Erythropoietin mRNA Expression Research Articles

Inhibition of Endogenous Hydrogen Sulfide Production Decreases Hypoxia Induced Erythropoietin Release By Hep3B Cells.

Introduction and Objective: Anemia of end stage renal disease (ESRD) affects over 90% of all hemodialysis patients and is a tremendous concern both for patients and health care providers. Natural erythropoietin (EPO) production is stimulated by local hypoxic conditions within the kidney tubular cells. Unfortunately over time, patients require exogenous EPO stimulating agents to treat their anemia, however they can become resistant to these agents thus necessitating increasing doses with resultant greater side effects. Novel therapeutics to overcome anemia of ESRD are necessary. Recent evidence suggests that endogenously produced hydrogen sulfide (H2S) may act as an oxygen sensor. Given the known involvement of other small molecules such as nitric oxide (NO) in natural EPO production and the observation of diminished urinary H2S levels in ESRD patients on hemodialysis, we postulated that H2S may be the primary mediator of EPO production during hypoxic conditions. Materials/Methods: Hep3B cells are a well described model for EPO production. These cells were incubated under hypoxic conditions (1% O2) for 24 hours at 37°C. During hypoxia cells were treated with varying concentrations of the H2S donor, GYY 4137, the substrate for H2S biosynthesis, L-cysteine, and various inhibitors of H2S production, including DL-propargylglycine (PAG) and hydroxylamine (HA). RNA was collected following the end of hypoxia and EPO expression was measured by real time RT-PCR. Results: In comparison to normoxic conditions, cells subjected to hypoxic conditions showed a significant increase in erythropoietin expression. Upon inhibiting endogenous H2S production using either HA and PAG, we found a significant decreases in EPO expression (p=0.013). Subsequent administration of GYY 4137 (500 uM) rescued the effect of these inhibitors, inducing a significant increase in EPO mRNA expression (p= 0.0012) compared to normoxic controls. Conclusions: These data are the first to show a novel influence of endogenous H2S on EPO production under hypoxic conditions. The use of novel oral H2S donors may represent a more efficacious and cost-effective alternative to expensive standard therapies in treatment of anemia of ESRD.

Reevaluation of erythropoietin production by the nephron

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.

Human cytomegalovirus inhibits erythropoietin production.

Anemia is a feature of CKD and a complication of renal transplantation, often caused by impaired production of erythropoietin. The kidney is a target organ for human cytomegalovirus (hCMV) in such patients, but it is not known whether hCMV effects erythropoietin production. We found that kidneys from patients with CKD were positive for hCMV protein and that blood levels of hCMV IgG inversely correlated with red blood cell count. In mice, systemic murine cytomegalovirus infection decreased serum erythropoietin levels. In human erythropoietin-producing cells, hCMV inhibited hypoxia-induced expression of erythropoietin mRNA and protein. hCMV early gene expression was responsible, as ultraviolet-inactivated virus had no effect and valganciclovir treatment showed that late gene expression was nonessential. Hypoxia-induced gene transcription is controlled by the transcription factors hypoxia-inducible transcription factor (HIF)-1α and HIF2α, which are constitutively produced but stable only under low oxygen conditions. We found that hCMV inhibited constitutive production of HIF2α mRNA. HIF2α is thought to be the master regulator of erythropoietin transcription. Single-cell analysis revealed that nuclear accumulation of HIF2α was inhibited in hCMV-infected cells, and the extent of inhibition correlated with hCMV protein expression. Our findings suggest that renal hCMV infection could induce or exacerbate anemia in patients.

Ciprofloxacin Enhances Stress Erythropoiesis in Spleen and Increases Survival after Whole-Body Irradiation Combined with Skin-Wound Trauma

Severe hematopoietic loss is one of the major therapeutic targets after radiation-combined injury (CI), a kind of injury resulting from radiation exposure combined with other traumas. In this study, we tested the use of ciprofloxacin (CIP) as a treatment, because of recently reported immunomodulatory effects against CI that may improve hematopoiesis. The CIP regimen was a daily, oral dose for 3 weeks, with the first dose 2 h after CI. CIP treatment improved 30-day survival in mice at 80% compared to 35% for untreated controls. Study of early changes in hematological parameters identified CI-induced progressive anemia by 10 days that CIP significantly ameliorated. CI induced erythropoietin (EPO) mRNA in kidney and protein in kidney and serum; CIP stimulated EPO mRNA expression. In spleens of CI mice, CIP induced bone morphogenetic protein 4 (BMP4) in macrophages with EPO receptors. Splenocytes from CIP-treated CI mice formed CD71+ colony-forming unit-erythroid significantly better than those from controls. Thus, CIP-mediated BMP4-dependent stress erythropoiesis may play a role in improving survival after CI.

