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.     

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.     

A Lentiviral Gene Therapy Strategy for the In Vitro Production of Feline Erythropoietin

Nonregenerative anemia due to chronic renal failure is a common problem in domestic cats. Unfortunately, administration of recombinant human erythropoietin often only improves anemia temporarily due to antibody development. In this in vitro study, feline erythropoietin cDNA was cloned from feline renal tissue and utilized in the construction of a replication-defective lentiviral vector. The native recombinant feline erythropoietin (rfEPO) sequence was confirmed by sequencing. Upon viral vector infection of human 293H cells, Crandall Renal Feline Kidney cell line and primary feline peripheral blood mononuclear cells, bioactive rfEPO protein was produced. The presence of cellular rfEPO cDNA was confirmed by standard PCR, production of abundant rfEPO mRNA was confirmed by real-time PCR, and secretion of rfEPO protein was demonstrated by Western blot analyses, while rfEPO protein bioactivity was confirmed via an MTT proliferation bioassay. This in vitro study demonstrates the feasibility of a replication-defective lentiviral vector delivery system for the in vitro production of biologically active feline erythropoietin. Anemic cats with chronic renal failure represent a potential in vivo application of a lentiviral gene therapy system.     

Lowered plasma erythropoietin in hypoxic rats with kidney tubule lesions

The role of the kidney tubules in the renal formation of erythropoietin is incompletely understood. Therefore, the capability to produce erythropoietin in response to hypoxia was studied in rats with tubular lesions. Nephron damage was induced in two different ways. First, rats were treated with the nephrotoxic aminoglycoside gentamicin (67.5 mg/kg and day) for 14 days. The animals were then subjected to simulated altitude (6,800 m) for 6 h. The resulting plasma erythropoietin concentration was significantly lower (0.5 IU/ml) than in saline treated control rats exposed to hypoxia (1.0 IU/ml). Second, unilateral hydronephrosis was induced by ureteral ligation. The contralateral kidney was removed immediately before the animals were exposed to simulated altitude for 6 h. The plasma erythropoietin concentration in the ureter-ligated rats did not increase above the value (0.3 IU/ml) in hypoxia exposed anephric rats. These results indicate that the production of erythropoietin is reduced following tubular injury. Tubule cells may directly produce the hormone or interfere with the O2-sensing mechanisms controlling its synthesis. The latter hypothesis would seem to be supported by our failure to demonstrate in vitro erythropoietin production by the two established kidney tubule cell lines, LLC-PK1 and PK-15.     

Is renal erythropoietin secretion consequence of local hypoxia coupled with local suppression of angiogenesis?

The kidney is a major source of systemic erythropoietin, despite itself showing little angiogenesis. Based on paper by Schumacher VA et-al. [J Am Soc Nephrol 2007;18:719-29] that renal synthesis of an inhibitory VEGF variant (VEGF165b) blocks local angiogenesis, we hypothesise that the chronic hypoxia in the renal medulla, due to limited perfusion through vasa recta stimulates continuous renal erythropoietin secretion.     

Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.

Regulation of the production of erythropoietin occurs in the kidney and liver largely through control of accumulation of erythropoietin mRNA. Erythropoietin mRNA was first detected in kidneys at 1.5 h postanemia and reached a plateau value at least 200-fold above the control value by 4 to 8 h. A 20-base sequence immediately upstream from the reported erythropoietin mRNA initiation site is complementary to a hypervariable sequence in 18S rRNA.     

Erythropoietin titers in response to anemia or hypoxia

The normal response to anemic or hypoxic hypoxia is synthesis and release of erythropoietin in accord with the concept that erythropoietin production is controlled by a renal oxygen sensor. In this study, erythropoietin production, as predicted, was abrogated in patients with renal impairment (55 cases), but normal in nonuremic individuals. Specifically, patients with rheumatoid arthritis (34 cases), sickle cell anemia (25 cases), aregenerative anemia (27 cases), and aplastic anemia (13 cases) had erythropoietin titers overlapping with those observed in simple anemia (61 cases) at corresponding hematocrits. The response of polycythemic laboratory animals to hypoxia is more difficult to fit within the concept of an oxygen sensor responsive both to anemic and hypoxic hypoxia. If the polycythemia was induced by hypertransfusion, erythropoietin production in response to hypoxia was, as predicted, less than that observed in normal animals. If, however the polycythemia was induced by previous exposure to hypoxia, the animals responded to hypoxia as though they were not polycythemic. An explanation for this challenging observation may provide a clue as to the operation of the oxygen sensor.     

