Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures

The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogeneous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxidase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus non-hypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin mRNA and by radioimmunoassay of secreted erythropoietin.     

The effect of haemorrhage on erythropoietin concentration in the mature ovine fetus.

This study examines the response in plasma erythropoietin values to haemorrhage of 20% of the estimated blood volume in chronically cannulated ovine fetuses, of gestational ages 128-144 days. Blood samples were collected at 0, 2, 4, 6 and 24h with respect to the haemorrhage. In 5 control experiments there was no significant change in plasma erythropoietin concentration, across this time period, values being 6.1 +/- 2.3 and 6.4 +/- 2.4 mU/ml at 0 and 24h respectively. Values are mean +/- SEM. Haemorrhage reduced the haematocrit and haemoglobin values, significantly, to 83 +/- 6% and 85 +/- 4% (n = 5) of the initial value, respectively, but did not cause a statistically significant increase in plasma erythropoietin concentrations (7.2 +/- 2.4 and 20.7 +/- 8.2 mU/ml; P = 0.131). A larger degree of haemorrhage, in four fetuses reduced the haematocrit to 64 +/- 2.8% of initial, over 24-54h and increased erythropoietin values very significantly (from 11.9 +/- 3.6 to 91 +/- 8.3 mU/ml; P = 0.001).     

Changes in globin messenger RNA content during erythroid cell differentiation.

Previous studies have shown that mouse fetal erythroid precursor cells isolated by an immunological technique synthesize little or no globin and contain little, if any, globin mRNA, as assayed in a cell-free system (translatable mRNA). After culture for 10 hours in the presence of erythropoietin, there is a marked increase in globin synthesis and in translatable globin mRNA. The present studies were designed to measure directly the content of globin mRNA sequences during erythroid cell differentiation, by molecular hybridization with 3H-labeled DNA complementary to globin mRNA. The results indicate that few, if any, globin mRNA sequences are present in the total RNA of erythroid precursor cells. There is little or no pool of untranslated globin mRNA in these cells. After 10 hours of culture with erythropoietin, there is an increase in globin mRNA content, as ;easured by a change in the Cot1/2 values obtained by cDNA: mRNA hybridization with (Co) representing the concentration of RNA. Between 0 and 22 hours of culture, there is a 250-fold rise, and between 22 and 44 hours, a further 2-fold increase in globin mRNA content. During the 44 hours in culture, the number of cells in culture increases 2- to 3-fold. The number of globin mRNA molecules rises in erythroid precursor cells to an average value of 1800 molecules/cell during 22 hours of culture. In cultures without added erythropoietin, the absolute number of cells decreases, however, cells presumably induced to differentiate by exposure to erythropoietin in vivo continue to differentiate in vitro, accumulating globin mRNA and initiating globin synthesis.     

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.     

Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition

Renal fibrosis and anaemia are two of the most relevant events in chronic kidney disease. Fibrosis is characterized by the accumulation of extracellular matrix proteins in the glomeruli and tubular interstitium. Anaemia is the consequence of a decrease in erythropoietin production in fibrotic kidneys. This work analyses the possibility that the accumulation of abnormal collagens in kidney interstitium could be one of the mechanisms responsible for erythropoietin decreased synthesis. In renal interstitial fibroblast grown on collagen I, erythropoietin mRNA expression and HIF‐2α protein decreased, whereas focal adhesion kinase protein (FAK) phosphorylation and proteasome activity increased, compared to cells grown on collagen IV. Proteasome inhibition or FAK inactivation in cells plated on collagen I restored erythropoietin and HIF‐2α expression. FAK inhibition also decreased the collagen I‐dependent proteasome activation. In a model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction in mice, increased collagen I protein content and an almost complete disappearance of erythropoietin mRNA expression were observed in the ureteral ligated kidney with respect to the contralateral control. Interestingly, erythropoietin synthesis was recovered in obstructed mice treated with proteasome inhibitor. These data suggest that reduced kidney erythropoietin synthesis could be caused by the accumulation of abnormal extracellular matrix proteins.     

