scholarly journals Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4097-4104 ◽  
Author(s):  
T Misaizu ◽  
S Matsuki ◽  
TW Strickland ◽  
M Takeuchi ◽  
A Kobata ◽  
...  
Keyword(s):  
Whole Body ◽  
Renal Handling ◽  
Sugar Chain ◽  
Erythroid Progenitor ◽  
Human Erythropoietin ◽  
Sugar Chains ◽  
Effective Transfer ◽  
Branched Structure

To elucidate the role of the branched structure of sugar chains of human erythropoietin (EPO) in the expression of in vivo activity, the pharmacokinetic profile of a less active recombinant human EPO sample (EPO-bi) enriched with biantennary sugar chains was compared with that of a highly active control EPO sample enriched with tetraantennary sugar chains. After an intravenous injection in rats, 125I-EPO-bi disappeared from the plasma with 3.2 times greater total body clearance (Cltot) than control 125I-EPO. Whole-body autoradiography after 20 minutes of administration indicated that the overall distribution of radioactivity is similar, but 125I-EPO-bi showed a higher level of radioactivity in the kidneys than control 125I-EPO. Quantitative determination of radioactivity in the tissues also indicated that radioactivity of 125I-EPO-bi in the kidneys was two times higher than that of control 125I-EPO. The difference in plasma disappearance between 125I-EPO-bi and control 125I-EPO was not observed in bilaterally nephrectomized rats. The distribution of 125I-EPO-bi to bone marrow and spleen was similarly inhibited by simultaneous injection of excess amounts of either the nonlabeled EPO-bi or control EPO. These results indicate that the low in vivo biologic activity of EPO-bi results from rapid clearance from the systemic circulation by renal handling. Thus, the well-branched structure of the N-linked sugar chain of EPO is suggested to play an important role in maintaining its higher plasma level, which guarantees an effective transfer to target organs and stimulation of erythroid progenitor cells.

Antiapoptotic properties of erythropoiesis-stimulating proteins in models of cisplatin-induced acute kidney injury

2008 ◽  
Vol 294 (6) ◽  
pp. F1354-F1365 ◽  
Author(s):  
Abdulla K. Salahudeen ◽  
Naeem Haider ◽  
John Jenkins ◽  
Manish Joshi ◽  
Harnish Patel ◽  
...  
Keyword(s):  
Serum Creatinine ◽  
Kidney Injury ◽  
Saline Group ◽  
Renal Protection ◽  
Erythroid Progenitor ◽  
Human Erythropoietin ◽  
Peptide Mass ◽  
Stat Pathway

Erythropoietin (Epo) induces erythrocytosis by suppressing erythroid progenitor cell apoptosis through the Janus-activated kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Since apoptosis contributes to cisplatin (CP)-induced nephrotoxicity and Epo receptors (EpoR) are expressed in the kidney, we examined the role of antiapoptosis in recombinant human erythropoietin (rHuEpo)-mediated renal protection. In human renal proximal tubular epithelial (RPTE) cells in culture, rHuEpo, but not inactive rHuEpo (I-rHuEpo), the receptor-binding sites of which are mutated, caused a significant reduction in CP-induced apoptosis at ≥100 U/ml. rHuEpo, but not I-rHuEpo, increased STAT5 and Akt/PKB phosphorylation, demonstrating functional EpoR expression on RPTE cells. Furthermore, the JAK2 inhibitor tyrphostin AG-490 attenuated rHuEpo protection, suggesting a role of the JAK-STAT pathway in rHuEpo-mediated antiapoptosis. In rats, intravenous administration of 5,000 U/kg rHuEpo, but not an equivalent peptide mass of I-rHuEpo, before a single 5.5 mg/kg iv injection of CP, significantly increased hematocrit (Hct) and reduced the CP-induced increase in serum creatinine. Serum creatinine on day 4 was 3.4 ± 0.3, 1.9 ± 0.3, and 3.5 ± 0.4 mg/dl in the CP, CP + rHuEpo, and CP + I-rHuEpo groups, respectively. Similarly, darbepoietin-α (DA), a hyperglycosylated analog of rHuEpo with prolonged in vivo activity when injected at 25 μg/kg iv before CP, significantly increased Hct and reduced serum creatinine. Renal clearance studies based on glomerular filtration rate and renal blood flow confirmed the significant renal protection by DA against CP. Tubular apoptosis and necrosis were significantly reduced in the kidneys of the CP + DA vs. the CP + saline group. Moreover, the equalization of Hct by venesection did not abrogate the DA-mediated renal protection. Administration of DA 48 h after CP injection also conferred significant renal protection. Thus our experiments confirm a role for erythropoiesis-stimulating proteins, including the new analog DA, in limiting CP-induced nephrotoxicity and suggest that antiapoptosis via the Epo-EpoR interaction is an important mechanism for renal protection.

