Rabbit Kidney Brush-border Membranes Research Articles

Oligomeric structure of a renal cystine transporter: implications in cystinuria

Homologous proteins (NBAT) which mediate sodium-independent transport of neutral as well as basic amino acids and cystine when expressed in Xenopus oocytes were recently cloned from mammalian kidneys. Mutations in human NBAT have been implicated in cystinuria. Here, we show that rat kidney and jejunal brush border membrane NBAT (85 kDa) is found in association with a 50 kDa protein. The association involves one or more interprotein disulfide bonds. Rabbit kidney brush border membranes and membranes of NBAT cRNA-injected Xenopus oocytes also contain such heterodimers. Our data suggest that the heterodimer is the minimal functional unit of NBAT-mediated amino acid transport and that the NBAT-associated 50 kDa protein could play a role in cystinuria.

Binding of nitrate to renal brush border membranes studied with 14N nuclear magnetic resonance (NMR)

Renal brush border membranes contain several anion exchangers that may play a role in electrolyte transport and pH regulation. To help characterize the types of exchangers present and the binding properties of these membranes, the binding of nitrate (NO 3 −) to highly purified rabbit kidney brush border membrane vesicles was studied. The method is based on the binding induced quadrupole relaxation of the 14N-NMR signal of nitrate [1,2]. Brush border membrane vesicles caused a relaxation of the 14N-NMR nitrate signal which could be characterized by relatively high affinity sites, K D = 6.7 ± 1.5 mM, as well as nonspecific interactions with the membranes, K D > 150 mM. The anion transport inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS) inhibited 51 ± 6% ( n = 4) of the relaxation due to the high affinity binding sites. The DNDS inhibition could be characterized by a K i of 10–80 μM. Both bicarbonate and formate (HCO 2 −) were found to partially inhibit the high affinity induced relaxation, with maximal inhibition of 37 ± 8% ( n = 3) and 30 ± 2% ( n = 3), respectively. The inhibitory effects of saturating concentrations of bicarbonate and formate were non-additive, suggesting the existence of a stilbene sensitive exchanger that can bind nitrate, as well as both bicarbonate and formate. This study indicates the usefulness of this new method for further investigation of anion exchangers on these and other membranes.

Reconstitution and characterization of a Na+/Pi co-transporter protein from rabbit kidney brush-border membranes.

A protein with Na+/Pi co-transporter activity has been extracted from rabbit brush-border membranes with chloroform/methanol and purified by hydroxyapatite chromatography. The protein has been incorporated by the dilution method into liposomes formed from different types and ratios of lipids. The greatest reconstitution has been achieved into liposomes prepared from cholesterol (20%), phosphatidylcholine (20%), phosphatidylethanolamine (30%) and phosphatidylserine (30%) (CH/PC/PE/PS). Pi uptake by these proteoliposomes had the following characteristics: (i) the initial rate was markedly greater in the presence of an inwardly directed Na+ gradient (600 pmol/10 s per mg) than with a K+ gradient (65 pmol/10 s per mg); (ii) maximal uptake was increased 8-fold above the equilibrium value ('overshoot') when a Na+ gradient was applied; (iii) Pi was not merely bound to proteoliposomes but was transported intravesicularly; and (iv) Na(+)-dependent Pi uptake was sensitive to the known phosphate transport inhibitors. This first successful attempt of reconstitution of Na+/Pi transport activity into proteoliposomes led us to isolate and characterize physico-chemically the protein responsible. Its isoelectric point was about 5.8, and urea/SDS gel electrophoresis revealed a broad band of molecular mass ranging from 63 to 66 kDa under both reducing and non-reducing conditions. In the native form, the molecular mass analysed by gel filtration was estimated to be 170 +/- 10 kDa, suggesting that the protein is a polymer, probably stabilized by hydrophobic bonds. Endoglycosidase F treatment decreased the molecular mass to approx. 50 kDa. It is postulated that this acidic glycoprotein might represent a subunit of the intact Na+/Pi co-transporter from rabbit kidney brush-border membranes.