Expression of the erythropoietin gene.

Injection of cobalt into rats resulted in erythropoietin (EPO) mRNA accumulation in the kidney. The same response was obtained upon bleeding. No EPO mRNA was detected in the spleen, salivary gland, or thymus following cobalt injection or bleeding. In some animals, but not in others, EPO mRNA was also expressed in the liver in response to cobalt injection. Time course studies showed that message appearance begins sometime between 3 and 6 h after cobalt injection. This correlated very well with the EPO concentration in the circulation; EPO levels in the circulation were the same as those of controls at 3 h but increased to six- to sevenfold that of controls by 6 h after cobalt injection. The mature EPO mRNA in the rat and mouse comigrated with the 18S rRNA, indicating that it is about 1,850 nucleotides in length.     

Expression of erythropoietin receptor mRNA in mouse brain hemispheres

Now there is a growing evidence that erythropoietin receptors (Epo-R) are present also in some nonhematopoietic tissues such as endothelial cells and fetal cells of neural origin, although the physiological role of Epo-R at these sites is unclear. There are some speculations that Epo-R may be expressed on cells only in the developing CNS. The objective of this study was to determine whether Epo-R mRNA may be expressed in the brain hemispheres of Balb/c mice of different age groups: 1) newborn mice, 2) young 2 months old mice, 3) old 1.8 year old mice. We also studied the in vivo effect of recombinant erythropoietin on the expression of Epo-R mRNA in the brain hemispheres of (CBA x C57BL)F1 mice by RT-PCR. We have detected the existence of Epo-R mRNA expression in brain hemispheres of all the groups, but in old mice this expression was significantly higher. We have discovered a decrease in Epo-R mRNA expression in brain hemispheres of (CBA x C57BL)F1 mice 24 h after in vivo administration of recombinant erythropoietin. The Epo-R mRNA expression in the left brain hemispheres of (CBA x C57BL)F1 was considerably higher than in the right one.     

Titration of mRNAs for cytochrome P-450c and P-450d under drug-inductive conditions in rat livers by their specific probes of cloned DNAs

By sequence analysis of the cloned cDNA or genomic DNA, we have recently deduced the complete primary structures of two forms of 3-methylcholanthrene-inducible cytochromes P-450 (P-450c and P-450d). Comparing these sequences, we identified two highly conserved regions, amino acid numbers from 35 to 200 and from 340 to 470. The nucleotide sequences corresponding to these homologous regions are also well conserved, whereas other regions have undergone considerable sequence divergence. In RNA blot analysis with unfractionated mRNA isolated from 3-methylcholanthrene-treated rat livers, Probe A (specific to P-450c sequence) hybridized with mRNA around 23 S, while Probe B (specific to P-450d sequence) hybridized with mRNA around 18 S. When common sequence between P-450c and P-450d was used as the probes (Probe C or D), two bands were clearly observed around 23 and 18 S mRNAs. With the common DNA sequence between P-450c and P-450d as a probe (93.7% homology), we studied the induction of specific mRNA for P-450c and P-450d by a single dose of several chemical compounds to rats. 3-Methylcholanthrene increased both P-450c and P-450d mRNA levels by 50 and 10 times above the control at 17 h after the administration, respectively. Despite the lower induction rates, the P-450d mRNA level was constantly higher than or at least similar to that of P-450c mRNA. beta-Naphthoflavone and Kaneclor KC 500 showed similar induction ability to 3-methylcholanthrene. On the other hand, isosafrole induced P-450d mRNA to a much greater extent than P-450c mRNA.     