Localization of the gene responsible for familial benign polycythemia to chromosome 11q23.

Familial benign polycythemia (FBP) (OMIM 263400) is a rare autosomal recessive condition characterized by erythrocytosis, normal leukocyte and platelet counts, normal uric acid level, and usually increased erythropoietin production. There is a high incidence of this disorder in Chuvashia (Russian Federation), probably due to a founder effect. In an attempt to locate the gene responsible for this disorder, we have carried out linkage studies in 12 Chuvash families, with 35 affected and 32 unaffected members. Linkage to the erythropoietin and erythropoietin receptor loci was excluded, and the FBP gene was assigned to the region of chromosome 11q23 between D11S4142 and D11S1356, with a maximal lod score of 6.61.     

Erythropoietin decreases cytotoxicity and nitric oxide formation induced by inflammatory stimuli in rat oligodendrocytes

In the present study, we investigated whether erythropoietin (Epo) has a protective effect against cytotoxicity induced by interferon-gamma (IFN-gamma ) and lipopolysaccharide (LPS) in primary rat oligodendrocyte cultures. The possible modulatory effect of erythropoietin on inducible nitric oxide synthase (iNOS) mRNA expression and nitrite production were also analyzed. Erythropoietin exerted a significant protective effect against IFN-gamma and LPS-induced oligodendrocyte injury as determined by lactate dehydrogenase assay. Treatment with erythropoietin inhibited the expression of iNOS mRNA and nitrite production resulting from proinflammatory stimulation by IFN-gamma and LPS. These results suggest that erythropoietin has protective effects against inflammatory oligodendrocyte injury in vitro and may play a protective role in neurological disorders characterized by oligodendrocyte death, such as multiple sclerosis.     

Intracellular accumulation of isoproterenol enhances the lactate production in the perfused rat heart

Effects of intracellular accumulation of isoproterenol (ISO) on lactate production were examined in perfused rat heart. The lactate production during ISO perfusion in rat heart was increased and subsequent addition of an inhibitor of catechol-O-methyl transferase (COMT) further enhanced the production, and the enhanced production was significantly reduced by uptake2 inhibitor. The perfusion with ISO free-medium in the heart with high intracellular accumulation of ISO produced lactate more than that in the low intracellular accumulation. The present experiments demonstrated that the enhanced lactate production is accompanied by intracellular accumulation of ISO in the perfused rat heart, and suggested that the accumulated ISO may activate intracellular beta-adrenoceptors in the rat heart.     

Effect of hyperoxia on erythropoietin biogenesis in the "endocrine" kidney model

The erythropoietin plasma level and RNA synthesis in both kidneys were studied in rats with the H. Selye "endocrine" kidney under 4-hour hyperoxia. It was shown that a short period of hyperoxia leads to a 2-fold decrease in erythropoietin plasma level and to the fall of RNA synthesis in the "endocrine" and intact kidneys. From the evidence obtained it is concluded that hyperoxia inhibits erythropoietin production in the kidneys. Changes in high-polymeric RNA synthesis suggest that DNA-dependent RNA synthesis is one of the mechanisms of the hormone biogenesis.     

Lack of maternal to fetal transfer of 125I-labelled erythropoietin in sheep.

We administered tracer quantities of biologically active 125I-labelled recombinant human erythropoietin by intravenous bolus injection to seven late gestation pregnant ewes. Maternal and fetal blood was sampled over the subsequent six hours and assayed for erythropoietin-specific radioactivity. Despite the expected increase in maternal plasma immunoprecipitable 125I-labelled erythropoietin radioactivity, fetal plasma levels remained unchanged throughout the study. In addition, erythropoietin receptors were not detected in ovine and human placental tissue. We conclude that biologically active 125I-labelled erythropoietin does not cross the placenta from mother to fetus in measurable quantities in sheep, and likely in humans. Thus, these data indicate the levels of erythropoietin measured in fetal plasma are reflective of fetal, and not maternal, erythropoietin production and elimination.     

Adenosine, AMP, cyclic AMP, theophylline and the action and production of erythropoietin.

Erythropoiesis, as measured by the uptake of 59Fe into plethoric mice, is stimulated by adenosine, AMP, cyclic AMP, and dibutyryl cyclic AMP, but not by cytidine, its nucleotides or cyclic GMP. This stimulation is erythropoietin dependent, because it is prevented by anti-erythropoietin. Theophylline neither stimulates erythropoiesis nor potentiates the action of erythropoietin on bone marrow cells in plethoric mice. Theophylline does potentiate the production of erythropoietin in rats following a frief hypoxic exposure but does not cause a similar increase in mice.     

