The objective of the proposed research is to define the pathogenic role that specific glomerular (ie., mesangial cells [MCs]) and tubular (i.e., tubule epithelial cells [TECs]) cells and the renin-angiotensin system (RAS) play in radiation induced kidney injury. We hypothesize that i] renal irradiation results in alterations in MC and TEC phenotype that lead to activation of the intra-renal RAS.
Specific aim (SA) 1 will test the hypothesis that radiation and/or Ang II lead to modulation of gene products associated with ECM accumulation and degradation, in part, by signaling mechanisms involving reactive oxygen species (ROS). We will determine if treating MCs and TECs with radiation, Ang II and other ROS generators in vitro leads to modulation of plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases-2 (TIMP-2) matrix metalloproteinase-2 (MMP-2), TGF-B and angiotensinogen (AGT, precursor of Ang II). We also will test whether addition of antioxidants or transfection with antioxidant enzymes blunts the radiation-and ANG II-induced modulation of these various gene products. Further, we will determine if the radiation-induced upregulation of PAI-1, MMP-2, TIMP-2, TGF-B and AGT expression observed in renal cells involves activation of the redox regulated transcription factors AP-1, AP-2 or NF-kB. The ability of Ang II to alter renal cell phenotype via ROS generation also will be studied. SA 2 will study the potential role of a glomerular-based intra-renal RAS by using adenovirus-based techniques to overexpress AGT in rat MCs. Using these transfected cells we will determine if overexpression of AGT can alter radiation-induced changes in MC expression of PAI-1, TIMP-2, MMP-2, and TGF-B. In addition, we will administer AGT transfected MCs to rat kidney glomeruli in vivo, to modulate intra-renal generation of Ang II in the irradiated kidney. SA 3 will use a unique double transgenic model produced by breeding mice that express human AGT only in proximal convoluted TECs with mice expressing human renin. Resultant double transgenics exhibit increased intra-renal Ang II synthesis; systemic levels are unaffected. Human AGT expression is androgen-dependent; expression in females is dependent on exogenous androgen administration. We will determine if irradiating kidneys of male double transgenic mice leads to increased ECM accumulation and exacerbation of functional and morphologic injury. Female mice will be used to investigate the temporal aspect of intra-renal RAS by administering androgens at various times/periods after irradiation. These studies offer the promise of developing rational therapeutic strategies directed at reducing the severity of this dose-limiting morbidity in human beings.
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