Hypoxia signalling in the control of erythropoietin gene expression in rat hepatocytes

This study aimed to investigate the sequence of events involved in the stimulation of erythropoietin (EPO) gene expression by hypoxia in hepatocytes. To this end, primary cultures of rat hepatocytes were kept at either high (40% O₂) or low (3% O₂) oxygen tensions for 2.5 h. Hypoxia increased EPO mRNA about fifteen-fold, whilst the divalent cation cobalt (50–100 μM) or the iron chelator desferrioxamine (10–200 μM) did not increase EPO mRNA levels. Addition of hydrogen peroxide (100–500 μM) to the culture medium did also not change EPO mRNA levels at high or low oxygen tension. Addition of catalase (50–200 μg/ml) to the culture medium resulted in a lower level of hypoxia-induced EPO mRNA. Inhibition of protein synthesis by cycloheximide (100 μM) completely abolished the increase of EPO mRNA in response to hypoxia. Hypoxia but not cobalt increased the appearance of the hypoxia-inducible factor 1 (HIF-1), and this increase was blunted by cycloheximide. Taken together, these findings suggest that a classic heme protein and a related oxygen-dependent production of oxygen radicals is less likely to be involved in the regulation of the EPO gene by oxygen in hepatocytes. On the other hand, intact protein synthesis is an absolute requirement for the hypoxia-induced appearance of HIF-1 and for hypoxia-stimulated expression of the EPO gene in hepatocytes. © 1996 Wiley-Liss, Inc.     

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.     

脑源性促红细胞生成素在短暂性前脑缺血沙土鼠海马的表达变化

目的 探讨短暂性前脑缺血鼠海马脑源性促红细胞生成素蛋白的表达变化,揭示脑缺血时中枢神经系统发生内源性脑保护的机制。方法 阻断沙土鼠双侧颈总动脉3．5min造成前脑缺血模型,再灌注1h,6h,12h,1d,3d,7d,应用免疫组织化学和免疫印迹法观察海马脑源性促红细胞生成素蛋白的表达变化。结果 脑缺血再灌注6h,可检测到脑源性促红细胞生成素的表达,再灌注12h,脑源性促红细胞生成素表达达到较高水平,以后随时间延长逐渐下调。结论 脑源性促红细胞生成素在脑缺血再灌注后的表达,可能是机体发生内源性脑保护的机制之一。     

Cloning of cDNA for mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E.

A cDNA coding for the lysosomal aspartic protease from the mosquito (mLAP) was cloned and sequenced. The mLAP cDNA is 1420 base pairs long with an open reading frame of 387 amino acids. The deduced amino acid sequence contains a signal pre-propeptide sequence of 18 amino acids followed by 369 amino acids with a 35-amino acid putative pro-enzyme domain in the NH2-terminal. The amino acid sequence of mLAP is 92 and 81% similar to human cathepsin D and cathepsin E, respectively. Typical cleavage sites for cathepsin D processing into light and heavy chains are lacking in mLAP. A single glycosylation site occurs in the mLAP sequence at a position corresponding to the first glycosylation site of cathepsins D. The mLAP sequence shares putative phosphorylation determinants, which in cathepsins D are linked to the formation of mannose 6-phosphate. In the mosquito fat body, lysosomal enzymes specifically degrade organelles involved in the biosynthesis and secretion of vitellogenin. The mLAP mRNA accumulates to its highest level 24 h after initiation of vitellogenin synthesis and 12 h before the peak of mLAP protein accumulation and its enzymatic activity. Translational regulation of mLAP mRNA may occur. The 5'-untranslated region of mLAP mRNA is similar to elements conferring negative translational control by steroids.     

The largest subunit of RNA polymerase II in Dictyostelium: conservation of the unique tail domain and gene expression.

cDNAs encoding the largest subunit of RNA polymerase II were isolated from a Dictyostelium cDNA library. A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced. Similar to other eukaryotic RNA polymerases II, the largest subunit of Dictyostelium RNA polymerase II contains a unique repetitive tail domain at its carboxyl-terminal region. It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein. RNA blots show that cDNA probes hybridized with a single mRNA species of approximately 6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons. Interestingly, expression of the largest subunit of RNA polymerase II appears to be under developmental regulation. The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h. Cells began to accumulate a higher level of the RNA polymerase II mRNA after 9 h of development. When cells were treated with low concentrations of cAMP pulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells.     

