Rat Renal Brush-border Membrane Vesicles Research Articles

Characterization of Na(+)-phosphate cotransporters in renal cortical endosomes

To determine the role of membrane recycling in proximal tubular P(i) transport, we studied the transport functions of simultaneously prepared rat renal cortical endosomal vesicles (EV) and brush-border membrane vesicles (BBMV). Initial P(i) uptake was Na+ gradient-dependent in both vesicles. Kinetic studies showed a lower apparent maximal rate (Vmax) for P(i) uptake in EV compared with BBMV (446 +/- 69 vs. 1493 +/- 117 pmol.mg protein-1 x 15 s-1, respectively; n = 4, P < 0.05), with no difference in the apparent Michaelis constant (Km). Endosomal Na(+)-P(i) cotransport was inhibited by phosphate analogues, phosphonoformic acid and arsenate, but not by the anion-exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Na(+)-P(i) cotransport was electroneutral in both vesicles, with a stoichiometry of 2 for BBMV and 1 for EV. The nonpermeant sulfhydryl reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), produced a marked inhibition of Na(+)-P(i) cotransport in EV but not in BBMV, suggesting accessible sulfhydryl groups, an "inside-out" orientation in EV, and "right-side-out" orientation in BBMV. The EV and the BBMV differed significantly in their phospholipid composition and lipid fluidity. The Na(+)-P(i) cotransporter protein (NaPi-2) abundance, determined by Western blots, was five times lower in EV than in BBMV (0.25 +/- 0.05 vs. 1.36 +/- 0.20 arbitrary units). Renal cortical endosomes contain Na(+)-P(i) cotransporters, albeit at a lower density, suggesting involvement of membrane recycling in the regulation of proximal tubular P(i) transport.

Transport mechanism of ceftibuten, a dianionic cephem, in rat renal brush-border membrane.

The uptake mechanism of ceftibuten by rat renal brush-border membrane vesicles was investigated. Uptake was found to be independent of a Na+ gradient and partially dependent on an inwardly directed H(+)-gradient. Competition experiments between ceftibuten and several compounds demonstrated that the peptide-like structural features of inhibitors are more essential than their charge properties for inhibiting uptake. Anionic compound, such as p-aminohippuric acid, also inhibited ceftibuten uptake by renal brush-border membrane vesicles in the presence of an H(+)-gradient. We conclude that ceftibuten, in spite of its anionic structure, is transported via the dipeptide transport systems, rather than the organic anion transport system.

Identification of organic cation transporter in rat renal brush-border membrane by photoaffinity labeling.

As an approach to identification of the organic cation transport system in brush-border membranes, we designed a photoaffinity probe, 1-cyano-2-(4-azido[3,5-3H]benzoylethyl)-3-[2-[[(5-methyl-4-imidazo lyl ) methyl]thio]ethyl]-guanidine ([3H]AMC) based on the molecular structure of cimetidine, which is taken up by the organic cation transport system in brush-border membrane vesicles. The effect of nonradioactive 1-cyano-2-(4-azidobenzoylethyl)-3-[2-[[(5-methyl-4- imidazolyl)methyl]thio]ethyl]guanidine (AMC) on tetraethylammonium uptake was investigated in rat renal brush-border membrane vesicles. We examined the photolysis of AMC in which the azido group was converted to an active nitrene group using UV light at a wavelength of 254 nm and established a half-life of 7 s. This half-life duration did not significantly impair brush-border membrane vesicles during the exposure to light for photo-labeling. Photoaffinity labeling of brush-border membrane vesicles from the rat renal cortex with [3H]AMC resulted in the covalent incorporation of radioactivity into membrane polypeptides; an apparent 36 kDa polypeptide was predominantly labeled. Photolabeling specificity was shown by a reduction in the labeling of the 36 kDa polypeptide in the presence of organic cations, cimetidine, tetraethylammonium and N-methylnicotinamide whereas the organic anion, fur osemide, had no effect on labeling patterns. These data demonstrate that AMC, as well as organic cations, cimetidine, tetraethylammonium and N-methylnicotinamide, interact with a common 36 kDa membrane polypeptide, which may be the transport system or one of its brush-border membrane components.

