Comparative effects of aminoglycosides on renal cortical and urinary phospholipids in the rat

We examined the relationship between the nephrotoxicity potential of four aminoglycosides and the capacity of the drugs to induce a renal cortical phospholipidosis. Sprague-Dawley rats were injected subcutaneously with neomycin, gentamicin, tobramycin, or netilmicin, 100 mg/kg per day, for 1 to 4 days, and phospholipid accumulation in the renal cortex and phospholipid excretion in the urine were measured. The rank order of the drug-induced renal cortical phospholipidosis was netilmicin greater than tobramycin greater than gentamicin greater than neomycin. This order is the reverse of the previously established nephrotoxicity potentials of these drugs. Conversely, the rank order according to peak urinary excretion of phospholipids was gentamicin greater than neomycin greater than tobramycin greater than netilmicin. The rank order of the total urinary phospholipid excretion during the 4 days of the study was neomycin greater than or equal to gentamicin greater than tobramycin greater than or equal to netilmicin. Urinary phospholipid excretion may prove to be a sensitive indicator of aminoglycoside nephrotoxicity.     

Inhibition of alkaline phosphatase activity and D-glucose uptake in rat renal brush-border membrane vesicles by aminoglycosides

The binding of aminoglycoside antibiotics to, and their effects on, the plasma membrane were studied using isolated rat renal brush-border membrane vesicles. Dibekacin was noted to bind with brush-border membrane vesicles having a single class of many binding sites. 3H-labeled dibekacin binding was inhibited competitively by unlabeled dibekacin, gentamicin or amikacin. The inhibition constants obtained from the Dixon plots followed the order of gentamicin approximately equal to dibekacin greater than amikacin. The alkaline phosphatase activity of brush-border membrane vesicles was inhibited by gentamicin significantly, as was also observed by a histochemical study. Sodium-dependent D-glucose uptake by brush-border membrane vesicles was significantly inhibited by the addition of gentamicin.     

Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore

Quantitative determination of rapid water and solute transport and solute reflection coefficients by light-scattering methods is complicated by dependence of vesicle or cell light scattering on nonvolume factors including solution refractive index, cell motion, and membrane aggregation. To overcome these difficulties, a fluorescence technique has been developed to measure accurately (1) osmotic water permeability (Pf), (2) solute permeability (Ps), and (3) solute reflection coefficient (sigma). The time course of vesicle volume is determined by the self-quenching of entrapped fluorescein sulfonate (FS), the best of a series of dyes screened for self-quenching, brightness, and vesicle loading/trapping. To validate the method, rabbit renal brush border vesicles (BBV) were loaded with 1-10 mM FS for 12 h at 4 degrees C and washed to remove extravesicular FS. FS leakage occurred over greater than 6 h at 4 degrees C and greater than 30 min at 23 degrees C. FS fluorescence vs vesicle volume was calibrated from the time course of fluorescence decrease (excitation 470 nm, emission greater than 515 nm) in response to a series of inward osmotic gradients in a stopped-flow apparatus. At 23 degrees C Pf was 0.005 +/- 0.001 cm/s, independent of osmotic gradient size, and inhibited 67% by 0.5 mM HgCl2. Urea Ps was 2 x 10(-6) cm/s with sigma 0.95-1.00 on the basis of the fluorescence time course analysis and the extravesicular [urea] required to obtain zero initial volume flow (null method) when vesicles were loaded with sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free energy potential for aggregation of mixed phosphatidylcholine/phosphatidylserine lipid vesicles in glucose polymer (dextran) solutions.

