Diluting segment in kidney of dogfish shark. I. Localization and characterization of chloride absorption

Single tubules, dissected from the peritubular sheath of the dorsal bundle zone of kidney of the dogfish shark, Squalus acanthias, were perfused in vitro at 17-18 degrees C. This segment is the largest of the five in the peritubular sheath and had average inner and outer diameters of 46.9 +/- 1.2 and 74.4 +/- 2.1 microns, respectively (n = 32). These values suggest that this is the intermediate IV segment. When perfused with symmetrical buffered elasmobranch saline, intermediate IV segments exhibited high rates of Cl- absorption (JCl, pmol.s-1.cm-2): 1,696 at an average perfusion rate (Vo) of 8.2 nl/min. Cl- absorption was highly flow dependent [1/JCl = 57.95(1/Vo) + 1.75; r = 0.71, P less than 0.01]. Maximal rates of Cl-absorption, calculated from reciprocal transformation of the flow dependence of JCl, yielded a value of 5,714 pmol.s-1.cm-2. In the presence of a 200-mosmol/kg transepithelial osmotic gradient, fluid absorption was negligible. The spontaneous transepithelial voltage (Vte, mucosal with respect to serosal compartment) averaged 8.0 +/- 1.0 mV (n = 26). Such active transport of Cl- in the absence of fluid movement and in the presence of a lumen-positive transepithelial voltage is characteristic of amphibian and mammalian diluting segments. Na(+)-to-Cl- permeability ratios (PNa/PCl) averaged 2.5 +/- 0.5, indicating that, as in mammalian thick ascending limbs, this segment is Na+ (cation) permselective. Vte was dependent on the presence of Na+ and Cl- in the external solutions and was reversibly abolished by isosmotic replacement with N-methyl-D-glucamine or with isethionate, respectively. Ouabain inhibited Vte but was not reversible within the time course of these experiments. Furosemide (10(-4) M), but not equimolar concentrations of amiloride or hydrochlorothiazide, added to the luminal perfusate inhibited both Vte and JCl. These results suggest that apical membrane Na+ entry in intermediate IV segments is mediated by Na(+)-K(+)-Cl- cotransport and is consistent with the existence of a functional role of urinary dilution in the reabsorption of urea in the elasmobranch kidney.