Isolated Rat Renal Brush Border Research Articles

Effect of PEGylation of N-WASP181-200 on the Inhibitory Potency for Renal Aminoglycoside Accumulation

We previously showed that a 20-residue basic peptide, N-WASP181-200 (NISHTKEKKKGKAKKKRLTK), inhibits renal accumulation of aminoglycoside antibiotics such as gentamicin and arbekacin. The aim of this study is to determine whether PEGylation of N-WASP181-200 enhances its inhibitory potency for renal accumulation of aminoglycosides. N-terminally PEGylated peptide (PEG1k-N-W) was synthesized by conjugating N-WASP181-200 with PEG of approximately 1 kDa using the Fmoc protection/deprotection method. PEG1k-N-W decreased gentamicin binding to isolated rat renal brush-border membrane in a concentration-dependent manner, but the in vitro inhibitory potency of PEG1k-N-W was weaker than that of N-WAP181-200. On the other hand, under in vivo conditions, PEG1k-N-W decreased the renal accumulation of arbekacin more potently than N-WASP181-200. When injected intravenously, PEG1k-N-W showed a 1.7-fold longer plasma half-life relative to N-WASP181-200. In addition, the stability of N-WASP181-200 in renal brush-borer membrane suspension was found to be increased by PEGylation. Our findings suggest that PEGylation of N-WASP181-200 is a useful strategy for reducing dosage of the concomitant with which to decrease renal accumulation in the kidney, leading to prevention of aminoglycoside-induced nephrotoxicity.

Inhibition of gentamicin binding to rat renal brush-border membrane by megalin ligands and basic peptides

Our previous studies showed that coadministration of cytochrome c and a 20-residue basic peptide, N-WASP181–200 (NISHTKEKKKGKAKKKRLTK, p I = 10.87) inhibits renal accumulation of gentamicin. In this study, we examined effects of ligands of megalin, an endocytic receptor involved in renal uptake of gentamicin, and basic peptides including N-WASP180–200 and its mutant peptides on gentamicin binding to isolated rat renal brush-border membrane (BBM). Gentamicin binding to BBM was inhibited by megalin ligands, basic peptide fragments of cytochrome c, and N-WASP181–200 in a concentration-dependent manner. Klotz plot analysis showed that N-WASP181–200 inhibited the binding of gentamicin in a competitive manner. By substituting glycines for lysines in N-WASP181–200 at positions 9 and 15, the inhibitory effect on gentamicin binding to BBM was reduced, which may be related to a decrease in the α-helix content in the peptide. Gentamicin binding to BBM treated with trypsin, in which megalin completely disappeared, was significantly but not completely decreased compared with the native BBM. In addition, treatment of BBM with trypsin led to a decrease in the inhibitory effect of N-WASP181–200 on gentamicin binding. These observations support that megalin ligands and basic peptides including N-WASP181–200 decrease renal accumulation of gentamicin by inhibiting its binding to BBM of proximal tubule cells, partly interacting with megalin. In addition, the α-helix conformation may play an important role in the inhibitory effect of N-WASP181-200 on the binding of gentamicin to BBM.

Effect of platinum coordination complex (PtCx) on citrate uptake by rat renal brush border membrane vesicles (BBMV): direct effect of cisplatin.

Platinosis (severe dermatitis and/or asthma) may be broadly defined as the effects of soluble platinum salts on people exposed to them occupationally. Platinum coordination complexes are widely used in the treatment of a variety of solid tumors. However, the clinical use of cisplatin (the most useful agent) is limited by the development of nephrotoxicity. High dose exposure of soluble platinum could possibly induce nephrotoxicity, occupationally. Urinary citrate is freely filtered at the glomerulus, and its reabsorption in the proximal tubule is the major determinant of rate. In this study, we exposed isolated rat renal brush border membrane vesicles (BBMV) to cisplatin and examined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. The preincubation of BBMV with 5 mM of cisplatin for 4 and 8 hours significantly inhibited citrate uptake compared with that of the control BBMV. These findings might contribute to nephrotoxicity in cisplatin therapy.

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

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.

Direct effect of inorganic mercury on citrate uptake by isolated rat renal brush border membrane vesicles.