Differential sensitivity of organic anion transporters in rat renal brush-border membrane to diethyl pyrocarbonate

The effect of various chemical modifiers on p- aminohippurate (PAH) uptake by a potential-sensitive system and by an anion exchanger was studied in rat renal brush-border membrane vesicles. Among various chemical modifiers, diethyl pyrocarbonate (DEPC) selectively inhibited potential-sensitive PAH uptake but not the uptake by the anion exchanger. The inhibitory effect of DEPC on potential-sensitive PAH uptake was not due to the facilitated dissipation of membrane potential, which was evidenced by the studies with a potential-sensitive fluorescence dye diS-C 3(5). The potential-sensitive PAH uptake was inhibited by DEPC in a concentration-dependent manner, and kinetic analysis showed that the decreased uptake of PAH in DEPC-treated vesicles was due to the decrease of V max . The inhibition of the PAH uptake was protected by the presence of organic anions during the DEPC treatment. These findings indicate that PAH transport by the potential-sensitive system and by the anion exchanger is mediated by structurally distinct transporters. Amino acid residue(s) modified by DEPC, most likely a histidine residue, should play an important role in the potential-sensitive transport of PAH in rat renal brush-border membrane.

Adenine nucleotides and adenosine metabolism in pig kidney proximal tubule membranes.

Exogenous adenosine triphosphate (ATP) added to brush-border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto-nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto-adenosine deaminase and ecto-adenosine monophosphate (AMP) nucleotidase was shown. The ecto-adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush-border membrane vesicles. Using orthovanadate, levamisole, and alpha, beta-methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto-AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto-AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine.

Thyroid hormone stimulates the Na(+)-PO4 symporter but not the Na(+)-SO4 symporter in renal brush border.

In our previous studies we established that thyroid hormones [L-thyroxine (T4) or 3,5,3'-triiodothyronine (T3)] elicit an increase in Na(+)-Pi symport in rat and mouse renal brush-border membrane (BBM) vesicles (BBMV), but the Na(+)-coupled symports of other solutes were not influenced. However, a recent report [H. S. Tenenhouse, J. Lee, and N. Harvey. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F420-F426, 1991]claimed that T3 increases to a similar degree both Na(+)-Pi symport and Na(+)-SO4 symport in murine renal BBM. Adult male rats were fed either normal (0.7% Pi; NPD) or high-phosphate (1.4% Pi; HPD) diet and received T3 (0.2 mg/100 g body wt ip) for 3 days before the kidneys were removed, BBMV were prepared, and the transport rates were determined. Although the Na(+)-Pi symport significantly increased (delta = +35%) in both NPD and HPD rats treated with T3, the Na(+)-35SO4 symport and Na(+)-D-[3H]glucose symports were not influenced by T3. Furthermore, treatment of NPD-fed mice with T3 using a similar protocol as rats resulted in a significant increase (delta = +26%) of Na(+)-(Pi)4 symport, but did not alter Na(+)-SO4 symport or Na(+)-glucose symport. Our findings thus document that T3 regulates selectively the Na(+)-Pi symporter in BBM without having any effect on other major divalent anions, such as SO4.

Isolation and characterization of renal cortical membranes using an aqueous two-phase partition technique

The aqueous two-phase partition technique is a simple, rapid and inexpensive method for the fractionation of membrane preparations. Aqueous two-phase partitioning separates according to surface properties such as charge and hydrophobicity, making it complementary to established centrifugation techniques, which separate on the basis of density. Although aqueous two-phase partitioning has been successfully applied to animal tissues, there are limited data on the functional properties of the isolated membranes. We have applied the aqueous two-phase partition technique to rat renal brush-border membrane vesicles and sheets. Our aim was to remove organelle contamination while maintaining the functional properties of the membranes. Evidence from marker enzyme analysis and electron microscopy supports the conclusion that renal brush-border membranes are fractionated separate from the mitochondria and endoplasmic reticulum. This separation procedure did not alter the Na(+)-dependent transport of brush-border membrane vesicles. Na(+)-D-glucose symporter and Na(+)-H+ antiporter activity in the fractionated preparation increased to the same extent as did the enrichment of enzyme markers for brush-border membranes.

