Erythropoietin Receptor Research Articles

Treatment of Chronic Kidney Diseases with Darpeboetin can Ameliorate Kidney Functions: A Possible Clinical Relevance

Background: Erythropoietin (EPO) has been used clinically for the treatment of anemia as it was the first characterized hematopoietic factor. Erythropoietin is produced primarily by the kidneys (renal cortical fibroblasts) which induces proliferation and differentiation of erythroid progenitor cells. Previous studies suggested that erythropoietin may directly stimulates the regeneration of damaged tubular cells due to presence of erythropoietin receptors on renal tubular epithelial, mesangial and endothelial cells. EPO directly prevent glomerulosclerosis as a consequence of amelioration of podocyte injury. Therefore, to improve kidney function in chronic kidney disease (CKD), it is of critical importance to investigate clinically the beneficial reno-protective effects of EPO in patients with CKD. Methodology: The study was conducted at King Abdulaziz Medical City and included patients with chronic kidney diseases. Patients were under medical treatment with Darpeboetin for the first time. The administration frequency of Darpeboetin was single dose weekly. Kidney function tests including serum creatinine, serum urea, alkaline phosphatase, serum calcium, serum phosphorus, serum albumin and albuminuria were collected from the hospital electronic medical record system (BestCareR). Test values before and after treatment were compared. of the patients before starting treatment and at the end of treatment. The week before starting treatment was considered week 0 (baseline) Results: The results indicated that, treatment with a weekly single dose of Darpeboetin reduces blood urea nitrogen (BUN p=0.002) and serum uric acid level (p=0.009). The effect of treatment on serum calcium and potassium, showed a significant elevation of serum potassium (P<0.001), serum calcium (P<0.001) and adjusted calcium (P<0.001) levels parallel with significant decrease in serum phosphorus (p=0.004). Also treatment of chronic kidney disease patients significantly ameliorates serum albumin (P<0.001). Conclusion: Treatment with Darpeboetin may confirm the reno-protective potency. Darpeboetin is a promising drug to prevent or attenuate tissues kidney damage especially in chronic kidney disease

Erythropoietin regulates developmental myelination in the brain stimulating postnatal oligodendrocyte maturation

Myelination is a process tightly regulated by a variety of neurotrophic factors. Here, we show—by analyzing two transgenic mouse lines, one overexpressing EPO selectively in the brain Tg21(PDGFB-rhEPO) and another with targeted removal of EPO receptors (EPORs) from oligodendrocyte progenitor cells (OPC)s (Sox10-cre;EpoRfx/fx mice)—a key function for EPO in regulating developmental brain myelination. Overexpression of EPO resulted in faster postnatal brain growth and myelination, an increased number of myelinating oligodendrocytes, faster axonal myelin ensheathment, and improved motor coordination. Conversely, targeted ablation of EPORs from OPCs reduced the number of mature oligodendrocytes and impaired motor coordination during the second postnatal week. Furthermore, we found that EPORs are transiently expressed in the subventricular zone (SVZ) during the second postnatal week and EPO increases the postnatal expression of essential oligodendrocyte pro-differentiation and pro-maturation (Nkx6.2 and Myrf) transcripts, and the Nfatc2/calcineurin pathway. In contrast, ablation of EPORs from OPCs inactivated the Erk1/2 pathway and reduced the postnatal expression of the transcripts. Our results reveal developmental time windows in which EPO therapies could be highly effective for stimulating oligodendrocyte maturation and myelination.

Dried blood spots for erythropoietin analysis: Detection of micro-doses, EPO c.577del variant and comparison with in-competition matching urine samples.

The abuse of recombinant erythropoietin (rEPO) and other erythropoietin (EPO) receptor agonists (ERAs) in sports prompted the need for sensitive detection methods of these substances. Dried blood spot (DBS) samples offer an easy solution for simultaneous collection of blood and urine during a doping control, but sensitivity issues are often presented as a challenge for routine EPO analysis from DBS. Its potential use for detecting rEPO micro-doses and the EPO gene c.577del variant thus needed further demonstration. Here, capillary blood collected from the arm skin of 111 athletes with Tasso-M20 (17.5μL/spot), collected during professional triathlon competitions, were analysed. Also, venous blood samples from healthy volunteers were used to prepare several spots of 20 μL on Mitra VAMS (from an rEPO micro-dose study) and Whatman filter paper (from an EPO gene variant study). Immunopurification of 2 spots with MAIIA EPO Purification Gel Kit and analysis with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE)/Western blot resulted in sensitive detection of (1) micro-doses of rEPO from Mitra VAMS, (2) endogenous EPO from Tasso-M20 in all in-competition subjects, and (3) the EPO c.577del variant from Whatman filter paper. Additionally, in-competition endogenous EPO was detected in DBS even when matching urine samples had undetectable EPO. In conclusion, this work demonstrated that DBS can be a useful complementary matrix to urine samples for EPO detection.

