Human Epo Research Articles

Enhanced analysis of equine plasma for the presence of recombinant human erythropoietin - Implementation of an improved workflow.

An improved screening workflow and a robust capillary flow LC-MS confirmatory method for the detection of recombinant human erythropoietin (rHuEPO) has been implemented to increase the sensitivity of rHuEPO detection and to reduce the number of suspect samples committed to confirmatory testing. The influence of repeated dosing of epoetin-β on the detection window of rHuEPO in equine plasma was assessed using the optimised method. Samples were initially assessed using an economical R&D Human EPO Duo-Set ELISA Development System. Samples indicating a result greater than the batch baseline were analysed using the complementary R&D Human EPO Quantikine IVD ELISA kit. All samples recording an abnormal screening result were subjected to confirmatory analysis. Confirmation of rHuEPO in plasma (≥2.5ml) in the range of 4-13 mIU/ml (n = 6) was achieved using immunoaffinity enrichment, tryptic digestion, and capillary flow LC-MS/MS. Four horses were administered a single dose of epoetin-β (10,000 IU) via the subcutaneous and intravenous routes, on two occasions, seven days apart. The excretion profile was rapid with epoetin-β detection times of 48 to 72 h following each administration, with no appreciable difference observed between the two routes of administration. This workflow has been shown as an effective anti-doping strategy related to rHuEPO misuse and supports the use of out-of-competition testing of horses in the 2 to 3-day period prior to race-day.

The Use of Erythropoietin in Cancer Treatment and Its Correlation with Ovarian Cancer

Erythropoietin is produced by the kidneys, which can stimulate the production of red blood cells, and has a better therapeutic and prognostic role in cancer anemia. Ovarian cancer, which originates from ovarian tissue, is one of the malignant tumors of the female reproductive system, with high lethality and not easy to detect. It has been proved that EPO in correlates with the growth of tumor cells, and its receptor EPOR and recombinant human EPO (rhEPO) are expressed in cancer cells. EPO can treat EPO tumors by restoring endothelial erythrocytes and promoting angiogenesis, while rhEPO mainly has a better ameliorative effect on cancer anemia induced by radiotherapy. However, it remains controversial and contradictory whether EPO has a concomitant promoting effect on tumor cells while relieving ischemia. In this article, we will introduce the sources of EPO and EPOR in vivo, analyze the pathways that promote erythropoiesis, and analyze their effects on tumor cells. It will also confirm the correlation between the two by analyzing the EPO content in ovarian cells. The intervention effect of rhEPO analogs on cancer is also briefly described. Since aberrant activation of the JAK/STAT signaling pathway may exist in ovarian cancer patients, in-depth study of such targets may become a new research direction for the treatment of ovarian cancer.

Stem Cell Secretome Modulated by Arsenicum album 30C Ameliorates Lipopolysaccharide-induced Cytokine Storm in Blood Mononuclear Cells in vitro.

