Changes of biomarkers for erythropoiesis, iron metabolism, and FGF23 by supplementation with roxadustat in patients on hemodialysis.

This study aimed to confirm changes in biomarkers of erythropoiesis and iron metabolism and serum fibroblast growth factor 23 (FGF-23) during darbepoetin-α treatment and then switching to the hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat. A total of 28 patients on hemodialysis who received weekly doses of darbepoetin-α were switched to roxadustat. Biomarkers for erythropoiesis and iron metabolism and intact and C-terminal FGF-23 were measured in blood samples collected before the HD session on days - 7 (darbepoetin-α injection), - 4, and - 2, and days 0 (switch to roxadustat treatment, three times weekly), 3, 5, 7, 14, 21, and 28. Erythropoietin and erythroferrone levels were elevated on day - 4 by darbepoetin-α injection and decreased to baseline levels at day 0. Levels of erythropoietin were not significantly increased by roxadustat supplementation, but erythroferrone levels were continuously elevated, similar to darbepoetin-α treatment. Hepcidin-25 and total iron binding capacity were significantly decreased or increased in patients treated with roxadustat compared with darbepoetin-α. Changes of intact and C-terminal FGF-23 levels were parallel to changes of phosphate levels during roxadustat treatment. However, the actual and percentage changes of intact FGF-23 and C-terminal FGF-23 in patients with low ferritin levels were greater than those in patients with high ferritin levels. Roxadustat might stimulate erythropoiesis by increasing iron usage through hepcidin-25, which was suppressed by erythroferrone in the physiological erythropoietin condition. Changes of intact FGF-23 and C-terminal FGF-23 levels might be affected by roxadustat in patients on hemodialysis, especially those with a low-iron condition.

Potential Interactions When Prescribing SGLT2 Inhibitors and Intravenous Iron in Combination in Heart Failure.

In patients with heart failure, sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to decrease hepcidin and ferritin and increase transferrin receptor protein, changes that are typically indicative of worsening absolute iron deficiency, as would be seen with poor dietary intake or gastrointestinal bleeding, neither of which is provoked by SGLT2 inhibitors. Therefore, 2 alternative conceptual frameworks may explain the observed pattern of changes in iron homeostasis proteins. According to the "cytosolic iron depletion hypothesis," the effect of SGLT2 inhibitors to decrease hepcidin and ferritin and increase transferrin receptor is related to a decline in cytosolic Fe2+ that occurs after drug-induced erythropoietin-related increase in iron use. Erythropoietin-mimetics (eg, darbepoietin) elicit this type of iron-deficiency pattern of response, and it is typically accompanied by erythropoietin resistance that is alleviated by intravenous iron supplementation. In contrast, according to the "cytosolic iron repletion hypothesis," the effect of SGLT2 inhibitors to decrease hepcidin and ferritin and increase transferrin receptor represents a direct action of these drugs: 1) to reverse inflammation-related increases in hepcidin and ferritin, and, thus, alleviate functional blocks on iron utilization; and 2) to increase in sirtuin-1 signaling, which suppresses hepcidin, accelerates the degradation of ferritin, and up-regulates transferrin receptor protein. Through either or both mechanisms, direct suppression of hepcidin and ferritin would be expected to increase cytosolic Fe2+, thus allowing an unattenuated erythrocytic response to erythropoietin without the need for intravenous iron supplementation. The totality of clinical evidence supports the "cytosolic iron repletion hypothesis" because SGLT2 inhibitors elicit a full and sustained erythrocytosis in response to erythropoietin, even in overtly iron-deficient patients and in the absence of intravenous iron therapy. Therefore, the emergence of an iron-deficiency pattern of response during SGLT2 inhibition does not reflect worsening iron stores that are in need of replenishment, but instead, represents potential alleviation of a state of inflammation-related functional iron deficiency that is commonly seen in patients with chronic heart failure. Treatment with intravenous iron may be unnecessary and theoretically deleterious.

Electroacupuncture attenuates ischemic injury after stroke and promotes angiogenesis via activation of EPO mediated Src and VEGF signaling pathways.

