The Critical Role of Src Homology Domain 2–Containing Tyrosine Phosphatase-1 in Recombinant Human Erythropoietin Hyporesponsive Anemia in Chronic Hemodialysis Patients

• Published in: Journal of the American Society of Nephrology Vol. 15; no. 12; pp. 3215 - 3224
• Main Authors: Akagi, Shigeru, Ichikawa, Haruo, Okada, Tatsuo, Sarai, Ai, Sugimoto, Taro, Morimoto, Hisanori, Kihara, Takashi, Yano, Ai, Nakao, Kazushi, Nagake, Yoshio, Wada, Jun, Makino, Hirofumi
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD Ovid Technologies (Wolters Kluwer Health) 01.12.2004; Lippincott Williams & Wilkins
• Subjects: Anemia; Anemia - drug therapy; Antigens, CD34; Antigens, CD34 - metabolism; Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Blotting, Western; Cell Division; Culture Media; Culture Media - pharmacology; DNA-Binding Proteins; DNA-Binding Proteins - metabolism; Erythroid Precursor Cells; Erythroid Precursor Cells - cytology; Erythroid Precursor Cells - drug effects; Erythroid Precursor Cells - enzymology; Erythroid Precursor Cells - metabolism; Erythropoietin; Erythropoietin - pharmacology; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Humans; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Kidney Failure, Chronic; Kidney Failure, Chronic - complications; Kidney Failure, Chronic - therapy; Medical sciences; Milk Proteins; Milk Proteins - metabolism; Nephrology. Urinary tract diseases; Nephropathies. Renovascular diseases. Renal failure; Oligodeoxyribonucleotides, Antisense; Pharmacology. Drug treatments; Phosphorylation; Protein Phosphatase 1; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein Tyrosine Phosphatases - genetics; Protein Tyrosine Phosphatases - metabolism; Protein-Tyrosine Kinases; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins; Proto-Oncogene Proteins - metabolism; Recombinant Proteins; Renal Dialysis; Renal failure; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; src Homology Domains; STAT5 Transcription Factor; Stem Cell Factor; Stem Cell Factor - pharmacology; Trans-Activators; Trans-Activators - metabolism; Transfection; Tyrosine; Tyrosine - metabolism; Nephrology; Erythropoietin; Hemodialysis; Phosphoprotein phosphatase; Phosphoric monoester hydrolases; Src Homology 2 domain; Esterases; Hemopathy; Glycoprotein hormone; Urology; Extrarenal dialysis; Complication; Recombinant protein; Kidney disease; Human; Urinary system disease; Enzyme; Anemia; Treatment efficiency; Cytokine; Antianemia agent; Chemotherapy; Chronic; Treatment; Polypeptide; Renal failure; Hydrolases; Protein-tyrosine-phosphatase
• ISSN: 1046-6673
• DOI: 10.1097/01.asn.0000145457.73744.24

The molecular mechanism of anemia that is hyporesponsive to recombinant human erythropoietin (rHuEPO) in hemodialysis patients without underlying causative factors has not been investigated fully in hematopoietic stem cell system. Circulating CD34+ cells (1 x 10(4)) were isolated from rHuEPO hyporesponsive hemodialysis patients (EPO-H; n = 9), patients who were responsive to rHuEPO (EPO-R; n = 9), and healthy control subjects (n = 9). The patients with known causes of EPO hyporesponsiveness were eliminated from the current study. The cells were cultured in STEM PRO 34 liquid medium, supplemented with rHuEPO, IL-3, stem cell factor, and granulocyte-macrophage colony stimulating factor for 7 d and then transferred to a semisolid methylcellulose culture medium for performing burst forming unit-erythroid (BFU-E) colony assay. Expression of src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was assessed with Western blot analysis. In EPO-H patients, SHP-1 antisense or scrambled S-oligos were included in the culture medium, and its effects were evaluated. The number of circulating CD34+ cells was not statistically different among the three groups, and their proliferation rates were similar for 7 d in culture. However, BFU-E colonies were significantly decreased in EPO-H patients compared with EPO-R and control groups. The mRNA and protein expression of SHP-1 and p-SHP-1 was significantly increased, whereas that of p-STAT5 was reduced in EPO-H patients. The inclusion of SHP-1 antisense S-oligo in culture suppressed SHP-1 protein expression associated with p-STAT5 upregulation, increase in p-STAT5-regulated genes, and partial recovery of BFU-E colonies. In EPO-H hemodialysis patients, the EPO signaling pathway is attenuated as a result of dephosphorylation of STAT5 via upregulation of SHP-1 phosphatase activity, and SHP-1 may be a novel target molecule to sensitize EPO action in these patients.