The therapeutic effectiveness of mesenchymal stem cells (MSCs) and their secretome can be enhanced by means of physical, chemical and biological preconditioning. Arsenicum album 30C (AA30) has been one of the leading homeopathic medicines used in prophylaxis against SARS-CoV-2 infection. This study aimed to investigate whether AA30 preconditioning could influence the growth factors and cytokine profile of the human dental pulp-derived MSC (DPD-MSC) secretome. Also, to test the efficacy of the AA30-preconditioned DPD-MSC secretome in ameliorating the lipopolysaccharide (LPS)-induced cytokine storm in human peripheral blood mononuclear cells (PBMCs) as an in-vitro cellular model. The cytotoxicity of AA30 was assessed in DPD-MSCs by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Growth factors and cytokine levels in the AA30-preconditioned DPD-MSC secretome were analysed by fluorescence-activated cell sorting (FACS) analysis. The angiogenic potential of the AA30-preconditioned DPD-MSC secretome was assessed by chick yolk-sac membrane (YSM) assay. Culture medium with 0.001% ethanol was used as vehicle control. The efficacy of the AA30-preconditioned DPD-MSC secretome in ameliorating the cytokine storm was assessed in LPS pre-treated PBMCs. The mRNA and protein expression of inflammatory markers such as IL-1β, IL-6 and IL-10 were analysed by using RT-PCR and FACS analysis respectively. AA30 did not exhibit cytotoxicity in the concentration range of 1% to 50%. Furthermore, the AA30-preconditioned DPD-MSC secretome exhibited a significant increase in the levels of angiogenic factors, such as human angiopoietin-2, EPO and PDGF-AA, and decreased levels of cytokines, such as TNF-α, CXCL-8 and IL-6. The AA30-preconditioned DPD-MSC secretome showed augmented angiogenesis compared to vehicle controls. The DPD-MSC secretome ameliorated LPS-induced mRNA and protein expression of IL-1β, IL-6 and IL-10 in PBMCs. The AA30-preconditioned DPD-MSC secretome augmented angiogenesis and ameliorated the LPS-induced cytokine storm in human PBMCs in vitro. Our data demonstrate that AA30 preconditioning enhances the therapeutic potency of MSCs and their secretome.

Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment.

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment.

Integration of CRISPR/Cas12a and Multiplexed RPA for Fast Detection of Gene Doping.

Fast and on-site detection is important for an effective antigene-doping strategy. However, the current gene doping (GD) evaluation methods require sophisticated instruments and laborious procedures, limiting their field applications. This study proposes a CRISPR/Cas12a-based detection platform (termed CasGDP) combining CRISPR/Cas12a and multiplexed Recombinase Polymerase Amplification (RPA) for rapid evaluation of GD. CasGDP showed high specificity for identifying the putative target genes such as EPO, IGF-1, and GH-1. By using fluorescence as the readout, the method achieved a limit-of-detection of 0.1 nM and 1 aM for unamplified and amplified target plasmids, respectively. Additionally, an in vitro GD cell model was successfully established with the human EPO gene (hEPO). The results indicated that the hEPO gene transfection promoted the hEPO protein expression. Furthermore, trace amounts of EPO transgene spiked in human serum were efficiently measured by CasGDP with fluorescence- and lateral flow device (LFD)-based readouts in 40 min. Finally, we designed a multiplexed microfluidic device and realized simultaneous detection of the three transgenes via LFD embedded in the device. To our knowledge, this is the first work that combines the CRISPR-based system and multiplexed RPA for GD detection. We anticipate CasGDP to be widely used as a rapid, sensitive, and robust tool for GD evaluation.

Therapeutic Effects of Mesenchymal Stem Cells Expressing Erythropoietin on Cancer-Related Anemia in Mice Model.

Cancer-related anemia (CRA) negatively influences cancer patients' survival, disease progression, treatment efficacy, and quality of life (QOL). Current treatments such as iron therapy, red cell transfusion, and erythropoietin-stimulating agents (ESAs) may cause severe adverse effects. Therefore, the development of long-lasting and curative therapies is urgently required. In this study, a cell and gene therapy strategy was developed for in vivo delivery of EPO cDNA by way of genetic engineering of human Wharton's jelly mesenchymal stem cells (hWJMSCs) to produce and secrete human EPO protein for extended periods after transplantation into the mice model of CRA. To evaluate CRA's treatment in cancer-free and cancerous conditions, first, a recombinant breast cancer cell line 4T1 which expressed herpes simplex virus type 1 thymidine kinase (HSV1-TK) by a lentiviral vector encoding HSV1-TK was developed and injected into mice. After three weeks, all mice developed metastatic breast cancer associated with acute anemia. Then, ganciclovir (GCV) was administered for ten days in half of the mice to clear cancer cells. Meanwhile, another lentiviral vector encoding EPO to transduce hWJMSCs was developed. Following implantation of rhWJMSCs-EPO in the second group of mice, peripheral blood samples were collected once a week for ten weeks from both groups. Analysis of peripheral blood samples showed that plasma EPO, hemoglobin (Hb), and hematocrit (Hct) concentrations significantly increased and remained at therapeutic for >10 weeks in both treatment groups. Data indicated that rhWJMSCs-EPO increased the circulating level of EPO, Hb, and Hct in both mouse subject groups and improved the anemia of cancer in both cancer-free and cancerous mice.

