Lumen LPS inhibits HCO 3 - absorption in medullary thick ascending limb through 2 TLR 4 - PI 3 K - Akt - mTOR - dependent inhibition of basolateral Na + / H + exchange 3 4

32 Sepsis and endotoxemia induce defects in renal tubule function but the mechanisms are 33 poorly understood. Recently we demonstrated that lipopolysaccharide (LPS) inhibits HCO3 34 absorption in the medullary thick ascending limb (MTAL) through activation of different Toll-like 35 receptor 4 (TLR4) signaling pathways in the basolateral and apical membranes. Basolateral 36 LPS inhibits HCO3 absorption through ERK-dependent inhibition of NHE3. Here, we examined 37 the mechanisms of inhibition by lumen LPS. Adding LPS to the lumen decreased HCO3 38 absorption by 29% in rat and mouse MTALs perfused in vitro. Inhibitors of phosphoinositide 339 kinase (PI3K) or its effectors Akt and mTOR eliminated inhibition of HCO3 absorption by lumen 40 LPS but had no effect on inhibition by bath LPS. Exposure to LPS for 15 min induced increases 41 in phosphorylation of Akt and mTOR in microdissected MTALs that were blocked by 42 wortmannin, consistent with activation of Akt and mTOR downstream of PI3K. The effects of 43 lumen LPS to activate Akt and inhibit HCO3 absorption were eliminated in MTALs from TLR4 44 and MyD88 mice but preserved in tubules lacking Trif or CD14. Inhibition of HCO3 absorption 45 by lumen LPS was eliminated under conditions that inhibit basolateral Na/H exchange and 46 prevent inhibition of HCO3 absorption mediated through NHE1. Lumen LPS decreased 47 basolateral Na/H exchange activity through PI3K. We conclude that lumen LPS inhibits HCO3 48 absorption in the MTAL through TLR4/MyD88-dependent activation of a PI3K-Akt-mTOR 49 pathway coupled to inhibition of NHE1. Molecular components of the TLR4-PI3K-mTOR 50 pathway represent potential therapeutic targets for sepsis-induced renal tubule dysfunction. 51 52