both animal and human studies when compared to the use of RHO or CAF individually.

Novel roles of cardiac-derived erythropoietin in cardiac development and function

The role of erythropoietin (EPO) has extended beyond hematopoiesis to include cytoprotection, inotropy, and neurogenesis. Extra-renal EPO has been reported for multiple tissue/cell types, but the physiological relevance remains unknown. Although the EPO receptor is expressed by multiple cardiac cell types and human recombinant EPO increases contractility and confers cytoprotection against injury, whether the heart produces physiologically meaningful amounts of EPO in vivo is unclear. We show a distinct circadian rhythm of cardiac EPO mRNA expression in adult mice and increased mRNA expression during embryogenesis, suggesting physiological relevance to cardiac EPO production throughout life. We then generated constitutive, cardiomyocyte-specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v-cre+/−; EPOΔ/Δ-CM). During cardiogenesis, cardiac EPO mRNA expression and cellular proliferation were reduced in EPOΔ/Δ-CM hearts. However, in adult EPOΔ/Δ- CM mice, total heart weight was preserved through increased cardiomyocyte cross-sectional area, indicating the reduced cellular proliferation was compensated for by cellular hypertrophy. Echocardiography revealed no changes in cardiac dimensions, with modest reductions in ejection fraction, stroke volume, and tachycardia, whereas invasive hemodynamics showed increased cardiac contractility and lusitropy. Paradoxically, EPO mRNA expression in the heart was elevated in adult EPOΔ/Δ-CM, along with increased serum EPO protein content and hematocrit. Using RNA fluorescent in situ hybridization, we found that Epo RNA colocalized with endothelial cells in the hearts of adult EPOΔ/Δ-CM mice, identifying the endothelial cells as a cell responsible for the EPO hyper-expression. Collectively, these data identify the first physiological roles for cardiomyocyte-derived EPO. We have established cardiac EPO mRNA expression is a complex interplay of multiple cell types, where loss of embryonic cardiomyocyte EPO production results in hyper-expression from other cells within the adult heart.

Effect of long-term treatment with trivalent chromium on erythropoietin production in HepG2 cells

Trivalent chromium (Cr(III)) is sometimes taken as a long-term supplement, but its effectiveness is unclear. Recently, Cr(III) reportedly modulates peroxisome proliferator-activated receptor gamma (PPARγ) expression. Our previous study reported that increased PPARγ after 24 h Cr(III) treatment promoted erythropoietin (EPO) production in HepG2 cells. In the current study, we analyzed 4-week Cr(III) treatment effects on PPARγ and EPO production in HepG2 cells. Long-term Cr(III) treatment resulted in significantly elevated mRNA expression levels of PPARγ and EPO compared to controls. Additionally, treatment with a PPARγ inhibitor suppressed EPO mRNA expression. Increased EPO mRNA expression due to stimulation with hypoxia or cobalt was unaffected by long-term Cr(III) treatment. Administration of lipopolysaccharide and pyocyanin which causes oxidative stress, promoted EPO production, but this effect was attenuated in cells treated with Cr(III). Long-term Cr(III) treatment increased hypoxia inducible factor (HIF)-1α and 2α mRNA expression and protein levels. Increased PPARγ, induced by long-term Cr(III) treatment, suppressed sirtuin1 (SIRT1) mRNA expression and increased EPO mRNA expression, suggesting that increased PPARγ attenuated the suppressive effect of SIRT1 on HIF. These results suggest that the sustained increase in PPARγ during long-term Cr(III) treatment maintains increased EPO production through a mechanism different from that observed under hypoxia.

Suppression of Indoxyl Sulfate Accumulation Reduces Renal Fibrosis in Sulfotransferase 1a1-Deficient Mice.