Interplay between Endothelin and Erythropoietin in Astroglia: The Role in Protection against Hypoxia

We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48–72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS). The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS) can be suggested.

Stimulating Effect of Ethanol on Erythropoietin Production in the Liver Cells

Increased erythropoietin (EPO) production is important for erythropoiesis as well as cell viability. The most effective factor for promoting EPO production is hypoxia, which alters the redox state and produces a reducing environment in the cell. In this study, we examined the influence of ethanol on EPO production in HepG2 cells to investigate the effect of increasing the free NADH/NAD+ ratio in the cytosol during normoxia. Ethanol treatment increased the lactate/pyruvate ratio, an index of the cytosolic redox state, in a dose-dependent manner, with maximal promotion of EPO production observed at 300 μM ethanol. These results suggest that altering the cytosolic NADH/NAD+ redox state to the same degree as hypoxia is effective in promoting EPO production. Ethanol (300 μM) increased mRNA expression and protein levels of sirtuin1, which is a transcription factor, related to both hypoxia inducible factor and cytosolic redox state, whereas 2000 μM ethanol did not produce these effects. Although the sirtuin1 inhibitorEX-527 did not affect the lactate/pyruvate ratio, EX-527 inhibited the induction of EPO mRNA expression by 300 μM ethanol. In rat primary hepatocytes and kidney cells, 300 μM ethanol increased sirtuin1 and EPO mRNA expression, as well as EPO concentrations in media. In conclusion, we showed low concentrations of ethanol promote EPO production by increasing sirtuin1 in HepG2 cells, as well as primary liver and kidney cells. The use of ethanol represents a hypoxia-independent method to promote EPO production.

Dynamics of Erythropoietic Markers in Response to Hypoxia in Rats

Purpose: Erythropoietin (EPO) regulates production of red blood cells in response to hypoxia. Decreased arterial oxygen saturation results in an increased production of EPO in various tissues including kidney as a major stimulation site leading to an increase in EPO plasma levels. This study aims to investigate the relationship between hypoxia and resultant erythropoietic response in rats. Methods: The in house hypoxia chamber was designed to regulate the fraction of inspired oxygen (FiO2) = 10% supplied to Wistar male rats (N=4) for 6 hours by mixing room air with N2 to maintain air pressure at 1 atm. Plasma and kidney samples were collected at various times during and after exposure to hypoxia. Kidney EPO mRNA, HIF-2α, plasma EPO concentration, reticulocyte counts and hemoglobin were measured. The time course of above markers and fold changes from pre-hypoxia values were used to quantify responses to hypoxia under the normoxic condition. Results: The arterial oxygen saturation of rats decreased from 95% to 60% during the hypoxia. Hypoxia resulted in 6-fold increase in kidney EPO mRNA expression, 10-fold increase in plasma EPO concentrations and 1.5-fold increase in reticulocytes count. The increase of HIF-2α expression is 2.4 fold after one hour hypoxia and returns below the baseline value after 1.5 hours. The result of DNA binding activity of HIF-2α is quite similar, which increased 1.5 fold at 0.5 hour and then went below baseline. Conclusion: After 6 hours hypoxia, the observed increase in the levels of kidney EPO mRNA, plasma EPO concentrations and reticulocytes count, and delays in peak response are consistent with times necessary for the stimulation for EPO mRNA transcription, synthesis of plasma EPO, and enhanced release of reticulocytes. The tolerance effect observed in HIF-2α time course implies existence of a counter-regulatory mechanism in a HIF-2α mediated signal for mRNA production.

Abstract 1623: PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells.

