Renal Brush-border Membrane Vesicles Research Articles

The presence of an Na+/spermine antiporter in the rat renal brush-border membrane.

This study was aimed at determining the driving force for spermine transport in rat renal proximal tubular brush-border membrane. The uptake of spermine and trientine, a spermine-like drug used for treating Wilson's disease, into rat renal brush-border membrane vesicles was significantly stimulated by an outwardly directed Na+ gradient. The Na+-dependent uptake was temperature dependent and saturable. A kinetic analysis of the initial uptake of spermine with an Na+ gradient gave a Km value of 1.44 microM and a Vmax value of 6.31 pmol (mg protein)(-1)/30s. The Na+ dependent uptake of [3H]spermine was inhibited by spermine, trientine and tetraethylene-pentamine. Substrates of the H+/organic cation transporter (cimetidine and tetraethyl-ammonium), physiological polyamines (putrescine and spermidine) with 2 or 3 amino groups and aminoglycosides (amikacin and tobramicin) with 4 or 5 cationic amines did not affect the uptake of spermine in the presence of an outwardly directed Na+ gradient. These results suggest that the renal tubular secretion of spermine is mediated by an Na+/spermine antiport system which is specific for a straight-chain polyamine compound with more than 4 amino groups.

Cadmium Binding and Sodium-Dependent Solute Transport in Renal Brush-Border Membrane Vesicles

Exposure to cadmium (Cd) impairs renal transport systems for glucose, amino acids, phosphate, and dicarboxylates. To investigate if these changes are directly related to a Cd binding to the renal brush-border membrane, Cd binding and the Na+-dependent uptakes ofd-glucose,l-alanine, phosphate, and succinate were determined in rat renal brush-border membrane vesicles (BBMV) exposed to CdCl2. Cd uptake by BBMV showed time and concentration dependence. Changes in medium osmolality had no effect on Cd uptake, indicating that the process primarily involves binding of Cd to the membrane. Scatchard analysis indicated the presence of two types of Cd binding sites, differing in affinity and number. Increasing the medium Cd concentration from 50 to 200 μM resulted in a progressive increase in Cd binding to the membrane and decrease in Na+-dependent transport ofd-glucose,l-alanine, inorganic phosphate, and succinate. In all cases, the inhibition of transport was directly proportional to the total amount of Cd binding to the membrane. These results suggest that, during chronic exposure to Cd, free Cd ions liberated in renal tubular cells may directly interact with brush-border membranes and impair Na+-dependent solute transports.

Maturational changes in rabbit renal brush border membrane vesicle urea permeability.

Urea transport in the proximal tubule is thought to occur by passive diffusion through the lipid bilayers of the cell membranes. The lipid composition of cell membranes changes during maturation and may directly affect urea permeability of proximal tubule membranes. The present study examined the maturation of urea transport in rabbit renal brush border membrane vesicles (BBMV). BBMV from adult and neonatal (9- to 11-d-old) New Zealand white rabbits were loaded with 500 mM urea and mixed with an iso-osmotic mannitol solution using a stop-flow instrument. Vesicle shrinkage, due to efflux of urea, was followed with light scattering and urea permeability was calculated from an exponential fit of the data. Urea permeability was significantly lower in the neonatal BBMV than the adult at 25 degrees C (0.34+/-0.04 x 10(-6) versus 0.56+/-0.03 x 10(-6) cm/sec;p < 0.001, n=7) and 37 degrees C (0.45+/-0.04 x 10(-6) versus 0.66+/-0.03 x 10(-6) cm/sec; p=0.001, n=7). There was no effect of 250 microM phloretin on urea permeability in either adult or neonatal BBMV at either temperature. The activation energy for urea diffusion was higher in the neonatal than the adult BBMV. Because the maturational increase in urea permeability could potentially be due to a sodium-dependent urea transporter in the adult BBMV, the sodium dependence of urea uptake in adult BBMV was examined. There was no difference in urea permeability in the presence or absence of 20 mM NaCl. Permeability of the lipid-soluble molecule, glycerol, was also found to be the same in the neonatal and adult BBMV. Urea transport in the apical membrane of neonatal and adult proximal tubules is not phloretin sensitive, a finding consistent with diffusion of urea via the lipid bilayer. The rate of urea diffusion is lower in neonatal membranes and may be an important factor in overall urea excretion. This may also play a role in developing and maintaining a high medullary urea concentration and thus the ability to concentrate the urine during renal maturation.

