Maleate injection causes dose-dependent injury in proximal tubular cells. This study sought to better define underlying pathogenic mechanisms and to test whether maleate toxicity recapitulates critical components of the hypoxic/ischemic renal injury cascade. CD-1 mice were injected with maleate or used as a source for proximal tubule segments (PTS) for in vitro studies. Maleate induced dose-dependent PTS injury [lactate deydrogenase (LDH) release, ATP reductions, nonesterified fatty acid (NEFA) accumulation]. These changes were partially dependent on maleate metabolism (protection conferred by metabolic inhibitors: succinate, acetoacetate). Maleate toxicity reproduced critical characteristics of the hypoxia/ATP depletion-induced injury cascade: 1) glutathione (GSH) conferred protection, but due to its glycine, not cysteine (antioxidant), content; 2) ATP reductions reflected decreased production, not Na-K-ATPase-driven increased consumption; 3) cell death was completely blocked by extracellular acidosis (pH 6.6); 4) intracellular Ca(2+) chelation (BAPTA) mitigated cell death; 5) maleate and hypoxia each caused plasma membrane cholesterol shedding and in both instances, this was completely glycine suppressible; 6) maleate + hypoxia caused neither additive NEFA accumulation nor LDH release, implying shared pathogenic pathways; and 7) maleate, like ischemia, induced renal cortical cholesterol loading; increased HMG CoA reductase (HMGCR) activity (statin inhibitable), increased HMGCR mRNA levels, and increased RNA polymerase II recruitment to the HMGCR locus (chromatin immunoprecipitation, ChIP, assay) were involved. These results further define critical determinants of maleate nephrotoxicity and suggest that it can serve as a useful adjunct for studies of ischemia/ATP depletion-induced, proximal tubule-specific, cell death.