Although electroacupuncture (EA) has been shown to be effective in the treatment of stroke, its mechanisms of action remain undefined. This study explored the therapeutic effects of EA in rats with cerebral ischemia-reperfusion injury (CIRI) and evaluated its possible mechanisms in promoting angiogenesis. To evaluate the effect of EA, we used 2, 3, 5-Triphenyl-2H-Tetrazolium Chloride (TTC) staining and behavior score to calculate the cerebral infarct volume and neurological deficit score after CIRI. Western blot (WB) analysis was employed to evaluate the expression of cluster of differentiation 34 (CD34), erythropoietin (EPO), vascular endothelial growth factor (VEGF) and phospho-Src (p-Src) in the brain of the rats with CIRI. On the other hand, we established an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model using brain microvascular endothelial cells (BMECs), and analyzed cell viability and expression of VEGF or p-Src using cell counting kit-8 (CCK-8) and WB, respectively. Our data showed that EA at the GV26 acupoint could significantly promote the expression of CD34, EPO, VEGF and p-Src in CIRI rats. Our CCK-8 results demonstrated that intervention with recombinant EPO and VEGF proteins remarkably improved the viability of BMECs after OGD/R, while a Src inhibitor, PP1, reversed this phenotype. The WB results showed that the recombinant EPO protein increased the expression of VEGF and p-Src, which was significantly inhibited by PP1. Taken together, our findings showed that EA at the GV26 acupoint can significantly attenuate ischemic injury after stroke and promote angiogenesis via activation of EPO-mediated Src and VEGF signaling pathways. Besides, the upregulation of VEGF may also be associated with the activation of Src by EPO.

Inflammation promotes erythropoietin induced vascular calcification by activating p38 pathway.

The current research aimed to verify the effects of erythropoietin (EPO) on vascular calcification under inflammatory conditions and the molecular regulator of vascular calcification induced by EPO. To induce vascular calcification and systemic chronic inflammation in SD rats, EPO was administered intraperitoneally, and 10% casein was injected subcutaneously. The administration period lasted for 20 consecutive weeks. Blood samples were subsequently collected to detect inflammatory factors and vascular calcification. Additionally, high-dose EPOs were applied to stimulate primary vascular smooth muscle cells (VSMCs), and vascular calcification was measured using alizarin red staining, alkaline phosphatase (ALP) activity, and calcium salt quantification. The probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) was employed to detect cellular reactive oxygen species (ROS) levels. The expressions of bone formation-related protein and anti-calcification protein matrix gla protein (MGP) were determined via Western blot. Compared with the control group, calcium deposits and vascular calcification were increased in the EPO group, tumor necrosis factor-alpha (TNF-α) group and TNF-α+ EPO group, whereas MGP was significantly reduced. Moreover, under the stimulation of TNF-α and EPO+TNF-α, pp38/p38 was increased substantially, the addition of p38 inhibitor SB203580 could significantly reduce calcium deposits and vascular calcification. In vivo experiment, compared with the EPO group, calcium salt deposition and vascular calcification were elevated in the EPO+casein group. The present results revealed that high-dose EPO could cause calcification of the abdominal aorta in rats. The inflammatory response aggravated the vascular calcification induced by EPO via activating p38 and ROS levels.

Is the erythropoietin-erythroferrone-hepcidin axis intact in human neonates?

In a two-part process, we assessed elements of the principal hormonal pathway regulating iron homeostasis in human neonates. Part 1: Quantifying erythropoietin (Epo), erythroferrone (ERFE), hepcidin, and relevant serum and erythrocytic iron-related metrics in umbilical cord blood from term (n = 13) and preterm (n = 10) neonates, and from neonates born to mothers with diabetes and obesity (n = 13); Part 2: Quantifying serum Epo, ERFE, and hepcidin before and following darbepoetin administration. Part 1: We measured Epo, ERFE and hepcidin in all cord blood samples. Epo and ERFE levels did not differ between the three groups. Preterm neonates had the lowest hepcidin levels, while neonates born to diabetic women with a very high BMI had the lowest ferritin and RET-He levels. Part 2: Following darbepoetin dosing, ERFE levels generally increased (p < 0.05) and hepcidin levels generally fell (p < 0.05). Our observations suggest that the Epo/ERFE/hepcidin axis is intact in the newborn period.