Renal fibrosis is the final manifestation of chronic kidney disease (CKD); its prevention is vital for controlling CKD progression. Indoxyl sulfate (IS), a typical sulfate-conjugated uremic solute, is produced in the liver via the enzyme sulfotransferase (SULT) 1A1 and accumulates significantly during CKD. We investigated the toxicopathological role of IS in renal fibrosis using Sult1a1-KO mice and the underlying mechanisms. The unilateral ureteral obstruction (UUO) model was created; kidney IS concentrations, inflammation, and renal fibrosis were assessed on day 14. After UUO treatment, inflammation and renal fibrosis were exacerbated in WT mice, with an accumulation of IS in the kidney. However, they were significantly suppressed in Sult1a1-KO mice. CD206+ expression was upregulated, and β-catenin expression was downregulated in Sult1a1-KO mice. To confirm the impact of erythropoietin (EPO) on renal fibrosis, we evaluated the time-dependent expression of EPO. In Sult1a1-KO mice, EPO mRNA expression was improved considerably; UUO-induced renal fibrosis was further attenuated by recombinant human erythropoietin (rhEPO). Thus, UUO-induced renal fibrosis was alleviated in Sult1a1-KO mice with a decreased accumulation of IS. Our findings confirmed the pathological role of IS in renal fibrosis and identified SULT1A1 as a new therapeutic target enzyme for preventing and attenuating renal fibrosis.

Placental Erythroferrone and Erythropoietin mRNA Expression is not Associated with Maternal or Neonatal Iron Status in Adolescents Carrying Singletons and Adult Women Carrying Multiples

BackgroundThe iron regulatory hormones erythroferrone (ERFE), erythropoietin (EPO), and hepcidin, and the cargo receptor nuclear receptor coactivator 4 (NCOA4) are expressed in the placenta. However, determinants of placental expression of these proteins and their associations with maternal or neonatal iron status are unknown. ObjectivesTo characterize expression of placental ERFE, EPO, and NCOA4 mRNA in placentae from newborns at increased risk of iron deficiency and to evaluate these in relation to maternal and neonatal iron status and regulatory hormones. MethodsPlacentae were collected from 114 neonates born to adolescents carrying singletons (14–18 y) and 110 neonates born to 54 adults (20–46 y) carrying multiples. Placental EPO, ERFE, and NCOA4 mRNA expression were measured by RT-qPCR and compared with maternal and neonatal iron status indicators (SF, sTfR, total body iron, serum iron) and hormones. ResultsPlacental ERFE, EPO, and NCOA4 mRNA were detected in all placentae delivered between 25 and 42 wk of gestation. Relationships between placental ERFE and EPO differed by cohort. In the multiples cohort, placental EPO and ERFE were positively correlated (P = 0.004), but only a positive trend (P = 0.08) was evident in the adolescents. Placental EPO and ERFE were not associated with maternal or neonatal iron status markers or hormones in either cohort. Placental NCOA4 was not associated with placental EPO or ERFE in either cohort but was negatively associated with maternal SF (P = 0.03) in the multiples cohort and positively associated with neonatal sTfR (P = 0.009) in the adolescents. ConclusionsThe human placenta expresses ERFE, EPO, and NCOA4 mRNA as early as 25 wk of gestation. Placental expression of ERFE and EPO transcripts was not associated with maternal or neonatal iron status. Greater placental NCOA4 transcript expression was evident in women and newborns with poor iron status (lower SF and higher sTfR, respectively). Further research is needed to characterize the roles of these proteins in the human placenta. Trial registration numberThese clinical trials were registered at clinicaltrials.gov as NCT01019902 (https://clinicaltrials.gov/ct2/show/NCT01019902) and NCT01582802 (https://clinicaltrials.gov/ct2/show/NCT01582802).

Quercetin’in HepG2 hücrelerinde, TNF-Alfa ve LPS ile indüklenen enflamasyonda, eritropoietin, HIF-1α, HIF-2α ve piroptoz ile ilgili genlerin ekspresyon seviyeleri üzerine etkileri

Purpose: TNF-α, which increases in the circulation as a result of systemic inflammation, suppresses the production of the erythropoiesis regulating hormone, erythropoietin (EPO) and causes inflammation anemia. The anti-inflammatory activity of quercetin has been reported in different studies. However, no study has been found showing the effect of this situation on EPO gene expression. For this purpose, we evaluated the effects of quercetin on inflammation and, more importantly, its effects on EPO, suppressed in the inflammatory environment and Hypoxia-inducible factors (HIF), which are stimulators of EPO synthesis. In addition, we aimed to evaluate the effect of quercetin on pyroptosis, which is defined as pro-inflammatory programmed cell death.
 Materials and Methods: We created inflammation models with TNF-α or LPS using HepG2 cells in vitro. In these inflammation models, we evaluated the effects of quercetin on proinflammatory mediators TNF-α, IL-1α, NF-κB gene expressions, EPO, HIF-1α, HIF-2α gene expressions, as well as pyroptosis-related caspase 1 and IL-18 gene expressions. 
 Results: Quercetin showed inflammatory effects by increasing TNF-α, IL-1α, and NF-κB mRNA levels. Consistent with this inflammatory effect, EPO mRNA expression was suppressed. HIF-1α and HIF-2α mRNA levels were increased. 
 Conclusion: These results suggested that the increase in HIFs could not prevent the suppressive effect of inflammatory cytokines and NF-κB on EPO production. However, it has been observed that it tends to suppress proinflammatory cell death by decreasing caspase 1 and IL-18 mRNA levels. These results show that quercetin may show conflicting effects, and further studies are needed to test its safety.