Studies of the antibody-dependent killing of schistosomula of Schistosoma mansoni employing haptenic target antigens. Analysis of mechanisms responsible for the rejection of parasites in vivo

Parasitology ◽  
1981 ◽  
Vol 83 (3) ◽  
pp. 543-558
Author(s):  
Gina Moser ◽  
F. von Lichtenberg ◽  
A. Sher
Keyword(s):  
Schistosoma Mansoni ◽  
Passive Immunization ◽  
Early Response ◽  
Challenge Infection ◽  
Delayed Hypersensitivity ◽  
Whole Body ◽  
Hypersensitivity Response ◽  
Time Points

SUMMARYSchistosomula, surface labelled with trinitrophenyl (TNP) target antigens were tested for their susceptibility to killing by humoral- or cell-mediated anti-TNP effector mechanisms in vivo. It was found that mice passively immunized with anti-TNP serum effectively rejected an intravenous (i.v.) challenge infection with TNP-labelled schistosomula. In contrast, mice which demonstrated a strong TNP-specific, delayed hypersensitivity response to the haptenated larvae as evidenced by ear swelling, were unable to eliminate the same challenge infection. Significant passive immunization against TNP-labelled schistosomula was shown to require microlitre quantities of anti-TNP serum and could be conferred with an IgG fraction purified from the serum. The role of cells in the antibody-dependent rejection of TNP-labelled schistosomula was investigated using histopathological methods. In passively immunized mice, haptenated larvae elicited neutrophil-enriched focal reactions in the lungs and showed evidence of degeneration as early as 2 h after injection. These cellular reactions were not observed in recipients which had received prior whole-body irradiation. Nevertheless, by 24 h TNP-labelled larvae were found to have been killed in the lungs of the irradiated mice despite the absence of significant cellular attack. The above observations suggest that the antibody-dependent destruction of haptenated schistosomula results from two overlapping responses, an early response mediated by radio-sensitive cells and a second, radio-resistant response manifesting its effects at later time points. Since mice genetically deficient in the fifth component of complement fail to develop the later response, it probably reflects the effect of the lytic pathway of complement on the parasite.

scholarly journals Ablation of 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) in Vascular Endothelial Cells Enhances Insulin Sensitivity by Reducing Visceral Fat and Suppressing Angiogenesis

2012 ◽  
Vol 26 (1) ◽  
pp. 95-109 ◽  
Author(s):  
Kazuhito Tawaramoto ◽  
Ko Kotani ◽  
Mitsuru Hashiramoto ◽  
Yukiko Kanda ◽  
Tomoki Nagare ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Insulin Sensitivity ◽  
Protein Kinase ◽  
Vascular Endothelial Cells ◽  
Whole Body ◽  
Vascular Endothelial ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Abstract The phosphatidylinositol 3-kinase signaling pathway in vascular endothelial cells is important for systemic angiogenesis and glucose metabolism. In this study, we addressed the precise role of the 3-phosphoinositide-dependent protein kinase 1 (PDK1)-regulated signaling network in endothelial cells in vivo, using vascular endothelial PDK1 knockout (VEPDK1KO) mice. Surprisingly, VEPDK1KO mice manifested enhanced glucose tolerance and whole-body insulin sensitivity due to suppression of their hepatic glucose production with no change in either peripheral glucose disposal or even impaired vascular endothelial function at 6 months of age. When mice were fed a standard diet at 6 months of age and a high-fat diet at 3 months of age, hypertrophy of epididymal adipose tissues was inhibited, adiponectin mRNA was significantly increased, and mRNA of MCP1, leptin, and TNFα was decreased in the white adipose tissue of VEPDK1KO mice in comparison with controls. Consequently, both the circulating adiponectin levels and the activity of hepatic AMP-activated protein kinase were significantly increased, subsequently enhancing whole-body insulin sensitivity and energy expenditure with increased hepatic fatty acid oxidation in VEPDK1KO mice. These results provide the first in vivo evidence that lowered angiogenesis through the deletion of PDK1 signaling not only interferes with the growth of adipose tissue but also induces increased energy expenditure due to amelioration of the adipocytokine profile. This demonstrates an unexpected role of PDK1 signaling in endothelial cells on the maintenance of proper glucose homeostasis through the regulation of adipocyte development.

scholarly journals Survival of recombinant erythropoietin in the circulation: the role of carbohydrates

Blood ◽  
1989 ◽  
Vol 73 (1) ◽  
pp. 84-89
Author(s):  
MN Fukuda ◽  
H Sasaki ◽  
L Lopez ◽  
M Fukuda
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Recombinant Erythropoietin ◽  
Lectin Affinity Chromatography ◽  
Glycoprotein Hormone ◽  
Human Erythropoietin

