The kidney is the main physiologic source of erythropoietin (EPO) in the adult and responds to decreases in tissue oxygenation with increased EPO production. Although studies in mice with liver-specific or global gene inactivation have shown that hypoxia-inducible factor 2 (Hif-2) plays a major role in the regulation of Epo during infancy and in the adult, respectively, the contribution of renal HIF-2 signaling to systemic EPO homeostasis and the role of extrarenal HIF-2 in erythropoiesis, in the absence of kidney EPO, have not been examined directly. Here, we used Cre-loxP recombination to ablate Hif-2␣ in the kidney, whereas Hif-2-mediated hypoxia responses in the liver and other Epo-producing tissues remained intact. We found that the hypoxic induction of renal Epo is completely Hif-2 dependent and that, in the absence of renal Hif-2, hepatic Hif-2 takes over as the main regulator of serum Epo levels. Furthermore, we provide evidence that hepatocyte-derived Hif-2 is involved in the regulation of iron metabolism genes, supporting a role for HIF-2 in the coordination of EPO synthesis with iron homeostasis. (Blood. 2010;116(16): 3039-3048) IntroductionThe glycoprotein erythropoietin (EPO) is essential for the regulation of red blood cell mass in response to changes in tissue oxygenation. EPO stimulates erythropoiesis by promoting erythroid precursor cell viability, proliferation, and differentiation, thus enhancing the oxygen-carrying capacity of blood. Its production is tightly regulated by developmental, tissue-specific, and physiologic cues. 1,2 Lack of Epo in the embryo, where it is produced by hepatocytes, leads to death from cardiac failure and anemia at embryonic day (E)13.5. 3 During late gestation, the site of EPO production switches from the fetal liver to the kidney, where fibroblast-like peritubular interstitial cells become the main physiologic source of EPO synthesis in adults. [4][5][6] Although the liver retains the ability to produce EPO in response to hypoxic stimuli, it does not contribute to the serum EPO pool under normoxic or mild hypoxic conditions. 7-9 Therefore, an impairment of renal EPO synthesis, which is typically associated with advanced chronic kidney failure, results in the development of anemia and is treated by administering recombinant EPO. 2,10 The primary physiologic stimulus of enhanced EPO gene transcription is tissue hypoxia, which can induce a several hundredfold increase in circulating serum EPO levels. 1 Although in vitro studies, using an 18-nucleotide fragment of the oxygen-sensitive 3Ј EPO regulatory element, suggested that hypoxia inducible factor-1 (HIF-1) regulates EPO in Hep3B cells, 11-13 recent genetic evidence indicates that Hif-2 has an important role in the maintenance of normal serum EPO levels. 14-16 HIF-1 and HIF-2 belong to the PER/arylhydrocarbon-receptor nuclear translocator (ARNT)/single minded family of hypoxia-regulated transcription factors and consist of an oxygen-sensitive ␣ subunit and a constitutively expressed ␤ subunit, also known as ARNT. Bo...

Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34+ bone marrow cells into CD41+ megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.

Eltrombopag (SB-497 115) is a first-inclass, oral, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), being developed as a treatment for thrombocytopenia of various etiologies. In this phase 1 placebo-controlled clinical trial in 73 healthy male subjects, eltrombopag was administered as oncedaily oral capsules for 10 days at doses of 5, 10, 25, 30, 50, and 75 mg. The pharmacokinetics of eltrombopag were dose dependent and linear, and eltrombopag increased platelet counts in a dosedependent manner. There were no apparent differences in the incidence or severity of adverse events in subjects receiving active or placebo study medication. These observations indicate that el- IntroductionThrombocytopenia, a reduction in platelet count, is a frequent finding in several medical disorders, such as aplastic anemia, some infections, myelodysplasia, idiopathic thrombocytopenic purpura (ITP), and chronic liver disease. Another etiology of clinically significant thrombocytopenia is the use of myelosuppressive chemotherapy or interferon antiviral treatment. Currently, there is an unmet need for thrombopoietic agents for the treatment of thrombocytopenia.Thrombopoietin (Tpo) is the key cytokine involved in thrombopoiesis, and is the endogenous ligand for the thrombopoietin receptor (TpoR) that is expressed on the surface of megakaryocytes, and megakaryocytic precursors. 1,2 Binding of Tpo to its receptor triggers the activation of the JAK-STAT pathway, leading to changes in gene expression that promote progression along the megakaryocytic pathway, ultimately leading to the release of platelets into the peripheral circulation. [3][4][5] Administration of recombinant Tpo to rodents, primates, and humans leads to significant increases in circulating platelet levels. [6][7][8][9][10][11] However, due to immunogenicity issues, forms of recombinant Tpo are no longer in clinical trials. Other molecules with Tpo-like activities are in clinical trials to treat thrombocytopenic patients. 13 Peptidyl thrombopoietic agents, such as AMG-531, have been shown to increase platelet counts in healthy volunteers 12 and patients with ITP 13 ; however, they are not orally bioavailable and they have the potential to be immunogenic. Nonpeptide, smallmolecule, TpoR agonists can be orally bioavailable and are less likely to induce an immune or injection site response. [14][15][16] In this report we describe a first-in-class, small-molecule, nonpeptide, orally bioavailable thrombopoietin receptor agonist, eltrombopag . Preclinical studies have shown that eltrombopag interacts selectively with TpoR, thereby activating intracellular signal transduction pathways, leading to increased proliferation and differentiation of human bone marrow progenitor cells. 17 Platelet counts increased when eltrombopag was administered as an oral suspension to chimpanzees. 18 Therefore, eltrombopag may be considered an oral platelet growth factor.This phase 1 clinical trial in healthy human subjects was conducted to assess the safety, tolerability, p...

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitortreated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

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