The energetics of lipid vesicle-vesicle aggregation in dextran (36,000 mol wt) solutions have been studied with the use of micromechanical experiments. The affinities (free energy reduction per unit area of contact) for vesicle-vesicle aggregation were determined from measurements of the tension induced in an initially flaccid vesicle membrane as it adhered to another vesicle. The experiments involved controlled aggregation of single vesicles by the following procedure: two giant (approximately 20 micron diam) vesicles were selected from a chamber on the microscope stage that contained the vesicle suspension and transferred to a second chamber that contained a dextran (36,000 mol wt) salt solution (120 mM); the vesicles were then maneuvered into position for contact. One vesicle was aspirated with sufficient suction pressure to create a rigid sphere outside the pipette; the other vesicle was allowed to spread over the rigid vesicle surface. The aggregation potential (affinity) was derived from the membrane tension vs. contact area. Vesicles were formed from mixture of egg lecithin (PC) and phosphatidylserine (PS). For vesicles with a PC/PS ratio of 10:1, the affinity showed a linear increase with concentration of dextran; the values were on the order of 10(-1) ergs/cm2 at 10% by weight in grams. Similarly, pure PC vesicle aggregation was characterized by an affinity value of 1.5 X 10(-1) ergs/cm2 in 10% dextran by weight in grams. In 10% by weight in grams solutions of dextran, the free energy potential for vesicle aggregation decreased as the surface charge (PS) was increased; the affinity extrapolated to zero at a PC/PS ratio of 2:1. When adherent vesicle pairs were transferred into a dextran-free buffer, the vesicles did not spontaneously separate. They maintained adhesive contact until forceably separated, after which they would not read here. Thus, it appears that dextran forms a "cross-bridge" between the vesicle surfaces.     

Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.

We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.     

Role of N-linked oligosaccharides in the transport activity of the Na+/H+ antiporter in rat renal brush-border membrane

The role of N-linked oligosaccharide side chains in the biogenesis and function of Na+-coupled transporters in renal luminal brush-border membrane (BBM) is not known. We examined the question of how in vivo inhibition by alkaloid swainsonine of alpha-mannosidase, a key enzyme in processing of glycoproteins in the Golgi apparatus, affects Na+/H+ antiport and Na+/Pi symport as well as activities of other transporters and enzymes in rat renal BBM. Administration of swainsonine to thyroparathyroidectomized rats, control or treated with 3,5,3'-triiodothyronine, markedly decreased the rate of Na+/H+ antiport, but had no effect on the rate of Na+/Pi symport across renal BBM vesicles (BBMV). Moreover, administration of swainsonine did not change activities of Na+ gradient, ([extravesicular Na+] greater than [intravesicular Na+])-dependent transport of D-glucose, L-proline, or the amiloride-insensitive 22Na+ uptake by BBMV; the activities of the BBM enzymes alkaline phosphatase, gamma-glutamyltransferase, or leucine aminopeptidase in BBMV were also not changed. The in vitro enzymatic deglycosylation of BBM by incubating freshly isolated BBMV with bacterial endoglycosidase F also resulted in a decreased rate of Na+/H+ antiport, but not Na+-coupled symports of Pi, L-proline, and D-glucose, or the activities of the BBM enzymes were not significantly affected. Similar incubation with endoglycosidase H was without effect on any of these parameters. Both the modification of BBMV glycoproteins by administration fo swainsonine in vivo as well as the in vitro incubation of BBMV with endoglycosidase F resulted in a decrease of the apparent Vmax of Na+/H+ antiport, but did not change the apparent Km of this antiporter for extravesicular Na+ and did not increase H+ conductance of BBM. Taken together, our findings suggest that intact N-linked oligosaccharide chains of the biantennary complex type in renal BBM glycoproteins are required, directly or indirectly, for the transport function of the Na+/H+ antiporter inserted into BBM of renal proximal tubules.     