Exploring the Functional Role of an Alternative mRNA Transcript of the EPO Gene

Erythropoietin (EPO) is the key regulator of erythropoiesis and stimulates the proliferation and differentiation of erythroid progenitor cells into mature erythrocytes. In a previous study, we identified a frame-shift mutation in the EPO gene that causes familial erythrocytosis with elevated erythropoietin (EPO) levels by initiating excess production of EPO protein from an alternative EPO mRNA [1]. This alternative non-coding mRNA is transcribed from a promoter (P2) located in intron 1, whereas the full length mRNA that encodes the EPO protein is transcribed from the P1 promoter located upstream of exon 1. Large parts of intron 1 sequences, including the P2 transcript are conserved among different species (Figure 1A), suggesting that the P2 transcript could have an as yet unknown physiological function. To study the putative function of the P2 transcript, we performed experiments in a human cell line, Hep3B, which can produce EPO. We used the CRISPR/Cas9 system to generate mutant Hep3B clones with targeted deletions in intron 1 of the EPO gene. Close to the P2 transcription start site in intron 1, we identified three GATA-consensus motifs that might act as promoter elements for the P2 transcript. In the first round of CRISPR/Cas9 mutagenesis, we targeted the first two GATA elements and expected to knock down the expression of the P2 transcript. Targeted Hep3B cells were sorted and deposited as single cells to derive clones that were genotyped by next generation sequencing. We obtained 2 clones with homozygous deletions in EPO intron 1 and one clone that was compound heterozygous (Figure 1A). In these Hep3B clones we assessed EPO mRNA expression by qPCR and EPO protein levels in the supernatant by ELISA (Figure 1B). Unexpectedly, the P2 transcript was still detectable in all three clones carrying deletions in intron 1 that eliminated GATA boxes 1 and 2 (data not shown). EPO P1 mRNA expression and EPO protein levels in the supernatant were slightly reduced in most of the clones. These data indicate that the GATA box 1 and 2 motifs are not required for generating the P2 transcript. We have now performed a second round of CRISPR/Cas9 mutagenesis. In one series we targeted all three GATA-motifs through a 186 bp deletion, and in a second series we targeted a highly conserved region upstream of the GATA boxes by a 93 bp deletion (Figure 1A). We obtained 4 clones homozygous for the 93 bp deletion and the clones for the 186 bp deletion are currently being expanded. The analysis of EPO mRNA and protein in these additions CRISPR/Cas9 clones will be presented. Our study shows that the GATA motifs 1 and 2 are not required for function of the P2 promoter in intron 1 of the EPO gene. However, deleting these two motifs in two different cell clones decreased the P1 full-length EPO mRNA expression and EPO protein production in Hep3B cells by 70.2% and 37.2%, respectively. Additional clones with deletion of all three GATA motifs and a highly conserved region upstream of the GATA boxes will likely provide more insights into the physiological role of the P2 transcript.

Flavonoids from Lycium barbarum Leaves Exhibit Anti-Aging Effects through the Redox-Modulation.

