Access to kidney transplantation as a cure for end-stage kidney disease is severely limited by donor organ shortage. Exacerbating this shortage is the need for re-transplantation resulting from (1) early graft failure due to poorly controlled alloimmunity; (2) late graft failure due to progressive organ fibrosis and occlusive vasculopathy, two characteristics of chronic allograft nephropathy (CAN). Our preliminary studies from murine models suggest that following organ anastomosis in kidney transplantation, the temporal process of initial recipient monocyte infiltration followed by their ensuing differentiation to pro-inflammatory macrophages and later macrophage maladaptive response to injuries plays a significant role in promoting alloimmunity and CAN. Specifically, following the initial recipient monocyte infiltration, a monocyte cell-surface receptor tyrosine kinase called AXL determines the ability of the infiltrating monocytes to differentiate to pro-inflammatory macrophages and promotes alloimmunity. Consequently, inhibiting AXL early post-transplantation significantly prolongs rejection-free allograft survival. Later, in response to injuries such as ischemia, a macrophage intracellular protein called Allograft Inflammatory Factor 1 (AIF-1) determines their maladaptive response to ischemia and promotes kidney fibrosis. Consequently, inhibiting AIF-1 during kidney ischemia significantly reduces long-term kidney fibrosis. These observations led us to hypothesize that in kidney transplantation, AXL determines early macrophage differentiation and function, whereas AIF-1 determines late macrophage response to injury. Thus, a potential therapeutic opportunity is to sequentially block AXL and AIF-1 in kidney transplant recipients to (1) inhibit early alloimmunity and (2) prevent late kidney fibrosis. In this new R01 application, we propose two specific aims that will: (1) determine mechanisms of AXL in early post-kidney transplant period on promoting alloimmunity. This aim will also determine extracellular and intracellular AXL signaling partners, and test clinical values of murine findings in human monocyte cell lines; (2) determine mechanisms of AIF-1 in late post-kidney transplant period on promoting maladaptive response to injuries and allograft fibrosis. This aim will also determine AIF-1 signaling components that control kidney resident macrophage functional outcome, and test clinical values of murine findings in human cell lines. Our experienced team of investigators include the PI Dr. Luo, an expert in transplant immunobiology who will direct all immunological studies in murine kidney transplant models, and the co-I Dr. Privratsky, an expert in acute and chronic kidney injury models who will direct all studies of post-kidney transplant injuries and measurement of long-term kidney allograft fibrosis and function. Our ultimate goal is to identify new temporal and pathway-specific targets for modulating macrophage functions post-kidney trans...