Changes in the surface charge properties of isolated cardiac sarcolemmal vesicles measured by light scattering. II. Characteristics of rabbit preparations

Numerous studies suggest that cation-sarcolemmal interactions play an essential role in the excitation/contraction/relaxation cycles of cardiac muscle cells. To help elucidate the molecular mechanisms involved in these processes the cation binding characteristics of isolated rabbit cardiac sarcolemmal vesicles were investigated. Cation-membrane interactions were studied by examining the cation-induced aggregation properties of the vesicles. The results obtained were qualitatively very similar to those previously reported for rat and canine cardiac sarcolemmal vesicle preparations (Leonards, K.S. (1988) Biochim. Biophys. Acta 938, 293-309), indicating that all three species have a shared set of basic membrane characteristics. Specifically the results indicate that cations, such as Ca2+, bind to the sarcolemmal surface, and suggest that two (or more) interacting sites are involved in the process. The selectivity series for the cation-induced aggregation of the sarcolemmal vesicles was: La3+ greater than or equal to Cd2+ much greater than Mn2+ greater than Ca2+ greater than Ba2+ = Sr2+ = Mg2+. Protons (H+) could also induce massive vesicle aggregation at pH 5.60-5.75. However, the results obtained also show that the interactions of cations with the rabbit cardiac sarcolemmal membrane surface are quantitatively distinct from those obtained in either rat or canine sarcolemmal vesicle preparations, thereby confirming the species specific nature of cation-sarcolemmal interactions in cardiac cells.     

Effect of Lipid Structure on the Capacity of Myelin Basic Protein to Alter Vesicle Properties: Potent Effects of Aliphatic Aldehydes in Promoting Basic Protein-Induced Vesicle Aggregation

The capacity of myelin basic protein or of poly-L-lysine to promote leakage of carboxyfluorescein from vesicles or the aggregation of vesicles was studied. The vesicles were composed of phosphatidylcholine as the sole or major lipid component. Addition of 10% sphingomyelin, 10% phosphatidylglycerol, 10% egg or bovine brain phosphatidylethanolamine, or 30% dodecanal had relatively little effect on the extent of carboxyfluorescein release in the presence of either myelin basic protein or poly-L-lysine. In contrast with these results, the extent of vesicle aggregation was very sensitive to lipid composition. Addition of 10% phosphatidylglycerol induced more aggregation than the other phospholipids tested. Admixing 10% of a partially degraded sample of bovine brain phosphatidylethanolamine also led to a large amount of aggregation induced by the myelin basic protein. This latter aggregation appeared more specific for the basic protein, as it occurred to a much smaller extent with poly-L-lysine. In general, the effects of the myelin basic protein on either carboxyfluorescein release or vesicle aggregation were similar to, although somewhat greater than, that of poly-L-lysine. The aggregation of vesicles containing degradation products of phosphatidylethanolamine can be ascribed largely to the presence of aliphatic aldehydes. The effect of aliphatic aldehydes was specific in that the aliphatic alcohol, hexadecanol, or the short-chain aldehydes, acetaldehyde or butyraldehyde, did not promote myelin basic protein-induced vesicle aggregation. In addition, poly-L-lysine was less effective than the basic protein in aggregating vesicles containing aliphatic aldehydes. (ABSTRACT TRUNCATED AT 250 WORDS)     

Stopped-flow studies of myelin basic protein association with phospholipid vesicles and subsequent vesicle aggregation

When mixed with vesicles containing acidic phospholipids, myelin basic protein causes vesicle aggregation. The kinetics of this vesicle cross-linking by myelin basic protein was investigated by using stopped-flow light scattering. The process was highly cooperative, requiring about 20 protein molecules per vesicle to produce a measurable aggregation rate and about 35 protein molecules per vesicle to produce the maximum rate. The maximum aggregation rate constant approached the theoretical vesicle-vesicle collisional rate constant. Vesicle aggregation was second order in vesicle concentration and was much slower than protein-vesicle interaction. The highest myelin basic protein concentration used here did not inhibit vesicle aggregation, indicating that vesicle cross-linking occurred through protein-protein interactions. In contrast, poly(L-lysine)-induced vesicle aggregation was easily inhibited by increasing peptide concentrations, indicating that it did cross-link vesicles as a peptide monomer. The myelin basic protein:vesicle stoichiometry required for aggregation and the low affinity for protein dimerization suggested that multiple protein cross-links were needed to form a stable aggregate. Stopped-flow fluorescence was used to estimate the kinetics of myelin basic protein-vesicle binding. The half-times obtained suggested a rate constant that approached the theoretical protein-vesicle collisional rate constant.     