Lycium barbarum leaves are a kind of vegetable, and modern nutrition studies have found that they have an anti-aging function. Our study aims to investigate the anti-aging effects of Lycium barbarum leaf flavonoid (LBLF) extracts and its underlying molecular mechanism. LBLFs were purified using D101 and polyamide resin, characterized by ultraperformance liquid chromatography coupled with mass spectrometry, and administered to hydrogen peroxide (H2O2)-treated human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. Appropriate enrichment conditions were optimized through dynamic adsorption and desorption experiments, the content of flavonoids reached 909.84 mg/g, rutin and kaempferol being the main ones. LBLFs attenuated H2O2-induced HUVEC apoptosis, decreased reactive oxygen species and malondialdehyde production levels, increased superoxide dismutase, glutathione peroxidase and catalase activities. Furthermore, pre-treatment with LBLF increased mRNA expression of erythropoietin (EPO) and heme oxygenase-1 (HO-1) via the mitogen-activated protein kinase (MAPK) signaling pathway in HUVECs. Compared with 100 µM rutin monomer, LBLF prolonged the lifespan of Caenorhabditis elegans, enhanced their mobility in middle life stages and upregulated expression of sod-2, gcs-1 and skn-1 genes, which indicated that the anti-aging effects of LBLF were due to its redox-modulation.

Steamed Panax notoginseng attenuates renal anemia in an adenine-induced mouse model of chronic kidney disease

Ethnopharmacological relevancePanax notoginseng (PN) (Burk.) F. H. Chen is a medicinal herb used to treat blood disorders since ancient times, of which the steamed form exhibits the anti-anemia effect and acts with a “blood-tonifying” function according to the traditional use. However, its pharmacological effect and mechanism on alleviating renal anemia (RA) are still unclear. Aims of the studyThe study aims to investigate the effect of steamed Panax notoginseng (SPN) to attenuate RA and its underlying mechanism based on the model of adenine-induced RA mice. Materials and methodsSeventy mice were randomly divided into seven groups of ten: the control group, model group, the erythropoietin (EPO) group, the Fufang E'jiao Jiang (FEJ) group, the high-dose steamed PN (H-SPN) group, the middle-dose steamed PN (M-SPN) group, and the low-dose steamed PN (L-SPN) group. The adenine induction RA model was applied to assess the “blood enriching” function of SPN. The blood routine indexes, erythrocyte fragility, pathologic morphology of kidney tissue and the expression levels of related cytokines and proteins in the mice were detected after 3-week administration with SPN and positive drugs. ResultsOur study provided evidences that SPN could ameliorate RA. Compared with the control group, SPN could attenuate RA by significantly increasing the numbers of peripheral blood cells (p < 0.01), improving the erythrocyte fragility (p < 0.01), and restoring the expression of EPO mRNA in the kidneys and EPO receptor mRNA in bone marrow nucleated cells. The expression of TGF-β1 mRNA was declined and the expression of HGF mRNA was significantly increased in a dose-dependent way after the treatment of SPN. Additionally, the expression of Bcl-2 and Bcl-2/Bax ratio in the kidneys were significantly increased. In contrast, there was a highly significant decrease in the expression of Bax (p < 0.01), following SPN treatment. ConclusionSPN could alleviate RA by promoting the overall hematopoiesis and inhibiting the progress of renal injury in mice.

The circadian clock regulates rhythmic erythropoietin expression in the murine kidney

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.

Effect of trivalent chromium on erythropoietin production and the prevention of insulin resistance in HepG2 cells

In erythropoietin (EPO)-producing HepG2 cells, we investigated the effect of trivalent chromium (Cr) on the promotion of EPO production and the induction of insulin resistance. Cr increased hypoxia-inducible factor (HIF)-1α protein, EPO mRNA expression and EPO protein levels in HepG2 cells. The effect of Cr on EPO production was inhibited by inhibition of proliferator-activated receptor γ (PPARγ). Insulin resistance was induced by culturing with insulin resistance induction medium supplemented with palmitic acid for 24 h. When Cr was added to the medium, the increase in glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1 mRNA expression levels and the decrease in the ratio of phosphorylated Akt to Akt protein were suppressed, and the induction of insulin resistance prevented. When a PPARγ inhibitor or siPPARγ was added together with Cr, the inhibitory effect of Cr on the induction of insulin resistance disappeared. In addition, pretreatment with siEPO suppressed the increase in EPO mRNA expression, and the inhibitory effect on the induction of insulin resistance due to the addition of Cr was significantly reduced. These results suggest that the inhibition of insulin resistance induction by Cr in HepG2 cells involves the promotion of EPO production mediated by PPARγ, in addition to other PPARγ-mediated activities.

