Suspensions of proximal tubular and distal tubular (DT) cells from rat kidney were treated with iodoacetate and potassium cyanide (IAA+KCN) as a model to assess injury from ATP depletion. Cells were also incubated under N2/CO2 to assess if they respond similarly to ATP depletion due to hypoxia. Based on cytotoxic effects of IAA (lactate dehydrogenase [LDH] release, protein sulfhydryl depletion) and inhibition of lactate formation, 20 microM IAA was chosen with 1 mM KCN to inhibit cellular ATP generation. DT cells exhibited significantly greater LDH release due to both IAA + KCN and hypoxia than PT cells. Mechanisms of cellular injury and the ability of various strategies to protect against (IAA+KCN)-induced injury were then studied in isolated renal DT cells to investigate factors responsible for the enhanced susceptibility of this renal cell population, about which little metabolic and toxicological information is known. IAA+KCN produced marked depletion of ATP, only minimal changes in cellular content of glutathione, but significantly decreased cellular content of glutathione disulfide, suggesting generation of a proreductant environment. Extracellular acidosis (pH 6.2 vs. 7.4) completely prevented the increase in LDH release during 2-hr incubations with IAA+KCN and partially prevented ATP depletion. Similarly, preincubation with glutathione, glycine, ATP, or adenosine significantly protected DT cells from injury. Complete restoration of cellular ATP content was not required for protection, although viability correlated better with cellular content of total adenine nucleotides. These studies are the first to explore cellular energetics and cytotoxicity in renal DT cells and demonstrate that these cells are highly sensitive to injury from ATP depletion due to either IAA+KCN or hypoxia.