Using Human Genetics to Develop Strategies to Increase Erythropoietic Output from Genome-Edited Hematopoietic Stem and Progenitor Cells

Genome editing is a powerful tool that enables precise changes in the genetic code of a cell. Genome-edited hematopoietic stem and progenitor cells (HSPCs) yield genome-corrected cells of all lineages, yet the only cell type of clinical relevance in red blood cell (RBC) disorders is the RBC. Most applications of genome editing have sought to correct disease-causing mutations of monogenic diseases. However, human genetic variation reveals variants that confer positive health benefits as well. In this study we took advantage of a naturally occurring human variant to increase erythropoietic output from genome-edited HSPCs. The variant leads to a condition termed congenital erythrocytosis (CE), a rare phenotype in which people have higher than normal levels of RBCs and consequently elevated hemoglobin. It is caused by truncations in the erythropoietin receptor (tEPOR) in which the intracellular inhibitory domain to erythropoietin (EPO) signaling is eliminated. The genetic cause of this phenotype was identified in a family of a Finnish Olympic gold medal-winning cross-country skier who was found to have levels of hemoglobin >50% higher than normal. One of the challenges in gene therapy is to achieve sufficient engraftment of genetically engineered cells to have a clinical effect. Here we sought to give edited cells a selective advantage such that low levels of engraftment might still result in a clinical benefit through use of genome editing to recreate the CE phenotype by engineering tEPOR into HSPCs in different ways. We hypothesized we could recapitulate the CE variant using CRISPR/Cas9 genome editing of HSPCs to create insertions/deletions (indels) at the EPOR locus near the site of the mutation ( EPOR c.1316G>A) and then transfer the cells into culture media that promotes erythroid differentiation (Fig. 1). We demonstrated increased erythropoietic output from cells containing truncating mutations in EPOR, measured by an increase in indel formation throughout erythroid differentiation (1.3-3.8-fold increase). Because not all indels created cause truncations in EPOR, we explored if we could amplify this effect by integrating an AAV6 repair template that introduces a stop codon into EPOR followed by a BGH-polyA tail and GFP marker driven by a constitutive UbC promoter. This strategy enriched for GFP + cells with the truncation even more dramatically as they developed during erythroid differentiation (2.5-4.6-fold increase). We also found a substantial increase in the total number of cells produced from the tEPOR edited condition at the end of differentiation (3.3 times greater fold increase compared to mock edited cells). Importantly, we observed no enrichment in cells kept in HSPC media or cultured in RBC media without EPO, indicating this effect is EPO-dependent (Fig. 2). Next, we investigated whether a tEPOR cDNA could be targeted to the CCR5 safe harbor locus under ubiquitous expression or to the HBA1 RBC-specific safe harbor site imparting high levels of erythroid specific expression. Integration of the tEPOR cDNA at both loci yielded strong enrichment of edited alleles in an EPO-dependent manner when put through erythroid differentiation (6.2-8.6-fold increase at CCR5 and 2.9-5.2 at HBA1). Lastly, we sought to pair the tEPOR cDNA with a clinical edit for β-thalassemia to boost production of disease-corrected RBCs. We showed that through use of bi-cistronic cassettes we were able to drive a >2-fold enrichment of edited alleles over the course of differentiation. Additionally, we were able to pair the tEPOR cDNA and clinical edit for β-thalassemia through multiplexed gene editing at EPOR and HBA1, respectively, and again demonstrated enrichment of edited alleles. Ultimately, we believe this work has the potential to amplify the efficacy of gene and cell therapies for blood disorders currently in the clinic. Expression of tEPOR could be integrated into any treatment for blood disorders that involve transplantation of HSPCs, including both allogeneic and autologous bone marrow transplants. Further, as low chimerism in the bone marrow could produce high levels of peripheral RBCs using our strategy, it has the potential to reduce the morbidity of myeloablation regimens currently required for cure of blood disorders. This work, in effect, has the potential to improve the safety and accessibility to currently available treatments for the millions of patients affected by RBC disorders worldwide.

