Rat Renal Basolateral Membrane Vesicles Research Articles

Kinetic characterization of Zinc transport process and its inhibition by Cadmium in isolated rat renal basolateral membrane vesicles: In vitro and In vivo studies

We firstly characterized zinc uptake phenomenon across basolateral membrane vesicles (BLMVs) isolated from normal rat kidney. The process was found to be time, temperature, and substrate concentration dependent, and displayed saturability. Zn(2+) uptake was competitively inhibited in the presence of 2 mM Cd with Ki of 3.9 mM. Zinc uptake was also inhibited in the presence of sulfhydryl reacting compound suggesting involvement of [-]SH groups in the transport process. Further, to elucidate the effect of in vivo Cd on zinc transport in BLMVs, Cd nephrotoxicity was induced by subcutaneous administration of CdCl(2) at dose of 0.6 mg/kg/d for 5 days in a week for 12 weeks. An indolent renal failure developed in Cd exposed rats was accompanied with a significantly high urinary excretion of Cd(2+), Zn(2+) and proteins. The histopathology and electron microscopy of kidneys of Cd exposed rats documented changes of proximal tubular degeneration. Notably, Cd content in renal cortex of Cd exposed rats was 215 microg/g tissue that was higher than the critical concentration of Cd in kidneys which was associated with significantly higher Zn and metallothionein (MT) contents. Zinc uptake in BLMVs isolated from kidneys of Cd exposed rats was significantly reduced. Further, kinetic studies revealed that decrease in zinc uptake synchronized with decrease in maximal velocity (V(max)) and increase in affinity constant which is suggestive of decreased number of active zinc transporters. Furthermore, conformational modulation of Zn transporter in BLM was further supported by observed variation in transition temperature for zinc transport in BLMVs isolated from Cd-exposed kidney.

Role of Organic Anion and Amino Acid Carriers in Transport of Inorganic Mercury in Rat Renal Basolateral Membrane Vesicles: Influence of Compensatory Renal Growth

Susceptibility to renal injury induced by inorganic mercury (Hg(2+)) increases significantly as a result of compensatory renal growth (following reductions of renal mass). We hypothesize that this phenomenon is related in part to increased basolateral uptake of Hg(2+) by proximal tubular cells. To determine the mechanistic roles of various transporters, we studied uptake of Hg(2+), in the form of biologically relevant Hg(2+)-thiol conjugates, using basolateral membrane (BLM) vesicles isolated from the kidney(s) of control and uninephrectomized (NPX) rats. Binding of Hg(2+) to membranes, accounted for 52-86% of total Hg(2+) associated with membrane vesicles exposed to HgCl(2), decreased with increasing concentrations of HgCl(2), and decreased slightly in the presence of sodium ions. Conjugation of Hg(2+) with thiols (glutathione, L-cysteine (Cys), N-acetyl-L-cysteine) reduced binding by more than 50%. Under all conditions, BLM vesicles from NPX rats exhibited a markedly lower proportion of binding. Of the Hg(2+)-thiol conjugates studied, transport of Hg-(Cys)(2) was fastest. Selective inhibition of BLM carriers implicated the involvement of organic anion transporter(s) (Oat1 and/or Oat3; Slc22a6 and Slc22a8), amino acid transporter system ASC (Slc7a10), the dibasic amino acid transporter (Slc3a1), and the sodium-dicarboxylate carrier (SDCT2 or NADC3; Slc13a3). Uptake of each mercuric conjugate, when factored by membrane protein content, was higher in BLM vesicles from uninephrectomized (NPX) rats, with specific increases in transport by the carriers noted above. These results support the hypothesis that compensatory renal growth is associated with increased uptake of Hg(2+) in proximal tubular cells and we have identified specific transporters involved in the process.

Uptake of dipeptide and β-lactam antibiotics by the basolateral membrane vesicles prepared from rat kidney

The transport of dipeptides and β-lactam antibiotics across the rat renal basolateral membrane was examined. The initial uptake of glycylsarcosine and cefadroxil by rat renal basolateral membrane vesicles was inhibited by the presence of all the di- and tripeptides and β-lactam antibiotics that were tested in this study. However, the uptake of both substrates was not inhibited by glycine, an amino acid. The initial uptake of zwitterionic β-lactam antibiotics, cefadroxil, cephradine, and cephalexin, was stimulated by preloaded glycylsarcosine (countertransport effect). On the other hand, the uptake of dianionic β-lactam antibiotics, ceftibuten and cefixime, was not affected. A concentration-dependent initial uptake of glycylsarcosine and cefadroxil suggested the existence of a carrier-mediated mechanism, whereas the transport of ceftibuten did not show any saturated uptake. The transporter that participates in the permeation of dipeptides and β-lactam antibiotics across basolateral membranes showed lower affinity than did PEPT1 and PEPT2. This is the first study that showed an evidence for a peptide transporter, expressed in the rat renal basolateral membrane, that recognizes zwitterionic β-lactam antibiotics using basolateral membrane vesicles isolated from normal rat kidney.

