PROJECT SUMMARY Chronic kidney disease (CKD) is a public health problem that afflicts more than 37 million Americans. The current therapeutic options for this progressive disorder are limited; therefore, novel therapeutic strategies are urgently needed. Common features of CKD are kidney inflammation and fibrosis. Inflammation often triggers fibrosis, and fibrosis is the end result of chronic inflammatory reactions. Renal inflammation is characterized by macrophage activation and proinflammatory molecule production. However, the molecular mechanisms underlying macrophage activation are not fully understood. Therefore, the long-term objective of this proposal is to understand the molecular mechanisms of macrophage activation so that effective strategies can be developed for the treatment of CKD. We have identified PU.1 as a critical factor in the regulation of kidney inflammation during the development of CKD. Specifically, we have demonstrated that PU.1 is induced in the kidney in experimental models of CKD and in human kidney with CKD and PU.1 is obligatory for macrophage activation and inflammatory molecule production and development of CKD. Genetic deletion or pharmacological inhibition of PU.1 prevents macrophage activation and inflammatory molecule production in macrophages in vitro, and pharmacological inhibition of PU.1 suppresses macrophage activation, inflammatory molecule production, and fibrosis development in the kidney with obstructive injury. Furthermore, the proinflammatory effect of PU.1 appears to be mediated by the NOD-like receptor family pyrin domain–containing 3 (NLRP3) inflammasome pathway. In this application, we propose to examine and characterize the role of PU.1-NLRP3 pathway in macrophage activation and polarization and proinflammatory and profibrotic molecule production to further understand the molecular mechanisms of inflammation and development of kidney fibrosis. Our central hypothesis is that PU.1 promotes NLRP3 expression and inflammasome activation in macrophages leading to proinflammatory and profibrotic molecule expression and development of kidney fibrosis. To test this hypothesis, we will pursue the following Specific Aims. Specific Aim 1 is to determine the role of PU.1 in macrophage activation and polarization in vitro and in vivo. Specific Aim 2 is to dissect the molecular mechanisms by which PU.1 promotes macrophage activation and polarization. Specific Aim 3 is to explore the therapeutic potential of a selective PU.1 inhibitor for the treatment of CKD. We plan to utilize molecular, genetic, and pharmacological approaches to examine the role of PU.1- NLRP3 pathway in macrophage activation and polarization and development of kidney fibrosis. Results from these studies will provide novel insights into the molecular mechanisms of kidney inflammation and could lead to the development of novel therapeutic strategies for the treatment of CKD.