Tamm-Horsfall protein (THP) is a glycoprotein expressed exclusively in thick ascending limbs (TAL) of the kidney. We recently described a novel protective role of THP against acute kidney injury (AKI) via downregulation of inflammation in the outer medulla. Our current study investigates the mechanistic relationships among the status of THP, inflammation, and tubular injury. Using an ischemia-reperfusion model in wild-type and THP-/- mice, we demonstrate that it is the S3 proximal segments but not the THP-deficient TAL that are the main targets of tubular injury during AKI. The injured S3 segments that are surrounded by neutrophils in THP-/- mice have marked overexpression of neutrophil chemoattractant MIP-2 compared with wild-type counterparts. Neutralizing macrophage inflammatory protein-2 (MIP-2) antibody rescues S3 segments from injury, decreases neutrophil infiltration, and improves kidney function in THP-/- mice. Furthermore, using immunofluorescence volumetric imaging of wild-type mouse kidneys, we show that ischemia alters the intracellular translocation of THP in the TAL cells by partially shifting it from its default apical surface domain to the basolateral domain, the latter being contiguous to the basolateral surface of S3 segments. Concomitant with this is the upregulation, in the basolateral surface of S3 segments, of the scavenger receptor SRB-1, a putative receptor for THP. We conclude that TAL affects the susceptibility of S3 segments to injury at least in part by regulating MIP-2 expression in a THP-dependent manner. Our findings raise the interesting possibility of a direct role of basolaterally released THP on regulating inflammation in S3 segments.