METHOD FOR RECOVERING ATP CONTENT AND MITOCHONDRIAL-FUNCTION AFTER CHEMICAL ANOXIA IN RENAL-CELL CULTURES

Cultured renal cells provide a highly reproducible and malleable model to study cellular responses to metabolic perturbations. Nevertheless, there is currently no good method to achieve metabolic inhibition and complete recovery in cultured cells. This study describes a specific method for reversibly inhibiting both glycolytic and oxidative metabol ism. Glycolysis was inhibited by removing all glycolytic substrates, a nd mito-chondrial respiration was inhibited with rotenone, a site I in hibitor of the electron transport chain. Within 30 min, ATP values wer e decreased by 98%. Glycolysis was restored through the reintroduction of glucose. Oxidative metabolism was restored by the addition of hept anoate, a short odd-chain fatty acid, which supplies reducing equivale nts to site II of the electron transport chain. Employing Madin-Darby canine kidney and LLC-PK1 cell lines, this protocol caused the immedia te and complete recovery of mitochondrial respiration and, by 60 min, the complete recovery of cellular ATP levels. Application of this prot ocol should allow the investigation of the cellular effects and altera tions that occur within cells recovering from sublethal energy depleti on.