Rusfertide Improves Markers of Iron Deficiency in Patients with Polycythemia Vera

Background: Polycythemia vera (PV) is a chronic myeloproliferative neoplasm characterized by erythrocytosis defined by an acquired increase in hemoglobin (Hb)/hematocrit (HCT). Disease complications are associated with increased blood viscosity, cardiovascular/thrombotic events and iron deficiency (Marchioli et al. N Engl J Med. 2013;368:22-33). Most patients with PV are iron deficient and therapeutic phlebotomy may exacerbate iron deficiency (Ginzburg et al. Leukemia. 2018;32:2105-16). Methods:PTG-300-08 (clinicaltrials.gov NCT04767802, PACIFIC study) is an open-label, 52-week, phase 2 study that investigated the efficacy and safety of rusfertide in Asian patients with poor hematocrit (HCT) control as noted by HCT >48%. Treatment was initiated with 40 mg rusfertide subcutaneously (SC) twice a week. Once HCT was <45%, the treatment was changed to 40 mg once a week with subsequent dose adjustments allowed to maintain HCT <45%. In addition to HCT and erythrocyte morphology (mean corpuscular volume, or MCV), biomarkers reflective of iron biology, such as hepcidin, erythroferrone (ERFE), erythropoietin (EPO), and soluble transferrin receptor (sTfR) were measured at baseline, Week 4, 16, and 32 following initiation of rusfertide treatment. To examine the effect of rusfertide on serum iron, patients were stratified by baseline ferritin levels to examine patients who were iron deficient (ferritin ≤20 µg/L) compared to patients who had normal iron (ferritin >20 µg/L). Results: Twenty patients (15 males, 5 females), average age 56.7 (SD, 9.12) years and average weight 65 (10.9) kg were enrolled. Thirteen patients (65%) were low risk and 7 (35%) were high risk, all based on age. No patients had a prior history of thromboembolic events. Mean duration since PV diagnosis was 4.2 (3.83) years. Rusfertide rapidly and robustly reduced mean HCT and sustained HCT <45% for the duration of treatment, and ferritin, TSAT and MCV increased (Table 1). At baseline, 30% of subjects had serum ferritin levels below 20 µg/L, with all subjects achieving and sustaining ferritin levels above 20 µg/L by Week 4 following initiation of rusfertide treatment. Table 1 also summarizes the biomarkers related to iron indices. Median hepcidin concentrations increased, median EPO concentration was below and remained below the limit of detection, and ERFE concentration decreased modestly with rusfertide dosing consistent with reduced erythropoietic drive. Among patients with evidence of baseline iron deficiency (ferritin <20 µg/L), rusfertide normalized iron levels while iron levels remained stable in rusfertide patients with normal iron levels at baseline (Figure 1). Similarly, patients with iron deficiency had low MCV at baseline (Figure 1) which increased following initiation of rusfertide treatment. In patients with normal iron levels, MCV remained stable in the normal range (>80 fl). Conclusions: Treatment with rusfertide resulted in rapid, robust, and sustained reduction in HCT levels in PV patients with elevated HCT levels at baseline. Treatment resulted in normalization of iron parameters as noted by increases in ferritin, and normalization of serum iron and MCV in patients who were iron deficient at baseline. Improvements in iron deficiency following rusfertide are suggestive of clinical benefits and merit further investigation. This analysis confirms the general understanding that managing iron deficiency in PV remains an unmet need.

Autoantibodies to Erythropoietin Receptor and Clinical Outcomes in Patients with Type 2 Diabetes and CKD: A Post Hoc Analysis of the CREDENCE Trial

Autoantibodies to Erythropoietin Receptor and Clinical Outcomes in Patients with Type 2 Diabetes and CKD: A Post Hoc Analysis of the CREDENCE Trial

Erythropoietin receptor is a risk factor for prognosis: A potential biomarker in lung adenocarcinoma