Recombinant human erythropoietin produced in transfected Chinese hamster ovary cells is glycosylated much the same way as the erythropoietin present in human urine. To determine the role of carbohydrates in the stability of recombinant human erythropoietin in vivo, [125I]-labeled recombinant erythropoietin was intravenously infused into rats. The erythropoietin was slowly cleared from the blood with a half-life of approximately two hours. Asialoerythropoietin, which was produced by treatment of recombinant human erythropoietin with sialidase, was found to be cleared rapidly from circulation within ten minutes. These data suggest that the galactose binding protein of hepatic cells is involved in the clearance of asialoerythropoietin. Erythropoietin also contains N-glycans with a few N-acetyllactosamine repeats, which can be enriched by tomato lectin affinity chromatography. The lectin-bound fraction was cleared to a larger extent than was the unfractionated erythropoietin, while the component that did not bind the lectin was found to be stable in the circulation. Authentic N-acetyllactosamine repeats (polylactosaminoglycans) prepared from erythrocytes were similarly rapidly cleared from the circulation to the liver, and this clearance was inhibitable with asialo-alpha 1- acid glycoprotein. These results suggest that (a) the sialic acid of the recombinant erythropoietin is necessary for this glycoprotein hormone to circulate stably and (b) glycoproteins with more than three lactosaminyl repeat units may be cleared by the galactose binding protein of hepatocytes.

scholarly journals Mesenchymal Igf2 is a major paracrine regulator of pancreatic growth and function

2019 ◽  
Author(s):  
Constanze M. Hammerle ◽  
Ionel Sandovici ◽  
Gemma V. Brierley ◽  
Nicola M. Smith ◽  
Warren E. Zimmer ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Whole Body ◽  
Imprinted Genes ◽  
Paracrine Signalling ◽  
Genetic Mechanisms ◽  
Pancreatic Epithelium ◽  
And Function ◽  
Maternal Glucose

AbstractThe genetic mechanisms that determine the size of the adult pancreas are poorly understood. Here we demonstrate that many imprinted genes are highly expressed in the pancreatic mesenchyme, and explore the role of Igf2 in-vivo. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy. Surprisingly, mesenchymal mass is unaffected, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Furthermore, increased IGF2 activity specifically in the mesenchyme, through Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Ex-vivo exposure of primary acinar cells to exogenous IGF2 increases cell proliferation and amylase production through AKT signalling. We propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.

BODY ORGAN CYTOCHROME OXIDASE ACTIVITY IN COLD- AND WARM-ACCLIMATED RATS

1963 ◽  
Vol 41 (9) ◽  
pp. 1847-1854 ◽  
Author(s):  
Ladislav Janský
Keyword(s):  
Steady State ◽  
Cytochrome Oxidase ◽  
Oxidase Activity ◽  
Whole Body ◽  
Cytochrome Oxidase Activity ◽  
Full Capacity ◽  
Body Organ ◽  
Total Body

The cytochrome oxidase activity was estimated in homogenates of the whole body and in nine body organs of cold- and warm-acclimated rats. The total body cytochrome oxidase activity expressed in terms of oxygen consumption was similar in cold- and warm-acclimated rats. In cold-acclimated animals the total cytochrome oxidase activity did not differ from maximal steady state metabolism measured in vivo, while in warm-acclimated rats the total cytochrome oxidase activity was almost twice as great as the maximal steady state metabolism. The results indicate that warm-acclimated rats do not utilize the full capacity of the cytochrome system and that cold-acclimation makes full exploitation of the oxidase capacity possible. In cold-acclimated rats the cytochrome oxidase activity of the muscles comprised 57% of the total, the liver 22.5%, and the skin 6%, with smaller roles for other organs. The role of the liver was greater in cold-acclimated than in warm-acclimated rats.

Cytoarchitecture of the Xenopus thymus following gamma-irradiation

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 95-105
Author(s):  
JH Russ ◽  
JD Horton
Keyword(s):  
Gamma Irradiation ◽  
Tolerance Induction ◽  
Cell Types ◽  
Whole Body ◽  
Thymic Stromal Cells ◽  
Normal Architecture

This paper describes in vitro and in vivo attempts to deplete the 4- to 8-month-old Xenopus laevis (J strain) thymus of its lymphocyte compartment. Gamma irradiation (2-3000 rad) of the excised thymus, followed by two weeks in organ culture, is effective in removing lymphocytes, but causes drastic reduction in size and loss of normal architecture. In contrast, in vivo whole-body irradiation (3000 rad) and subsequent in situ residence for 8-14 days proves successful in providing a lymphocyte-depleted froglet thymus without loss of cortical and medullary zones. In vivo-irradiated thymuses are about half normal size, lack cortical lymphocytes, but still retain some medullary thymocytes; they show no signs of lymphocyte regeneration when subsequently organ cultured for 2 weeks. Light microscopy of 1 micron, plastic-embedded sections and electron microscopy reveal that a range of thymic stromal cell types are retained and that increased numbers of cysts, mucous and myoid cells are found in the thymus following whole-body irradiation. In vivo-irradiated thymuses are therefore suitable for implantation studies exploring the role of thymic stromal cells in tolerance induction of differentiating T lymphocytes.