K306 ラット腎刷子縁膜のクエン酸輸送系に対する重金属の影響の検討

K306 ラット腎刷子縁膜のクエン酸輸送系に対する重金属の影響の検討

Effects of interactions between drugs on the renal excretion of trientine in rats--acetazolamide and furosemide increase trientine excretion.

To elucidate the effects of drug interactions on the urinary excretion of trientine in rats. Trientine and various other drugs were intravenously administered to rats and the urinary excretion of trientine was investigated. To clarify the mechanisms of drug-drug interactions, we also investigated the effects of various drugs on spermine uptake by rat renal brushborder membrane vesicles. Cimetidine, a substrate of the H+/organic cation antiporter, and aminoglycoside antibiotics did not affect trientine excretion, while acetazolamide and furosemide, which increase the concentration of sodium ions in renal proximal tubules, increased the excretion of trientine. However, trichlormethiazide, which acts in renal distal tubules, did not affect trientine excretion. Acetazolamide and furosemide did not directly affect the Na+/spermine transporter because these diuretics had no effect on the uptake of spermine into the rat renal brush-border membrane vesicles. There is no interaction between trientine and the substrate of the H+/organic cation antiporter or aminoglycoside antibiotics. However, drugs that change the concentration of sodium ions in renal proximal tubules, such as diuretics, can increase the trientine excretion since the increase in the luminal concentration of sodium ion accelerates the Na+/spermine antiporter.

Similar effects of cis-diamminedichloroplatinum(II) and cis-diammine-1,1-cyclobutanedicarboxylatoplatinum(II) on sodium-coupled glucose uptake in renal brush-border membrane vesicles.

cis-Diamminedichloroplatinum(II) (cDDP) has been shown to interfere with reabsorption processes in renal tubular epithelia, leading to polyuria, magnesium and sodium wasting and glucosuria. cDDP inhibits the Na+-coupled uptake of methyl-alpha-D-glucopyranoside (MGP) in renal proximal tubular cells in primary culture. cis-Diammine-1,1-cyclobutane dicarboxylatoplatinum(II) (CBDCA) produces tubular injury qualitatively similar to that of cDDP with a reduced severity. CBDCA inhibits Na+-coupled MGP uptake in renal proximal tubular cells in primary culture at concentrations 20- to 30-times higher than those of cDDP. The Na+/glucose cotransport protein possesses sulphydryl groups (SH) essential for its activity. Platinum complexes have strong affinity for SH groups. We compared the direct effects of cDDP (0.04-1.0 mM) and CBDCA (1-30 mM) on Na+-coupled MGP uptake in rabbit renal brush-border membrane (BBM) vesicles. cDDP and CBDCA inhibited Na+-coupled MGP uptake in a concentration-dependent manner, mainly through a decrease in Vmax of the cotransport protein. These effects were associated with platinum binding to BBM and decreases in protein-bound SH groups. CBDCA altered Na+-coupled MGP uptake at concentrations 30-times higher than those of cDDP. When BBM vesicles were preincubated with cDDP or CBDCA, diethyldithiocarbamate (an antidote against cDDP-induced nephrotoxicity) partly restored Na+-coupled MGP uptake and reduced the amount of platinum bound to BBM, but did not restore protein-bound SH groups. These findings strongly suggest that the inhibition of Na+-coupled MGP uptake by cDDP and CBDCA is mainly mediated by direct chemical binding of platinum to essential SH groups of the cotransport protein but may also involve other nucleophilic groups, such as the SCH3 group of methionine residues.

4-aminomethylbenzoic acid is a non-translocated competitive inhibitor of the epithelial peptide transporter PepT1.