Possible mechanisms by which silkworm faeces extract ameliorates adenine-induced renal anaemia in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Silkworm faeces are the dry faeces of the insect Bombyx mori (Linnaeus) and have historically been used in traditional Chinese medicine to treat blood deficiency and rheumatic pain. Silkworm faeces extract (SFE) is derived from silkworm faeces. AIM OF THE STUDY: Clinical observations of patients in the Department of Nephrology have shown that SFE effectively improves renal anaemia. However, the molecular mechanism remains unclear. This article mainly explores the regulatory effects of SFE on erythropoietin (EPO) and hepcidin to identify the molecular mechanism of SFE. MATERIALS AND METHODS: A rat model of renal anaemia was established by feeding rats food containing 0.75% adenine. SFE was orally administered to the rats, while recombinant human erythropoietin (rhEPO) was used as a positive control drug. Haematological parameters and inflammation levels were compared between rats from each group, and pathological kidney sections from each rat were observed. The serum EPO and hepcidin levels were detected using enzyme-linked immunosorbent assay (ELISA) kits, while Western blot analyses were performed to detect the levels of proteins involved in the EPO-related hypoxia-inducible factor 2α (HIF-2α)/prolyl hydroxylase 2 (PHD2) signalling pathway and hepcidin-related BMP6/SMAD4 and interleukin-6 (IL-6)/STAT3 signalling pathways. RESULTS: SFE significantly ameliorated haematological parameters, renal function, and inflammation levels in the rats. A mechanistic study showed that SFE promoted EPO expression by upregulating HIF-2α expression and inhibiting the expression of NF-κB and GATA2 both in vivo and in vitro. In particular, SFE inhibited PHD2 expression, resulting in a decrease in the enzymatic reaction of HIF-2α to increase EPO expression. Furthermore, SFE inhibited hepcidin expression by blocking the BMP6/SMAD4 and IL-6/STAT3 pathways. CONCLUSIONS: SFE regulated iron metabolism by inhibiting hepcidin and simultaneously promoted EPO synthesis to improve renal anaemia in rats.

Discovery of Clinical Candidate (5-(3-(4-Chlorophenoxy)prop-1-yn-1-yl)-3-hydroxypicolinoyl)glycine, an Orally Bioavailable Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)glycine inhibitors of prolyl hydroxylase (PHD) are described. These compounds showed potent in vitro inhibitory activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin (EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25 mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with oral bioavailabilities of 55.7 and 54.0%, respectively, and shows excellent safety profiles even at a high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated in a phase I clinical trial for anemia.

A biomathematical model of human erythropoiesis and iron metabolism.

Anaemia therapy or perisurgical support of erythropoiesis often require both, EPO and iron medication. However, excessive iron medication can result in iron overload and it is challenging to control haemoglobin levels in a desired range. To support this task, we develop a biomathematical model to simulate EPO- and iron medication in humans. We combine our previously established model of human erythropoiesis including comprehensive pharmacokinetic models of EPO applications with a newly developed model of iron metabolism including iron supplementation. Equations were derived by translating known biological mechanisms into ordinary differential equations. Qualitative model behaviour is studied in detail considering a variety of interventions such as bleeding, iron malnutrition and medication. The model can explain time courses of erythrocytes, reticulocytes, haemoglobin, haematocrit, red blood cells, EPO, serum iron, ferritin, transferrin saturation, and transferrin under a variety of scenarios including EPO and iron application into healthy volunteers or chemotherapy patients. Unknown model parameters were determined by fitting the predictions of the model to time series data from literature. We demonstrate how the model can be used to make predictions of untested therapy options such as cytotoxic chemotherapy supported by iron and EPO. Following our ultimate goal of establishing a model of anaemia treatment in chronic kidney disease, we aim at translating our model to this pathological condition in the near future.

Acai Extract Transiently Upregulates Erythropoietin by Inducing a Renal Hypoxic Condition in Mice.

