Rat Kidney Brush-border Membrane Vesicles Research Articles

Characterization of D-fructose transport by rat kidney brush-border membrane vesicles: changes in hypertensive rats.

D-fructose transport was characterized in renal brush-border membrane vesicles (BBMVs) from both spontaneously hypertensive rats (SHR) and normotensive genetic control Wistar-Kyoto (WKY) rats. Kinetic studies indicated that the maximal rate (Vmax) of D-fructose transport was significantly lower in SHR compared with WKY rats. No differences were observed in the Michaelis constant (Km) or the diffusion constant (Kd) between the two groups of animals. D-fructose inhibited its own transport, whereas the presence of D-glucose, D-galactose, phlorizin, and cytochalasin B did not inhibit the transport of D-fructose in either animal group. To explain the reduction in D-fructose transport in SHR, the density of the D-fructose transporter, GLUT5, was analyzed by Western blot. GLUT5 levels were lower in SHR, a reduction similar to that of the Vmax. Thus, there appears to be a high-affinity, low-capacity, GLUT5-type fructose carrier in the apical membranes of rat kidney cortex, and the decrease in the Vmax of D-fructose transport in renal BBMVs from hypertensive rats correlates well with a reduction in the expression of GLUT5 protein.

Characterization of L-carnitine transport into rat skeletal muscle plasma membrane vesicles.

Transport of L-carnitine into skeletal muscle was investigated using rat sarcolemmal membrane vesicles. In the presence of an inwardly directed sodium chloride gradient, L-carnitine transport showed a clear overshoot. The uptake of L-carnitine was increased, when vesicles were preloaded with potassium. When sodium was replaced by lithium or cesium, and chloride by nitrate or thiocyanate, transport activities were not different from in the presence of sodium chloride. However, L-carnitine transport was clearly lower in the presence of sulfate or gluconate, suggesting potential-dependent transport. An osmolarity plot revealed a positive slope and a significant intercept, indicating transport of L-carnitine into the vesicle lumen and binding to the vesicle membrane. Displacement experiments revealed that approximately 30% of the L-carnitine associated with the vesicles was bound to the outer and 30% to the inner surface of the vesicle membrane, whereas 40% was unbound inside the vesicle. Saturable transport could be described by Michaelis-Menten kinetics with an apparent Km of 13.1 microM and a Vmax of 2.1 pmol.(mg protein-1).s-1. L-Carnitine transport could be trans-stimulated by preloading the vesicles with L-carnitine but not with the carnitine precursor butyrobetaine, and was cis-inhibited by L-palmitoylcarnitine, L-isovalerylcarnitine, and glycinebetaine. On comparing carnitine transport into rat kidney brush-border membrane vesicles and OCTN2, a sodium-dependent high-affinity human carnitine transporter, cloned recently from human kidney also expressed in muscle, the Km values are similar but driving forces, pattern of inhibition and stereospecificity are different. This suggests the existence of more than one carnitine carrier in skeletal muscle.

Inhibition of the hydrolytic and transpeptidase activities of rat kidney γ‐glutamyl transpeptidase by specific monoclonal antibodies

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.

Proton/solute cotransport in rat kidney brush-border membrane vesicles: relative importance to both d-glucose and peptide transport

