Over 600,000 Americans live with end-stage renal disease (ESRD), and ∼468,000 of them are dialysis patients who depend on a functional vascular access to extend their lives. A mature arteriovenous (A-V) fistula is the preferred dialysis access due to its higher patency rates and lower medical costs compared to synthetic grafts and central venous catheters. However, ~40% of newly created fistulas fail to mature, i.e., they are not usable for dialysis because stenosis prevents them from reaching the necessary blood flow. There is paucity of research into the mechanisms underlying postoperative stenosis in A-V fistulas, despite its negative impact on morbidity, mortality, and quality of life of these patients. This translational proposal establishes the mechanistic relationship among the CXCL12 and PU.1/calprotectin signaling network, inward remodeling, and fistula outcomes in order to design targeted therapies to prevent maturation failure. Our proposal is built on strong scientific premises that suggest a mechanistic relationship between postoperative accumulation of calprotectin in A-V fistulas caused by ectopic expression of the transcription factor PU.1 and inward remodeling that causes fistula failure. Our overarching hypothesis is that smooth muscle cell (SMC)-derived calprotectin increases the risk for stenosis and failure in newly created A-V fistulas. Our mechanistic hypothesis is that elevated CXCL12 level in hemodialysis patients leads to ectopic expression of PU.1 in the vasculature and the subsequent accumulation of calprotectin in SMCs. The released calprotectin exacerbates inflammation, fibrosis, and intimal hyperplasia (IH) after fistula creation. We will test our hypothesis in three specific aims and five experimental layouts that will prove: 1) the contribution of calprotectin to fistula inward remodeling; 2) the underlying mechanisms by which CXCL12 and PU.1 increases calprotectin and the risk of A-V fistula failure; and 3) the relationship between PU.1 and fistula maturation outcomes in a human cohort. We will combine fine microsurgical techniques and knockout mice to successfully achieve our goals. We will also interrogate a human biorepository of 100 randomly selected patients undergoing creation of two-stage brachiobasilic transposition fistulas to search for associations between the levels of PU.1 after venous remodeling and inadequate maturation. In conclusion, with the successful accomplishment of this proposal, we are paving the way for the design of new drugs and cell type-specific interventions to effectively target A-V fistula fibrosis and IH and reduce vascular access complications.