Acai (Euterpe oleracea Mart. Palmae, Arecaceae) is a palm plant native to the Brazilian Amazon. It contains many nutrients, such as polyphenols, iron, vitamin E, and unsaturated fatty acids, so in recent years, many of the antioxidant and anti-inflammatory effects of acai have been reported. However, the effects of acai on hematopoiesis have not been investigated yet. In the present study, we administered acai extract to mice and evaluated its hematopoietic effects. Acai treatment significantly increased the erythrocytes, hemoglobin, and hematocrit contents compared to controls for four days. Then, we examined the hematopoietic-related markers following a single injection. Acai administration significantly increased the levels of the hematopoietic-related hormone erythropoietin in blood compared to controls and also transiently upregulated the gene expression of Epo in the kidney. Furthermore, in the mice treated with acai extract, the kidneys were positively stained with the hypoxic probe pimonidazole in comparison to the controls. These results demonstrated that acai increases the erythropoietin expression via hypoxic action in the kidney. Acai can be expected to improve motility through hematopoiesis.

Regulation of muscle and metabolic physiology by hypothalamic erythropoietin independently of its peripheral action.

OBJECTIVE: The glycoprotein hormone erythropoietin (EPO) is required for erythropoiesis, and the kidney is the primary site of adult EPO synthesis. Limited evidence has suggested that EPO could be detectable in the brain under certain conditions, but it remains unknown if the brain might have its own EPO system for biological functions that are independent of peripheral EPO production and action. We performed this study to address this question using mice under normal conditions versus pathophysiological conditions including aging and dietary obesity. METHODS: EPO expression was measured in different brain regions as well as in the cerebrospinal fluid. Hypothalamic ventricular EPO was administered to physiologically examine possible therapeutic effects on the conditions of aging and dietary obesity. Body weight, body composition, insulin tolerance, and glucose tolerance were measured to assess the central effects of EPO on metabolic physiology, and muscle strength and histology were analyzed to assess the central effects of EPO on muscle function. In addition, ß2-adrenergic receptor knockout bone marrow transplant was employed to determine the potential role of bone marrow in linking the brain to some of these peripheral functions. RESULTS: This study revealed that EPO is expressed in the ventromedial hypothalamus in addition to a few other brain regions and is present in the cerebrospinal fluid. Unlike blood EPO concentration, which increased with aging and dietary obesity, hypothalamic EPO decreased in these disease conditions. Therapeutically, aged mice were chronically treated with EPO in the hypothalamic ventricle, showing an increase in lean mass, while body weight and fat mass decreased as a result of a moderate reduction of food intake. Both muscle and metabolic functions were improved by this central treatment, and mechanistically, adrenergic signals to the bone marrow played a role in conveying hypothalamic EPO to these peripheral actions. Dietary obesity was also studied, showing that hypothalamic EPO treatment caused a reduction in food intake and obesity, leading to improved metabolic functions related to decreased fat as well as increased lean mass. CONCLUSIONS: Hypothalamic EPO plays a role in the central regulation of muscle and metabolic physiology, while its decline contributes to aging and obesity physiology in a manner that is independent of peripheral EPO.

Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.

The blood hormone erythropoietin (EPO), upon binding to its receptor (EpoR), modulates high-fat diet-induced (HFD-induced) obesity in mice, improves glucose tolerance, and prevents white adipose tissue inflammation. Transgenic mice with constitutive overexpression of human EPO solely in the brain (Tg21) were used to assess the neuroendocrine EPO effect without increasing the hematocrit. Male Tg21 mice resisted HFD-induced weight gain; showed lower serum adrenocorticotropic hormone, corticosterone, and C-reactive protein levels; and prevented myeloid cell recruitment to the hypothalamus compared with WT male mice. HFD-induced hypothalamic inflammation (HI) and microglial activation were higher in male mice, and Tg21 male mice exhibited a lower increase in HI than WT male mice. Physiological EPO function in the brain also showed sexual dimorphism in regulating HFD response. Female estrogen production blocked reduced weight gain and HI. Targeted deletion of EpoR gene expression in neuronal cells worsened HFD-induced glucose intolerance in both male and female mice but increased weight gain and HI in the hypothalamus in male mice only. Both male and female Tg21 mice kept on normal chow and HFD showed significantly improved glycemic control. Our data indicate that cerebral EPO regulates weight gain and HI in a sex-dependent response, distinct from EPO regulation of glycemic control, and independent of erythropoietic EPO response.

Effects of oral iron and calcium supplement on the pharmacokinetics and pharmacodynamics of molidustat: an oral HIF-PH inhibitor for the treatment of renal anaemia.