We have determined the relative importance of the transmembrane proton electrochemical gradient to the transport of d-[ 14C]glucose and [ 14C]glycylsarcosine (gly-sar) in rat kidney brush-border membrane vesicles (BBMV) from superficial renal cortex. Electrogenic [ 14C]gly-sar transport was first optimised by imposing a pH gradient (pH o = 5.7, pH i = 8.4) and an interior negative p.d. (using outwardly directed K + gradient plus valinomycin). Under identical conditions (pH o = 5.7, pH i = 8.4), an acceleration f of initial d-[ 14C]glucose (at 100 μM) transport by 2.0 ± 0.7-fold was observed compared to no proton gradient (pH o = 8.4, pH i = 8.4). This increase was due primarily to an effect of external protons, since acidic conditions (pH o = pH i = 5.7) also resulted in ann acceleration of d-glucose influx (2-fold). The increase in d-glucose transport in the presence of external acidity was reduced by the uncoupler FCCP, even in the absence of a proton gradient. Furthermore, the increased d-glucose transport with external acidity in the presence of a proton gradient was insensitive to a K + gradient-driven diffusion potential in the presence of valinomycin. In no instance was an overshoot accumulation of d-[ 14C]glucose observed in H + gradient conditions. H +-stimulated d-[ 14C]glucose transport showed a linear dependence on d-glucose concentration up to 20 mM d-glucose, unlike electrogenic Na +-dependent d-glucose transport, whose K m was 1.77 ± 0.35 mM. In contrast, the initial rate of [ 14C]gly-sar (100 μM) transport by the renal H +/di-tripeptide transporter was accelerated 15.7 ± 3.3-fold and stimulated a marked overshoot of 5.1 ± 0.4-fold over equilibrium values. Conversely, the electrogenic, Na +/glucose transporter could be readily demonstrated, whilst [ 14C]gly-sar transport could not be energised by an inward Na + gradient. The absence of electrogenic d-glucose transport in H + gradient conditions is clear evidence against H +/glucose cotransport in Na +-free conditions mediated by SGLT2 (sodium-glucose transporter, renal cortex). Furthermore, since a proton gradient does not increase brush-border membrane d-glucose uptake in Na +-rich media, it is unlikely that in vivo renal d-glucose transport mediated via SGLT2 may be energised by the transmembrane proton gradient.

Comparison of GABA uptake by brain and kidney preparations

The uptake ofγ-aminobutyric acid (GABA) was measured in rat cerebral cortical synaptosomes and rat kidney brush-border membrane vesicles (BBMV). Three GABA uptake systems (Km = 1.3, 50 and 3246µM respectively) were present in synaptosomes, but only two uptake systems (Km = 11 and 1203µM respectively) were detectable in BBMV. The uptake systems in the two types of tissue preparations were similar in that every system was inhibited byp-hydroxymercuribenzoate and by the action of neuraminidase, thereby indicating that, irrespective of the tissue, both sulfhydryl and sialyl groups were necessary components of all the GABA uptake systems. In contrast, differences were observed between synaptosomal and BBMV uptake systems with respect to their sensitivity to inhibition by GABA structural analogs. While the synaptosomal GABA uptake systems were strongly inhibited by nipecotic acid, diaminobutyric acid,β-alanine and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), none of these compounds significantly inhibited the BBMV uptake systems. It is therefore concluded that the GABA uptake systems in brain and kidney tissues are quite different entities. While the role of GABA transport in the inactivation of the neurotransmitter function of GABA is well documented, the role of the kidney GABA uptake system is unclear, and the possibility exists that the latter systems are present in kidney tissues primarily to transport compounds other than GABA. The present study does however highlight a difference in drug susceptibility of the brain and kidney uptake systems, a phenomenon which may have some therapeutic relevance with respect to the use of GABA analogs as anticonvulsant agents.

Brush-border membrane cation conducting channels from rat kidney proximal tubules

This is a description and kinetic characterization of cation channels from rat kidney brush-border membrane vesicles and from apical membranes of proximal tubule cells in culture. Channel activity was demonstrated and characterized in both artificial phospholipid bilayers and in tissue culture. Intermediate conductance, approximately 50 pS, cation-selective channels were observed by both methods. Channels were characterized by a Na permeability (PNa)/K permeability (PK) of 1-5:1. Open-channel current-voltage curves were linear in symmetric 300 mM NaCl. In tissue culture the gating kinetics are described by two open-time constants and two closed-time constants. Channel activity was neither voltage nor Ca2+ dependent and the probability of being in the open state ranged from 0.6 to 0.95. In tissue culture experiments the channel demonstrated nonstationary gating activity. A second, 15-pS cation channel, seen in planar bilayers, demonstrated a higher selectivity for Na+ with a (PNa/PK ratio of greater than 10).