Renal Oxygen Sensing Mechanisms May Contribute to Maintaining Cerebral Perfusion During Acute Anemia

Introduction The integrative physiological response to anemia is complex and incompletely understood. We utilized data from studies of acute anemia in rodents to determine the relationship between changes in blood oxygen content (CaO2) and the heterogenous response of the kidney, heart and brain. We hypothesize that renal hypoxia sensing mechanisms contribute to adaptive physiological responses to maintain cerebral oxygen delivery (DO2) during acute anemia. Methods With animal care committee approval, we synthesized novel and published data from 5 previously published studies. Outcomes included: assessment of the relationship between CaO2 and microvascular renal and brain PO2 (phosphorescence quenching of oxyphor G4); cardiac output (CO) and renal and cerebral blood flow (ultrasound Doppler); hypoxia induced cellular responses (brain and kidney erythropoietin (EPO) mRNA and serum protein levels (ELISA)). Statistical analysis (SigmaPlot 14) was performed by ANOVA, Holm-Sidak and Mann-Whitney rank sum test when appropriate. Significance was assigned at p<0.05. Results In two models of anemia (hemodilution and RBC antibody mediated), acute reductions in blood CaO2 were associated with larger decreases in renal microvascular PO2, relative to brain microvascular PO2 (p<0.05). After acute hemodilution, there was a strong relationship between CaO2 and renal microvascular PO2 (r2=0.75). The magnitude of reduction in renal microvascular PO2 correlated with the degree of renal EPO mRNA expression and serum EPO protein levels. The magnitude of the increase in EPO mRNA was much larger in the kidney than in the brain (p<0.03). While no change in renal blood flow was observed in either model, a significant increase in common carotid and internal carotid blood flow was observed in both models (p<0.012). When DO2 was assessed, the kidney DO2 was reduced at all levels of anemia (p<0.01) whereas brain tissue DO2 was maintained in mild and moderate (Hb 90 and 70 g/L) (p=0.44) anemia but reduced in severe anemia (Hb 50 g/L) (p<0.02). The role of active cardiovascular increases in brain blood flow and maintained DO2 during anemia was impaired by systemic beta blockade, suggesting that active cardiovascular mechanisms are required to maintain optimal brain DO2 during anemia. Discussion Our analysis demonstrated: evidence of quantitative renal PO2 sensing of changes in CaO2; the clamping of renal blood flow (reduced DO2) during anemia may be a central mechanism allowing for sensing of changes in CaO2; reduced arterial CaO2 resulted in a local renal hypoxia response (increased serum EPO) and may have initiated the cardiovascular response to increase cerebral blood flow and maintain cerebral DO2. Inhibition of the adrenergic system impaired these responses and resulted in reduced brain DO2. Understanding the heterogeneous adaptive responses to acute anemia may inform clinical practice and optimize management of acutely anemia patients.

Prolonged astrocyte-derived erythropoietin expression attenuates neuronal damage under hypothermic conditions

BackgroundHypoxic-ischemic encephalopathy (HIE) has a high morbidity rate and involves severe neurologic deficits, including cerebral palsy. Therapeutic hypothermia (TH) has been shown to decrease the mortality rate and provide neuroprotection in infants with HIE. However, death and disability rates in HIE infants treated with TH remain high. Although the cellular mechanism of the neuroprotective effect of TH remains unclear, astrocytic erythropoietin (EPO) is known to be a key mediator of neuroprotection under hypoxic conditions. In the present study, we investigated the hypothermia effect on EPO expression in astrocytes and determined whether hypothermia attenuates neuronal damage via EPO signaling.MethodsAstrocytes derived from rat cerebral cortex were cultured under oxygen/glucose deprivation (OGD). The expression of EPO and hypoxia-inducible factor (HIF), a transcription factor of EPO, was assessed. After OGD, astrocytes were cultured under normothermic (37 °C) or hypothermic (33.5 °C) conditions, and then EPO and HIF expression was assessed. After OGD, rat cortical neurons were cultured in astrocyte-conditioned medium (ACM) derived from the hypothermic group, and neuronal apoptosis was evaluated.ResultsOGD induced EPO mRNA and protein expression, although at lower levels than hypoxia alone. HIF-1α and HIF-2α protein expression increased under hypoxia alone and OGD, although OGD increased HIF-2α protein expression less than hypoxia alone. EPO gene and protein expression after OGD was significantly higher under hypothermia. Moreover, expression of HIF-1α and HIF-2α protein was enhanced under hypothermia. In the presence of ACM derived from hypothermic astrocytes following OGD, the number of cleaved caspase 3 and TdT-mediated dUTP nick-end labeling-positive apoptotic neurons was lower than in the presence of ACM from normothermic astrocytes following OGD. Blockade of EPO signaling using anti-EPO neutralization antibody attenuated the anti-apoptotic effect of ACM derived from hypothermic astrocytes following OGD.ConclusionsHypothermia after OGD stabilized HIF-EPO signaling in astrocytes, and upregulated EPO expression could suppress neuronal apoptosis. Investigating the neuroprotective effect of EPO from astrocytes under hypothermic conditions may contribute to the development of novel neuroprotection-based therapies for HIE.