Lung cancer has the highest mortality rate of all cancers, and LUAD's survival rate is particularly poor. Erythropoietin receptor (EPOR) can be detected in lung adenocarcinoma (LUAD), however, the expression levels and prognostic value of EPOR in LUAD are still unclear. In our study, clinicopathological data of 92 LUAD patients between January 2008 and June 2016, multiple bioinformatics databases and immunohistochemistry were used to explore the EPOR expression, the mutant genes affecting EPOR expression, and the correlation of EPOR expression with oxidative stress - related genes, prognosis, immune microenvironment. All statistical analyses were performed in the R version 4.1.1. The study found that EPOR expression might be down-regulated at the mRNA levels and significantly up-regulated at the protein levels in LUAD, which indicates that the mRNA and protein levels of EPOR are inconsistent. The muTarget showed that the expression of EPOR was significantly different between the mutant group and the wild group of 15 genes, including DDX60L and C1orf168. Importantly, we found that EPOR was associated with VEGF and HIF family members, and had significant positive correlation with oxidative stress - related genes such as CCS, EPX and TXNRD2. This suggests that EPOR may be involved in the regulation of oxidative stress. The Kaplan-Meier Plotter and PrognoScan databases consistently concluded that EPOR was associated with prognosis in LUAD patients. Our clinicopathological data showed that high EPOR expression was associated with poorer overall survival (29.5 vs 46 months) and had a good predictive ability for 4-year and 5-year survival probability. EPOR is expected to be a potential new prognostic marker for LUAD.

Clinical Experience of a Long-acting Pegylated Erythropoietin-Stimulating Agent in Pediatric Chronic Kidney Disease.

Management of anemia of chronic kidney disease (CKD) often includes subcutaneous or intravenous administration of erythropoietin-stimulating agents (ESAs). Mircera, a pegylated continuous erythropoietin receptor agonist, has a longer duration of action and requires less frequent administration than other ESAs. Pediatric experience with Mircera is limited. We retrospectively reviewed our long-term experience of Mircera in a national pediatric nephrology center. Patients were identified via an electronic patient record database. Data collected included demographics (sex, age, etiology of CKD, CKD stage, dialysis modality), dosing information, and laboratory data-hemoglobin (Hb), parathormone (PTH), ferritin, hematinics prior to commencing Mircera and all subsequent values associated with dose adjustments. Seventy-seven patients aged 2 to 18 years, with CKD stages 2 to 5T had received at least 1 dose of Mircera, with 75 patients having sufficient data and a total of 1473 doses. No patients discontinued Mircera owing to adverse effects. One patient experienced a potential severe adverse drug reaction. Mircera was effective in improving or maintaining Hb ≥10.0 g/dL in most (58/75, 77.3%) patients. The median dose to achieve Hb ≥10.0 g/dL was 2.1 µg/kg/4 wk. Most doses (1039, 71.5%) were administered 4-weekly. The doses (161, 11.1%) that were administered 6-weekly remained efficacious. Thirty-two patients started Mircera with Hb <10.0 g/dL; 26 (81%) achieved Hb ≥10.0 g/dL within a median time of 4 months. Mircera was less effective if given every 8 weeks, or in the presence of hyperparathyroidism or hyperferritinemia. Mircera appears safe and effective in pediatric patients with CKD.

Recombinant Erythropoietin Induces Oligodendrocyte Progenitor Cell Proliferation After Traumatic Brain Injury and Delayed Hypoxemia

Traumatic brain injury (TBI) can result in axonal loss and demyelination, leading to persistent damage in the white matter. Demyelinated axons are vulnerable to pathologies related to an abnormal myelin structure that expose neurons to further damage. Oligodendrocyte progenitor cells (OPCs) mediate remyelination after recruitment to the injury site. Often this process is inefficient due to inadequate OPC proliferation. To date, no effective treatments are currently available to stimulate OPC proliferation in TBI. Recombinant human erythropoietin (rhEPO) is a pleiotropic neuroprotective cytokine, and its receptor is present in all stages of oligodendroglial lineage cell differentiation. Therefore, we hypothesized that rhEPO administration would enhance remyelination after TBI through the modulation of OPC response. Utilizing a murine model of controlled cortical impact and a primary OPC culture in vitro model, we characterized the impact of rhEPO on remyelination and proliferation of oligodendrocyte lineage cells. Myelin black gold II staining of the peri-contusional corpus callosum revealed an increase in myelinated area in association with an increase in BrdU-positive oligodendrocytes in injured mice treated with rhEPO. Furthermore, morphological analysis of OPCs showed a decrease in process length in rhEPO-treated animals. RhEPO treatment increased OPC proliferation after in vitro CSPG exposure. Erythropoietin receptor (EPOr) gene knockdown using siRNA prevented rhEPO-induced OPC proliferation, demonstrating that the rhEPO effect on OPC response is EPOr activation dependent. Together, our findings demonstrate that rhEPO administration may promote myelination by increasing oligodendrocyte lineage cell proliferation after TBI.