Tracking the ancestral functions of erythropoietin: neuroprotection & mitochondria

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis; now, it is known that EPO in mammal's brain plays key roles in the development, maintenance, protection, and repair of the nervous system. Also, EPO in mammals contributes to the efficient use of oxygen through the regulation of mitochondrial bioenergetic. Remarkably, a similar neuroprotective impact of recombinant human EPO (rhEPO) has been found in the brain of grasshoppers, raising questions about the evolutive origin of the EPO and its generic molecular function. The objective of this study is to show that the neuroprotective effect of rhEPO in insects involves the regulation of mitochondrial functions. The experiments were performed in crickets (Acheta domesticus). These insects were exposed under normoxia and hypoxia (5 days; 6% O2) conditions. Before experimentation, the animals were treated with EPO (30 IU/ml ‐ intra‐lymphatic injection) or PBS, as a control. The brains of the crickets were removed, and then we determined the mitochondrial respiration and production of mitochondrial ROS using our system oxygraphy ‐ 2K (ORORBOROS). Our results show that compares to normoxia; hypoxia significantly reduces mitochondrial respiration of complexes 1 and 1&2. On the other hand, the treatment of EPO in hypoxia, despite significantly increasing these parameters, does not recover the levels of mitochondrial respiration under normoxic conditions. In addition, the activity of complex IV (an indicator of the number of mitochondria) does not vary significantly between any of the treatments. Furthermore, we observed that while hypoxia did not significantly affect H2O2 production, the treatment with EPO increased ROS production under normoxic but not hypoxic conditions. Our data suggest that rhEPO regulates in some way the mitochondrial respiration and ROS production in the brain of crickets. Considering that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing, which could cause great oxidative stress due to the change in the metabolism of these animals, this molecule would have appeared as a regulator of mitochondrial functions.

A Reference Standard for Analytical Testing of Erythropoietin

PurposeErythropoietin (EPO) is a 165 amino acid protein that promotes the proliferation of erythrocytic progenitors. A decrease in endogenous EPO production causes anemia that can be treated with recombinant Human EPO (rHuEPO).ObjectiveTo ensure the safety and efficacy of the rHuEPO, manufacturers must use analytical methods to demonstrate similarity across batches and between different products. To do this they need reference standards to validate their equipment and methods.MethodWe used peptide mapping, size-exclusion chromatography, glycoprofiling, and isoelectric focusing to analyze a rHuEPO reference standard.ResultsCharacterization demonstrates that our rHuEPO reference standard meets the criteria for quality.ConclusionThe rHuEPO reference standard is fit for purpose as a tool for validating system suitability and methods.

Defining the specificity of recombinant human erythropoietin confirmation in equine samples by liquid chromatography‐tandem mass spectrometry

The proteotypic human EPO peptides YLLEAK (T4), SLTTLLR (T11), TITADTFR (T14), and VYSNFLR (T17) are often used to confirm the presence of recombinant human EPO (rhEPO) in equine samples. Each of these peptides contains one or more isomeric leucine or isoleucine amino acids, raising the possibility that a simple leucine/isoleucine substitution could lead to a false identification when compared with a rhEPO reference standard. To examine this possibility variants of these four peptides were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These studies indicate that confirmation of rhEPO in equine samples by immuno-affinity capture and LC-MS/MS analysis is true and accurate. It was also found that chromatography played a greater role in determining LC-MS/MS specificity than tandem mass spectrometry and that, in the case of more hydrophilic peptides, the accuracy of peptide identification could be enhanced by the inclusion of 13 C and 15 N labelled peptide internal standards.