Palmitate transport by rat renal basolateral membrane vesicles in the presence of albumin.

Long chain fatty acids are an important metabolic fuel for renal proximal tubules. Because of their insolubility, they are carried in the blood primarily bound to albumin. Processes involved in the cellular uptake of albumin-bound ligands are incompletely understood. To determine how fatty acids enter renal tubular cells when substrate was presented primarily bound to albumin (molar ratio albumin:palmitate = 1.0), [3H]palmitate uptake by basolateral membrane vesicles (BLMV) isolated from rat renal cortex was measured by a rapid filtration technique. The pattern of uptake was strikingly affected by buffer composition. In low-ionic-strength buffer (300 mM mannitol, 20 mM Tris-N-hydroxethylpiperazine-N'-2-ethanesulfonic acid [HEPES]), [3H]palmitate uptake plateaued in less than 1 min. The calculated volume of distribution of [3H]palmitate indicated extensive binding. In high-ionic-strength buffer with an inwardly directed NaCl, sodium gluconate, or KCl concentration gradient (100 mM) or equal intravesicular and extravesicular NaCl concentration (100 mM), uptake was biphasic with peak values 1.4- to 3-fold higher than plateau values. To test whether uptake could be specifically inhibited, phloretin (200 microM), probenecid (500 microM), or 4,4'-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 500 microM) was added to NaCl uptake buffer. These agents were without effect on initial uptake but inhibited the normal biphasic pattern. The biphasic uptake pattern suggested binding followed by translocation to the interior of BLMV with subsequent mediated efflux. Medicated palmitate efflux was demonstrated in BLMV preloaded with [3H]-palmitate in NaCl buffer by: (1) phloretin inhibition of efflux and (2) trans-stimulation of efflux by p-aminohippurate. Results suggest that the translocation of fatty acids derived from albumin:fatty acid complexes occurs only in the presence of high external ion concentration. Subsequent equilibration of fatty acids between albumin, BLMV, and vesicle interior is mediated by the organic anion transport system.

Uric acid transport in rat renal basolateral membrane vesicles

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 α-ketoglutarate/urate; Cl −/urate and OH −(HCO 3 −)/urate as well as a mediated conductive mechanism was assessed from tracer flux measurements. In the presence of an inwardly directed Na + gradient an α-ketoglutarate dependent concentrative accumulation of PAH but not urate was observed suggesting an absence of the mediated exchange of α-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 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 −(HCO 3 −)/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 −, α-ketoglutarate or HCO 3 −. This finding, when considered together with existing evidence supporting the presence of transport pathways mediating the active uptake of urate across the brush-border membrane, suggests the transtubular reabsorptive flux of urate in rat proximal tubules occurs by active uptake at the apical membrane and passive efflux at the basolateral membrane.

Renal palmitate transport: possible sites for interaction with a plasma membrane fatty acid-binding protein

In previous studies from this laboratory, a mediated transport system for long chain fatty acids was observed in rat renal basolateral membrane vesicles. Transport was measured in the absence of albumin and indicated the presence of a Na+ independent anion exchange mechanism. The present experiments were done to characterize renal transport of fatty acids derived from fatty acid-albumin complexes. 3H-palmitate uptake by brush border (BBMV) and basolateral membrane vesicles (BLMV) isolated from rat renal cortex was determined using a rapid filtration technique. All incubation media contained 100 microM 3H-palmitate complexed to 100 microM bovine serum albumin. Up to 65% of initially bound fatty acid-albumin complexes were displaceable by washing with solution containing 0.1% albumin. Total palmitate uptake was measured as the remaining non-displaceable radioactivity. In BBMV in low ionic strength (300 mM mannitol) or ionic buffers (100 mM mannitol + 100 mM NaCl or KCl), total palmitate uptake at 15 sec did not differ from equilibrium (60 min) values of 10-11 nmoles/mg protein. Uptake was primarily due to binding. A similar pattern was seen with BLMV in 300 mM mannitol buffer. In BLMV in 100 mM NaCl or KCl buffers, equilibrium uptake was 10-fold lower than at 15 sec. This suggests binding followed by cation-dependent translocation. If a putative FABPPM is involved in transport only, its presence should be confined to BLMV.