Thiol redox and phosphate transport in renal brush-border membrane. Effect of nicotinamide

In the present study, the effect of thiol redox and its possible role in the inhibitory effect of nicotinamide on renal brush-border membrane (BBM) phosphate uptake was examined. Addition of thiol reducing agent, dithiothreitol (DTT, 5 mM), caused an increase, while addition of thiol oxidant, diamide (DM, 5 mM) caused a reversible decrease in sodium-dependent BBM phosphate uptake. Kinetic analyses revealed an increase in both Vmax and Km by DTT, and a decrease in Vmax by DM. These results suggest that thiol redox influences BBM phosphate uptake with sulfhydryl (SH) groups relate to its capacity and disulfide (SS) groups to its affinity for phosphate. Since changes in cytosolic NAD levels may affect BBM thiol redox through changes in redox states of NADP and glutathione systems, we have examined such possibility by studying the effect of nicotinamide (NM). Incubation of proximal tubules with NM (10 mM) induced an oxidative effect on redox states of cytosolic NAD, NADP systems as inferred from decreased cellular lactate/pyruvate, malate/pyruvate, respectively. Measurements of cytosolic glutathiones and BBM thiols also revealed that NM pretreatment shifted the cytosolic glutathione redox (GSH/GSSG) and BBM thiol redox (SH/SS) toward more oxidized state. On the other hand, incubation of proximal tubules with NM suppressed phosphate uptake by the subsequently isolated BBM vesicles. The lower phosphate uptake by NM-pretreated BBM vesicles was reversed by DTT and was resistant to the inhibitory effect of DM. These results thus suggest that BBM thiol oxidation may be involved in the inhibitory effect of NM on BBM phosphate uptake.     

Osmoelastic coupling in biological structures: decrease in membrane fluidity and osmophobic association of phospholipid vesicles in response to osmotic stress

Poly(ethylene glycol)- (PEG-) induced change in membrane fluidity and aggregation of phospholipid vesicles were studied. A threshold concentration of PEG was required to induce the aggregation. This concentration increased with a decrease in the molecular weight of PEG, e.g., from 5% (w/w) with PEG 6000 (PEG with an average molecular weight of 7500) to more than 30% (w/w) with PEG 200. The aggregation was reversible upon dilution of PEG if the initial PEG concentration was smaller than a certain value, e.g., 22% (w/w) for PEG 6000. Addition of PEG caused a decrease in membrane fluidity of the vesicles detected by fluorescence anisotropy of diphenylhexatriene and by electron spin resonance of a spin-labeled fatty acid. The anisotropy change of diphenylhexatriene fluidity change had an inflection point at approximately 5% (w/w) of PEG 6000, which might suggest that the aggregation would make the decrease of membrane fluidity smaller. Transfer of lipid molecules between phospholipid vesicles was enhanced by the PEG-induced aggregation. The enhancement occurred not only upon direct addition of PEG to the suspending medium, but also upon dialysis of the vesicle suspension against a high concentration of PEG. All these features are consistent with osmoelastic coupling in the phospholipid membranes and the subsequent osmophobic association of the vesicles. The imbalance of osmolarity between the region adjacent to the vesicle surface (exclusion layer) and the bulk aqueous phase, which results from the preferential exclusion of PEG from the exclusion layer in the case of direct addition of PEG, exerts an osmotic stress on the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. I. Effect of vesicle size on the kinetics of aggregation between a fatty acid conjugate of lectin and a liposomal asialoganglioside

