Recycling Information Research Articles

Ion exchangers mediating NaCl transport in the renal proximal tubule.

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.

Mechanism of H(+)-coupled formate transport in rabbit renal microvillus membranes.

It has been proposed that a major fraction of Cl- absorption in the mammalian proximal tubule occurs by Cl-/formate exchange across the apical membrane with recycling of formate by nonionic diffusion. The purpose of this study was to characterize the mechanism of formate recycling in rabbit renal microvillus membrane vesicles. Formate uptake was stimulated by an inside-alkaline pH gradient. When external pH (pH alpha) was varied at constant internal pH (pHi), the initial rate of formate uptake was less than predicted for nonionic diffusion of formic acid at constant formic acid permeability. When pHi was varied at constant pHi, the initial rate of formate uptake exhibited cooperative and saturable kinetics with respect to pHi, in contrast to the pHi independence predicted for nonionic diffusion. pH gradient-stimulated [14C]formate uptake was stimulated by internal formate, indicating formate/formate exchange. pH gradient-stimulated formate influx was sensitive to inhibition by 1 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid but not by furosemide or hydroxycinnamate. We conclude that pH gradient-stimulated formate uptake takes place by a carrier-mediated process of H(+)-formate cotransport, OH-/formate exchange, or facilitated formic acid diffusion, rather than solely by passive nonionic diffusion through the lipid bilayer.

Role of ion exchangers in mediating NaCl transport in the proximal tubule

The reabsorption of NaCl in the proximal tubule occurs passively through the paracellular pathway, and actively by a transcellular route. Transcellular NaCl transport involves Na(+)-coupled Cl- entry across the apical membrane by two mechanisms involving Cl(-)-organic anion exchange. One mechanism is Cl(-)-formate exchange with recycling of formate from lumen to cell by H(+)-coupled formate transport in parallel with Na(+)-H+ exchange. A second mechanism is Cl(-)-oxalate exchange with recycling of oxalate from lumen to cell by oxalate-sulfate exchange in parallel with Na(+)-sulfate cotransport. Cl- exit across the basolateral membrane is most likely mediated by Cl- channels. Apical membrane Na(+)-H+ exchange is involved in mediating both NaHCO3 and NaCl reabsorption in the proximal tubule. Immunocytochemical studies indicate that NHE3 is the principal Na(+)-H+ exchanger isoform expressed on the brush border membrane. Detection of NHE3 in a subapical, intracellular, vesicular compartment in proximal tubule cells is consistent with its possible regulation by membrane trafficking. That NHE3 is the isoform responsible for apical membrane Na(+)-H+ exchange activity is supported by studies of inhibitor sensitivity, and by studies demonstrating increased expression of NHE3 protein in association with enhanced Na(+)-H+ exchange activity during renal maturation and in response to glucocorticoids and metabolic acidosis.