悬浮培养CHO 细胞生产重组人促红细胞生成素条件的优化*

目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO)。方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量。结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础。结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本。     

Immunoreactive erythropoietin concentration and haemoglobin type in the perinatal period in sheep of various haemoglobin genotypes

This study examines the hypotheses that (i) erythropoietin (the hormone responsible for red blood cell production) is higher in the fetus (at low PO2) than in the neonate (at high PO2); and (ii) that the level of erythropoietin in the neonate is influenced by the presence of high oxygen affinity haemoglobin. Haematocrit (PCV), PO2 and plasma immunoreactive erythropoietin were measured in 4 chronically cannulated fetal sheep (120 days to birth, n = 22) and in 7 neonatal lambs until 233 days post-conception (75-85 days after birth, n = 83). The percentage of globin chains (alpha, gamma, beta A, beta B, beta C) was quantitated by gel electrophoresis. Plasma erythropoietin values, in 4 fetuses were 9.8 +/- 1.3 mU/ml at 120-132 days of gestation, declined significantly (P less than 0.01) to 5.2 +/- 0.4 at 133 days until birth, then increased significantly (P less than 0.001) to 24.2 +/- 5.5(5), 26.3 +/- 7.3 (6), and 24.8 +/- 8.5 (6), respectively in weeks 1, 2 and 3 of postnatal life. By weeks 5-8 the erythropoietin was 13 +/- 0.6 (4) mU/ml. PO2 was 17.4 +/- 0.9 mmHg before birth and 88.0 +/- 10.7 in the first week after birth. PCV was constant until three weeks after birth and then declined. Fetal haemoglobin had virtually disappeared from the circulation by 166 days (3 weeks after birth); in the 4 heterozygotes (beta A beta B) beta C was expressed transiently, with a maximum value of 4%, whilst in the homozygote lamb (beta A beta A) the maximum beta C was 12%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dietary Protein-induced Changes of the Erythropoietin Level in Rat Serum

Erythropoietin, a glycoprotein, is the primary regulator of erythropoiesis. The most convenient and sensitive assay for active erythropoietin is to measure its stimulatory effect on in vitro ³H-thymidine incorporation into DNA of erythropoietin-responsive cells. An attempt with this method to estimate the erythropoietin level in rat serum, however, was unsuccesful because of the presence of inhibitory substance(s) and non-erythropoietic factor(s) stimulating ³H-thymidine incorporation. Pretreatment of the serum by heating, extraction of erythropoietin from denatured-protein aggregates, and subsequent concentration of erythropoietin in the extract with alcohol precipitation made it possible to measure the serum erythropoietin levels. Rabbit anti-erythropoietin antibody was used for a quantitative estimation of erythropoietin in the concentrated extracts. Erythropoietin levels in sera of rats fed on varied amounts of casein for 7 days were measured with these procedures to find if the impairment of erythropoiesis upon protein deprivation was due to changes in the erythropoietin level. We found that the level in protein-deprived rats was less than 1/8 that of 20% casein-fed rats, a level undetectable by the present assay, and that the serum erythropoietin increased as the protein content in the diet was increased up to 20%, then leveled off. The erythropoietin in serum decreased rapidly after protein deprivation; the level at 12hr after deprivation began was about 1/5 that in 20% casein-fed rats. Thus, the depression of erythropoiesis upon protein deprivation is primarily caused by the lowered level of erythropoietin.     

The extracellular domain of the human erythropoietin receptor: expression as a fusion protein in Escherichia coli, purification, and biological properties

We developed an efficient production system of the soluble extracellular domain of the human erythropoietin receptor (sEPO-R) and characterized the binding of erythropoietin (EPO) with the purified recombinant protein. The sEPO-R, fused to the maltose binding protein (MBP), was expressed as a soluble protein in the periplasm of Escherichia coli (E. coli) and did not accumulate in inclusion bodies. After lysis of the bacteria by an osmotic shock, the fusion protein was purified by affinity chromatography on amylose followed by size exclusion chromatography (SEC). Specific binding of 125I-labelled EPO to the sEPO-R was demonstrated by competitive and saturation binding assays. A single affinity class (Kd = 0.25 nM) of the binding site was evident by Scatchard analysis. This value is similar to the Kd observed between EPO and the EPO-R of high affinity present on human erythroid progenitors. The complex has a molecular size corresponding to a 1:1 complex of EPO and the fusion protein.