Mechanism of acridine orange interaction with phospholipids and proteins in renal microvillus vesicles.

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.

Neutral maltase/glucoamylase from rabbit renal cortex

Maltase activity (EC 3.2.1.20) was solubilized from rabbit kidney brush-border membrane by using 1.0% Triton X-100 and purified 230-fold with an overall recovery of 30%. The purification procedure makes use of heat precipitation, chromatography on DE-52 DEAE-cellulose and gel filtration on Sephacryl S-300. Rabbit kidney brush border exhibited glucoamylase activity with a maltase/glucoamylase ratio of 1.5:1 to 2.0:1. During purification the maltase and glucoamylase activities behaved identically. The Mr of the complex is 590,000, and it appears to be composed of eight identical subunits linked by disulphide bridges.

Determination of brush border membrane vesicle orientation using monoclonal antibodies recognizing extracytoplasmic and cytoplasmic domains of neutral endopeptidase-24.11

A method for determination of the orientation and integrity of brush border membrane vesicles is described. The method takes advantage of the availability of two monoclonal antibodies, 23B11 and 2B12, which recognize a cytoplasmic and an extracytoplasmic domain, respectively, of the neutral endopeptidase-24.11. Specific binding of the antibodies to intact kidney brush border vesicles or to vesicles permeabilized by digitonin is detected by fluorescence using an anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate. The method allows discrimination between right side out, inside out, and unsealed vesicles. It requires limited amounts of material and can be completed the day of the brush border vesicle preparation. Application to rabbit kidney brush border membranes freshly prepared led to values of 89, 8, and 3% for right side out, inside out, and unsealed vesicles, respectively. Storage at low temperature was associated with a marked increase in the proportion of unsealed vesicles.

Monoclonal antibodies as probes for the transmembrane structure of neutral endopeptidase 24.11 (‘enkephalinase’)

The neutral endopeptidase (EC 3.4.24.11) (‘enkephalinase’) is a membrane-bound metalloendopeptidase that is present in large amounts in the microvilli of the kidney proximal tubules. By immunizing mice with purified rabbit kidney brush-border membranes, we have obtained four different monoclonal antibodies that recognize this enzyme in dot-blot and Western-blot assays and can be used for immunoprecipitation of neutral endopeptidase from crude kidney solubilizates. One of these monoclonal antibodies (2B12) allows the labeling of proximal tubule cells with colloidal gold particles. This monoclonal antibody also binds to native brush-border membrane vesicles (which are mostly in the right-side-out configuration) and recognizes an epitope which is destroyed after reduction and alkylation of the protein. By contrast, all three other monoclonal antibodies (21G10, 23B11 and 22E2) compete for another epitope of neutral endopeptidase that is not exposed at the extracytoplasmic surface either in intact cells or in sealed brush-border vesicles. Permeabilization of the vesicles with digitonin, however, restores the full binding activity. Binding of these antibodies is not altered by prior reduction and alkylation of the protein. Taken together, these results strongly suggest that the 2B12 monoclonal antibody binds a conformational epitope located on the ectodomain of the enzyme, whereas the three others (21G10, 23B11 and 22E2) bind to a common or to overlapping epitopes located on the cytosolic domain. These results also demonstrate unambiguously the transmembrane nature of neutral endopeptidase.

Phospholipid asymmetry in renal brush-border membranes

The topological distribution of phospholipids between the inside and the outside of rabbit kidney brush-border membranes has been investigated by incubating membrane vesicles with sphingomyelinase, phospholipases A 2 from bee venom and hog pancreas, phospholipases C and D, and trinitrobenzene sulfonate. Orientation and integrity of vesicles upon phospholipase treatment was determined by using two monoclonal antibodies recognizing an extracytoplasmic and a cytoplasmic domain, respectively, of the neutral endopeptidase (EC 3.4.24.11). It is shown that the transbilayer distribution of phospholipids is highly asymmetrical in kidney brush-border membranes: sphinomyelin accounted for 75% of the phospholipids present in the external leaflet, whereas phosphatidylethanolamine and phosphatidylserine plus phosphatidylinositol were found to comprise the majority of the inner layer of the membrane.

