Abstract

The main objective of this proposal will be to elucidate the cellular origin of renal erythropoietin and the mechanisms that regulate its secretion. The kinetics of erythropoietin production will be studied in animals exposed to hypoxia or injected with cobalt chloride. Erythropoietin will be measured by bio and radioimmunoassays and the sites of production will be identified by monospecific antibodies. Isolated perfused kidneys, renal cell fractions, primary renal cultured cells and established cell lines will be studied physiologically and biochemically for their production of erythropoietin. Erythropoietin will be labelled endogenously with radioactive aminoacids and erythropoietin will be identified by immunoprecipitation followed by electrophoresis and fluorography of the precipitated material. Messenger-RNA will be isolated from whole kidneys and isolated cells and translated in vitro. Inhibitors of RNA synthesis, protein synthesis inhibitors and inhibitors of glycosylation will be utilized in order to determine the renal synthesis and secretion of erythropoietin. Specific c-DNA Epo probes will be utilized for identification of Epo mRNA extracted from tissue homogenates and also for in-situ hybridization in tissue slices. The elucidation of the mechanism that controls erythropoietin synthesis and secretion will increase our understanding of the pathophysiology of the anemia of chronic renal disease and secondary polycythemias.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK034642-04
Application #
3232927
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1986-08-01
Project End
1991-02-28
Budget Start
1989-08-01
Budget End
1991-02-28
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
Thomas Jefferson University
Department
Type
Schools of Medicine
DUNS #
061197161
City
Philadelphia
State
PA
Country
United States
Zip Code
19107

  Publications

Minchenko, A; Caro, J (2000) Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: role of hypoxia responsive element. Mol Cell Biochem 208:53-62
Srinivas, V; Zhu, X; Salceda, S et al. (1998) Hypoxia-inducible factor 1alpha (HIF-1alpha) is a non-heme iron protein. Implications for oxygen sensing. J Biol Chem 273:18019-22
Salceda, S; Beck, I; Srinivas, V et al. (1997) Complex role of protein phosphorylation in gene activation by hypoxia. Kidney Int 51:556-9
Salceda, S; Caro, J (1997) Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 272:22642-7
Salceda, S; Beck, I; Caro, J (1996) Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia. Arch Biochem Biophys 334:389-94
Minchenko, A; Salceda, S; Bauer, T et al. (1994) Hypoxia regulatory elements of the human vascular endothelial growth factor gene. Cell Mol Biol Res 40:35-9
Minchenko, A; Bauer, T; Salceda, S et al. (1994) Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest 71:374-9
Schuster, S J; Caro, J (1993) Erythropoietin: physiologic basis for clinical applications. Vox Sang 65:169-79
Beck, I; Weinmann, R; Caro, J (1993) Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells. Blood 82:704-11
Beck, I; Ramirez, S; Weinmann, R et al. (1991) Enhancer element at the 3'-flanking region controls transcriptional response to hypoxia in the human erythropoietin gene. J Biol Chem 266:15563-6

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