Role of hepcidin as a biomarker for iron status and its effect on anemia management in patients with chronic kidney disease (stage II-Iv) after HCV treatment

Abstract Background Anemia is a severe complication of chronic kidney disease (CKD) that is seen in more than 80% of patients with impaired renal function. Although there are many mechanisms involved in the pathogenesis of anemia of renal disease, the primary cause is the inadequate production of erythropoietin by the damaged kidneys. Aim of the work to assess hepcidin level in non dialysis patients (CKD stage 4 &5) treated from Hepatitis C virus and its relation to iron parameters. Patients and Methods This study was conducted on 20 CKD patients (stage 4 and 5) treated from hepatitis C virus. All candidates included in this study subjected to careful history taking, full clinical examination and investigations (including complete blood count, renal chemistry, HCVAb, serum iron, total iron binding capacity, TSAT%, ferritin and hsCRP. Serum hepcidin was analyzed by ELISA technique. Results Serum hepcidin was 26.35±7.26; 40% in stage III, 37.8% in stage IV and 22.2% in stage V. There was statistically significant difference between GFR stages according to Hb., Drug intake ACE inhibitor/ARB, Plt., Creatinine, BUN, Iron, TIBC, Ferritin, T SAT%, CRP and Serum Hepcidin. We showed significant correlations between serum hepcidin and TIC, Iron, TIBC, Ferritin and TSAT%. Conclusion Median hepcidin value is elevated in nondialysis CKD patients due to increased inflammation and decreased clearance of hepcidin. Furthermore, iron status modifies serum hepcidin level and its association with Hb. Increased hepcidin level leads to iron-restricted erythropoiesis and recombinant human EPO (rhEPO) resistance by inhibiting iron absorption from gut and iron recycling from macrophages. Hence, elevated hepcidin can predict need for parenteral iron to overcome hepcidin-mediated iron-restricted erythropoiesis and need for relatively higher rhEPO doses to suppress hepcidin.

Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector.

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.