Occupational exposure to mercury has long been associated with renal proximal injury and an increased incidence of proteinuria, as has such exposure to cadmium. Renal citrate excretion is very important with respect to acid-base balance since the metabolism of citrate generates three bicarbonate ions. In this study, we exposed isolated rat renal brush border membrane vesicles (BBMV) to mercury (Hg2+) and examined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration method. The preincubation of BBMV with 0.5 and 2 mM HgCl2 for 1 min significantly inhibited citrate uptake compared with that of BBMV without Hg preincubation. The analysis of the time course of citrate uptake during a 30-min preincubation of BBMV with 0.1 mM Hg2+ also revealed a significant reduction in the uptake compared with that of the control BBMV without preincubation. These findings indicate that the preincubation of BBMV with mercury results in a time- and concentration-dependent inhibition of citrate uptake.

Citrate uptake by isolated rat renal brush border membrane vesicles in cadmium-intoxicated rats.

Epidemiological Studies have reported that the formation of renal stones is often observed in workers exposed to cadmium (Cd). Citrate is known to be a protective factor against renal stone formation. We previously reported that direct exposure to high-level cadmium impaired the citrate uptake by the brush border membrane vesicles (BBMV), which may mean that the formation of renal stones from exposure to Cd could be prevented. In the present study, to clarify the mechanism of Cd-induced renal stone formation, the characteristics of BBMV-induced citrate uptake were determined in Cd-intoxicated rats. Cd intoxication was induced by daily subcutaneous injections of CdCl2 at a dose of 2 mg Cd/kg body wt per day for 17 to 20 days. The BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. The citrate uptake per mg protein was not significantly different between the two groups. The volume of BBMV was significantly reduced (50%) in Cd-intoxicated rats compared with that of control rats. Percentages of the equilibrium value were also significantly inhibited in Cd-intoxicated rats.

Effect of ethanol on folate binding by isolated rat renal brush border membranes

Deficiency of folic acid, an essential vitamin involved in critical metabolic pathways, occurs in several conditions, including alcoholism. In humans and animal models, chronic ethanol consumption leads to decreased plasma levels and increased urinary levels of folate. An isolated perfused rat kidney model has shown that ethanol produces increased urinary excretion of folate, suggesting a direct effect of ethanol on the kidney. Because the folate binding protein, located in the brush border membrane (BBM) of proximal tubule cells, is thought to be involved in renal folate reabsorption, the effects of ethanol on BBM binding of folate were assessed. Binding studies were conducted using isolated rat kidney cortex BBM preparations, incubated with 3H-labeled 5-methyltetrahydrofolate (5-CH 3H 4PteGlu) at varying concentrations (0.1–100 nM). Ethanol at 500 mg/dl did not significantly affect [ 3H]5-CH 3H 4PteGlu binding in BBM. The structural analogue, folic acid, decreased [ 3H]5-CH 3H 4PteGlu binding under similar conditions. Because of the lack of effect of ethanol on binding to isolated BBM, the effects of ethanol probably occur at other steps in the renal uptake and metabolism of folate.

Direct effect of cadmium on citrate uptake by isolated rat renal brush border membrane vesicles

High incidence of multiple kidney stone formation has been observed among workers exposed to cadmium (Cd). Citrate is known to be a protective factor against renal stone formation. To study the direct effect of cadmium on citrate uptake by the renal brush border membrane, we exposed isolated rat renal brush border membrane vesicles (BBMV) to cadmium and determined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. Preincubation of BBMV with 2 and 10 mM CdCl 2 for 1 min significantly inhibited citrate uptake compared with that of BBMV without Cd. Analysis of the time course of citrate uptake during 30-min preincubation of BBMV with 0.5 mM Cd also revealed significant reduction of the uptake compared with that of the control BBMV without preincubation. These findings indicate that preincubation of BBMV with cadmium results in time-dependent and concentration-dependent inhibition of citrate uptake.

Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles

The shared transport system for uptake of l-cystine and l-lysine was examined in isolated rat renal brush-border membrane vesicles for the ionic requirements for activation of the system. No requirement for sodium was seen for either cystine or lysine influx. However, the efflux of lysine from the vesicle was stimulated by Na +. Therefore, the transport system appears to be asymmetric in its requirement for sodium. Two different divalent cations were used in the membrane isolations which resulted in different responses of cystine uptake to the electrogenic movement of K + out of the vesicle. Membranes prepared by Mg-aggregation showed no stimulation of cystine influx by the imposition of a transient interior negative potential while vesicles prepared by Ca-aggregation did respond to electrogenic stimulation by an outwardly directed K-diffusion potential in the presence of valinomycin. Lysine influx was stimulated by electrogenic potassium efflux in both Mg-prepared and Ca-prepared membranes. No difference in sodium requirement for cystine influx was seen between the vesicles isolated by different cation-aggregation methods.

