Alterations in epithelial cell polarity and in the subcellular distributions of epithelial ion transport proteins are key molecular consequences of acute kidney injury and intracellular energy depletion. AMP-activated protein kinase (AMPK), a cellular energy sensor, is rapidly activated in response to renal ischemia, and we demonstrate that its activity is upregulated by energy depletion in Madin-Darby canine kidney (MDCK) cells. We hypothesized that AMPK activity may influence the maintenance or recovery of epithelial cell organization in mammalian renal epithelial cells subjected to energy depletion. MDCK cells were ATP depleted through a 1-h incubation with antimycin A and 2-deoxyglucose. Immunofluoresence localization demonstrated that this regimen induces mislocalization of the Na-K-ATPase from its normal residence at the basolateral plasma membrane to intracellular vesicular compartments. When cells were pretreated with the AMPK activator metformin before energy depletion, basolateral localization of Na-K-ATPase was preserved. In MDCK cells in which AMPK expression was stably knocked down with short hairpin RNA, preactivation of AMPK with metformin did not prevent Na-K-ATPase redistribution in response to energy depletion. In vivo studies demonstrate that metformin activated renal AMPK and that treatment with metformin before renal ischemia preserved cellular integrity, preserved Na-K-ATPase localization, and led to reduced levels of neutrophil gelatinase-associated lipocalin, a biomarker of tubular injury. Thus AMPK may play a role in preserving the functional integrity of epithelial plasma membrane domains in the face of energy depletion. Furthermore, pretreatment with an AMPK activator before ischemia may attenuate the severity of renal tubular injury in the context of acute kidney injury.