A Gain-of-Function Mutation in EPO Gene Causes Familial Erythrocytosis

Mutations in the erythropoietin-receptor gene (EPOR) are well known causes of inherited forms of primary erythrocytosis that is characterized by low serum erythropoietin (Epo) levels. Secondary erythrocytosis with elevated Epo serum levels is mainly caused by mutations in genes involved in oxygen sensing pathway. Epo is the primary regulator of erythropoiesis, but mutations in the EPO gene have so far not been described as a cause or erythrocytosis. Here we studied a Norwegian family with autosomal dominant erythrocytosis and elevated Epo serum levels.We performed genome-wide linkage analysis using SNP arrays on 16 informative family members including 10 affected individuals and identified a co-segregating region on chromosome 7q22.1 with a LOD score of 3.3. Targeted sequencing of all 215 genes within the co-segregating region revealed only a heterozygous single base deletion in exon 2 of EPO as the sole candidate gene mutation. ThisΔG deletion creates a frame-shift that truncates the Epo signal peptide and generates a novel peptide, terminating after additional 51 amino acids. Thus, contrary to the expectation based on the erythrocytosis phenotype, the EPO ΔG deletion was predicted to be a loss-of-function mutation.Since we had no access to patient's kidney or liver tissues where Epo is produced, we used the CRISPR technique to introduce the ΔG mutation into the EPO gene into Hep3B cells, a human hepatoma cell line that expresses EPO . We obtained two Hep3B single-cell derived clones (ΔG1 and ΔG4) that were homozygous for the EPO ΔG mutation and measured Epo protein in the culture supernatants. We found that these supernatants contained 8-10 times more Epo than parental Hep3B cells or controls, as determined by ELISA (Figure 1A), and were also capable of stimulating the growth of an Epo-dependent cell line (BaF3- EpoR), demonstrating biological activity (Figure 1B). Thus, the ΔG deletion introduced into the EPO gene locus in Hep3B cells behaved as a gain-of-function mutation.To decipher the mechanism of how the ΔG mutation was able to produce the excess of Epo protein, we searched for alternative EPO mRNAs by 5'-RACE. In addition to the expected transcripts originating from the physiologic promoter (P1) upstream of exon 1, we discovered two types of alternative mRNAs that initiate in intron 1 of EPO. Both initiatied from a putative alternative promoter (P2) in intron 1 that contains two GATA motifs. In addition to Hep3B ΔG clones, P2 transcripts were also detected in parental Hep3B cells and also in mRNA from normal kidney and liver.To determine which transcripts are capable of producing Epo, we transfected HEK293 cells with cDNAs representing P1 or P2 transcripts with or without the ΔG mutation. Supernatants of cells transfected with P2 ΔG transcripts contained more Epo than cells transfected with the wildtype P1 cDNA. These supernatants also stimulated the growth of Epo dependent BaF3- EpoR cells and supported erythroid colony formation of progenitors from human peripheral blood, demonstrating that P2 ΔG transcripts produce biologically active Epo protein.Analysis of the P2 mRNA sequences shows that the normally non-coding P2 transcripts through the ΔG single base deletion connect to the Epo coding sequence creating a functional signal peptide with a novel N-terminus, but retaining the physiologic Epo signal peptide cleavage site. These P2 ΔG transcripts are more abundant in liver and produce an excess of biologically active Epo by escaping the oxygen sensing regulation. Our data demonstrate for the first time mutation in EPO as a cause of familial erythrocytosis and provide an explanation of how the ΔG results in a gain-of-function mutation (Figure 1C).Figure 1 legend: A) Production of Epo protein by He3B cells carrying the ΔG mutation. Supernatants of parental Hep3B or CRISPR-modified Hep3B clones were collected and Epo protein was measured by Epo ELISA. The levels of Epo are given as a mean ± SD (n=3), p < 0.001 = *** as determined by one-way ANOVA. B) Biological activity of Epo produced by parental Hep3B or CRISPR-modified Hep3B cells measured by a proliferation assay with BaF3-EpoR cell line. BaF3-EpoR cells were grown in serial dilutions of supernatants and cell numbers were assessed by Cell Titer-Glo Assay, (n=3). Serial dilutions of 2.5 IU/ml of recombinant human Epo were used for reference. C) Model and proposed mechanism for EPO overproduction by a single nucleotide deletion in exon 2 of the EPO gene. [Display omitted] DisclosuresWaage:Celgene, Takeda: Honoraria. Skoda:Novartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Consultancy, Speakers Bureau.

Study Of The Influence Of Hypoxia On The Formation Of Erythrocytes From Induced Pluripotent Human Stem Cells

The production of erythrocytes from human hematopoietic stem cells (HSC) is considered one of the solutions to the deficiency of donor blood in transfusion medicine. The disadvantage of using HSC is the difficulty in multiplying in the required amount for use in transfusion. In this regard, pluripotent stem cells that can be multiplied in sufficient quantity may become a source of functional erythrocytes. The aim of this work was to study the effects of hypoxia on the formation of erythrocytes from human induced pluripotent stem cells (iPSCs) in ex vivo conditions. For the obtaining the erythroid bodies (EB), human iPSCs were cultured in induction medium containing human plasma and SCF, VEGF, BMP-4, TPO, EPO, interleukins IL-3 and IL-6 (20 days). EB treated with collagenase and cultured in a 96-well suspension culture plate under normoxia (21% O2) and hypoxia (2% O2) conditions in IMDM medium containing 20% ​​human plasma, SCF, IL-3, Epo, insulin and heparin. Erythroid maturation was evaluated by Giemsastaining and flow cytometry analysis ofCD235α. As a result of PSC differentiation, EBs with spherical morphology and sizes from 120 to 300 µm were obtained. Analysis for erythroid maturation showed that the number of mature erythrocytes with expression of CD235α after hypoxia averaged 56%, and after normoxia 19.3% of the total number of analyzed cells. The size of erythrocytes formed from iPSCs averaged 7 μm, which corresponds to the mature form of this type of cells. Thus, the obtained results of the study indicate that hypoxia plays a significant role in erythropoiesis, increasing the level of erythrocyte formation from human iPSCs.