PURPOSE: The present studies assessed the drug-drug interaction of molidustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor, with iron and calcium supplements, which are common medications in patients with anaemia due to chronic kidney disease (CKD). METHODS: Forty-two healthy men received molidustat alone (fasted or fed) or combined with oral iron(II) or calcium(II), given immediately before or between 4 h before and 1 h after molidustat in three randomized, open-label, crossover studies (12-15 participants per study). Molidustat AUC and Cmax were assessed as the main pharmacokinetic parameters, and endogenous erythropoietin (EPO) was measured to evaluate pharmacodynamics. RESULTS: Depending on prandial state, concomitant intake of iron(II) reduced molidustat AUC and Cmax by 50-75% and 46-84%, respectively, and EPO AUC(0-24) and Cmax by 31-44% and 36-48%, respectively. The influence of iron(II) declined with increasing the time interval to the intake of molidustat, with reductions in molidustat AUC and Cmax of 9% and 10%, respectively, when iron(II) intake occurred 4 h before molidustat. Accordingly, effects on endogenous EPO were less pronounced with increased time separation between oral iron(II) and molidustat intake. Calcium(II) reduced molidustat AUC and Cmax by 15% and 47%, respectively, without influence on EPO response. All treatments were well tolerated. CONCLUSIONS: In contrast to concomitant oral intake of calcium, the effect of oral iron supplements on molidustat pharmacokinetics and pharmacodynamics should be considered, and the two agents should be administered with an appropriate time separation.

Effect of iron chelation therapy on EPO-STAT5 signalling pathway and EPO resistance in iron-overloaded low-risk myelodysplastic syndrome patients.

Objectives: Background/aims: We aim to explore low-risk MDS patients' ESA response and the difference between iron-overloaded (IO) group and the control group in the expression of SOCS1, STAT5 and BCL2L1 which play a key role to EPO-STAT5 signal pathway.Methods: 56 low-risk MDS patients were divided into experimental group, IO patients; control group, non-IO patients. Among experimental group, 28 IO patients were treated with iron chelation therapy (ICT). SOCS1, phosphorylated STAT5 (p-STAT5) and BCL2L1 protein concentration in bone marrow supernatant have been analyzed by ELISA, STAT5a+b protein concentration in bone marrow mononuclear cells (BMMC) have been analyzed by Western blot, and mRNA expression of them have been detected in BMMC by RQ-PCR. The percentage of CD71+ cells in BMMC, apoptotic rate of CD71+ cells and ROS expression in CD71+ cells were detected by Flow cytometry.Results: Compared with the control group, the sEPO concentration, the efficacy of ESA and the expression of SOCS1, apoptotic rates of CD71+ cells and ROS expression in CD71+ cells in IO group were increased, the expression of STAT5 and BCL2L1 was reduced. Interestingly, after receiving ICT, some patients with EPO resistance have responded again to ESA treatment, with the decrease of the expression of SOCS1, apoptotic rates of CD71+ cells, ROS expression in CD71+ cells and the increase of the expression of STAT5 and BCL2L1.Conclusion: Iron overload can increase EPO resistance and the expression of SOCS1, inhibit the expression of STAT5 and BCL2L1. ICT could allivation of EPO resistance.

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.

An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals.

Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)-II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.

