Effect of α-ketoglutarate on organic anion transport in single rabbit renal proximal tubules

Abstract

The effect of exogenous α-ketoglutarate (αKG) and the peritubular Na+-dicarboxylate (Na-DC) cotransporter on organic anion/dicarboxylate (OA/DC) exchange in S2 segments of single, nonperfused rabbit proximal tubules was measured using 1 μM fluorescein (FL), a model OA, and epifluorescence microscopy. The effect of different transmembrane distributions of 10 μM αKG on peritubular FL uptake was measured at 37°C using bicarbonate-buffered, nutrient-containing buffers, which are conditions similar to those found in vivo. Compared with FL uptake in the absence of exogenous αKG, preloading tubules with αKG ( trans-configuration) or acute exposure to αKG ( cis-configuration) increased FL uptake 62% and 54%, respectively, whereas a cis-trans-configuration of αKG increased FL uptake by 76%. The cis-stimulation of FL uptake by αKG was rapid, within 5–7 s. This stimulation was blocked 96% by simultaneous exposure to 2 mM Li+, indicating that stimulation of transport was secondary to the uptake of exogenous αKG. In the absence of exogenous αKG, selective inhibition of Na-DC cotransport using 2 mM Li+ or 1 mM methylsuccinate decreased FL uptake by 25% (effects that were reversible but not additive), suggesting that the Na-DC cotransporter recycles endogenous αKG that has left the cell in exchange for FL and that this activity supports ∼25% of baseline activity of the OA/DC exchanger. With recycling of αKG accounting for ∼25% of FL uptake and with accumulation of exogenous αKG accounting for another ∼75% increase in FL uptake, Na-DC cotransport appears to directly support (25% + 75%)/175%, or ∼57%, of total FL transport.

Interaction of the Metal Chelator 2,3-Dimercapto-1-propanesulfonate with the Rabbit Multispecific Organic Anion Transporter 1 (rbOAT1)

“…Emitted light (collected as photons per second) was measured at 1-s intervals using a photomultiplier tube (model HC120; Hamamatsu, Bridgewater, NJ) coupled to a 520-nm long-pass filter (Omega Optical). The signal from tubule autofluorescence at this wavelength was Ͻ1% of the signal arising from exposure to 1 M FL (Welborn et al, 1998).…”

“…S 2 segments of proximal tubules, ϳ1.5 mm in length, were isolated from kidneys of New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station, TN) applying previously published methods (Welborn et al, 1998). Tubules were dissected in the lid of a plastic Petri dish that was kept on ice and contained dissection buffer (110 mM NaCl, 25 mM NaHCO 3 , 5 mM KCl, 2 mM Na 2 HPO 4 , 1.8 mM CaCl 2 , 1 mM MgSO 4 , 10 mM sodium acetate, 8.3 mM D-glucose, 5 mM L-alanine, 4 mM lactate, and 0.9 mM glycine, adjusted to pH 7.4 with HCl or NaOH, and gassed continuously with 95% O 2 /5% CO 2 to maintain the pH; osmolality was ϳ290 mOsmol/kg H 2 O).…”

“…Measurements of FL uptake into isolated proximal tubules were performed using the technique of Welborn et al (1998). Tubules were observed with an Olympus IMT microscope equipped with a 40ϫ oil-immersion fluor objective (1.3 numerical aperture; Nikon, Tokyo, Japan).…”

“…The kidney is also a site for accumulation of toxic heavy metals, including mercury, cadmium, and arsenic (Welborn et al, 1998). Several mechanisms have been proposed for the entry of these toxins, especially mercury, into the proximal tubule cell (Zalups, 2000).…”

Specificity of the fluorescein transport process in Malpighian tubules of the cricketAcheta domesticus

“…Quantification of fluorescein uptake by fluorescence microscopy Fluorescein (FL) accumulation in mid-tubule sections of individual Malpighian tubules was assessed by the technique of Welborn et al (1998). Tubules were stuck on coverslips coated with Cell-Tak (BD Biosciences; Bedford, MA, USA), which reduced movement of the tubules during experiments, and then covered with a Coverwell Perfusion chamber (0.5·mm deep, 4·mm ϫ 32·mm; Electron Microscope Sciences, Washington, PA, USA).…”