ATP depletion by iodoacetate and cyanide in renal distal tubular cells.

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.

Hypoxia and oxygen dependence of cytotoxicity in renal proximal tubular and distal tubular cells.

Ischemia and hypoxia are major causes of renal failure and altered oxygen supply may affect renal responses to toxic chemicals. In vitro experiments were designed to evaluate the susceptibility of isolated proximal tubular (PT) and distal tubular (DT) cells from rat kidney to brief periods of oxygen deprivation and to assess how variations in oxygen supply affect chemical-induced cytotoxicity. Isolated cells were incubated for 1 hr in either oxygen (95% O2/5% CO2), air (21% O2), or nitrogen (95% N2/5% CO2) atmosphere. PT cells exhibited no injury due to brief oxygen deprivation whereas DT cells exhibited moderate, but significant injury, indicating that DT cells are more susceptible than PT cells to hypoxic injury. The cytotoxicity of chemicals that alter cellular redox status [i.e. tert-butyl hydroperoxide (tBH), menadione, methyl vinyl ketone] and the cytotoxicity of "chemical hypoxia" [i.e. KCN + iodoacetic acid] were greatest in air, intermediate in oxygen, and lowest in nitrogen. In contrast, the cytotoxicity of the alkylating agent N-dimethylnitrosamine was independent of oxygen concentration and the cytotoxicity of p-aminophenol was related directly to oxygen concentration. The mechanism of the oxygen dependence of chemical injury was investigated further, employing tBH as a model toxicant. tBH metabolism was oxygen independent in both PT and DT cells. Depletion of cellular protein sulfhydryl groups by tBH increased with increasing oxygen concentration and lipid peroxidation due to tBH was inhibited in nitrogen but was not different in air as compared with oxygen. Although these processes may contribute to the much lower toxicity in nitrogen as compared with oxygen, it does not explain the higher toxicity in air as compared with that in oxygen. Other processes that predominate at lower oxygen concentrations but that only produce injury if enough oxygen is present are likely to be responsible for the enhanced susceptibility of both PT and DT cells to oxidants in air as compared with oxygen.