Abstract PDGF signaling system contributes to tumor angiogenesis and vascular remodeling. Here, we show PDGF-BB markedly induces erythropoietin (EPO) mRNA and protein expression by targeting the PDGFR-β+ stromal and perivascular compartments. In mouse tumor models, PDGF-BB-induced EPO promotes tumor growth via two mechanisms: 1) paracrine stimulation of tumor angiogenesis by directly inducing endothelial cell proliferation, migration, sprouting and tube formation; and 2) endocrine stimulation of extramedullary hematopoiesis leading to increased oxygen perfusion and protection against tumor-associated anemia. Similarly, delivery of an adenovirus-PDGF-BB to tumor-free mice markedly increases EPO production and hematopoietic parameters. An EPO-specific antibody attenuates PDGF-BB-induced tumor growth, angiogenesis and hematopoiesis. At the molecular level, we show that the PDGF-BB-PDGFR-β signaling system activates EPO promoter via in part transcriptional regulation of ATF3 by possible association with c-Jun and SP1. These findings uncover a novel mechanism of PDGF-BB-induced tumor growth, angiogenesis and hematopoiesis. Citation Format: Yuan Xue, Sharon Lim, Yunlong Yang, Zongwei Wang, Lasse Dahl Ejby Jensen, Eva–Maria Hedlund, Patrik Andersson, Masakiyo Sasahara, Ola Larsson, Dagmar Galter, Renhai Cao, Kayoko Hosaka, Yihai Cao. PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1623. doi:10.1158/1538-7445.AM2013-1623

Testosterone administration inhibits hepcidin transcription and is associated with increased iron incorporation into red blood cells

Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown. Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferrin saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound (58) Fe, the amount of (58) Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to bone morphogenetic protein (BMP)-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. In conclusion, testosterone inhibits hepcidin transcription through its interaction with BMP/Smad signaling. Testosterone administration is associated with increased iron incorporation into red blood cells.

Hormone supplying renal cell sheet in vivo produced by tissue engineering technology.

Regenerative medicine is a new medical field and is expected to have a profoundly positive effect in curing difficult-to-treat diseases. Cell sheet fabrication is an important tissue engineering technology used in regenerative medicine. This study investigated the creation of a hormone-releasing tissue using cell sheet technology, which could be utilized in future therapy for chronic renal disease. Renal cell sheets were fabricated on a temperature-responsive cell culture surface with primary renal cells from adult porcine kidney. These sheets contained various kinds of renal cells that showed cyst-like formation. An important renal function is the synthesis of 1,25-dihydroxyvitamin D3, and this was confirmed in the cell sheets in vitro. Erythropoietin (EPO) production is another important renal function. This ability was also observed in the renal cell sheets in vitro, and then again after transplantation in a nude rat. In particular, the relative expression of EPO mRNA increased more under cell sheet culture conditions compared with exponential cell growth conditions. Histological analysis of the implanted renal cell sheets showed them to be Dolichos biflorus agglutinin-positive and to have regenerated renal tubular-like morphology. These results indicated that both functional and morphological regenerative renal tissues were fabricated by cell sheet technology. This study introduces a hormone-supplying treatment for renal dysfunctional diseases using engineered renal tissues. Moreover, since our renal cell sheets developed renal tubular-like structures in vivo, it holds promise for fabricating artificially engineered true renal tissue in the future.

Hepatic confinement of newly produced erythrocytes caused by low-temperature exposure inXenopus laevis

Diminished erythrocyte count and erythropoiesis have been reported during hypothermia in some ectothermic animals. In this study, the African clawed frog, Xenopus laevis, was used to investigate the cause of hypothermia-induced anemia. We developed a new model of hypothermia at 5°C and monitored blood cell count and erythropoiesis on several days. Erythrocyte count declined by 30% on the first day following cold exposure (5°C) and mRNA expression of hemeoxygenase-1 was enhanced 10-fold; accumulation of iron as a result of heme degradation was observed in the liver. One day after low-temperature exposure, erythropoietin mRNA expression was elevated in the liver and lung compared with that at normal temperature (22°C) by qRT-PCR analysis. Examination of liver sections (i.e. the erythropoietic organ) showed an increase in o-dianisidine-positive erythrocytes in the hepatic sinusoid 5 days after the onset of low-temperature exposure compared with normal liver. Peripheral erythrocyte count remained low, indicating that newly produced erythrocytes did not migrate from the liver to the circulation during hypothermia. In conclusion, this study reveals hypothermic anemia as being associated with hepatic erythrocyte destruction; prolonged anemia during low-temperature exposure is concomitant with newly produced erythrocytes being confined to the liver and may lead to new insights into vertebrate hematopoiesis.