Transport of bestatin in rat renal brush-border membrane vesicles

Bestatin [(2 S,3 R)-3-amino-2-hydroxy-4-phenylbutanoyl- l-leucine] is a dipeptide, comprising l-leucine and an unusual β-ammo acid. We studied its transport mechanism in rat renal brush-border membrane vesicles. Uptake of cephradine, an aminocephalosporin, by isolated brush-border membrane vesicles was trans-stimulated and cis-inhibited by bestatin, indicating that these drugs are transported via the same transport system(s). The uptake of bestatin was trans-stimulated by preloading the vesicles with glycylsarcosine, and was cis-inhibited by substrates for the H +/dipeptide cotransport system. Bestatin inhibited tetraethylammonium (an organic cation) uptake, and bestatin uptake was cis-inhibited by substrates for the H +/organic cation antiport system. In addition, bestatin uptake was stimulated by an outward H + gradient (the driving force for the H +/organic cation antiport system). These findings suggest that bestatin, in spite of being a dipeptide, is transported via not only the H +/dipeptide cotransport system but also the H +/organic cation antiport system in rat renal brush-border membrane.

Co-expression of an anion conductance pathway with Na(+)-glucose cotransport in rat renal brush-border membrane vesicles.

Brush-border membrane vesicles were prepared from superficial rat renal cortex by a Mg(2+)-precipitation technique. The initial (20 s) [14C]glucose uptake rate from solutions containing 100 mmol/l Na (salt) was found to be dependent upon the anion composition of the medium; in comparison to gluconate-containing medium (0.46 +/- 0.05 nmol/mg protein), Cl- accelerated the initial rate to 1.47 +/- 0.21 nmol/mg protein (n = 4 preparations, +/- SEM). This enhancement was reduced by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.5 mmol/l), but was unaffected by 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS, 0.5 mmol/l). When membrane vesicles were pre-equilibrated with 100 mmol/l K (salt) and 100 mmol/l mannitol and glucose uptake was measured from a solution containing 100 mmol/l Na gluconate and 100 mmol/l mannitol in the presence of 80 mumol/l valinomycin (to generate an outward K+ diffusion electrical p. d.), it was found that intravesicular KCl depressed the initial glucose uptake compared to K gluconate. NPPB (0.5 mmol/l) increased the initial glucose uptake with intravesicular KCl towards values seen in K gluconate vesicles. In conditions where the only driving force for glucose uptake was established by an inward anion gradient (Nao = Nai) it was found that inward Cl- gradients could drive uphill glucose transport and that this was sensitive to NPPB (0.5 mmol/l), but insensitive to DIDS. We conclude that a Cl- conductance co-exists with Na-cotransport in rat renal brush-border membrane vesicles prepared from superficial renal cortex and this may function to regulate the activity of electrogenic transport systems at this membrane.

P-Aminohippurate transport in rat renal brush-border membranes: a potential-sensitive transport system and an anion exchanger.

Transport mechanisms of p-aminohippurate (PAH) were investigated in rat renal brush-border membrane vesicles. The uptake of PAH was stimulated by an inside-positive membrane potential created by K+ and valinomycin. This potential-stimulated uptake of PAH was inhibited by various anion transport inhibitors and was saturable. In addition, PAH uptake in the presence of valinomycin was linearly increased in proportion to log[K+]out/[K+]in. On one hand, PAH uptake was stimulated by [14C]PAH/PAH or [14C]PAH/Cl- exchange, and the [14C]PAH/PAH exchange was insensitive to the membrane potential. The uptake by the exchanger was also inhibited by anion transport inhibitors, but the potential-stimulated uptake of PAH was more sensitive to furosemide and 4,4'-diisothiocyano-2,2'-disulfonic stilbene. On the contrary, [14C]PAH/PAH exchange was more sensitive to urate than the potential-stimulated uptake of PAH. These findings indicate that PAH is transported by two distinct transport systems in rat renal brush-border membranes, a potential-sensitive transport system and an anion exchanger.

Increased Na(+)-dependent D-glucose transport and altered lipid composition in renal cortical brush-border membrane vesicles from bile duct-ligated rats.