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.     

Cloning and characterization of a hsp70 gene from Asiatic hard clam Meretrix meretrix which is involved in the immune response against bacterial infection

In the present study, a 71.43 kDa heat shock protein cDNA was cloned from Asiatic hard clam Meretrix meretrix. The cDNA was 2292 bp, containing an open reading frame (ORF) of 1959 bp, which encodes a protein of 652 amino acids with a theoretical molecular weight of 71.43 kDa and an isoelectric point of 5.32. Based on the amino acid sequence analysis and phylogenetic analysis, this hsp70 cDNA is a member of cytoplasmic hsc70 (constitutive genes) subfamily in the hsp70 family, and is designated as MmeHsc71. Quantitative RT-PCR was carried out to compare the spatial and temporal expression patterns of MmeHsc71 in the mRNA level between control clams and Vibrio parahaemolyticus-infected clams. Spatially, MmeHsc71 mRNA was found in all tested tissues, including foot, hepatopancreas, mantle and gill. MmeHsc71 mRNA expression level in hepatopancreas and gill displayed a significant increase in vibrio-challenged clams at 24h post-infection compared to control clams (P < 0.05). Temporally, there was a significant increase of MmeHsc71 mRNA level in hepathopancreas of vibrio-challenged clams compared to control clams at 6, 12, and 24h post-challenge, respectively. The result of quantitative immunofluorescence also indicated that there was obvious increase of MmeHsc71 in hepatopancreas of vibrio-challenged clams compared to control clams in protein level at 24h post-infection. The results suggested that MmeHsc71 may play an important role in mediating the immune responses of M. meretrix to bacterial challenge.     

Effect of erythropoietin levels on mortality in old age: the Leiden 85-plus Study

Background

The production of erythropoietin is triggered by impaired oxygen delivery to the kidney, either because of anemia or hypoxemia. High erythropoietin levels have been shown to predict the risk of death among patients with chronic heart failure. We investigated the prognostic value of elevated erythropoietin levels on mortality among very elderly people in the general population.

Methods

The Leiden 85-plus Study is a population-based prospective follow-up study involving 599 people aged 85 years in Leiden, the Netherlands, enrolled between September 1997 and September 1999. Erythropoietin levels were determined at age 86. For this analysis, we included 428 participants with a creatinine clearance of at least 30 mL/min. Mortality data, recorded until Feb. 1, 2008, were obtained from the municipal registry.

Results

During follow-up, 324 (75.7%) participants died. Compared with participants whose erythropoietin levels were in the lowest tertile (reference group), those whose levels were in the middle tertile had a 25% increased risk of death (hazard ratio [HR] 1.25, 95% confidence interval [CI] 0.95–1.64), and those whose levels were in the highest tertile had a 73% increased risk (HR 1.73, 95% CI 1.32–2.26) (p value for trend < 0.01). The association between erythropoietin levels and mortality remained largely unchanged after we adjusted for sex, creatinine clearance, hemoglobin level, comorbidity, smoking status and C-reactive protein level, and was similar for deaths from cardiovascular and noncardiovascular causes.

Interpretation

Among people aged 85 years and older, elevated erythropoietin levels were associated with an increased risk of death, independent of hemoglobin levels.Decreased oxygen availability in the kidney triggers the peritubular capillary lining cells within the kidney to produce erythropoietin — the principal regulator of red blood cell mass.¹ Impaired oxygen delivery to the kidney can result from various pathophysiologic mechanisms, such as anemia, hypoperfusion due to renal arteriosclerosis, decreased renal blood flow or heart failure, and decreased oxygen saturation due to diseases such as chronic obstructive pulmonary disease.^1–5Studies involving patients with chronic heart failure have shown that high erythropoietin levels predict poor survival.^6–11 However, whether this prognostic value is limited to patients with heart failure or whether it is generalizable to very elderly people regardless of specific pathologic conditions has not been studied. We conducted this study to investigate whether high levels of erythropoietin predict mortality among very elderly people in the general population.