Exogenous erythropoietin administration attenuates intermittent hypoxia-induced cognitive deficits in a murine model of sleep apnea

BackgroundIn rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Such findings are markedly attenuated in rodents exposed to sustained hypoxia 9SH) of similar magnitude. The hypoxia-sensitive gene erythropoietin (EPO) has emerged as a major endogenous neuroprotectant, and could be involved in IH-induced neuronal dysfunction.Methods and ResultsIH induced only transiently increased expression of EPO mRNA in hippocampus, which was continued in (SH)-exposed mice. IH, but not SH, adversely affected two forms of spatial learning in the water maze, and increased markers of oxidative stress. However, on a standard place training task, mice treated with exogenously administered EPO displayed normal learning, and were protected from the spatial learning deficits observed in vehicle-treated (C) littermates exposed to IH. Moreover, anxiety levels were increased in IH as compared to normoxia, while no changes in anxiety emerged in EPO-treated mice. Additionally, C mice, but not EPO-treated IH-exposed mice had significantly elevated levels of NADPH oxidase expression, as well as increased MDA and 8-OHDG levels in cortical and hippocampal lysates.ConclusionsThe oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by imbalances between EPO expression and increased NADPH oxidase activity, and thus pharmacological agents targeting EPO expression in CNS may provide a therapeutic strategy in sleep-disordered breathing.

Simvastatin downregulates the expression of hepcidin and erythropoietin in HepG2 cells

Statin therapy may improve responsiveness to erythropoietin-stimulating agents in patients with end-stage renal disease. Although statins increase hepatic iron uptake and storage capacity in cholestatic rats, the underlying mechanisms are unclear. Therefore, we examined the effects of a statin (simvastatin) on the expression of hepcidin, erythropoietin receptor (EPOR) and erythropoietin (EPO) in cultured HepG2 cells. HepG2 cells (6-6.5 × 10(5) cells) were seeded in 6-cm dishes and incubated overnight. The cells were then treated with 0, 0.5, 1, 3, 5, or 10 μM simvastatin, and the mRNA expression of hepcidin, EPOR, and EPO was determined. Data were collected from three independent experiments. The cDNA extracted from the cells was used as a template for real-time polymerase chain reaction, and each sample was tested in duplicate. Significant differences (P < 0.05) among groups were determined using one-way analysis of variance with Fisher's least significant difference post hoc test. Data were adjusted using Bonferroni's method. The relative mRNA expression of hepcidin in HepG2 cells treated with 0.5, 1, 3, 5, and 10 μM simvastatin, relative to the control group, was 0.7273, 0.3303, 0.2418, 0.4131, and 0.4064, respectively. The relative mRNA expression of EPOR was 0.5196, 0.2319, 0.2398, 0.4253, and 0.1245, respectively, while that of EPO was 0.9751, 0.4712, 0.4613, 0.4875, and 0.1654. There was a reverse dose-dependent effect of simvastatin. These results suggest that statins increase erythropoiesis by targeting hepcidin and iron regulatory pathways, independent of erythropoietin.

Salidroside stimulates the accumulation of HIF-1α protein resulted in the induction of EPO expression: A signaling via blocking the degradation pathway in kidney and liver cells

Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance to mountain sickness in preventing hypoxia; however, the functional ingredient responsible for this adaptogenic effect has not been revealed. Here, we have identified salidroside, a glycoside predominantly found in Rhodiola, is the chemical in providing such anti-hypoxia effect. Cultured human embryonic kidney fibroblast (HEK293T) and human hepatocellular carcinoma (HepG2) were used to reveal the mechanism of this hematopoietic function mediated by salidroside. The application of salidroside in cultures induced the expression of erythropoietin (EPO) mRNA from its transcription regulatory element hypoxia response element (HRE), located on EPO gene. The application of salidroside stimulated the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein, but not HIF-2α protein: the salidroside-induced HIF-1α protein was via the reduction of HIF-1α degradation but not the mRNA induction. The increased HIF-1α could account for the activation of EPO gene. These results supported the notion that hematopoietic function of Rhodiola was triggered, at least partially, by salidroside.

