Oxalate Exposure Research Articles

Oxalate toxicity in LLC-PK1 cells: Role of free radicals

Oxalate, the most common constituent of kidney stones, is an end product of metabolism that is excreted by the kidney. During excretion, oxalate is transported by a variety of transport systems and accumulates in renal tubular cells. This process has been considered benign; however, recent studies on LLC-PK1 cells suggested that high concentrations of oxalate are toxic, inducing morphological alterations, increases in membrane permeability to vital dyes and loss of cells from the monolayer cultures. The present studies examined the basis for oxalate toxicity, focusing on the possibility that oxalate exposure might increase the production/availability of free radicals in LLC-PK1 cells. Free radical production was monitored in two ways, by monitoring the reduction of nitroblue tetrazolium to a blue reaction product and by following the conversion of dihydrorhodamine 123 (DHR) to its fluorescent derivative, rhodamine 123. Such studies demonstrated that oxalate induces a concentration-dependent increase in dye conversion by a process that is sensitive to free radical scavengers. Specifically, addition of catalase or superoxide dismutase blocked the oxalate-induced changes in dye fluorescence/absorbance. Addition of these free radical scavengers also prevented the oxalate-induced loss of membrane integrity in LLC-PK1 cells. Thus it seems likely that free radicals are responsible for oxalate toxicity. The levels of oxalate that induced toxicity in LLC-PK1 cells (350 microM) was only slightly higher than would be expected to occur in the renal cortex. These considerations suggest that hyperoxaluria may contribute to the progression of renal injury in several forms of renal disease.

Effect of Oxalate on Function of Kidney Mitochondria

The effects of oxalate on kidney mitochondria were evaluated in vitro to test whether oxalate exposure leads to derangement(s) in mitochondrial function that could in turn promote the formation of kidney stones. Our previous studies demonstrated that oxalate is transported across the mitochondrial membrane via the dicarboxylate carrier. The present studies indicated that oxalate competitively inhibits the uptake and oxidation of exogenous malate and succinate in isolated mitochondria but has no effect on mitochondrial respiration in the presence of a mixture of glutamate plus malate or glutamate plus pyruvate. Oxalate attenuates the increase in mitochondrial respiration produced by the uncoupler CCCP or by the Ca2+ ionophore A23187, and the latter effect is more pronounced in kidney than in liver mitochondria. The apparent Ki of oxalate for the response to Ca2+ ionophore is 1.9 ± 0.3mM in kidney and 6.1 ± 0.2mM in liver mitochondria. Similarly, the ability of oxalate to attenuate calcium-induced swelling of mitochondria is more dramatic in kidney than in liver mitochondria (apparent KiS of 1.7 ± 0.1 and 18.2 ± 0.7mM, respectively). Oxalate has no effect on the rate of calcium uptake by energized mitochondria or on the rate of ruthenium red-insensitive calcium efflux from mitochondria in either tissue. The above findings indicate that oxalate interacts with the inner mitochondrial membrane or with processes controlling membrane integrity to a greater extent in kidney than liver mitochondria. The effects of oxalate on membrane permeability or integrity may be more important than its effects on mitochondrial energy production or calcium sequestration in the pathogenesis of calcium oxalate microlith formation in the kidney.