Taurine transport by rabbit kidney brush-border membranes: coupling to sodium, chloride, and the membrane potential.

Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na+-D-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na+ gradient (K+ diffusion potential) and could be completely inhibited by other beta-amino acids. It displayed a specific anion requirement (Cl- greater than or equal to Br- much greater than SCN- greater than I- greater than NO-3). At chemical Na+ equilibrium, Cl- gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl- gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl- as well as Br-. The Cl- stoichiometry was found to be one, whereas taurine transport, in the presence of Cl-, was sigmoidally related to the Na+ concentration, resulting in a coupling ratio of 2 to 3 Na+: 1 taurine. Upon Cl- replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na+ concentration (up to 150 mM). These results suggest a 2 to 3 Na+ :1 Cl- :1 taurine cotransport mechanism driven mainly by the Na+ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl- appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.

Atrial peptide inactivation by rabbit‐kidney brush‐border membranes

Atriopeptin (AP) 24, containing amino acids Ser103-Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103-Ser104, Cys105-Phe106 and Ser123-Phe124. des-Ser103-AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at Cys105-Phe106 was inactive. Regarding the proteolytic cleavage at Ser123-Phe124, there was an accumulation of the C-terminal tripeptide, Phe-Arg-Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino- and carboxy-terminal protected peptide, acetyl-AP24-amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was Cys105-Phe106. We conclude that the Cys-Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.

Inactivation of atrial natriuretic factor by the renal brush border

Atrial natriuretic factor (ANF), a 28-amino-acid peptide secreted from the mammalian heart, is known to be cleared rapidly from the circulation. In vitro and in vivo studies implicate the kidney as an important site for clearance and subsequent degradation of atrial natriuretic factor. We have observed that atrial natriuretic factor is inactivated rapidly by rabbit kidney brush-border membranes. The rate of degradation of ANF measured by the loss of bioactivity followed a similar time-course to the decrease in peptide peak area measured by high-performance liquid chromatography. Interestingly, inactivation of ANF produced only a single major degradation product, which was isolated and purified. Sequence analysis revealed that the product had the same sequence of amino acids as ANF with the Cys-7-Phe-8 bond cleaved and the disulfide bridge between Cys-7 and Cys-23 remaining intact. As the renal brush border contains an abundance of proteolytic activities, it is surprising that this peptide is cleaved primarily at a single peptide bond.

CAMP-associated inhibition of Na+-H+ exchanger in rabbit kidney brush-border membranes.

Adenosine 3',5'-cyclic monophosphate (cAMP) inhibits the rate of bicarbonate reabsorption and the rate of Na+-H+ exchange transport in the apical membrane of the proximal convoluted tubule. To study the relation between cAMP, cAMP-dependent protein kinase, and Na+-H+ exchange transport, brush-border membrane vesicles from the rabbit kidney were phosphorylated in vitro. The rate of proton gradient-stimulated amiloride-inhibitable 22Na+ uptake was measured as an index of Na+-H+ exchange transport activity. The inclusion of cAMP (10(-6) M) in a phosphorylating solution containing ATP decreased the 10-s uptake of amiloride-sensitive sodium from 2.25 +/- 0.21 nmol/mg protein in controls to 1.94 +/- 0.19 (P less than 0.001). Incubation of vesicles in the presence of purified catalytic subunit of cAMP-dependent protein kinase inhibited the amiloride-sensitive uptake of 22Na+ at 10 s from 2.35 +/- 0.49 nmol/mg protein to 2.05 +/- 0.44 (P less than 0.005). The inhibitory effect of both cAMP and catalytic subunit of cAMP-dependent protein kinase was blocked by the specific thermostable protein inhibitor of the kinase. These studies demonstrate that activation of endogenous membrane-bound cAMP-dependent protein kinase or exposure to exogenous catalytic subunit of cAMP-dependent protein kinase inhibits the rate of Na+-H+ exchange transport in the brush-border membrane of the rabbit kidney.

