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.

Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

To develop a technique for measuring Na(+)-H+ exchange activity and intracellular pH (pH(i)) “on line” in resistance vessels, we utilized strips of rat mesenteric arteries loaded with the pH-sensitive dye 2',7”-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Strips were held at a fixed length within a 3-ml cuvette, and fluorescence emission was monitored at 530 nm. The spectrofluorimeter was monitored in the ratio mode, and the excitation wavelength was alternated between 440 and 505 nm. Tissues were maintained by perfusing with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid containing buffers. The introduction of ammonium chloride produced a rapid alkalinization. Washout of ammonium caused rapid acidification. Restoration of pH(i) was Na+ dependent and inhibited by dimethyl amiloride (concentration that produces half-maximal inhibition, K0.5 = 30 microM), features characteristic of Na(+)-H+ exchange. Further studies assessed the transport rate of the exchanger, which averaged 0.19 +/- 0.02 pH U/min (means +/- SE, n = 8). An estimate of the dependence of Na(+)-H+ exchange on external Na+ gave an apparent Michaelis constant for external Na+ of 10 mM and an apparent maximal velocity of 0.1 mM H+/s. Intracellular H+ was found to have a cooperative effect (Hill coefficient = 4) on Na(+)-H+ exchange.

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