Aqueous extract from You-Gui-Yin ameliorates cognitive impairment of chronic renal failure mice through targeting hippocampal CaMKIIα/CREB/BDNF and EPO/EPOR pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: You-Gui-Yin (YGY) is a traditional Chinese recipe used for reinforcing kidney essence which is recorded in Jingyue Quanshu written by Zhang Jingyue in Ming dynasty. According to traditional Chinese medicine theory, kidney essence is associated with brain and without sufficient kidney essence, cognitive impairment may occur. AIM OF THE STUDY: In this study, we aimed to investigate the effect of YGY extract on cognitive impairment of chronic renal failure (CRF) mice and explore the mechanisms involved. MATERIALS AND METHODS: Aqueous extract of YGY was prepared from crude drugs and was quality controlled by high-performance liquid chromatography (HPLC). CRF was induced by 0.2% adenine in mice and CRF mice were intragastrically administered with 1.5 g kg-1, 3.0 g kg-1, and 6.0 g kg-1 of YGY extract. Mice were identified with CRF by determining several biochemical and physiological indexes, including creatinine clearance rate, serum creatinine, serum urea nitrogen, serum Ca, serum P, serum Mg, body weight and body temperature. Morris water maze and novel object recognition tests were conducted for evaluation of cognitive function. In addition, changes of CaMKIIα/CREB/BDNF and EPO/EPOR pathways in hippocampus were examined by detecting the protein expressions of CaMKIIα, p-CaMKIIα (Thr286), CREB1, p-CREB1 (Ser133), BDNF, EPO, EPOR, p-EPOR (Tyr485), STAT5, and AKT1 using western blotting assays. Also, the primary EPO-producing cells in brain (i.e. astrocytes) and EPO expression regulator HIF-2α were checked by fluorescence microscopy and western blotting assay, respectively. RESULTS: Nine components in YGY extract were figured out and monitored with their contents by HPLC for the quality control of YGY extract. Biochemical and physiological measurements validated the success of induction of CRF in mice, and YGY extract significantly retarded the CRF progression and ameliorated the CRF-induced cognitive impairment. The behavioral tests showed that compared with normal control mice, CRF mice had impaired cognitive function. However, treatment of YGY extract significantly ameliorated the cognitive impairment of CRF mice. Additionally, decreased expressions of hippocampal CaMKIIα, p-CaMKIIα (Thr286), CREB1, p-CREB1 (Ser133), and BDNF were observed in the hippocampus of CRF mice, but YGY extract significantly restored these protein expressions. Moreover, hippocampal EPO, EPOR, p-EPOR (Tyr485), STAT5, AKT1, and HIF-2α, as well as the number of astrocytes in CA1 zone of hippocampus were also decreased in CRF mice, while YGY extract prominently promoted the expressions of these proteins and increased the number of astrocytes. CONCLUSIONS: All the data in this study suggested that YGY extract ameliorated the cognitive impairment of CRF mice, and this amelioration was related to up-regulating the CaMKIIα/CREB/BDNF and EPO/EPOR pathways.

VEGF-loaded mineral-coated microparticles improve bone repair and are associated with increased expression of epo and RUNX-2 in murine non-unions.

A poor vascular supply of the fracture gap is a key factor for the development of atrophic non-unions. Mineral-coated microparticles (MCM) represent a sophisticated carrier system for the delivery of vascular endothelial growth factor (VEGF). Hence, we investigated whether VEGF-loaded MCM improve bone repair in non-unions. For this purpose, we analyzed binding and release kinetics of MCM for VEGF in vitro. Moreover, we applied VEGF-loaded or -unloaded MCM in a murine non-union model in vivo and studied the process of bone healing by means of biomechanical, radiological, histomorphometric, and Western blot techniques. MCM-free non-unions served as controls. The binding efficiency of MCM for VEGF was 46 ± 3% and the release profile revealed an initial minor burst release followed by a sustained release over a 50-day study period, thus, mimicking the physiological expression profile of VEGF during bone healing. In vivo, bone defects treated with VEGF-loaded MCM exhibited a higher bending stiffness, a higher fraction of bone volume/tissue volume and a larger callus area on days 14 and 70 when compared to the other groups. Western blot analyses on day 14 revealed a higher expression of VEGF, erythropoietin (EPO), and runt-related transcription factor 2, but not of EPO-receptor in bone defects treated with VEGF-loaded MCM. These findings demonstrate that the use of MCM for VEGF delivery shows great potential due to the ability to maintain protein stability and functionality in vivo. Moreover, the application of VEGF-loaded MCM represent a promising strategy for the treatment of non-unions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Hepcidin in chronic kidney disease anemia.