Two types of phospholipid vesicles capable of mutual recognition have been tailor-made to serve as a model system for the study of carbohydrate-mediated cellular adhesion. One of the vesicles contained a fatty acid conjugate of a galactose specific lectin (lectin vesicle) and the other an asialoganglioside with a reactive terminal galactose residue (galactose vesicle). The kinetics of aggregation of these two types of vesicles was followed by monitoring time-dependent change in turbidity. A 10-100-fold enhancement in the forward rate constant (kf ranging from 7.1 x 10(5) to 4.5 x 10(7) M-1.s-1 at 27 degrees C) was observed when compared with that for the lectin-galactose system in solution (kf being 4.5 x 10(5) M-1.s-1), reported in the literature. A study of the influence of vesicle size on the rate of aggregation showed that enhancement depended on the curvature of the galactose vesicle rather than the density of asialoganglioside suggesting a possible diffusion in the plane of the membrane. The ratio, kf/kd is found to be approx. 10(10) M-1 indicating that the formation of multiple bonds plays a role for stable adhesion.     

Inhibition of superoxide-dependent processes by aminoglycoside antibiotics

The ability of various antibiotics to inhibit superoxide anion(O-2)-mediated formation of adrenochrome from adrenaline and recovery of cytochrome c by xanthine oxidase was studied. In the adrenaline system (pH 10.2), aminoglycosides might be arranged, according to the inhibitory effect, in the following order: monomycin greater than gentamicin greater than kanamycin greater than lincomycin greater than streptomycin. In the xanthine oxidase system (pH 7.8), that order was the following: monomycin greater than gentamicin greater than lincomycin greater than greater than kanamycin. It was suggested that the antibiotic inhibition of the O-2-dependent processes at the essential sites of metabolism and/or the antibiotic involvement into the process of free radical oxidation initiated by O-2 in the cells might be one of the mechanisms of the drug action and toxicity with respect to the host.     

Effect of ischemia-reperfusion on the renal brush-border membrane sodium-dependent phosphate cotransporter NaPi-2

To understand the mechanisms underlying ischemia-reperfusion-induced renal proximal tubule damage, we analyzed the expression of the Na+-dependent phosphate (Na+/Pi) cotransporter NaPi-2 in brush border membranes (BBM) isolated from rats which had been subjected to 30 min renal ischemia and 60 min reperfusion. Na+/Pi cotransport activities of the BBM vesicles were also determined. Ischemia caused a significant decrease (about 40%, P < 0.05) in all forms of NaPi-2 in the BBM, despite a significant increase (31+/-3%, P < 0.05) in the Na+/Pi cotransport activity. After reperfusion, both NaPi-2 expression and Na+/Pi cotransport activity returned to control levels. In contrast with Na+/Pi cotransport, ischemia significantly decreased Na+-dependent glucose cotransport but did not affect Na+-dependent proline cotransport. Reperfusion caused further decreases in both Na+/glucose (by 60%) and Na+/proline (by 33%) cotransport. Levels of NaPi-2 were more reduced in the BBM than in cortex homogenates, suggesting a relocalization of NaPi-2 as a result of ischemia. After reperfusion, NaPi-2 levels returned to control values in both BBM and homogenates. These data indicate that the NaPi-2 protein and BBM Na+/Pi cotransport activity respond uniquely to reversible renal ischemia and reperfusion, and thus may play an important role in maintaining and restoring the structure and function of the proximal tubule.     

ATP-driven copper transport across the intestinal brush border membrane

The divalent metal ion transporter DMT1 is localized in the brush border membrane (BBM) of the upper small intestine and has been shown to be able to transport Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. Belgrade rats have a glycine-to-arginine (G185R) mutation in DMT1, which affects its function. We investigated copper transport with BBM vesicles of Belgrade rats loaded with calcein, which exhibits fluorescence quenching by various metal ions. Transport of copper was disrupted in unenergized BBM vesicle of b/b Belgrade rats, as had been described for iron transport, while +/b vesicles exhibited normal transport by DMT1. When either b/b or +/b vesicles were loaded with ATP and magnesium, similar high-affinity accumulation of copper was observed in both types of vesicles. Thus, brush border membranes possess an ATP-driven, high-affinity copper transport system which could serve as the primary route for copper uptake by the intestine.     