1. 4-Aminomethylbenzoic acid, a molecule which mimics the special configuration of a dipeptide, competitively inhibits peptide influx in both Xenopus Laevis oocytes expressing rabbit PepT1 and through PepT1 in rat renal brush border membrane vesicles. 2. This molecule is not translocated through PepT1 as measured both by direct HPLC analysis in PepT1-exp ressing oocytes and indirectly by its failure to trans-stimulate labelle d peptide efflux through PepT1 in oocytes and in renal membrane vessicle s. 3. However 4-aminiomethylbenzoic acid does reverse trans-stimulation through expressed PepT1 of labelled peptid efflux induced by unlabelled peptide. Quantitatively this reversal is compatible with 4-aminomethyl benzoic acid competitively binding to the external surface of PepT1. 4. 4-Aminomethylbenzoic acid (the first molecule discovered to be a non-translocated competitive inhibitor of proton-coupled oligopeptide transport) and its derivatives may thus be particularly useful as experimental tools.

PH-Dependent transport of cadmium in rat renal brush border membrane vesicles: cadmium efflux via H +-antiport

The effects of pH on the Cd efflux across the renal brush border membrane were investigated using isolated rat renal brush border membrane vesicles (BBMV). BBMV equilibrated at pH 5.5 or 7.5 (pH in) were mixed with an experimental buffer at pH 5.5 or 7.5 (pH out) containing CdCl 2. After the incubation with CdCl 2, BBMV were washed with a chelating agent, EGTA, to remove Cd bound to the outer surface of BBMV. The initial accumulation of Cd in BBMV incubated for 1, 3 or 5 min at pH in=5.5 and pH out=7.5 (outwardly directed H +-gradient) was significantly higher than that at pH in=pH out=7.5, but the equilibrated Cd accumulation incubated for over 15 min was almost the same. FCCP, a protonophore, diminished the increasing effect of the H +-gradient on the initial Cd accumulation, whereas valinomycine, a K + ionophore, did not. Kinetic analysis of the Cd accumulation incubated with different concentrations of CdCl 2 suggests that Cd is taken up into BBMV via the unsaturable component and saturable component and the outwardly directed H +-gradient increases the V max value of the saturable component. The BBMV were incubated with CdCl 2 at different temperatures under the outwardly directed H +-gradient, and the increase in Cd accumulation due to the gradient was analyzed by an Arrhenius plot. The plot of increased Cd accumulation was nonlinear, suggesting the involvement of a carrier-mediated process. These findings suggest that the outwardly directed H +-gradient increases the Cd efflux across the renal brush border membrane via H +-antiport system.

S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types.

Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Proton-coupled oligopeptide transport by rat renal cortical brush border membrane vesicles: a functional analysis using ACE inhibitors to determine the isoform of the transporter

We demonstrate that the angiotensin-converting enzyme inhibitors enalapril and captopril inhibit the transport of D-Phe-L-Gln into PepT1-expressing Xenopus oocytes and into rat renal cortical brush border membrane vesicles (BBMV). The kinetics of inhibition are competitive. Enalapril and captopril are not substrates for PepT2 (Boll et al., Proc. Natl. Acad. Sci. 93 (1996) 284–289). Therefore we conclude that in rat renal cortical BBMV this neutral dipeptide is transported via PepT1.

Maturational changes in rabbit renal brush border membrane vesicle osmotic water permeability.

We have recently shown that the osmotic water permeability (Pf) of proximal tubules from neonatal rabbits is higher than that of adults (AJP 271:F871-F876, 1996). The developmental change in Pf could be due to differences in one or more of the components in the path for transepithelial water transport. The present study examined developmental changes in water transport characteristics of the proximal tubule apical membrane by determining Pf and aquaporin 1 (AQP1) expression in neonatal (10-14 days old) and adult rabbit renal brush border membrane vesicles (BBMV). AQP1 abundance in the adult BBMV was higher than the neonatal BBMV. At 25 degrees C the Pf of neonatal BBMV was found to be significantly lower than the adult BBMV at osmotic gradients from 50 to 250 mOsm/kg water. The activation energy for osmotic water movement was higher in the neonatal BBMV than the adult BBMV (9.19 +/- 0.37 vs. 5.09 +/- 0.57 kcal . deg-1 . mol-1, P < 0.005). Osmotic water movement in neonatal BBMV was inhibited 17.9 +/- 1.3% by 1 mm HgCl2 compared to 34.3 +/- 3.8% in the adult BBMV (P < 0.005). These data are consistent with a significantly greater fraction of water traversing the apical membrane lipid bilayer in proximal tubules of neonates than adults. The lower Pf of the neonatal BBMV indicates that the apical membrane is not responsible for the higher transepithelial Pf in the neonatal proximal tubule.

