A stopped flow capillary perfusion method to evaluate contraluminal transport parameters of methylsuccinate from interstitium into renal proximal tubular cells

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Abstract
In order to study the transport of dicarboxylic acids through the contraluminal cell membrane of proximal tubular cells,3H-methylsuccinate has been synthetized by catalytic hydration of methylfumarate. As the chromatography of radioactive material excreted in the urine after i.v. injection of3H-methylsuccinate shows, no metabolite is detectable during the first 3 min. After 10 min, less than 10% of the excreted radiolabel is metabolized. To measure the contraluminal influx of3H-methylsuccinate from the interstitium into cortical tubular cells, the renal vessels were clamped so that the proximal tubular lumina collapsed. Then Ringer solution was injected into the blood capillaries. It contained different concentrations of3H-methylsuccinate and14C-inulin as extracellular space marker. After contact times between 1 and 10 s, this fluid was withdrawn from the capillaries and the disappearance of3H-methylsuccinate relative to14C-inulin was measured. The morphological compartments in the outer cortex of the clamped glutaraldehyde-fixed kidney were evaluated by a stereological method. For proximal tubular cells a ratio of extracellular water space to intracellular space of 1:3.1 and a ratio extracellular water space to free cell water space of 1:2 was found. It was tested whether the experimental disappearance curves with 4 different starting concentrations of3H-methylsuccinate fit with the data from four model calculations. It was found that the data and the conditions of transport are consistent with the predictions of a facilitated diffusion model. In this model, a transport coefficient occurs which depends on the concentration of3H-methylsuccinate following saturation kinetics. The calculated parameters wer:K m for3H-methylsuccinate=0.12 mmol/l,J max=0.50 pmol/s ·cm (related to tubular length in cm). Furthermore, equations are given to calculate inhibitory constantsK i of competing dicarboxylic acids.