Active Hexose Correlated Compound (AHCC) Alleviates Gemcitabine-Induced Hematological Toxicity in Non-Tumor-Bearing Mice

Active hexose correlated compound (AHCC) is known as a dietary supplement derived from an extract of a basidiomycete mushroom. The present study was conducted to evaluate the role of AHCC in alleviating the side effects, particularly hematological toxicity, in non-tumor-bearing mice receiving monotherapy with gemcitabine (GEM). The results from the GEM treatment groups with and without AHCC administration were compared to control group that received vehicle alone. The GEM alone treatment reduced peripheral leukocytes and hemoglobin, and bone marrow cell viability in spite of no influence on body weight, food consumption, and renal and hepatic parameters. Supplementation with AHCC significantly alleviated these side effects. The colony forming assay of bone marrow cells revealed that AHCC improved reduction of colony forming unit-granulocyte macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) related to GEM administration. However, when mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) was examined using a quantitative reverse transcription polymerase chain reaction (RT-PCR), AHCC showed no effect for the mRNA levels of their hematopoietic growth factors. These results support the concept that AHCC can be beneficial for cancer patients with GEM treatment through alleviating the hematotoxicity.

General Anesthetics Inhibit Erythropoietin Induction under Hypoxic Conditions in the Mouse Brain

BackgroundErythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes.Methodology/Principal FindingsBALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10–1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2α protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2α protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2α protein and EPO mRNA.Conclusions/SignificanceTaken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.

PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells

The platelet-derived growth factor (PDGF) signaling system contributes to tumor angiogenesis and vascular remodeling. Here we show in mouse tumor models that PDGF-BB induces erythropoietin (EPO) mRNA and protein expression by targeting stromal and perivascular cells that express PDGF receptor-β (PDGFR-β). Tumor-derived PDGF-BB promoted tumor growth, angiogenesis and extramedullary hematopoiesis at least in part through modulation of EPO expression. Moreover, adenoviral delivery of PDGF-BB to tumor-free mice increased both EPO production and erythropoiesis, as well as protecting from irradiation-induced anemia. At the molecular level, we show that the PDGF-BB-PDGFR-bβ signaling system activates the EPO promoter, acting in part through transcriptional regulation by the transcription factor Atf3, possibly through its association with two additional transcription factors, c-Jun and Sp1. Our findings suggest that PDGF-BB-induced EPO promotes tumor growth through two mechanisms: first, paracrine stimulation of tumor angiogenesis by direct induction of endothelial cell proliferation, migration, sprouting and tube formation, and second, endocrine stimulation of extramedullary hematopoiesis leading to increased oxygen perfusion and protection against tumor-associated anemia.

Indoxyl sulfate, a representative uremic toxin, suppresses erythropoietin production in a HIF-dependent manner

Advanced chronic kidney disease (CKD) patients encounter anemia through insufficient erythropoietin (EPO) production by peritubular fibroblasts. Recent studies showed an increase in EPO production by pharmacological activation of hypoxia-inducible transcription factors (HIFs) in dialysis patients, suggesting that desensitization of the oxygen-sensing mechanism is responsible for the development of renal anemia. Our recent work demonstrated that indoxyl sulfate (IS), a uremic toxin, dysregulates oxygen metabolism in tubular cells. Here we provide evidence of an additional property that IS impairs oxygen sensing in EPO-producing cells. HepG2 cells were stimulated with cobalt chloride (CoCl2) or hypoxia under varying concentrations of IS. EPO mRNA was evaluated by quantitative PCR. Nuclear accumulation of HIF-α was evaluated by western blotting. Transcriptional activity of HIF was checked by hypoxia-responsive element (HRE)-luciferase reporter assay. The impact of IS was further evaluated in vivo by administering rats with indole, a metabolic precursor of IS, and subjecting them to CoCl2 stimulation, in which renal EPO mRNA as well as plasma EPO levels were measured by quantitative PCR and enzyme-linked immunosorbent assay, respectively. Although IS induced cellular toxicity at relatively high concentrations (⩾2.5 mM), EPO mRNA expression was significantly suppressed by IS at concentrations below cytotoxic ranges. In HepG2 cells, IS treatment decreased nuclear accumulation of HIF-α proteins and suppressed HRE-luciferase activity following hypoxia. Furthermore, administration of rats with indole suppressed renal EPO mRNA expression and plasma EPO levels, corroborating in vitro findings. Results of the present study provide a possible connection between a uremic toxin and the desensitization of the oxygen-sensing mechanism in EPO-producing cells, which may partly explain inadequate EPO production in hypoxic kidneys of CKD patients.