Urate and antioxidants inhibit Na +-adenosine transport in rat renal brush border membranes

The effects of urate and antioxidants were evaluated on sodium-dependent [ 3H]adenosine transport (Na +/ADO) in rat renal brush border membrane vesicles (BBMV). Na +/ADO was estimated for a range of adenosine concentrations of 1–10 μmol/l in BBMV preincubated with urate (0.5–50 μmol/l). Michaelis-Menten kinetics showed a significant increase in K m values from 2.48±0.49 μmol/l in control to 20.58±4.56 μmol/l with 50 μmol/l urate; V max (243±15 pmol/mg protein×min) was not modified. Menadione (10 μmol/l) significantly increased the Na +/ADO activity, from 17.57±5.50 in the control, to 27.70±7.60 pmol/mg prot.×min (a 1.60 times increase, p<0.05). This stimulation was prevented when BBMV were preincubated with either 1 μmol/l α-tocopherol (trolox) or urate. Similarly conjugated dienes and malonaldehyde were stimulated in a dose-dependent fashion by menadione and the effect was inhibited with 10 μmol/l trolox. The antioxidants probucol, captopril and allopurinol inhibited in a concentration-dependent manner the Na +/ADO (IC 50 were 79±8, 100±9 and 89±9 nM, respectively). This effect might be specific on K m of the Na +/ADO, since 1 μmol/l trolox (IC 50=1000±20 nM), inhibited V max but not K m of the Na +/glucose transport. Our results suggest that the Na +/ADO in BBMV is modified by agents that affect the redox status of the membranes.

P3B66 - Similar interactions of cisplatin and carboplatin with the Na +/glucose cotransporter in renal brush-border membrane vesicles

P3B66 - Similar interactions of cisplatin and carboplatin with the Na +/glucose cotransporter in renal brush-border membrane vesicles

Influence of substrate structure on turnover of the organic cation/H+ exchanger of the renal luminal membrane.

We examined the influence of organic cation (OC) structure on the rate of turnover of the OC/H+ exchanger in rabbit renal brush-border membrane vesicles (BBMV). The rate of efflux of [14C]tetraethylammonium ([14C]TEA) from BBMV, measured in the presence of an inwardly directed chemical gradient for test agent, provided an indirect measure of activity of the OC/H+(OC) exchanger. The trans-stimulation of [14C]TEA efflux from BBMV was a saturable function of increasing extravesicular concentration of both unlabeled TEA and tetramethylammonium (TMA), with an apparent Michaelis constant (Kt) for the interaction of these compounds with the OC/H+(OC) exchanger of 25 microM and 1 mM, respectively. The effect on [14C]TEA efflux of saturating extravesicular concentrations of a series of n-tetraalkylammonium compounds was examined. Whereas the short-chain compounds TMA and TEA markedly stimulated [14C]TEA efflux (by 830% and 690%, respectively), the long-chain compounds tetrapropylammonium and tetrabutylammonium were less effective, increasing efflux by only 40% and 120%, respectively. When the exchanger was saturated with tetrapentylammonium, mediated efflux of [14C]TEA was reduced. Increasing alkyl chain length was also correlated with an increase in the inhibitory effect (as measured by the apparent inhibition constant, Ki, or the IC50 value) that these compounds had against transport of [14C]TEA by the OC/H+(OC) exchanger; i.e., there was a correlation between decreasing IC50 and decreasing turnover of the OC/H+(OC) exchanger. This same correlation was observed for a broader set of test agents of diverse molecular structure, including a series of n-tetraalkylammonium and -phosphonium compounds and the OCs, choline, N1-methyl nicotinamide, 1-methyl-4-phenylpyridinium, and amiloride. Because high affinity of substrates for the OC/H+(OC) exchanger is correlated with increasing substrate hydrophobicity, we conclude that the interaction of hydrophobic OCs with the renal OC/H+(OC) exchanger results in the formation of a substrate-exchanger complex that has a comparatively low rate of turnover.

