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Pathways for p-aminohippurate (PAH) transport across the basolateral membrane of rabbit proximal tubule cells were investigated from studies of [3H] PAH uptake in membrane vesicles isolated by Percoll-density gradient centrifugation. The 10-s uptake of PAH was not significantly different when measured in the absence of cation gradients or in the presence of inwardly directed Na, Li, K or choline gradients that suggests the absence of a mechanism mediating Na-PAH cotransport. A probenicid-sensitive, trans-stimulation of [3H] PAH uptake was observed in the presence of an outward PAH gradient. PAH gradient-driven [3H] PAH uptake was cis-inhibited by glutarate, alpha-ketoglutarate, adipate and sebacate and outward gradients of alpha-ketoglutarate trans-stimulated probenicid-sensitive PAH uptake. A concentrative accumulation of PAH was measured in the presence of an inward Na gradient and the dicarboxylates glutarate or alpha-ketoglutarate. Compared to the absence of a pH gradient, an inside alkaline pH gradient induced an increased PAH uptake both in the presence and absence of CO2/HCO3. Inside-negative and inside-positive voltage differences were observed to stimulate and inhibit alpha-ketoglutarate gradient-driven PAH uptake, respectively. alpha-Ketoglutarate gradient-driven PAH uptake was progressively reduced in the presence of increasing penicillin concentration and an outward gradient of alpha-ketoglutarate induced an increased level of [14C] penicillin uptake. These results suggest the presence of a probenicid-sensitive organic anion exchange mechanism as a pathway for PAH and penicillin transport across the basolateral membrane of rabbit proximal tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathways for urate transport across the basolateral membrane of rat proximal tubule cells were investigated using membrane vesicles isolated from rat renal cortex. The presence of an anion exchange mechanism(s) operative in the mode of alpha-keto-glutarate/urate; Cl-/urate and OH-(HCO3-)/urate as well as a mediated conductive mechanism was assessed from tracer flux measurements. In the presence of an inwardly directed Na+ gradient an alpha-ketoglutarate dependent concentrative accumulation of PAH but not urate was observed suggesting an absence of the mediated exchange of alpha-ketoglutarate for urate. The imposition of an outwardly directed Cl- gradient stimulated urate uptake in the absence but not the presence of conditions designed to minimize membrane potential development suggesting an indirect electrostatic coupling of urate uptake to a Cl- gradient-induced diffusion potential. Conditions favoring the development of an inside-positive K+ diffusion potential was observed to induce an inhibitor-sensitive, concentrative accumulation of urate in the absence of Cl-. The stimulation of urate uptake measured in the presence of an inside-alkaline pH gradient was not of sufficient magnitude to suggest the apparent conductive urate uptake was secondary to a membrane voltage induced, inside alkaline pH gradient and the operation of an OH-(HCO3-)/urate exchanger. The evidence obtained from the present investigation suggests rat basolateral membrane urate transport occurs by a mediated, conductive mechanism and is not coupled to Cl-, alpha-ketoglutarate or HCO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of chronic acid loading on the activity of luminal membrane Na(+)-H+ exchange and basolateral membrane Na+/HCO3- cotransport and Cl(-)-HCO3- exchange was investigated using membrane vesicles isolated from rat renal cortex. Na(+)-H exchange activity was increased approx. 50% in brush-border membranes isolated from acidemic compared to control kidneys. Na+/HCO3- cotransport and Cl(-)-HCO3- exchange activity was increased approx. 45% and 100%, respectively, in basolateral membranes isolated from acidemic kidneys. The increased Na+/HCO3- cotransport activity resulted from an increased apparent maximal rate of transport (Vmax) with no change in affinity (Km) for Na+. In contrast to acid/base transport activities chronic acid loading had no effect on the activity of basolateral membrane Na+/dicarboxylate cotransport. These results suggest proximal tubule cells coordinately increased luminal and basolateral membrane acid/base transport activities to accommodate an adaptive increase in the capacity for transcellular bicarbonate reabsorption.

Transepithelial acidification in the proximal tubule occurs by the simultaneous actions of the Na(+)-H+ exchanger in the brush border and the basolateral Na(+)-HCO3- cotransporter. The presence of these systems has been demonstrated for cultured cells; however, their contributions to the transepithelial movement of acid equivalents has not been confirmed in monolayers. To examine transepithelial acidification by intact cells, tubules were grown on membrane filters. Confluent cultures developed a transepithelial pH gradient within 6 h by decreasing the pH of medium in the apical chamber (6.66 +/- 0.03) while raising the basolateral pH to 7.40 +/- 0.02. Cells maintained on plastic did not acidify the medium during this time. Amiloride (10-100 microM) inhibited development of the gradient only when placed in the top chamber. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10-100 microM), which inhibits basolateral Na(+)-HCO3- cotransport, decreased the gradient only when added to the bottom. These results demonstrate that cultured proximal tubule cells can develop a transepithelial pH gradient and that the polarized distribution of the transport systems is maintained in vitro.

Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.