Gentamicin inhibits Na +-dependent d-glucose transport in rabbit kidney brush-border membrane vesicles

We studied the effect of gentamicin on Na +-dependent d-glucose transport into brush-border membrane vesicles isolated from rabbit kidney outer cortex (early proximal tubule) and outer medulla (late proximal tubule) in vitro. We found the same osmotically active space and nonspecific binding between control and gentamicin-treated brush-border membrane vesicles. There was no difference in the passive permeability properties between control and gentamicin-treated brush-border membrane vesicles. Kinetic analyses of d-glucose transport into 1 mM gentamicin-treated brush-border membrane vesicles demonstrated that gentamicin decreased V max in the outer cortical preparation, while it did not affect V max in the outer medullary preparation. With regard to K m, there was no effect of gentamicin in any vesicle preparation. When brush-border membrane vesicles were incubated with higher concentrations of gentamicin, Na +-dependent d-glucose transport was inhibited dose-dependently in both outer cortical and outer medullary preparations. Dixon plots yield inhibition constant K i = 4 mM in the outer cortical preparation and K i = 7 mM in the outer medullary preparation. These results indicate that the Na +-dependent d-glucose transport system in early proximal tubule is more vulnerable to gentamicin toxicity than that in late proximal tubule.

Reconstitution of the partially purified renal phosphate (Pi) transporter.

Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.

Interaction of guanidinium and guanidinium derivatives with the Na+/H+ exchange system.

Guanidinium, a small organic monovalent cation that is permeant through voltage-dependent cationic channels cannot be transported by the cardiac Na+/H+ exchange system. Yet it recognizes the exchanger and is able to block its activity (K0.5 = 30 mM). Guanidinium derivatives that do not belong to the amiloride series and which possess potent antihypertensive properties also block the activity of the Na+/H+ exchange system in various cell types with a greater potency than unsubstituted guanidinium. The most potent compound found, guanochlor, has an affinity for the exchanger ranging between 0.5 microM and 6 microM in different systems and is more potent than amiloride in all systems studied. Guanochlor has the same action as amiloride derivatives on the cardiac cells; it prevents intracellular pH recovery in cardiac cells that have been acidified and also antagonizes the effect of ouabain on 45Ca2+ uptake by chick cardiac cells. Guanochlor does not compete with [3H]ethylpropylamiloride for its binding to the Na+/H+ exchange system of rabbit kidney brush border membrane. It is suggested that guanochlor recognizes a binding site on the Na+/H+ exchanger that is distinct from the amiloride binding site.

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na +-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M r ≅ 165000 which is apparently a dimer of M r ≅ 85 000. When reconstituted and tested for transport, this protein showed Na +-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.

Phosphate-binding proteolipid from brush border.

A proteolipid that binds inorganic phosphate with high affinity and specificity has been extracted from rabbit kidney brush-border membranes. This proteolipid has been partially purified by chromatography on LH-20. The molecular weight of the proteolipid is approximately 3000 as determined by urea-sodium dodecyl sulfate gel electrophoresis. This proteolipid can bind and transport phosphate into an organic phase. The K0.5 for phosphate binding is 8 microM with a Hill coefficient of 1.92. Arsenate inhibits phosphate binding in a competitive manner with a KI of 27.5 microM. The aminoreactive reagent 2,4-dinitrofluorobenzene inhibits phosphate binding to the proteolipid. Similarly, 2,4-dinitrofluorobenzene inhibited Na+-driven Pi uptake in renal brush-border membrane vesicles. In contrast to the mitochondrial phosphate binder, this proteolipid is not inhibited by sulfhydryl reagents. We suggest that this molecular species is a likely candidate for involvement in phosphate uptake in the renal tubule.