Cardiac‐derived Erythropoietin: A Novel Therapeutic Strategy to Treat Myocardial Infarction?

BackgroundIn response to hypoxia, the kidney is considered the major source for erythropoietin (EPO) – a protein responsible for stimulating hematopoiesis. Interestingly, recombinant human EPO (rhEPO) also has known anti‐apoptotic, cardioprotective, and inotropic effects. Preclinically, supraphysiological concentrations of rhEPO, given at the time of permanent coronary artery occlusion, is effective at reducing apoptosis in the area‐at‐risk, infarct size, and left ventricular remodeling and functional deficits. Clinically, researchers have encountered significant translational difficulties using EPO post‐myocardial infarction, as the hematopoietic effect of chronic rhEPO dosing limits its therapeutic use in patients.Emerging findings demonstrate that EPO mRNA expression occurs in non‐renal tissues, including the liver, bone, and reproductive organs, yet the evidence is divided with regards to the heart. Our preliminary data shows that cardiac EPO expression is upregulated during embryonic development, suggesting it has a paracrine role in cardiac development. Therefore, whether the adult heart produces EPO under a stress (e.g., myocardial infarction) and has physiological relevance remains unknown. Notably, in humans, serum EPO levels are elevated at 3 days post‐myocardial infarction, which indicates that the injured/hypoxic heart may produce EPO in vivo. Accordingly, our objective was to improve our understanding of the regulation and physiological significance of cardiac‐derived EPO using a murine model of myocardial infarction. It was hypothesized that a myocardial infarction would increase cardiac EPO mRNA expression, which may serve as a paracrine factor to preserve cardiac structure and function following an ischemic injury.Methods and ResultsMale CD1 mice were subjected to permanent ligation of the left anterior descending coronary artery to induce a myocardial infarction. At 12 h post‐surgery, hearts were harvested for qPCR analyses, which showed a significant upregulation in EPO mRNA expression. At 2, 4, and 9 weeks post‐myocardial infarction (when hearts were anoxic), hematocrit was significantly elevated, compared to age‐matched shams, indicating that serum EPO levels were still increased at these timepoints. To investigate whether cardiac EPO is driven solely by hypoxia, we subjected mice to severe hypoxia (9% O2) for 24 h and evaluated EPO mRNA expression in the heart and kidney. Indeed, EPO expression was significantly increased in the kidney, while we observed a very modest increase in the heart.ConclusionsHere we show that the heart is a significant non‐renal source of EPO post‐myocardial infarction. Further, profound hypoxia does not significantly drive cardiac‐derived EPO expression, suggesting it is regulated by a hypoxia‐independent mechanism post‐injury. Taken together, endogenous cardiac EPO production may be elevated to provide paracrine cardioprotective support following a myocardial infarction.Support or Funding InformationCanadian Institutes of Health Research. Natural Sciences and Engineering Research Council of Canada.

HIF‑1α attenuates neuronal apoptosis by upregulating EPO expression following cerebral ischemia‑reperfusion injury in a rat MCAO model.

