|Abstract||Albuminuria, a hallmark of chronic kidney disease (CKD), plays a crucial role in the etiology of CKD. High albumin levels are thought to induce endoplasmic reticulum (ER) stress and activate the AKT pathway, and lead to inactivation of AMP-activated kinase (AMPK), an energy-sensing molecule within cells. This in turn, activates mTOR (mammalian target of rapamycin), which inhibits autophagy and induces epithelial-to-mesenchymal transition (EMT), and ultimately results in accelerated renal cell apoptosis. Thus, the objectives of this study are: 1) to investigate the effects of restoration of AMPK signaling in renal cells using metformin on ER stress, AKT, EMT, autophagy and apoptosis that are thought to mediate renal cell injury during proteinuria, and 2) to dissect the AMPK- and non-AMPK mediated effects of metformin using an in vitro model of albumin-induced renal cell injury. Normal rat kidney proximal tubular (NRK-52E) cells grown to 60% confluency were exposed to 10 and 15 mg/ml of albumin for 72 hours in the presence or absence of 1 mM Metformin and/or 0.5 μM compound C, (an AMPK inhibitor). Cells were assessed for alterations in AMPK, AKT and mTOR pathways, and the markers of ER stress, EMT, autophagy and apoptosis. Metformin treatment significantly induced phosphorylation of AMPK, and attenuated the phosphorylation of AKT and mTOR markers in NRK-52E cells exposed to albumin. More importantly, metformin treatment prevented albumin-mediated induction of α-SMA (an EMT marker) and the expressions of pro-apoptotic ER stress marker CHOP and apoptotic caspases -12 and -3 but augmented the levels of autophagy markers (P-ULK-1 and LC3-II) in renal cells. Blockade of metformin-induced AMPK activation with compound C blunted the ER defense response and autophagy but had no effect on the markers of EMT (α-SMA) and apoptosis (caspase-12 and BAX) in renal cells exposed to albumin. Our studies suggest that metformin protects renal cells against proteinuric cytotoxicity via suppression of AKT and mTOR activation, EMT and apoptosis, and augmentation of autophagy and ER defense response in renal tubular cells. Studies with Compound C reveal that metformin’s effects on autophagy and ER chaperone expression were AMPK-mediated whereas its effects on EMT and apoptosis were AMPK-independent.