Indirect coupling to Na+ of p-aminohippuric acid uptake into rat renal basolateral membrane vesicles.

Experiments with basolateral membrane vesicles prepared from rat kidney cortex were performed to study the mechanism by which p-aminohippuric acid (PAH) is taken up across the contraluminal membrane and is concentrated in proximal tubule cells. An inward Na+ gradient failed to stimulate [3H]PAH uptake compared with K+ or Li+ and did not cause intravesicular PAH accumulation above equilibrium distribution. In the absence of Na+, the dicarboxylates glutarate and suberate cis-inhibited and trans-stimulated [3H]PAH uptake, indicating a common transport system. In the presence of Na+, 10 microM glutarate in the incubation medium did not cis-inhibit, but rather stimulated [3H]PAH uptake and caused PAH accumulation above equilibrium distribution ("overshoot"). Li+ diminished this stimulation, but was without effect on [3H]PAH/PAH- and [3H]PAH/glutarate exchange. The data indicate the coexistence of a Na+ -coupled, Li+-sensitive transport system for dicarboxylates and a Li+ -insensitive PAH/dicarboxylate exchanger in the basolateral membrane. We propose that dicarboxylates are cotransported with Na+ into the cell and subsequently exchange for extracellular PAH at the basolateral membrane. PAH uptake is thereby indirectly coupled to Na+ via the Na+/dicarboxylate cotransporter.

Role of chloride on carrier-mediated transport of [formula omitted] in rat renal basolateral membrane vesicles

Effect of inorganic anions on p- amino [ 3H]hippurate transport in renal basolateral membranes has been studied using the vesicles preloaded with unlabeled p- aminohippurate (countertransport condition). The uptake of p- amino [ 3H]hippurate was stimulated by the outward gradient of unlabeled p- aminohippurate and the labeled substrate was accumulated into the vesicles against its concentration gradient in the presence of Cl −. The substitution of SCN − and SO 4 2− for Cl − in both sides of the vesicles depressed the initial rate and the overshoot magnitude of p- amino [ 3H]hippurate uptake. These results suggest that Cl − may play an important role for the carrier-mediated transport system of organic anion in renal basolateral membranes.

Electro-osmosis and the reabsorption of fluid in renal proximal tubules.

The lateral intercellular spaces (LIS) are believed to be the final common pathway for fluid reabsorption from the renal proximal tubule. We postulate that electrogenic sodium pumps in the lateral membranes produce an electrical potential within the LIS, that the lateral membranes bear a net negative charge, and that fluid moves parallel to these membranes because of Helmholtz-type electro-osmosis, the field-induced movement of fluid adjacent to a charged surface. Our theoretical analysis indicates that the sodium pumps produce a longitudinal electric field of the order of 1 V/cm in the LIS. Our experimental measurements demonstrate that the electrophoretic mobility of rat renal basolateral membrane vesicles is 1 micron/s per V/cm, which is also the electro-osmotic fluid velocity in the LIS produced by a unit electric field. Thus, the fluid velocity in the LIS due to electro-osmosis should be of the order of 1 micron/s, which is sufficient to account for the observed reabsorption of fluid from renal proximal tubules. Several experimentally testable predictions emerge from our model. First, the pressure in the LIS need not increase when fluid is transported. Thus, the LIS of mammalian proximal tubules need not swell during fluid transport, a prediction consistent with the observations of Burg and Grantham (1971, Membranes and Ion Transport, pp. 49-77). Second, the reabsorption of fluid is predicted to cease when the lumen is clamped to a negative voltage. Our analysis predicts that a voltage of -15 mV will cause fluid to be secreted into the Necturus proximal tubule, a prediction consistent with the observations of Spring and Paganelli (1972, J. Gen. Physiol., 60:181).

Sodium-dependent dicarboxylate transport in rat renal basolateral membrane vesicles.