Gamma-Aminobutyric acid uptake by rat kidney brush-border membrane vesicles.

Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for gamma-aminobutyric acid (GABA), a high affinity system (Km = 10.9 microM) and a low affinity system (Km = 1203 microM). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, beta-alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5 c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMV uptake systems were clearly different from the GABA transport systems present in brain tissue.

Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index

Solute reflection coefficients σ of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of σ. We determined σ NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone ( M r 40 000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a σ NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again σ NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.

Folate binding and transport by rat kidney brush-border membrane vesicles

[ 3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [ 3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with K m values of 6.7 · 10 −7 and 11.2 · 10 −7 M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this.

Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane.

We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L-[3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1-diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system.

Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles

Solute reflection coefficients, σ i , of rat kidney brush-border membrane vesicles were determined by the comparison of water flows induced by equiosmolal gradients of sucrose and NaCl, KCl or mannitol. The values of 0.53 for σ NaCl and 0.56 for σ KCl when compared with 0.92 for σ mannitol suggested some interactions between salt and water pathways. Altering the membrane proteins with 0.4 mM HgCl 2 decreased the osmotic water permeability of the vesicles by 70 to 80% and brought σ NaCl and σ KCl to a value not different from 1. This argued in favor of water protein pathways in the luminal membrane of kidney proximal cells which are partly accessible to NaCl and KCl.

Transport of glutamine by rat kidney brush-border membrane vesicles.

Transport of glutamine by brush-border vesicles prepared from the renal cortex was studied. The transport system had both Na+-dependent and Na+-independent components. The presence of Na+ in the incubation resulted in an 'overshoot' at 30s at which time the rates of transport were approx. 8 times the values obtained in the absence of Na+. Variation of the glutamine concentration showed that the system obeyed Michaelis-Menten kinetics with Km and Vmax. values for the Na+-dependent system of 0.86 mM and 9.6 nmol/min per mg of protein respectively. Vesicles obtained from chronically acidotic rats showed similar kinetic characteristics. The Km and Vmax. values for the Na+-dependent system were 0.76 mM and 9.6 nmol/min per mg of protein respectively. There was increased uptake of glutamine by vesicles from acidotic rats and this increase was associated with increased activity of gamma-glutamyltransferase in these preparations. Vesicles from acidotic rats, however, showed no increase in glucose transport and no increase in the activity of maltase, another brush-border enzyme.

L-proline transport by newborn rat kidney brush-border membrane vesicles

The transport of L-proline was studied in brush-border membrane vesicles isolated from the kidneys of newborn rats. In contrast with the rapid initial uptake with an 'overshoot' observed in adult vesicles, uptake by the newborn vesicle was slow, showed no 'overshoot', and proline continued to accumulate at a time when the adult vesicle had already equilibrated. L-Proline transport in the newborn rat occurs by Na+-dependent and independent mechanisms. There appeared to be essentially no uptake by anti-luminal vesicles isolated from newborn rat kidney. These observations may help to explain the prolinuria that occurs in the newborn animal.

On the maleic acid induced Fanconi syndrome: Effects on transport by isolated rat kidney brushborder membrane vesicles

The association of maleic acid with isolated rat kidney brushborder membranes and the effect of maleic acid on uptake of amino acids and α-methyl-D-glucoside by brushborder membrane vesicles has been studied. Although maleic acid associates with the membrane, no alteration of sodium dependent transport of these substrates into the vesicles was observed. These findings indicate that the maleic acid induced Fanconi syndrome in the rat with aminoaciduria and glycosuria is not due to a direct, adverse effect on the renal tubule brushborder membrane per se .