Continuous Erythropoietin Receptor Activator for the Treatment of Chronic Dialysis Patients with Renal Anemia in Daily Clinical Practice in Poland: A Non-Interventional, Multi-Center, Pragmatic NAVIGO Trial

Background: Renal anemia is one of the most common complications of chronic kidney disease (CKD). This real-life study assessed the effectiveness of methoxy polyethylene glycol-epoetin beta, a continuous erythropoietin receptor activator (C.E.R.A.), for the treatment of CKD-associated anemia in patients receiving dialysis in daily clinical practice. Methods: 247 patients receiving chronic intermitted dialysis in 26 centers in Poland with CKD-associated symptomatic anemia, ESA-naïve, and with balanced iron stores in the investigators’ opinion were enrolled this real-life study. Over 12 months, the following data were collected: hemoglobin (Hb) concentration and dosage, route of administration and dosing scheme of C.E.R.A., dialysis adequacy, adverse events, iron therapy, and blood transfusions. Results: During the treatment, a Hb concentration of ≥10 g/dL was noted in 90.9% of hemodialysis patients (n = 224) and 96.0% of peritoneal dialysis patients (n = 23). At baseline, 7.8% of patients had a Hb concentration of 10–12 g/dL, which increased to 63.3% after 12 months. The median time when Hb concentration was maintained within 10–12 g/dL was 115.2 (interquartile range 49.1–188.7) days. A Hb concentration ≥12 g/dL was observed after 7 months of treatment in a maximum of 24.1% of hemodialysis patients, and 31.8% of peritoneal dialysis patients. The median time elapsed between the start of treatment and the first Hb concentration >10 g/dL was 42.0 (21.0–78.2) days. C.E.R.A. was well tolerated. Conclusions: C.E.R.A. corrects CKD-associated anemia in dialysis patients, and maintains Hb levels within the recommended target range. The study also confirmed the acceptable safety profile of the drug.

Achieving routine application of dried blood spots for erythropoietin receptor agonist analysis in doping control: low-volume single-spot detection at minimum required performance level.

Background: Erythropoietin receptor agonists (ERAs) are substances prohibited in sports and currently monitored in urine and blood. There is a great interest in new matrices like dried blood spots (DBSs). Method: A direct method for the detection of ERAs in DBSs using one single spot of 25μl has been optimized and validated. Results: Limits of detection close or equal to those required by the World Anti-Doping Agency for serum/plasma samples were achieved, using a volume 20-times lower. All analytes were stable for at least 90days at room temperature. Conclusion: Method performance was comparable to the requirements established for blood samples and, thus, monitoring of ERAs is reliable in DBSs in the context of doping control.

Real-world evidence on the dosing and safety of C.E.R.A. in pediatric dialysis patients: findings from the International Pediatric Dialysis Network registries

BackgroundThis retrospective real-world study used data from two registries, International Pediatric Peritoneal Dialysis Network (IPPN) and International Pediatric Hemodialysis Network (IPHN), to characterize the efficacy and safety of continuous erythropoietin receptor activator (C.E.R.A.) in pediatric patients with chronic kidney disease (CKD) on peritoneal dialysis (PD) or hemodialysis (HD).MethodsIPPN and IPHN collect prospective data (baseline and every 6 months) from pediatric PD and HD centers worldwide. Demographics, clinical characteristics, dialysis information, treatment, laboratory parameters, number and causes of hospitalization events, and deaths were extracted for patients on C.E.R.A. treatment (IPPN: 2007–2021; IPHN: 2013–2021).ResultsWe analyzed 177 patients on PD (median age 10.6 years) and 52 patients on HD (median age 14.1 years) who had ≥ 1 observation while being treated with C.E.R.A. The median (interquartile range [IQR]) observation time under C.E.R.A. exposure was 6 (0–12.5) and 12 (0–18) months, respectively. Hemoglobin concentrations were stable over time; respective means (standard deviation) at last observation were 10.9 (1.7) g/dL and 10.4 (1.7) g/dL. Respective median (IQR) monthly C.E.R.A. doses at last observation were 3.5 (2.3–5.1) µg/kg, or 95 (62–145) µg/m2 and 2.1 (1.2–3.4) µg/kg, or 63 (40–98) µg/m2. Non-elective hospitalizations occurred in 102 (58%) PD and 32 (62%) HD patients. Seven deaths occurred (19.8 deaths per 1000 observation years).ConclusionsC.E.R.A. was associated with efficient maintenance of hemoglobin concentrations in pediatric patients with CKD on dialysis, and appeared to have a favorable safety profile. The current analysis revealed no safety signals.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