Inactivation of erythropoietin leads to defects in cardiac morphogenesis

Development ◽  
1999 ◽  
Vol 126 (16) ◽  
pp. 3597-3605
Author(s):  
H. Wu ◽  
S.H. Lee ◽  
J. Gao ◽  
X. Liu ◽  
M.L. Iruela-Arispe
Keyword(s):  
Cardiac Development ◽  
Fetal Liver ◽  
Ventricular Myocardium ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitor ◽  
Cardiac Morphogenesis ◽  
Proliferation And Differentiation ◽  
Proliferation Assays

Erythropoietin is an essential growth factor that promotes survival, proliferation, and differentiation of mammalian erythroid progenitor cells. Erythropoietin(−/−) and erythropoietin receptor(−/−) mouse embryos die around embryonic day 13.5 due, in part, to failure of erythropoiesis in the fetal liver. In this study, we demonstrated a novel role of erythropoietin and erythropoietin receptor in cardiac development in vivo. We found that erythropoietin receptor is expressed in the developing murine heart in a temporal and cell type-specific manner: it is initially detected by embryonic day 10.5 and persists until day 14.5. Both erythropoietin(−/−) and erythropoietin receptor(−/−) embryos suffered from ventricular hypoplasia at day 12–13 of gestation. This defect appears to be independent from the general state of hypoxia and is likely due to a reduction in the number of proliferating cardiac myocytes in the ventricular myocardium. Cell proliferation assays revealed that erythropoietin acts as a mitogen in cells isolated from erythropoietin(−/−) mice, while it has no effect in hearts from erythropoietin receptor(−/−) animals. Erythropoietin(−/−) and erythropoietin receptor(−/−) embryos also suffered from epicardium detachment and abnormalities in the vascular network. Finally, through a series of chimeric analysis, we provided evidence that erythropoietin acts in a manner which is non-cell-autonomous. Our results elucidate a novel role of erythropoietin in cardiac morphogenesis and suggest a combination of anemia and cardiac failure as the cause of embryonic lethality in the erythropoietin(−/−) and erythropoietin receptor(−/−) animals.

Targeting Hepcidin with Antisense Oligonucletides Improves Anemia Endpoints in Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 269-269 ◽  
Author(s):  
Jeffrey R. Crosby ◽  
William A. Gaarde ◽  
Jarrett Egerston ◽  
Robert McKay ◽  
Yingqing Sun ◽  
...  
Keyword(s):  
Serum Iron ◽  
Cell Function ◽  
Iron Absorption ◽  
Mrna Levels ◽  
Erythroid Progenitor ◽  
Primary Mouse ◽  
Progenitor Cell Proliferation ◽  
Hepcidin Mrna

Abstract Anemia is one of the more common blood disorders and is associated with a number of diseases, including chronic kidney disease, chronic inflammation, and certain types of cancer. Under these conditions iron is essential as it is required for erythroid progenitor cell proliferation and red cell function. Hepcidin is a liver-derived growth factor that regulates iron absorption in the GI tract and iron absorption and release in tissues. Furthermore, hepcidin overexpression has been strongly linked mechanistically as a mediator of decreased iron availability and anemia. We have utilized an antisense approach to investigate the role of hepcidin in animal models of anemia and as a potential therapeutic approach for the treatment of this disorder in humans. Second-generation 2′-O-methoxyethyl chimeric antisense oligonucleotides (ASOs) were screened in isolated primary mouse hepatocytes, followed by in vivo screening in mice, for the ability to reduce hepcidin mRNA levels. ASO treatment resulted in a reduction of hepcidin mRNA in liver which was associated with a significant increase in serum iron levels. The best hepcidin ASO was then tested in a mouse model of turpentine induced hypoferremia and anemia to determine the role of hepcidin in regulating serum iron and anemia endpoints. Mice were treated (I.P. twice/weekly) with hepcidin or control ASO for two weeks at varying doses prior to a single subcutaneous injection of turpentine. Turpentine treatment 16 hours post-injection produced a significant reduction in serum iron levels and at two weeks resulted in reduced RBC numbers, hematocrit and hemoglobin levels. Treatment with the hepcidin ASO resulted in a dose dependent improvement in all of these endpoints while the control oligonucleotide had no effect. Studies are in progress to further characterize the pharmacological activity of hepcidin ASO in additional models of anemia and results from these on-going studies will also be presented.

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