Dicarboxylate transport in basolateral membrane vesicles prepared from rat kidney cortex was studied using 3H-methylsuccinate as a substrate. A sodium gradient (out greater than in) simulated methylsuccinate uptake and led to a transient overshoot. Lithium inhibited methylsuccinate uptake in the presence of sodium. The dependence of methylsuccinate uptake on sodium concentration indicated the interaction of more than one sodium ion with the transporter. Half-maximal stimulation was observed at 24 mmol/l sodium. Sodium-driven methylsuccinate uptake was electrogenic carrying a net positive charge. The basolateral dicarboxylate transport system exhibited an optimum at pH 7.0-7.5. In contrast, the sodium-dependent dicarboxylate transport system of brush border membranes depended much less on pH and had no optimum in the tested range. Cis-inhibition studies showed a preference of the system for dicarboxylates in the trans-configuration (fumarate) over cis-dicarboxylates (maleate). Citrate was accepted but oxalate and L-glutamate were not. DIDS exhibited a small inhibition. Among the monocarboxylates, gluconate and pyruvate inhibited methylsuccinate uptake whereas probenecid and p-aminohippurate (1 mmol/l) were without effect. The data indicate the presence of a sodium-dependent transport system in the basolateral membrane which accepts tricarboxylic acid cycle intermediates. This system is most likely not identical to the transport system responsible for organic anion secretion.

Mechanism of L-malate transport in rat renal basolateral membrane vesicles.

The transport of L-malate was evaluated in basolateral and brush border membrane vesicles isolated from the rat renal cortex. In both types of membrane vesicles, an inwardly directed sodium gradient stimulated transport and caused an overshoot of the equilibrium concentration. In basolateral membrane vesicles, the presence of sodium, but without a sodium gradient, stimulated the uptake of L-malate. Sodium gradient-stimulated L-malate uptake was stimulated and inhibited by maneuvers designed to render the inside of the basolateral membrane vesicles more electronegative or electropositive, respectively. The tricarboxylic acid cycle intermediates citrate, alpha-ketoglutarate, succinate, fumarate, L-malate, and oxaloacetate and the monocarboxylic acid pyruvate inhibited the sodium gradient-stimulated uptake of L-[14C]malate in basolateral membrane vesicles. Maleate, gluconate, formate, acetate, L-lactate, and beta-hydroxybutyrate did not inhibit uptake. These results indicate that basolateral membrane vesicles from the rat renal cortex contain a sodium-L-malate cotransport system that may be shared by the other tricarboxylic acid cycle intermediates and by pyruvate. Such a transport system, together with a similar sodium-tricarboxylic acid cycle intermediate cotransport system located in the brush border membrane, would ensure high concentrations of tricarboxylic acid cycle intermediates within the cells of the proximal tubule.

Properties of an anion exchanger in rat renal basolateral membrane vesicles.

Sulfate uptake in basolateral membrane vesicles from rat kidney cortexes was studied to test for the presence of an anion exchanger in these membranes. A pH difference (pHout = 6.4, pHin = 8.4) and a sodium gradient (out greater than in, pHout = pHin = 7.4) provided the driving force for intravesicular sulfate accumulation above the equilibrium content ("overshoot"). Sulfate uptake in vesicles preloaded with 25 mM of sulfate, thiosulfate, phosphate, chloride, or bicarbonate, or 10 mM of formate, acetate, L-lactate, pyruvate, p-aminohippurate, or oxalate was stimulated with respect to uptake in unpreloaded vesicles. Probenecid inhibited these trans stimulations. When the uptake of 0.1 mM labeled sulfate was determined in the presence of 5 mM of various anions in the incubation medium, cis inhibitions were observed when sulfate was driven by a proton gradient (out greater than in), a sodium gradient (out greater than in), or a p-aminohippurate gradient (in greater than out). All anions that trans stimulated sulfate uptake also showed a cis inhibition. Only small cis inhibitions were seen with maleate and succinate. No inhibition by any of the anions was found when sulfate uptake was studied in the absence of driving cation or anion gradients. Our data indicate a common exchanger for inorganic and organic anions that can additionally be driven by a sodium gradient and a pH difference. This exchanger may be involved in reabsorption and secretion of anions in the proximal tubule of the rat kidney.

Glutamine uptake by rat renal basolateral membrane vesicles.

The presence of a sodium-dependent, saturable uptake process is described in basolateral membranes of rat renal cortex for L-glutamine. Concentration-dependence studies indicate the presence of multiple transport systems with Km1 of 0.032 mM and V1 of 0.028 nmol/mg of protein per min, and Km2 of 17.6 mM and V2 of 17.6 nmol/mg of protein per min. Lysine completely inhibits the high-affinity, low-capacity Km system and partially inhibits the low-affinity, high-capacity system. Cystine and other dibasic amino acids also affect glutamine uptake.