Lack of cisplatin-ranitidine kinetic interactions: In vivo study in children, and in vitro study using dog renal brush border membrane vesicles

The interactions between cisplatin and organic ions have been extensively investigated in animal models for the potential to reduce cisplatin cellular uptake and resultant nephrotoxicity. To further investigate the beneficial interaction clinically, we studied the effects of the organic cation, ranitidine, on the renal handling of cisplatin in children. In parallel, we examined the effects of cisplatin on the uptake kinetics of organic cations and anions by brush border membrane vesicles (BBMV) prepared from dog renal cortex. The results indicate that: 1) there is no measurable effect of ranitidine on renal clearance of cisplatin in children; and 2) BBMV uptake of anionic p-aminohippurate, but not cationic N-methylnicotinamide, is inhibited by cisplatin at concentrations of < 1 mM. These findings suggest that cisplatin may not share transport systems with organic cations to a clinically significant degree. Assuming that renal tubular transport is a prerequisite for cisplatin nephrotoxicity, the lack of apparent kinetic interactions between cisplatin and organic cations may preclude clinical use of organic cations as a modality to prevent cisplatin nephrotoxicity.

Membrane Topology of NHE3: EPITOPES WITHIN THE CARBOXYL-TERMINAL HYDROPHILIC DOMAIN ARE EXOPLASMIC

Experimental data indicate that the relatively hydrophilic carboxyl-terminal domains of Na+-H+ exchangers mediate the regulation of transporter activity through interactions with cytoskeletal effectors. It has therefore been assumed that this entire domain lies on the cytoplasmic surface of the plasma membrane. The purpose of the present study was to determine the membrane orientation of the COOH-terminal 131 amino acids of Na+-H+ exchanger isoform NHE3 by use of three monoclonal antibodies that recognize at least two distinct epitopes within this region. Enzyme-linked immunosorbent assay studies demonstrated binding of these monoclonal antibodies (mAbs) to intact right-side-out renal brush border membrane vesicles in the absence of detergent. Moreover, when coupled to an affinity matrix to isolate membrane vesicles, the anti-NHE3 mAbs bound structures that were morphologically identical to intact microvilli. To confirm the identity of the exoplasmic antigen bound by the antibodies, immunoprecipitation studies were performed. Intact right-side-out brush border membrane vesicles were incubated with the mAbs in the absence of detergent. The membranes were pelleted, supernatant with unbound antibody was removed, the pellet was solubilized, and then immunoprecipitation with secondary antibody was performed. Immunoblot analysis indicated that NHE3 was precipitated after binding of the mAbs to intact membranes. Finally, the localization of the mAb epitopes was determined using high resolution immunocytochemistry. Ultrathin cryosections of rat kidney were labeled with the mAbs and bound antibody detected with the colloidal gold technique. Labeling was restricted to the exoplasmic surface of microvilli of the proximal tubule. Taken together, these findings indicate that epitopes within the carboxyl terminus of the Na+-H+ exchanger isoform NHE3 are exposed to the outside of the plasma membrane.

Characterization of the chloride conductance in porcine renal brush-border membrane vesicles.

The chloride conductance in brush-border membrane vesicles prepared from pig kidney cortex was investigated using a light-scattering assay, anion-diffusion-potential-dependent Na+-D-glucose cotransport and 36Cl- influx. K+-diffusion-potential-driven salt exit from, or entry into, the vesicles was slow in the presence of gluconate, SO42- and F-, intermediate with Cl- and Br-, and fast with I-, NO3-, and SCN-. Stimulation of Na+-D-glucose uptake followed a similar anion sequence. Conductive Cl- flux had a low activation energy and was inhibited by suphhydryl reagents, the stilbene disulphonates 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) and 4, 4'-diisothiocyanato-2,2'-disulphonate (DIDS), and the arylaminobenzoates diphenylamine-2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Intravesicular Ca2+ and extravesicular nucleotides were without effect on conductive Cl- flux. These characteristics tentatively exclude some known Cl- channels and leave members of the ClC family as possible candidates responsible for the Cl- conductance in brush-border membranes.