Subchronic Arsenite Exposure Induced Atrophy and Erythropoietin Sensitivity Reduction in Skeletal Muscle Were Relevant to Declined Serum Melatonin Levels in Middle-Aged Rats.

Arsenic is a kind of widespread environmental toxicant with multiorgan-toxic effects, and arsenic exposure is associated with the occurrence and development of many chronic diseases. The influence of environmental arsenic exposure on skeletal muscle, which is a vital organ of energy and glucose metabolism, has received increasing attention. This study aimed to investigate the types of inorganic arsenic-induced skeletal muscle injury, and the potential regulatory effects of melatonin (MT) and erythropoietin (EPO) in young (3-month-old) and middle-aged (12-month-old) rats. Our results showed that 1 mg/L sodium arsenite exposure for 3 months could accelerate gastrocnemius muscle atrophy and promote the switch of type II fibers to type I fibers in middle-aged rats; however, it did not cause significant pathological changes of gastrocnemius muscle in young rats. In addition, arsenite could inhibit serum MT levels, and promote serum EPO levels but inhibit EPO receptor (EPOR) expression in gastrocnemius muscle in middle-aged rats, while serum MT levels and EPOR expression in gastrocnemius muscle showed an opposite effect in young rats. Importantly, exogenous MT antagonized the arsenite-induced skeletal muscle toxic effect and restored serum EPO and gastrocnemius muscle EPOR expression levels in middle-aged rats. There was a positive correlation among gastrocnemius muscle index, serum MT level, and gastrocnemius muscle EPOR protein level in arsenite-exposed rats. This study demonstrated that inorganic arsenic could accelerate skeletal muscle mass loss and type II fiber reduction in middle-aged rats, which may be related to decreased MT secretion and declined EPO sensitivity in skeletal muscle.

Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis.

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.

Mitochondrial modulation of amplified preconditioning influences of remote ischemia plus erythropoietin against skeletal muscle ischemia/reperfusion injury in rats

AimsSkeletal muscle ischemia and reperfusion (S-I/R) injury is relieved by interventions like remote ischemic preconditioning (RIPC). Here, we tested the hypothesis that simultaneous exposure to a minimal dose of erythropoietin (EPO) boosts the protection conferred by RIPC against S-I/R injury and concomitant mitochondrial oxidative and apoptotic defects. Main methodsS-I/R injury was induced in rats by 3-h right hindlimb ischemia followed by 3-h of reperfusion, whereas RIPC involved 3 brief consecutive I/R cycles of the contralateral hindlimb. Key findingsS-I/R injury caused (i) rises in serum lactate dehydrogenase and creatine kinase and falls in serum pyruvate, (ii) structural deformities like sarcoplasm vacuolations, segmental necrosis, and inflammatory cells infiltration, and (iii) decreased amplitude and increased duration of electromyography action potentials. These defects were partially ameliorated by RIPC and dose-dependently by EPO (500 or 5000 IU/kg). Further, greater repairs of S-I/R-evoked damages were seen after prior exposure to the combined RIPC/EPO-500 intervention. The latter also caused more effective (i) preservation of mitochondrial number (confocal microscopy assessed Mitotracker red staining) and function (citrate synthase activity), (ii) suppression of mitochondrial DNA damage and indices of oxidative stress and apoptosis (succinate dehydrogenase, myeloperoxidase, cardiolipin, and cytochrome c), (iii) preventing calcium and nitric oxide metabolites (NOx) accumulation and glycogen consumption, and (iv) upregulating EPO receptors (EPO-R) gene expression. Significancedual RIPC/EPO conditioning exceptionally mends structural, functional, and neuronal deficits caused by I/R injury and interrelated mitochondrial oxidative and apoptotic damage. Clinically, the utilization of relatively low EPO doses could minimize the hormone-related adverse effects.