Erythrocyte membranes of patients with liver disease are characteristically enriched in cholesterol, a change known to impair several carrier-mediated membrane transport functions. In the present study we have assessed whether experimental liver disease can affect the membrane lipid composition and transport function of kidney epithelial cells. Small (about 5%) but significant (P less than 0.01) increases were found in the cholesterol-to-phospholipid molar ratio (C/PL) of rat renal cortical brush-border membrane (BBM) vesicles 3, 8, and 15 days after bile duct ligation which correlated closely with increased fluorescence polarization, i.e., decreased membrane fluidity (r = 0.75, P less than 0.001; n = 27). A lipoprotein-mediated pathogenesis was suggested by the close relationship between BBM C/PL and plasma C/PL (r = 0.69, P less than 0.001). The mean high-affinity Na(+)-coupled D-glucose uptake by BBM vesicles was higher 1, 3, 8, and 15 days after ligation than in non-operated rats, significantly so at 3 and 8 days (611 +/- 37 and 593 +/- 22 vs. 507 +/- 21 pmol/mg protein per 4 sec; P less than 0.05), and was positively correlated with BBM C/PL (r = 0.58, P less than 0.01) and fluorescence polarization (r = 0.41, P less than 0.05). Brief incubation of BBM vesicles from normal rats with cholesterol-rich phospholipid liposomes simultaneously increased BBM C/PL and Na(+)-dependent D-glucose uptake. Stimulation of BBM Na(+)-glucose cotransport in ligated rats was not due to delayed dissipation of the Na+ gradient or to a more rapid development of membrane potential. High-affinity Na(+)-dependent D-glucose uptake kinetics in 3-day bile duct-ligated rats showed a lower Kt, without an alteration in maximum velocity, Vmax, compared to sham-operated animals (0.298 +/- 0.015 vs. 0.382 +/- 0.029 mM; P less than 0.05), whilst the binding dissociation constant, Kd of high-affinity phlorizin binding sites was reduced by ligation (0.453 +/- 0.013 vs. 0.560 +/- 0.015 microM; P less than 0.001). We conclude that an early effect of bile duct ligation is to enrich renal cortical brush-border membranes in cholesterol, thereby decreasing membrane fluidity and stimulating Na(+)-dependent D-glucose uptake by increasing the affinity of the carrier.

Effect of a rickets-inducing diet and vitamin D metabolites [25-OH-D 3 and 1,25(OH) 2D 3] on transmembrane transport systems in rat kidney

We previously demonstrated that feeding rats Steenbock and Black's rickets-inducing diet (deficient in vitamin D and with an altered Ca:P ratio) produces remarkable changes in the metabolic picture of the intestinal mucosa, kidney, and liver and in the transmembrane transport systems of D-glucose in intestinal and renal brush-border membrane vesicles. We have now investigated the effect of both 25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3 on Na +-dependent D-glucose and citrate transport and on calcium, phosphorus, and citrate content in rat serum and kidney. Na +-dependent D-glucose uptake, which decreased in rachitic rat jejunum brush-border membrane vesicles, returned to control values after 25-hydroxyvitamin D 3 or 1,25-dihydroxyvitamin D 3 administration, while in kidney normal D-glucose transport was restored only after 1,25-dihydroxyvitamin D 3 treatment. Na +-dependent citrate uptake was lowered in rachitic rat renal brush-border membrane vesicles and both 25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3 proved to be ineffective in restoring to normal values. The in vitro addition to vesicle preparations of calcium or phosphate, citrate, 1,25-dihydroxyvitamin D 3, or valinomycin did not show a selective influence on D-glucose and citrate transport.

Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles

The shared transport system for uptake of l-cystine and l-lysine was examined in isolated rat renal brush-border membrane vesicles for the ionic requirements for activation of the system. No requirement for sodium was seen for either cystine or lysine influx. However, the efflux of lysine from the vesicle was stimulated by Na +. Therefore, the transport system appears to be asymmetric in its requirement for sodium. Two different divalent cations were used in the membrane isolations which resulted in different responses of cystine uptake to the electrogenic movement of K + out of the vesicle. Membranes prepared by Mg-aggregation showed no stimulation of cystine influx by the imposition of a transient interior negative potential while vesicles prepared by Ca-aggregation did respond to electrogenic stimulation by an outwardly directed K-diffusion potential in the presence of valinomycin. Lysine influx was stimulated by electrogenic potassium efflux in both Mg-prepared and Ca-prepared membranes. No difference in sodium requirement for cystine influx was seen between the vesicles isolated by different cation-aggregation methods.

