2 3 High mobility group box 1 inhibits HCO 3 - absorption in medullary thick ascending 4 limb through a basolateral receptor for advanced glycation end products pathway 5 6

33 High mobility group box 1 (HMGB1) is a damage-associated molecule implicated in 34 mediating kidney dysfunction in sepsis and sterile inflammatory disorders. HMGB1 is a nuclear 35 protein released extracellularly in response to infection or injury, where it interacts with TLR4 36 and other receptors to mediate inflammation. Previously we demonstrated that LPS inhibits 37 HCO3 absorption in the medullary thick ascending limb (MTAL) through a basolateral TLR438 ERK pathway. Here we examined whether HMGB1 could inhibit HCO3 absorption through the 39 same pathway. Adding HMGB1 to the bath decreased HCO3 absorption by 24% in isolated, 40 perfused rat and mouse MTALs. In contrast to LPS, inhibition by HMGB1 was preserved in 41 MTALs from TLR4 mice and was unaffected by ERK inhibitors. Inhibition by HMGB1 was 42 eliminated by the RAGE antagonist FPS-ZM1 and by neutralizing anti-RAGE antibody. Confocal 43 immunofluorescence showed expression of RAGE in the basolateral membrane domain. 44 Inhibition of HCO3absorption by HMGB1 through RAGE was additive to inhibition by LPS 45 through TLR4 and to inhibition by Gram-positive bacterial molecules through TLR2. Bath 46 amiloride, which selectively prevents inhibition of MTAL HCO3 absorption mediated through 47 NHE1, eliminated inhibition by HMGB1. We conclude that HMGB1 inhibits MTAL HCO3 48 absorption through a RAGE-dependent pathway distinct from TLR4-mediated inhibition by LPS. 49 These studies provide new evidence that HMGB1-RAGE signaling acts directly to impair the 50 transport function of renal tubules. They reveal a novel paradigm for sepsis-induced renal tubule 51 dysfunction, whereby exogenous pathogen-associated molecules and endogenous damage52 associated molecules act directly and independently to inhibit MTAL HCO3 absorption through 53 different receptor signaling pathways. 54 55