Intranasal erythropoietin protects granular cells and reduces astrogliosis in the dentate gyrus after ischemic damage, an effect associated with molecular changes in erythropoietin and its receptor

Within the hippocampus, the CA1 and dentate gyrus (DG) regions are considered the most and the least susceptible to damage by cerebral ischemia, respectively. In addition, it has been tested that rHuEPO exhibits neuroprotective properties. This work investigates the effect of different intranasal doses of rHuEPO, applied in different ischemic post-damage times in the DG, and the effect of the rHuEPO on astroglial reactivity after cerebral ischemia. Additionally, an effective dose for neuroprotection and an administration time was used to evaluate gene and protein expression changes of EPO and EPOR in the DG region. We observed a considerable loss of cells on the granular layer and an increased number of GFAP immunoreactive cells in this region only 72 h after the onset of ischemia/damage. When rHuEPO was administered, the number of morphologically abnormal cells and immunoreactivity decreased. In the analysis of protein and gene expression, there is no correlation between expression level of these molecules, although the rHuEPO amplifies the response to ischemia of EPO and EPOR gene for each evaluated time; in the case of the protein only at 2 h this effect was observed. We demonstrated the susceptibility of the DG to ischemia; so granular cells damage was observed, moreover of the astrocytic response, which is accompanied by molecular changes in signaling mediated by rHuEPO intranasal administration.

EpoR Activation Stimulates Erythroid Precursor Proliferation by Inducing Phosphorylation of Tyrosine-88 of the CDK-Inhibitor p27Kip1.

Erythrocyte biogenesis needs to be tightly regulated to secure oxygen transport and control plasma viscosity. The cytokine erythropoietin (Epo) governs erythropoiesis by promoting cell proliferation, differentiation, and survival of erythroid precursor cells. Erythroid differentiation is associated with an accumulation of the cyclin-dependent kinase inhibitor p27Kip1, but the regulation and role of p27 during erythroid proliferation remain largely unknown. We observed that p27 can bind to the erythropoietin receptor (EpoR). Activation of EpoR leads to immediate Jak2-dependent p27 phosphorylation of tyrosine residue 88 (Y88). This modification is known to impair its CDK-inhibitory activity and convert the inhibitor into an activator and assembly factor of CDK4,6. To investigate the physiological role of p27-Y88 phosphorylation in erythropoiesis, we analyzed p27Y88F/Y88F knock-in mice, where tyrosine-88 was mutated to phenylalanine. We observed lower red blood cell counts, lower hematocrit levels, and a reduced capacity for colony outgrowth of CFU-Es (colony-forming unit-erythroid), indicating impaired cell proliferation of early erythroid progenitors. Compensatory mechanisms of reduced p27 and increased Epo expression protect from stronger dysregulation of erythropoiesis. These observations suggest that p27-Y88 phosphorylation by EpoR pathway activation plays an important role in the stimulation of erythroid progenitor proliferation during the early stages of erythropoiesis.

239-LB: Erythropoietin Alters Fat Mass and Gut Microbial Composition

Erythropoietin is an essential hormone for promoting erythropoiesis. Erythropoietin also stimulates angiogenesis, affects fat metabolism and can promote survival in cells expressing erythropoietin receptor. Here, we found that erythropoietin reduces fat mass, decreasing epididymis fat pad mass and subcutaneous fat pad mass of Tg6 male mice that express high transgenic erythropoietin. However, how erythropoietin activity in the gut improves fat mass and glucose metabolism is not known. Tg6 male mice have increased microvilli height and intestine length. High erythropoietin changes the gut microbiota by altering β-diversity that includes increasing the abundance of Bifidobacterium and reducing the Lactobacillaceae in Tg6 male mice. EPO decreased lactate dehydrogenase (LDHa) expression and reduces lactate production from intestinal endothelial cells that affects the gut microbiota components. Accompanying the altered microbiome are increased intestinal short-chain fatty acid levels that can affect fat metabolism. Transplantation of fecal pellet from Tg6 male mice into antibiotic treated male wild type mice reduces fat mass and mimics the lean phenotype of Tg6 mice. Conversely, transplanting feces from WT mice into Tg6 mice resulted in increased fat mass in Tg6 mice. Wild type male mice treated with erythropoietin show improved glucose tolerance and also exhibit increased intestine length and changes in the gut microbiota with increase in Bifidobacterium and decrease in Lactobacillaceae, analogous to changes in Tg6 male mice. Our results on erythropoietin activity in the gut to change gut morphology and remodel the gut microbiome provide a new mechanism by which EPO affects fat metabolism. Key words: EPO; EPOR; gut microbial composition; fat mass Disclosure W. Yin: None. H. Rogers: None. X. An: None. M. Gassmann: None. C. T. Noguchi: None.

