Date of Award

5-2008

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Jonathan Himmelfarb

Second Committee Member

Leif Oxburgh

Third Committee Member

John Vella

Abstract

Erythropoietin (EPO) is emerging as a potential therapy for ischemic tissue injury as it has been shown in animal models to reduce ischemic injury in the brain, heart and kidneys. EPO’s cytoprotective properties in all tissues have been conducted primarily in vivo and the mechanisms of EPO mediated cytoprotection, especially in the kidney, remain undefined. Most of what is known about EPO signaling has come from studies of erythroid progenitor cells in the bone marrow. In erythropoiesis, ligand binding of EPO to its cognate receptor causes the autophosphorylation of Janus kinase 2 (JAK2) and subsequent phosphorylation of the eight tyrosines in the cytoplasmic domain. STAT5 is recruited and phosphorylated at Y343, dimerizes and translocates to the nucleus for transcription of B cell lymphoma 2 (Bcl2) family of genes which inhibit apoptosis of erythroid progenitors. Although the JAK2/Y343/STAT5 signaling axis is considered the major pathway for erythropoiesis, recent studies indicate this axis is dispensable for steady-state erythropoiesis but required for erythropoiesis under stress conditions. EPO cytoprotection against ischemic injury appears to involve JAK2 phosphorylation and activation of downstream cell survival pathways such as PI3K/AKT and NF-kB. The role of STAT proteins, however, has not been investigated. As STAT5 appears to play a crucial role in cell survival under stress conditions in erythroid progenitors, this study investigated the hypothesis that the JAK2/Y343/STAT5 signaling axis may also be required for EPO conferred cytoprotection in kidney injury. To test this hypothesis an in vitro model of reproducible ischemic injury in primary mouse renal tubule epithelial cells was established using EPO receptor mutant strains with differential STAT5 signaling capabilities. Using this model the results demonstrate for the first time that 1) EPO protects against mild-moderate but not severe necrotic ischemic injury in the kidney, 2) the JAK2/Y343/STAT5 signaling pathway is required for EPO mediated protection against ischemic kidney injury and 3) Pim-3, a STAT5 target cell survival gene is up regulated in response to EPO under non injury conditions in the kidney in vitro and in vivo.

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