Effect of repeated hyperbaric oxygen preconditioning on expression of hypoxia-inducible factor-1 alpha and erythropoietin following spinal ischemia-reperfusion in rats

Objective To investigate the effect of repeated hyperbaric oxygen preconditioning (HOP) on the expression of hypoxia-inducible factor-1 alpha (HIF-1a) and erythropoietin (EPO) following spinal ischemiareperfusion (I/R) in rats. Methods Forty-eight male SD rats weighing 250-300 g were randomly divided into 3 groups (n = 16 each): sham operation group (S group); I/R group and repeated HOP group (HOP group). The animals in HOP group were pretreated with O2 ( ＞ 98 % ) at 2.5 ATA I h/d for 5 consecutive days at 24 h before spinal cord I/R. The animals were anesthetized with intraperitoneal pentobarbital sodium 35 mg/kg. Spinal cord ischemia was produced by cross-clamping of abdominal aorta distal to renal artery for 20 min in I/R and HOP groups. Hind-limb motor function was assessed and scored st 48 h of reperfusion. The animals were then sacrificed and the L5-7 segment of the spinal cord was removed for determination of the expression of HIF-1 α and EPO mRNA and protein and microscopic examination. Results Compared with group S, the hind-limb motor function scores and the number of normal motor neurons in the anterior horn of the spinal cord were significantly decreased, and the expression of HIF-1α and EPO mRNA and protein was up-regulated in I/R and HOP groups ( P ＜ 0.05). Compared with group I/R, the hind-limb motor function scores and the number of normal motor neurons in the anterior horn of the spinal cord were significantly increased, and the expression of HIF-1α and EPO mRNA and protein was up-regulated in group HOP ( P ＜ 0.05). The spinal cord injury was attenuated in group HOP compared with group I/R. Conclusion Repeated HOP can reduce the spinal cord I/R injury though up-regulating the expression of HIF1α and EPO in rats. Key words: Hyperbaric oxygenation; Ischemic preconditioning; Hypoxia-inducible factor 1, alpha subunit; Erythropoietin; Reperfusion injury; Spinal cord

Prenatal Glucocorticoid Overexposure Causes Permanent Increases in Renal Erythropoietin Expression and Red Blood Cell Mass in the Rat Offspring

Glucocorticoids promote maturation of fetal systems, including erythropoiesis, in preparation for extrauterine life. However, recent studies have shown that prenatal glucocorticoid excess can cause long-term deleterious cardiometabolic and other consequences to the offspring. Here, we examined the effect of prenatal treatment with the synthetic glucocorticoid dexamethasone (DEX) during the last week of gestation on red blood cell (RBC) mass in the rat offspring. DEX-treated offspring at 9 months of age had significantly higher RBC count (9.4 ± 0.1 vs. 8.8 ± 0.2 × 10(12) liter; P = 0.02), hematocrit (50.0 ± 0.5 vs. 46.7 ± 0.7%; P=0.004), hemoglobin (17.3 ± 0.2 vs. 16.2 ± 0.2 g/dl; P = 0.02) and number of reticulocytes (258.2 ± 8.8 vs. 235.7 ± 5.6 × 10(9) liter; P = 0.04), compared with offspring of vehicle-treated control pregnancies. White blood cells and platelets were unaltered. Renal mRNA expression and plasma concentrations of erythropoietin, the main regulator of erythropoiesis, were increased by nearly 100% in both newborn and adult DEX-treated rats (P < 0.01). This increase was accompanied by marked elevation in renal expression of hepatocyte nuclear factor 4α mRNA, whereas other erythropoietin-regulating transcription factors, such as hypoxia-inducible factor 1, hypoxia-inducible factor 2, and GATA2 were unchanged. These data indicate that RBC mass can be programmed by prenatal glucocorticoid excess, and if extrapolatable to humans, provide a novel mechanism for fetal origins of polycythemia and its associated complications.