Riboflavin transport by rabbit renal brush border membrane vesicles

The present study examined riboflavin (RF) uptake by isolated rabbit renal brush border membrane (BBM). RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfaces. The process of RF uptake was saturable as a function of substrate concentration with an apparent K mof 25.7 ± 7.6 μM and V maxof 75.6 ± 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and its structural analogues, lumiflavin and lumichrome, inhibited the uptake of [ 3H]RF significantly, indicating the involvement of a carrier-mediated process in RF uptake by renal BBM. RF uptake by renal BBM was partly Na +-dependent so that when Na + was replaced by potassium, choline, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60% of the control. This Na +-dependency was unlikely to be due to Na +-cotransport mechanism because RF uptake occurred without the characteristic ‘overshoot’ phenomenon as for other Na +-cotransport systems and the elimination of transmembrane Na +-gradient by preloading Na + to the intravesicular space did not affect RF uptake. In contrast, removal of Na + eliminated the binding component of RF uptake, suggesting the requirement of Na + for RF binding to BBM. The RF uptake was not affected when extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [ 3H]RF uptake was found when the transmembrane electrical potential was altered by either the presence of anions with different membrane permeability ( Cl − = NO 3 − SO 4 −gluconate −) or by using nigericin (10 μg/mg protein) with an outwardly or inwardly directed transmembrane K + gradient. The uptake of RF by BBM vesicles was, however, inhibited by probenecid and organic anion transport inhibitors, 4,4-diiso-thiocyanatostilbene-2,2-disulfonic acid (DIDS, 1 mM) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mM). In summary, these results demonstrate the existence of a membrane-associated, and organic anion inhibitor-sensitive, carrier system for RF uptake by renal BBM.

Renal brush border sodium-sulfate cotransport in guinea pig: effect of age and diet.

Renal adaptation to changes in inorganic sulfate intake and age was studied by comparing sulfate uptake by proximal tubule brush border membrane vesicles (BBMV) from guinea pigs of different ages on relatively high- or low-sulfate diets. Adult (> 60 days) or young guinea pigs (< 25 days) were fed either a control diet (0.28% sulfur content), a sulfur-free diet, or a high-sulfate diet. After 5 days on the diet, BBMV were obtained and kinetic analysis of 35sulfate uptake was determined. In adult guinea pigs, the low-sulfate diet produced a significant increase in apparent maximal velocity (Vmax). In young guinea pigs, a lower sulfate intake did not appreciably increase Vmax, but a high-sulfate intake produced a reduction in Vmax. The affinity for sulfate (Km) was not changed in either age group. The dietary sulfate intake did not alter sodium gradient dependent-D-glucose or 32phosphate Vmax. In conclusion, our data indicate that renal inorganic sulfate BBMV uptake is regulated and responds to conditions of increased need (i.e., during the growth phase in young animals and during periods of decreased sulfate availability in adult animals) by increasing BBMV Vmax.

Effect of Cadmium on Na–PiCotransport Kinetics in Rabbit Renal Brush-Border Membrane Vesicles

Chronic exposure to cadmium impairs various renal functions, including phosphate (Pi) transport. To further investigate the mechanism of cadmium-induced alterations in renal Pitransport, kinetics of Na+-dependent Piuptake were studied in renal cortical brush-border membrane vesicles (BBMV) exposed to CdCl2in vitro.BBMVs isolated from rabbit renal outer cortex were preincubated in a buffer containing CdCl2(50 μmin most cases) for 60 min at 37°C and then tested for Piuptake at 25°C. CdCl2treatment resulted in a marked attenuation of Na+-dependent Piuptake with no changes in Na+-independent Piuptake and membrane permeability to Na+. CdMt treatment induced no changes in Pitransport. The inhibition required preincubation of vesicles with CdCl2for more than 30 min and was not reversed by extravesicular EDTA, suggesting that cadmium affects the transport system at the internal side of the membrane. Kinetic analysis indicated that two sodium ions and one phosphate ion interact with a carrier, and this stoichiometry was not altered by cadmium treatment. Cadmium treatment did not change the apparentKmfor Na+(KNa) and that for phosphate (KPi), but it markedly reduced theVmaxof the Na+-dependent Pitransport. These results indicate that exposure of proximal tubular brush border membranes to cadmium impairs the Pitransport capacity, probably by reducing the effective number of Na–Picotransporter units without altering substrate affinities of the carrier.