Renal tubular mechanisms of organic solute transport

Until recently the kidney was not among the organs favored for research on the molecular mechanisms of transport processes. The key experiments for exploring the molecular events of sugar and amino acid transport, for instance, were performed on intestinal [1, 2] and Ehrlich ascites tumor cells [3, 4]. The interest of the scientist studying molecular transport in the kidney rose rapidly, however, since the organ served as a source for transport enzymes such as the Na-K-ATPase [5] or the glucose binding protein [6] and especially since it became possible to obtain closed vesicles from either cell side of the proximal tubule [7, 8]. With these vesicles the transcellular transport steps could be studied separately without interference by cellular metabolism. Previously or simultaneously performed microperfusion [9, 10] and electrophysiological studies [11, 12] served as a basis for, or were at least complementary to, experiments with tubular membrane vesicles. At once some similarities with the corresponding intestinal transport processes became apparent [13]. Although our knowledge is growing rapidly so that many more answers can be expected in the near future, the main principle for transtubular hexose and amino acid transport is already evident: co-transport with sodium (secondary active transport) at one cell side. The transport step at the other cell side proceeds also by a carrier, but with a different specificity.The situation with organic acids, if they are not transported by nonionic diffusion, and with the organic bases is less clear, but a similar principle, possibly a whole chain of countertransport processes, may also hold for them.Because in this review, results gained largely from double-perfused kidney tubules and from membrane vesicles are reported, the corresponding methods and their potency should be discussed.The method of luminal, combined with simultaneous peritubular, capillary microperfusion [9] allows one to measure the specificity and even kinetics of the overall transtubular organic solute transport and can give a strong indication whether there is dependence on other transport processes, such as that of Na+ or H+. The same method can also be used for electrical measurements [11]. Provided that the respective organic solute transport causes a change in the electrical potential difference, it is even possible to localize the event to either cell side by introducing an intracellular electrode.The main parameter measured in connection with the organic solute transport is the steady state electro-chemical potential difference, Δη, at zero net flux of solutes and water. Under these conditions active transport, Jact,1 equals passive backflux, the latter being proportional to the permeability coefficient, P, times the electrochemical potential difference, Δη. Δη is the sum of the transtubular concentration difference, Δc, and the electrical potential difference (Δη) which is brought to the proper dimension by multiplying with zF/RT and the mean transtubular concentration Ci. The equation Jact = ? · (Ac + zF/RT d Af) shows that A? is a measure of the active transport rate provided that ? remains constant. Af can be measured directly or it can be evaluated by the distribution of an electrolyte which is not transported actively and for which the equation —Af = Ac RT/Ci zF holds. Af vanishes in the case of nonelectrolytes, and is otherwise small across the proximal convolution so that it can be neglected, especially if Ac is large and if the electrolyte is univalent or only partially dissociated. Since almost all Ac measurements of organic solutes are made with isotopes instead of being based on chemical analyses, it is important that the measurements be performed when the net flux of isotope is also zero, that is, when the specific activities in the luminal and capillary perfusate are the same. Another prerequisite is that the substance must not be metabolized or that its metabolic rate must be small compared with its active transport rate.For electrical measurements, the concentration of the organic solutes within the luminal or peritubular perfusate has to be changed rapidly [14, 14a]. For this reason double or multibarrelled perfusion pipettes are used. On evaluating the potential changes across either cell side, it is important to realize that a potential change caused solely across one cell side will also show up at the other cell side because of the large par-acellular shunt conductance [14, 14a]. This large shunt conductance is also the reason why considerable changes in the intracellular potential as measured across one cell side can hardly be seen with transtubular measurements.The most critical aspects of transport studies with membrane vesicles [15] are the purity of the vesicular preparation and the tightness of the vesicles. Furthermore, fast and complete removal of the incubation fluid without backleak of solutes already taken up by the vesicles is essential. The homogeneity of the brush border preparation from the proximal convolution can readily be checked by determination of enzymes such as alkaline phosphatase and the disaccharidases which are located only in the brush border and not in other kidney membranes. A marker enzyme for the basolateral membrane of the proximal tubule with a specificity similar to that for the brush border, unfortunately, does not exist. The cortical membrane fraction which contains the Na-K-ATPase seems however, to include little Na-K-ATPase activity which is not located within the basolateral cell membranes of the proximal convolution [16]. This subject as well as the various separation procedures were recently reviewed by Kinne [15]. So far, differential gradient centrifugation and additional free-flow electrophoresis seem to give the purest membrane preparations. The tightness of the vesicles for molecules of the size of hexoses is readily tested by demonstrating that the intravesicular space varies inversely with the concentration of these substances in the incubation medium [7, 8]. For removal of the extravesicular test substances after incubation, the Millipore filter technique and washing with ice-cold Ringer's solution has proved to be satisfactory[7].The strongest indication for carrier-mediated transport through the vesicular membranes is the demonstration of an isotope counterflux near equilibrium, i.e., the fact that preloading of the vesicles with unlabelled substance enhances the uptake of the same, but labelled, compound. Furthermore, a carrier-mediated co-transport is indicated, if a mutual enhancement of the uptake of an electrolyte and a nonelectrolyte is observed, as in the case of Na+ and hexoses [7, 13]. A third important feature is whether the uptake is influenced by the transvesicular electrical potential difference, the sign and the magnitude of which can be varied at will [17]. The most common procedure for this is to use one cation with anions of different permeabilities or to increase significantly and selectively the permeability for some other ion also present in the solutions by adding ionophores, such as the K+ ionophore valinomycin or the ionophore monactin. With proper modification of the experimental procedures it is not too difficult to discriminate the mode of transport that prevails for a given substance: simple diffusion, carrier-mediated transport, co-transport, countertransport or ATP-driven transport