Trans-stimulation effect on H(+)-organic cation antiport system in rat renal brush-border membranes

Trans-stimulation effect on tetraethylammonium (an organic cation) transport was examined in rat renal brush-border membrane vesicles. The uptake of [14C]tetraethylammonium at pH 6.0-8.5 was stimulated by preloading the membrane vesicles with unlabeled tetraethylammonium. When the uptake was measured in preloaded membrane vesicles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, stimulation was observed at high pH but not at low pH. These results suggest that the mechanisms of the trans-stimulation effect on organic cation transport are different depending on the pH. When pH is low, the trans-stimulation is due to generation of an outward H+ gradient, which in turn stimulates [14C]tetraethylammonium uptake by H(+)-[14C]tetraethylammonium exchange. In contrast, when pH is high, the stimulation is due to direct exchange of tetraethylammonium for [14C]tetraethylammonium.

Effects of cysteine derivatives of styrene on the transport of p-aminohippurate ion in renal plasma membrane vesicles.

The effects of cysteine conjugates of styrene, e.g. S-1/2-(phenyl-hydroxyethyl) cysteine (PEC) and its N-acetyl derivative (NAPEC) on the transport of p-amino-hippurate (PAH) ion in plasma membranes were studied in vitro using isolated rat renal brush-border membrane (BBM) and basolateral membrane (BLM) vesicles. The uptake of PAH was significantly inhibited by both PEC and NAPEC in both the membrane vesicles, as verified by decrease of the membrane/medium concentration ratio of PAH as the concentration of either PEC or NAPEC in the medium increased. These results show that both PEC and NAPEC are capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. The inhibition of PAH uptake in BBM vesicles due to 10 mM PEC or NAPEC was found to be nearly competitive, almost similar to probenecid, whereas in BLM vesicles such inhibition was found to be partially noncompetitive, as verified by the double reciprocal plots. Both PEC and NAPEC showed dose-dependent inhibition of the specific activity of the marker enzyme in each membrane, e.g. gamma-glutamyl transferase in BBM and Na(+)-K(+)-ATPase in BLM vesicles. However, no such inhibition was noticed with probenecid. The in vitro pretreatment with probenecid prevented the inhibition of gamma-glutamyl transferase activity in BBM due to PEC or NAPEC, but such was not the case for the Na(+)-K(+)-ATPase activity in BLM. In conclusion, the data suggest that the transport of cysteine or N-acetylcysteine conjugates of styrene by renal proximal tubular cells across both the membrane vesicles accompanied by the inhibition of the membrane-specific enzymes may lead to cellular dysfunction and consequently to the initial development of their nephrotoxicity.

The influence of pH on cystine and dibasic amino acid transport by rat renal brushborder membrane vesicles

The uptake of cystine and lysine by rat renal brushborder membrane vesicles was examined at various intravesicular and extravesicular hydrogen ion concentrations to discern whether ionic species are determinants of specificity for the shared transport system and whether hydrogen ion gradients play a role in determining uptake values. When intravesicular and extravesicular pH are identical, the highest uptake of cystine occurred at pH 7.4, with lesser uptake at pH 6.0 and 8.3. Since cystine is electroneutral at pH 6.0 and 90% anionic at pH 8.3, it appears that neither form of the amino acid is a preferred species for transport. A similar relationship between pH and uptake occurs for lysine, which is cationic at pH below 8.5. This suggests that pH affects the functioning of the membrane carrier system independent of ionic species of the substrate. There is no apparent relationship of cystine uptake to hydrogen ion gradients across the membrane. Over the range of extravesicular pH studied, optimal cystine uptake occurred whenever the intravesicular pH was 7.4. Competitive interactions between unlabeled amino acids and labeled cystine were not affected by the extravesicular pH and, therefore, did not seem determined by the ionic species of cystine.