Chronic kidney disease (CKD) is associated with several complications that worsen with progression of disease; anemia, disturbances in iron metabolism and inflammation are common features. Inflammatory response starts early, releasing pro-inflammatory cytokines, acute phase reactants and hepcidin. Hepcidin production is modulated by several factors, as hypoxia/anemia, erythropoietin and erythropoiesis products, transferrin saturation (TSAT) and liver iron levels, which are altered in CKD. Treatment of CKD anemia is based on pharmaceutical intervention, with erythropoietic stimulating agents and/or iron supplementation; however, in spite of the erythropoietic benefits, this therapy, on a regular basis, involves risks, namely iron overload. To overcome these risks, some therapeutic approaches are under study to target CKD anemia. Considering the actual alerts about risk of iron overload in dialysis patients, inhibition of hepcidin, the central key player in iron homeostasis, could be a pivotal strategy in the management of CKD anemia.

Upregulation of endogenous erythropoietin expression by DLBS6747, a bioactive fraction of Ipomoea batatas L. leaves, via increasing HIF1α transcription factor in HEK293 kidney cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ipomoea batatas L., locally known as ubi jalar, is widely used in Indonesia and other countries as a folk remedy for various chronic diseases, including anemia-associated chronic kidney disease by increasing hematological parameters such as packed cell volume, white blood cells and platelet counts. AIM OF THE STUDY: The aim of this study is to evaluate the effect of DLBS6747, a bioactive fraction of I. batatas L. leaves, on increasing EPO expression through the upregulation of HIF1α. MATERIALS AND METHODS: Effect of DLBS6747 on EPO expression and its transcription factor, HIFs, was evaluated in normoxia and hypoxia conditions. Effect of DLBS6747 on several genes involved in EPO expression were evaluated in a time-course manner using conventional and real-time PCR, while the protein level were revealed using western blot and ELISA. The involvement of HIF1α was also confirmed by HIF1α siRNA. RESULTS: Administration of DLBS6747 increased transcriptional activity of EPO through the regulation of its transcriptional factors, which include HIF1α, HIF2α and NFᴋB. The effect was found to be dependent on oxygen availability, wherein DLBS6747-increased EPO expression was found to be more significant in hypoxic condition. In normoxia and hypoxia, 40 µg/mL DLBS6747 increased HIF1α and HIF2α expressions at mRNA level, wherein the peak appeared in 12 h treatment (up to 7.9- and 8.6-folds, respectively). On the other hand, increased protein level was only found in hypoxia, where the highest HIF1α expression was observed at 6 h (7.5-folds increase) and started to decrease after the hours, while HIF2α was found to be increased time-dependently (up to 13.8-folds in 24 h). The mechanism of action of DLBS6747 as erythropoietin stimulating agent is more likely to affect the regulation of HIF1α, as confirmed by HIF1α siRNA which showed that DLBS6747 failed to increase EPO expression during co-incubation with HIF1α siRNA. DLBS6747 treatment also decreased NFᴋB time-dependently in normoxia, while no NFᴋB was detected in hypoxia, which revealed mimicking hypoxia activity of DLBS6747 to increase EPO expression. CONCLUSION: These findings showed convincing evidences that DLBS6747 increases endogenous EPO production primarily via upregulation of its transcription factors, especially HIF1α, in human embryonic kidney HEK293 cells. This is the first molecular report that reveals the mechanism of action of natural-based erythropenia drug in different oxygen availability.

HIF stabilizers in the management of renal anemia: from bench to bedside to pediatrics.

Anemia is a common complication of chronic kidney disease (CKD) in adult and pediatric patients. It has traditionally been treated with erythropoietin therapy and iron supplementation, with great success. With the discovery of the major transcription factor hypoxia inducible factor (HIF) for the erythropoietin gene in 1992, molecules were created that inhibit the HIF prolyl-hydroxylase enzyme. This new class of drug-called HIF stabilizers, or HIF prolyl-hydroxylase inhibitors-prevents the proteasomal degradation of HIF-α, thereby inducing upregulation of the erythropoietin gene. This new strategy for treating CKD anemia is already in phase III clinical trials in adults, and the potential advantages of this therapy are that it is orally active (thereby avoiding injections), and patients are exposed to lower circulating levels of erythropoietin. The long-term safety of this strategy, however, requires elucidation in these trials, particularly since there are many other hypoxia-sensitive genes, notably, angiogenic factors such as vascular endothelial growth factors (VEGF), as well as glycolytic enzymes. As with all new therapies, it is only once a positive benefit: risk profile has been ascertained in adults that the treatment will translate across into pediatrics. Specific issues in the pediatric CKD population are discussed in this review.