Deregulating iron-erythropoiesis regulation: transferrin receptor 2 as potential target for treating anemia in CKD

Both insufficient kidney production of erythropoietin and inflammation-mediated reduction of transferrin-bound iron are major factors in anemia of chronic kidney disease. Improved therapies for anemia in chronic kidney disease may involve modifying regulators of erythropoiesis and iron availability. Olivari etal. show in a mouse model of chronic kidney disease that transferrin receptor 2 in hepatocytes, where it is required for hepcidin production, and in erythroid cells, where it downregulates erythropoietin receptor activity, is a potential therapeutic target.

#4927 DARBEPOETIN ALFA ONCE IN 4 WEEKS CORRECTS ANEMIA IN PATIENTS WITH CHRONIC KIDNEY DISEASE NOT ON DIALYSIS

Abstract Background and Aims In anemia management in patients with CKD not on dialysis (CKD ND), darbepoetin alfa (DA), which has shorter half-life but lower cost than continuous erythropoietin receptor activator (C.E.R.A.), is preferred in actual clinical practice in Korea. The current study evaluated the efficacy and safety of DA every 4 weeks (Q4W) compared to C.E.R.A. Q4W administration for in erythropoiesis-stimulating agent (ESA)-naïve patients with CKD ND. Method In this randomized, prospective, noninferiority study, 40 ESA-naïve patients with CKD ND were randomized 1:1 to receive either DA or C.E.R.A. Q4W during a 12-week correction period and 24-week efficacy evaluation period (EEP). Two primary efficacy end points were analysed: 1) the mean difference in the changes in haemoglobin (Hb) levels between baseline and EEP and 2) the Hb response rates defined as the proportion of patients who reached the target Hb level range (10-11 g/dL) during correction period. The non-inferiority margin is pre-defined as: 1) The lower limit of the 95% confidence interval (CI) is &amp;gt;-0.75 g/dL for the mean difference in the changes in Hb levels and 2) &amp;gt;60% for Hb response rates. Safety profiles including changes in blood pressure (BP), laboratory safety parameters, and frequency of all adverse events (AEs) were monitored and analysed during study period. Results The mean difference in the changes in Hb levels between the two groups was [0.375 g/dL; 95% confidence interval (CI), -0.446 to 1.196)] and [-0.070 g/dL; 95% CI, -0.730 to -0.590] in the intent-to-treat (ITT) and per-protocol (PP) population, respectively (Fig. 1). The Hb response rates in correction period were comparable between DA and C.E.R.A. group; [100%; 95% CI, 81.4 to 100] versus [94.1%; 95% CI, 71.3 to 99.8)] and [100%; 95% CI, 79.4 to 100] versus [100%; 95% CI: 88.2 to 100)] in ITT and PP population, respectively (Fig. 2). The mean estimated glomerular filtration rate, systolic and diastolic BP, sodium, and potassium level over time were not different during total study period between two groups (P = 0.264, 0.999, 0.823, 0.941, and 0.978). All of the AEs in each group were reported to be mild to moderate in intensity. Peripheral edema, neck pain, herpes zoster, and dyspnea occurred in DA group (20%) and urinary tract infection, dyspnea, and femur neck fracture occurred in C.E.R.A. group (15%). There were no serious AEs which led to discontinuation of the treatment. All AEs were mild to moderate in severity, and they are probably not associated with the study drugs and were successfully cured. The reasons for discontinuation of the drugs were starting HD or follow-up loss, but not due to the administered study drugs. No subjects received RBC transfusion or iron replacement therapy in both groups. There was no death during the study period. Conclusion These study results suggest that DA Q4W has non-inferiority in anemia correction efficacy and similar safety profiles compared to C.E.R.A. Q4W in ESA-naïve patients with CKD ND.