Locust Hemolymph Conveys Erythropoietin-Like Cytoprotection via Activation of the Cytokine Receptor CRLF3.

The cytokine receptor-like factor 3 (CRLF3) is an evolutionary conserved class 1 cytokine receptor present in all major eumetazoan groups. Endogenous CRLF3 ligands have not been identified and the physiological responses mediated by mammalian CRLF3 are poorly characterized. Insect CRLF3 is activated by erythropoietin (Epo) and several related molecules that protect mammalian neurons from stress-induced apoptosis. However, insects neither express Epo nor “classical” Epo receptor. Cell-protective effects of insect hemolymph have been described for several species. In this study, we explored the possibility that the endogenous CRLF3 ligand is contained in locust hemolymph. PCR analyses confirmed expression of crfl3-transcripts in neurons and hemocytes of Locusta migratoria and Tribolium castaneum. Survival of locust hemocytes in primary cultures was significantly increased by supplementation of culture medium with locust hemolymph serum. Locust primary neuron cultures were also protected by locust hemolymph, though preceding exposure to fetal bovine serum changed the hemolymph dose-dependency of neuroprotection. Direct comparison of 10% hemolymph serum with recombinant human Epo in its optimal neuroprotective concentration revealed equivalent anti-apoptotic effects on hypoxia-exposed locust neurons. The same concentration of locust hemolymph serum also protected hypoxia-exposed T. castaneum neurons. This indicates that the neuroprotective factor in locust hemolymph is sufficiently conserved in insects to allow activation of neuroprotective receptors in different species. Locust hemolymph-induced neuroprotection in both L. migratoria and T. castaneum was abolished after RNAi-mediated suppression of crlf3-expression. In summary, we report the presence of a conserved endogenous cytokine in locust hemolymph that activates CRLF3 and connected anti-apoptotic processes in hemocytes and neurons. Identification and characterization of the CRLF3 ligand will promote knowledge about cytokine evolution and may unravel cell-protective agents with potential clinical application.

Regulated Basal, Inducible, and Tissue-Specific Human Erythropoietin Gene Expression in Transgenic Mice Requires Multiple cis DNA Sequences

Erythropoietin (Epo) gene expression in kidney and liver is inducible by anemia. To localize the sequences necessary for regulated expression of the Epo gene, we constructed transgenic mice containing five human Epo gene constructs and examined Epo expression under basal conditions and with anemia. Mice containing the Epo gene with 0.3 kb of 5' flanking sequence, 0.7 kb of 3' flanking sequence, and either all introns or only intron I alone were polycythemic, had Epo expression in various tissues (including non-Epo-producing tissues), and induction only in liver. In contrast, mice containing the Epo gene with 8.5 kb of 3' flanking sequence and either 9.5 or 22 kb of 5' flanking sequence had basal expression at low levels in appropriate tissues and were less likely to be markedly polycythemic. Mice with the smaller of these two constructs had induction only in the liver, whereas those with the larger construct had induction in the kidney and liver. These studies indicate that sequences sufficient for induction in the liver are located in close proximity to the Epo gene, including the immediate 5' and 3' flanking sequence and the first intron. They also indicate that sequences required for induction in the kidney are located more than 9.5 kb 5' to the gene. Furthermore, comparison of these and prior transgenic studies suggest that sequences that limit the basal expression of the Epo gene are located downstream of the gene. We conclude that multiple cis DNA sequences are required for regulated Epo gene expression.

Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis

BackgroundRenal tubulointerstitial fibrosis (TIF) plays an important role in the progression of chronic kidney disease (CKD) and its pathogenesis involves epithelial-to-mesenchymal transition (EMT) upon renal injury. Recombinant human erythropoietin (rhEPO) has been shown to display novel cytoprotective effects, in part by inhibiting transforming growth factor (TGF)-β1-induced EMT. Here, we evaluated the inhibitory effects of microparticles (MPs) derived from human EPO gene-transfected kidney mesenchymal stem cells (hEPO-KMSCs) against TGF-β1-induced EMT in Madin-Darby canine kidney (MDCK) cells and against TIF in mouse kidneys with unilateral ureteral obstruction (UUO).MethodsEMT was induced in MDCK cells by treatment with TGF-β1 (5 ng/mL) for 48 h and then inhibited by co-treatment with rhEPO (100 IU/mL), mock gene-transfected KMSC-derived MPs (MOCK-MPs), or hEPO-KMSC-derived MPs (hEPO-MPs) for a further 48 h. UUO was induced in FVB/N mice, which were then treated with rhEPO (1000 IU/kg, intraperitoneally, every other day for 1 week), MOCK-MPs, or hEPO-MPs (80 μg, intravenously). Alpha-smooth muscle actin (α-SMA), fibronectin, and E-cadherin expression were evaluated in MDCK cells and kidney tissues, and the extent of TIF in UUO kidneys was assessed by immunohistochemical staining.ResultsTGF-β1 treatment significantly increased α-SMA and fibronectin expression in MDCK cells and decreased that of E-cadherin, while co-treatment with rhEPO, MOCK-MPs, or hEPO-MPs markedly attenuated these changes. In addition, rhEPO and hEPO-MP treatment effectively decreased phosphorylated Smad2 and Smad3, as well as phosphorylated p38 mitogen-activated protein kinase (MAPK) expression, suggesting that rhEPO and rhEPO-MPs can inhibit TGF-β1-induced EMT via both Smad and non-Smad pathways. rhEPO and hEPO-MP treatment also significantly attenuated the extent of renal TIF after 1 week of UUO compared to MOCK-MPs, with hEPO-MPs significantly reducing myofibroblast and F4/80+ macrophage infiltration as well as EMT marker expression in UUO renal tissues in a similar manner to rhEPO.ConclusionsOur results demonstrate that hEPO-MPs modulate TGF-β1-induced EMT in MDCK cells via the Smad2, Smad3, and p38 MAPK pathways and significantly attenuated renal TIF in UUO kidneys.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre‐purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis‐stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG‐bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400‐fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

P1379PLATELET/LYMPHOCYTE RATIO AS A MARKER OF ERYTHROPOIETIN RESPONSIVENESS IN END-STAGE RENAL DISEASE

Abstract Background and Aims Neutrophil/Lymphocyte Ratio (NLR) and Platelet/Lymphocyte Ratio (PLR) are closely associated with increased inflammation in end-stage renal disease, which often contributes to the severity of anemia in these patients. Erythropoiesis stimulating agents (ESA) have become a standard treatment of anemia in hemodialysis patients. Since some patients do not respond well to erythropoietin therapy (EPO) the aim of this study is to investigate if NLR and PLR as markers of increased inflammation, could be associated with resistance to EPO therapy. Method A total of 90 patients (36 females, 54 males; mean age 60,45 ±11,58) undergoing maintenance hemodialysis and who received recombinant human EPO therapy were examined. Patients' clinical characteristics, laboratory data, dialysis adequacy and the applied doses os EPO were examined in a period of 3 months. EPO hyporesponsiveness index (EHRI) was calculated as the weekly dose of EPO divided by kilograms of body weight divided by the hemoglobin level. Results Obtained results show a statistically significant correlation of moderate-intensity between EHRI and NLR ( r = 0.497, p &amp;lt; 0.01) as well as a negative correlation of moderate-intensity between EHRI and hemoglobin levels (Hgb) (r = -0.403, p &amp;lt; 0.01). When it comes to the connection of NLR and PLR with logarithmically converted EHRI values, the results show that there is no statistically significant correlation between NLR and EHRI. Comparison of PLR among 25th, 50th and 75th percentile of EHRI showed that PLR levels increased going from the 25th towards the 75th percentile (p &amp;lt; 0.01). Post hoc analysis indicated that there is also a statistically strong connection for the 25th i 50th percentile (&amp;lt;0 .05) and furthermore for the 50th and 75th percentile (&amp;lt; 0.05). Conclusion PLR was found to be superior to NLR in terms of evaluating ESA therapy resistance. PLR could be used as a predictor of ESA therapy response.

Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles

Plant-based expression system has many potential advantages to produce biopharmaceuticals, but plants cannot be directly used to express human glycoproteins because of their differences in glycosylation abilities from mammals. To exploit plant-based expression system for producing recombinant human erythropoietin (rhuEPO), we glycoengineered tobacco plants by stably introducing seven to eight mammalian genes including a target human EPO into tobacco in order to generate capacities for β1,4-galactosylation, bisecting N-acetylglucosamine (GlcNAc) and sialylation. Wild type human β1,4-galactosyltransferase gene (GalT) or a chimeric GalT gene (ST/GalT) was co-expressed to produce rhuEPO bearing β1,4-galactose-extended N-glycan chains as well as compare their β1,4-galactosylation efficiencies. Five mammalian genes encoding enzymes/transporter for sialic acid biosynthesis, transport and transfer were co-expressed to build sialylation capacity in plants. The human MGAT3 was co-expressed to produce N-glycan chains with bisecting GlcNAc. Our results demonstrated that the above transgenes were incorporated into tobacco genome and transcribed. ST/GalT was found to be more efficient than GalT for β1,4-galactosylation. Furthermore, co-expressing MGAT3 generated N-glycans likely bearing bisected GlcNAc. However, our current efforts did not result in generating sialylation capacity. Created transgenic plants expressing EPO and ST/GalT could be used to produce rhuEPO with high proportion of β1,4-galactose-extended N-glycan chains for tissue protective purposes.

Tracking the ancestral functions of erythropoietin: neuroprotection &amp; mitochondria

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis, allowing erythroid progenitor cells to survive and mature through their antiapoptotic action. We now know that Epo in mammals has also other functions in the brain, playing key roles in the development, maintenance, protection, and repair of the nervous system. However, the recombinant human Epo (rhEpo) has neuroprotective effects in orthoptera insects (such as grasshoppers), and this effect appears to be mediated by the cytokine receptor‐like factor 3 (CRLF3), raising interesting questions about the evolutionary origin of the Epo signaling pathway and its role in invertebrate species. Taking into account that: 1) Epo in mammals modulates the mitochondrial oxidative phosphorylation and the production of reactive oxygen species (ROS) in several tissues, including the brain; and 2) that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing and required the implementation of antioxidant systems at the cellular level; here we tested the hypothesis that activation of the “Epo‐like” system in the brain domestic crickets (Acheta domesticus) exposed to 6 % of hypoxia during 5 days, modulates mitochondrial functions for preventing against oxidative damages. To do so, we used our oxygraph‐2K system (OROBOROS) that measures the mitochondrial bioenergetics in saponin‐permeabilized tissue of 2 mg weight. Our preliminary results showed that rhEpo increased the survival of domestic crickets exposed to hypoxia by 20%. We showed also that, in normoxic animals, rhEpo increased the mitochondrial O2 consumption rate (OCR), but in hypoxic animals, rhEPO limited the increase of mitochondrial OCR. In parallel, rhEpo significantly decreased the production of ROS in hypoxia. These preliminary results suggest that rhEpo significantly improves cricket’s survival under hypoxia, by promoting a robust antioxidant effect through mitochondria. Our data also suggest that a neuroprotective “Epo‐like” endogenous molecule evolved during the “Cambrian explosion” from a urbilaterian (common to vertebrates and invertebrates) ancestor.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada