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Abstract

The question was investigated whether the chloride reabsorption in the cortical thick ascending limb (cTAL) of rabbit kidney is primary or secondary active, i.e. whether it depends on sodium. Isolated cTAL segments were perfused in vitro at high flow rates (10–20 nl·min⁻¹) with identical modified Ringer's solutions on both sides of the epithelium. The modified Ringer's solution contained sodium and/or organic cations (tris-hydroxymethyl-aminomethane, tetraethylammonium, choline) and 150 mmol·l⁻¹ chloride. Transepithelial electrical potential difference (PD) and transepithelial specific resistance (R_T) were directly measured, and used to calculate the short circuit current (I_sc) under three types of experimental conditions. In group 1 (n=8), the tubules were first perfused with solutions containing 150 mmol·l⁻¹ sodium. The PD was +9.6±0.4 mV (relative to the tubule lumen). Then sodium was replaced by an organic cation whereby the PD fell slightly to +8.2±1.6 mV. This PD disappeared when furosemide was added to the lumen perfusate, ouabain added to the bath, or temperature decreased to 295°K (22° C). To test whether the removal of sodium was successfull in this series, the sequence of perfusions was reversed in the second group (n=31). First the perfusion system was thoroughly rinsed with a sodium-free solution. Then a tubule was mounted and perfused under seemingly sodium-free conditions. The PD was only + 2.6 ±0.3 mV, corresponding to an Isc of 29±3 μA·cm⁻². When sodium was gradually added, PD and I_sc increased steeply with a half maximal response at 3.4 mmol·l⁻¹. The maximal I_sc was 258 μA·cm⁻². In the last series (n=13), the efforts to make the system sodium-free were even more rigorous. The perfusion system in this series was made from sodium-free glass and the bath exchange rate was increased. The PD in the absence of sodium was +0.6±0.2 mV. These data indicate that the apparent sodium-independence in series 1 as well as in previous reports on this issue is artefactual and is caused by a small amount of sodium which is left in the system after the replacement by sodium-free solutions. A kinetic analysis of series 2 and 3 revealed that all active chloride reabsorption by the cortical thick ascending limb of Henle's loop is sodium-dependent suggesting that sodium and chloride are cotransported in the luminal membrane of this nephron segment.