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements.

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of antiserum to a 99 kDa polypeptide on the uptake of taurocholic acid by rat ileal brush border membrane vesicles

A 99 kDa polypeptide in rat ileal brush border membrane (BBM), regarded as a component of the active bile acid transport system on account of photoaffinity labeling, has been purified by affinity chromatography and preparative gel electrophoresis and utilized as an immunogen for raising polyclonal antibody. Immune serum, but not preimmune serum, specifically recognized a single band of 99 kDa protein on immunoblots of ileal and renal BBM. In contrast, no reactivity was observed with proteins in jejunal BBM. This polyclonal antibody, compared with preimmune serum and anticytosolic bile acid binding protein (14 kDa) serum, significantly inhibited the Na+ dependent uptake of [3H] taurocholate by BBM vesicles (p less than 0.01). [14C] D-glucose uptake by BBM vesicles was not influenced by the immune serum (p less than 0.01). Thus, these studies provide further support for the specific role of a 99 kDa protein in ileal BBM bile acid transport.     

Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane.

Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid (p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the Vmax but not the apparent Km of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the Vmax, but not the apparent Km, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.     

Renal handling and acute urinary electrolyte effects of aminoglycoside antibiotics: Use of a solitary renal autotransplant in the conscious sheep

Renal handling of the aminoglycoside antibiotics gentamicin and tobramycin were studied before and after one hour of constant intravenous infusions adjusted to maintain a concentration of 15 μg/mL. A solitary renal autotransplant model in four conscious volume replete 40 Kg sheep was used. This unique surgical preparation allows sampling of renal arterial and renal venous blood as well as urine drained through an exteriorized parotid-ureteral fistula. This surgical preparation has considerable potential in renal pharmacology since it uses a conscious, large animal. Baseline studies in this preparation demonstrated normal, ⁵¹CrEDTA and ¹²⁵I PAH, clearances which were unaffected by the drugs. Aminoglycoside binding to pooled sheep sera was 11% at physiologic PH. calcium and magnesium concentrations. A–V difference was 1.3 ± .3 μg/mL and extraction by the kidney was 9 ± 3.2% with no differences between gentamicin and tobramycin. Clearance of gentamicin was 84% and tobramycin 86% of GFR. There was no evidence of tubular injury as evidenced by unchanged urinary beta-2 microglobulin excretion. Serum Na, K, Ca and Mg did not change over the course of the study. Both drugs caused a prompt decrease in absolute and fractional sodium excretion while only gentamicin produced a kaliuresis. Early aminoglycoside effects on electrolyte balance may be an eventual determinant of nephrotoxic potential rather than differences in renal drug handling.Nephrotoxicity is a major side effect of aminoglycoside antibiotic therapy. Although gentamicin and tobramycin have similar pharmacokinetics, including renal cortical accumulation, both double blind clinical studies (1) and experimental data (2) have shown that gentamicin is more frequently associated with renal dysfunction. Recent studies in the dog have suggested that hypokalemia due to renal potassium wasting is a risk factor predisposing to nephrotoxicity (3). In clinical usage aminoglycosides may induce hypokalemia and hypocalcemia, perhaps resulting from drug-induced magnesium depletion (4). Previous studies reporting data concerning the acute effects of aminoglycosides on renal function and electrolyte excretion have used anesthetized animals (5) or isolated perfused kidney preparations (6). The present experiments utilize a unique surgical preparation in which a solitary kidney is autotransplanted to the neck of a sheep followed by a contral ateral nephrectomy. Urine flow is exteriorized through a uretero-parotid-cutaneous fistula thus providing a conscious animal with ready access to renal arterial and renal venous blood. Our results show that renal handling of gentamicin and tobramycin do not differ during short-term constant infusions. Both drugs caused a decrease in sodium excretion while gentamicin caused a larger increase in fractional and absolute potassium excretion. This raises the possibility that nephrotoxic properties of aminoglycosides may be secondary to their effects on electrolytes.     

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.