Hypoxia-inducible factor-1α (HIF-1α) is a key transcriptional factor in response to hypoxia and is involved in ischemic stroke. In the present study, the potential for HIF-1α to inhibit neuronal apoptosis through upregulating erythropoietin (EPO) was investigated in a transient middle cerebral artery occlusion (tMCAO) rat stroke model. For this purpose, a recombinant adenovirus expressing HIF-1α was engineered (Ad-HIF-1α). Control adenovirus (Ad group), Ad-HIF-1α (Ad-HIF-1α group) or Ad-HIF-1α in addition to erythropoietin mimetic peptide-9 (EMP9), an EPO-receptor (-R) antagonist (Ad-HIF-1α+EMP9 group), were used for an intracranial injection into rat ischemic penumbra 1 h following MCAO. All rats demonstrated functional improvement following tMCAO, while the improvement rate was faster in rats treated by Ad-HIF-1α compared with all other groups. The EPO-R inhibitor partially reversed the benefits of Ad-HIF-1α. Apoptosis induced by tMCAO was significantly inhibited by Ad-HIF-1α (P<0.05). The expression of HIF-1α, evaluated by immunohistochemistry either in neurons or astrocytes, was upregulated by Ad-HIF-1α. Both EPO mRNA and protein expression were increased by Ad-HIF-1α, however, there was no significant change of EPO-R either on an mRNA level or protein level. Furthermore, EMP9 did not change the EPO expression which was upregulated by Ad-HIF-1α. Activated caspase 3 in neurons was suppressed by Ad-HIF-1α. Activated caspase 3 downregulated by HIF-1α was partially blocked by EMP9. Altogether, the present data demonstrated that HIF-1α attenuates neuronal apoptosis partially through upregulating EPO following cerebral ischemia in rat. Thus, upregulating HIF-1α subsequent to a stroke may be a potential treatment for ischemic stroke.

Tectorigenin, an isoflavone aglycone from the rhizome of Belamcanda chinensis, induces neuronal expression of erythropoietin via accumulation of hypoxia-inducible factor-1α.

Traditional Chinese medicines (TCMs) have been demonstrated as an important source for potential drug discovery. Flavonoids are regarded as the most common active components in TCMs because of their beneficial functions in the brain and erythropoietic system. Erythropoietin (EPO), a glycoprotein hormone, has been well-studied for its neuroprotective function. The blood circulating EPO is not able to cross the blood brain barrier, and thus there is mounting demand to search for compounds that can induce endogenous cerebral EPO. Here, tectorigenin, an active compound in the rhizome of Belamcanda chinensis (L.) DC., significantly induced the expression of EPO mRNA via accumulation of hypoxia-inducible factor (HIF)-1α in cultured neuron-like NT2/D1 cells and rat cortical neurons. Furthermore, tectorigenin induced transcription of HIF-1α and reduced degradation of HIF-1α-OH, a hydroxylated form of HIF-1α, in the culture. Thus, the upregulation of HIF-1α was assumed to play a significant role in regulating EPO during the treatment of tectorigenin in cultured neurons. Hence, we reported the neuroprotective function of tectorigenin through upregulation of EPO in neurons, which could be a good candidate in developing drugs or food supplements for the treatment of brain disorders.

Effects of sorbitol and lactate on erythropoietin production in HepG2 cells

Promotion of erythropoietin (EPO) production is important for erythropoiesis as well as cell viability. The most effective inducing factor for EPO production is hypoxia. Hypoxia inducible factor (HIF), a regulator of EPO production, is increased under hypoxic conditions and is also affected by various regulators such as sirtuin1 (SIRT1). SIRT1 is regulated by the cytoplasmic redox state, which is thought to affect EPO production. Therefore, we investigated the effects of sorbitol and lactic acid, which serve as substrates for cellular respiration and bring cells into a reduced state, on EPO production in HepG2 cells. The addition of low-concentration sorbitol to HepG2 cells produced a mildly reduced state similar to that of hypoxia and increased NAD+, SIRT1, and HIF-α, and EPO mRNA expression. On the other hand, lactate suppressed EPO mRNA expression at all concentrations. Inhibition of lactate production from pyruvate abolished the effect of low sorbitol concentrations on EPO mRNA expression. When low-concentration sorbitol and a reducing agent were administered simultaneously, the effect of increasing EPO mRNA expression disappeared. It was suggested that SIRT1 and EPO production increased under conditions where lactate production was not suppressed, even under mildly reduced conditions similar to hypoxia.

TP0463518, a Novel Prolyl Hydroxylase Inhibitor, Specifically Induces Erythropoietin Production in the Liver.

Prolyl hydroxylase (PHD) 1/2/3 pan inhibitors are known to potentially induce erythropoietin (EPO) production in both the kidney and liver. The 2-[[1-[[6-(4-chlorophenoxy)pyridin-3-yl]methyl]-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl]amino]acetic acid (TP0463518) is a novel PHD 1/2/3 pan inhibitor; however, the main source of EPO production after TP0463518 administration remained to be investigated. We examined the effect of TP0463518 in inducing EPO production in the kidney and liver by measuring the hypoxia-inducible factor 2α (HIF-2α), EPO mRNA, and serum EPO levels in normal and bilaterally nephrectomized rats. Furthermore, we examined whether liver-derived EPO improved anemia in 5/6 nephrectomized (5/6 Nx) rats. TP0463518 scarcely increased the HIF-2α and EPO mRNA expression levels in the kidney cortex, whereas oral administration of TP0463518 at 40 mg/kg dramatically increased the HIF-2α level from 0.27 to 1.53 fmol/mg and the EPO mRNA expression level by 1300-fold in the livers of healthy rats. After administration of TP0463518 at 20 mg/kg, the total EPO mRNA expression level in the whole liver was 22-fold that in the whole kidney. In bilaterally nephrectomized rats, TP0463518 raised the serum EPO concentration from 0 to 180 pg/ml at 20 mg/kg. Furthermore, repeated administration of TP0463518 at 10 mg/kg increased the reticulocyte count in 5/6 Nx rats on day 7 and raised the hemoglobin level on day 14. The present study revealed that TP0463518 specifically induced EPO production in the liver and improved anemia. The characteristic feature of TP0463518 would lead to not only a more detailed understanding of the PHD-HIF2α-EPO pathway in erythropoiesis, but a new therapeutic alternative for renal anemia. SIGNIFICANCE STATEMENT: Prolyl hydroxylase (PHD) 1/2/3 pan inhibitors are known to potentially induce erythropoietin (EPO) production in both the kidney and liver; however, their effects on renal EPO production have been shown to vary depending on the experimental conditions. The authors found that 2-[[1-[[6-(4-chlorophenoxy)pyridin-3-yl]methyl]-4-hydroxy-6-oxo-2,3-dihydropyridine-5-carbonyl]amino]acetic acid (TP0463518), a PHD 1/2/3 pan inhibitor, specifically induced EPO production in the liver and that the liver-derived EPO was pharmacologically effective. Investigation of the effects of TP0463518 may pave the way for the development of a new therapeutic alternative for renal anemia patients.

The EPO-FGF23 Signaling Pathway in Erythroid Progenitor Cells: Opening a New Area of Research.

We provide an overview of the evidence for an erythropoietin-fibroblast growth factor 23 (FGF23) signaling pathway directly influencing erythroid cells in the bone marrow. We outline its importance for red blood cell production, which might add, among others, to the understanding of bone marrow responses to endogenous erythropoietin in rare hereditary anemias. FGF23 is a hormone that is mainly known as the core regulator of phosphate and vitamin D metabolism and it has been recognized as an important regulator of bone mineralization. Osseous tissue has been regarded as the major source of FGF23. Interestingly, erythroid progenitor cells highly express FGF23 protein and carry the FGF receptor. This implies that erythroid progenitor cells could be a prime target in FGF23 biology. FGF23 is formed as an intact, biologically active protein (iFGF23) and proteolytic cleavage results in the formation of the presumed inactive C-terminal tail of FGF23 (cFGF23). FGF23-knockout or injection of an iFGF23 blocking peptide in mice results in increased erythropoiesis, reduced erythroid cell apoptosis and elevated renal and bone marrow erythropoietin mRNA expression with increased levels of circulating erythropoietin. By competitive inhibition, a relative increase in cFGF23 compared to iFGF23 results in reduced FGF23 receptor signaling and mimics the positive effects of FGF23-knockout or iFGF23 blocking peptide. Injection of recombinant erythropoietin increases FGF23 mRNA expression in the bone marrow with a concomitant increase in circulating FGF23 protein. However, erythropoietin also augments iFGF23 cleavage, thereby decreasing the iFGF23 to cFGF23 ratio. Therefore, the net result of erythropoietin is a reduction of iFGF23 to cFGF23 ratio, which inhibits the effects of iFGF23 on erythropoiesis and erythropoietin production. Elucidation of the EPO-FGF23 signaling pathway and its downstream signaling in hereditary anemias with chronic hemolysis or ineffective erythropoiesis adds to the understanding of the pathophysiology of these diseases and its complications; in addition, it provides promising new targets for treatment downstream of erythropoietin in the signaling cascade.