Inhibition of the rat renal Na +/H + exchanger by β-adrenergic antagonists

The beta-adrenergic antagonists, alprenolol and propranolol, inhibit the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Half-maximal inhibition occurs at 86 microM alprenolol and 36 microM propranolol. Similar to amiloride and Na+, propranolol protects the Na+/H+ exchanger from irreversible inhibition by the carboxyl group reagent, N,N'-dicyclohexyl-carbodiimide (DCCD). Protection is incomplete, depends on propranolol concentration, and reaches a maximum at 0.4 mM propranolol. With a comparable dose dependence, propranolol protects a 65 kDa band from labeling with [14C]DCCD. The data indicate that beta-adrenergic antagonists specifically interact with the proximal tubular Na+/H+ exchanger.

Dopamine inhibits Na(+)-H+ exchanger activity in renal BBMV by stimulation of adenylate cyclase

To determine a renal tubular mechanism for the natriuretic effect of dopamine (DA) and DA-1 agonists, we measured Na(+)-H+ exchange activity (amiloride sensitive) in rat renal cortical brush-border membrane vesicles (BBMV). Renal cortical tissues were preincubated with ligands before BBMV preparation to study Na(+)-H+ exchange activity in the absence of the added ligands that may compete for ion binding sites of the exchanger. DA and DA-1 agonist-inhibited Na(+)-H+ exchange activity was concentration and time dependent. The inhibitory effect was not due to increased permeability, collapse of the proton gradient, or change in vesicle size and did not extend to Na(+)-glucose symport. DA-2 agonists had no effect, whereas alpha-adrenergic agonists increased Na(+)-H+ exchange activity. Kinetic analysis revealed that the DA-1 agonist inhibited Na(+)-H+ exchange activity by a noncompetitive process. 2',5'-Dideoxyadenosine inhibited adenylate cyclase activity and reversed the inhibitory effect of DA-1 agonist on the exchanger. H4, an isoquinoline sulfonamide, which inhibits protein kinase A, also reversed the inhibitory effect of DA-1 agonist on the exchanger. Thus the DA-1 agonist-mediated inhibition of Na(+)-H+ exchange activity in BBMV is a receptor-mediated adenylate cyclase-linked process.

Transport characteristics of renal brush border Na(+)- and K(+)-dependent uridine carriers

The uptake of uridine into rat renal brush-border membrane vesicles is mediated by Na(+)- and K(+)-dependent concentrative transport processes. At a 100 mM extravesicular cation concentration the apparent Km values were 9.7 +/- 4.2 and 28 +/- 5 microM, and Vmax values were 28 +/- 4 and 7 +/- 1 pmol.mg protein-1.s-1 for the Na(+)- and K(+)-dependent systems, respectively. Uracil, D-ribose, and D-glucose failed to inhibit the uptake processes, indicating that these carriers are specific for nucleosides. Other purines and pyrimidines inhibited uridine uptake competitively, although these two transport systems seem to favor adenosine and pyrimidines as permeants. Evidence is also given that transport is rheogenic, involving a net transfer of positive charge. The Na+:uridine and K+:uridine coupling stoichiometry was found to be 1:1 and 3:2, respectively. Both systems can also be driven by an anion gradient with apparent NO3- affinity (KNO3-) values of 42 +/- 13 and 163 +/- 54 mM for Na(+)- and K(+)-dependent systems, respectively.

Role of N-acetylneuraminic acid in rat renal brush-border membrane vesicle aggregation by aminoglycosides

Interaction between aminoglycosides (AGs) and rat renal brush-border membrane (BBM) vesicles was investigated by the aggregation technique. The order of aggregation was gentamicin > dibekacin ≑ netilmicin > amikacin , and this order corresponds to the strength of the nephrotoxicity of the aminoglycosides in vivo rather than the number of amino groups in the aminoglycosides. BBM vesicles were aggregated through ionic interaction, as evident from the finding that aggregation ceased to occur at alkaline pH. By addition of N-acetylneuraminic acid (NANA) to the incubation medium, the vesicle aggregation induced by gentamicin was significantly inhibited. To affect the liberation of the NANA residue from BBM vesicles, the vesicles were treated with neuraminidase, resulting in an about 60% release with a significant decrease in the uptake of gentamicin into the vesicles. The decrease in the degree of vesicle aggregation was in proportion to the amount of NANA liberated. It follows from the findings that the NANA residue may in some way be responsible for the accumulation of aminoglycosides in renal proximal tubular cells.