Effects of cysteine derivatives of styrene on the transport of p-aminohippurate ion in renal plasma membrane vesicles.

The effects of cysteine conjugates of styrene, e.g. S-1/2-(phenyl-hydroxyethyl) cysteine (PEC) and its N-acetyl derivative (NAPEC) on the transport of p-amino-hippurate (PAH) ion in plasma membranes were studied in vitro using isolated rat renal brush-border membrane (BBM) and basolateral membrane (BLM) vesicles. The uptake of PAH was significantly inhibited by both PEC and NAPEC in both the membrane vesicles, as verified by decrease of the membrane/medium concentration ratio of PAH as the concentration of either PEC or NAPEC in the medium increased. These results show that both PEC and NAPEC are capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. The inhibition of PAH uptake in BBM vesicles due to 10 mM PEC or NAPEC was found to be nearly competitive, almost similar to probenecid, whereas in BLM vesicles such inhibition was found to be partially noncompetitive, as verified by the double reciprocal plots. Both PEC and NAPEC showed dose-dependent inhibition of the specific activity of the marker enzyme in each membrane, e.g. gamma-glutamyl transferase in BBM and Na(+)-K(+)-ATPase in BLM vesicles. However, no such inhibition was noticed with probenecid. The in vitro pretreatment with probenecid prevented the inhibition of gamma-glutamyl transferase activity in BBM due to PEC or NAPEC, but such was not the case for the Na(+)-K(+)-ATPase activity in BLM. In conclusion, the data suggest that the transport of cysteine or N-acetylcysteine conjugates of styrene by renal proximal tubular cells across both the membrane vesicles accompanied by the inhibition of the membrane-specific enzymes may lead to cellular dysfunction and consequently to the initial development of their nephrotoxicity.

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. 1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the nitration method. 3. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant K d of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.

Binding and degradation of 125I-insulin by isolated rat renal brush border membranes: evidence for low affinity, high capacity insulin recognition sites.

The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane. 125I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time- and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of 125I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane associated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of 125I-insulin by BBV, but these processes were not appreciably affected by the insulin-like growth factors IGF-I and IGF-II or by cytochrome c and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of 125I-insulin with BBV was studied at various medium osmolarities (300-1100 mosM) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of 125I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink 125I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an 125I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of 125I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10(-5) M. These results indicate the presence of low-affinity, high-capacity binding sites for 125I-insulin on renal brush border membranes which can clearly distinguish insulin from the insulin-like growth factors and other low molecular weight proteins and polypeptides, but which do not differentiate insulin from its analogues as do the biological receptors for the hormone. The properties and location of these binding sites make them attractive candidates for the sites at which insulin is reabsorbed in the renal tubule.

Interactions of cyclosporine with renal proximal tubule cells and cellular membranes.

Cyclosporine-induced nephrotoxicity is a limiting factor in the clinical use of cyclosporine. Since the manner in which cyclosporine interacts with proximal tubule cells and their membranes may provide insight into the cellular pathophysiology of cyclosporine toxicity, experiments were undertaken to characterize the interactions of cyclosporine with proximal tubule cells, renal brush border membranes, and renal cortical mitochondria. Cyclosporine bound to isolated rat renal brush border membranes in a saturable manner with a Kd of 0.38 microM and an nmax of 0.33 nmoles/mg protein. Scatchard analysis suggested that the interaction of cyclosporine at low concentrations with brush border membranes was consistent with a partitioning process rather than binding to a specific membrane component. Cyclosporine inhibited rat renal cortical mitochondrial respiration in a dose-dependent manner, with 8 microM as a threshold dose. This inhibitory effect was greater for respiration supported by succinate than pyruvate-malate. TMPD-ascorbate-supported respiration was unaffected. Suspensions of rabbit renal proximal tubule segments were incubated in vitro with 0.5-500 microM 3H-cyclosporine to measure the kinetics of cyclosporine uptake. Uptake was rapid (80% after 10 min) and saturable at 100 microM, with 9 nmoles cyclosporine/mg protein accumulated. Incubation of suspensions of enriched in rabbit renal proximal tubule segments with 10 microM cyclosporine in vitro for 2 hr with or without 22.5 min of hypoxia, or for 16 hr without hypoxia, had no effect on a variety of quantitative metabolic parameters of cell injury, including basal and uncoupled tubule respiratory rates and tubule K+, Ca++ and adenine nucleotide levels. These results demonstrate that cyclosporine interacts with critical renal membrane components at low concentrations but this interaction does not result in proximal renal tubular cell injury acutely in vitro.

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 ≅ dibekacin > 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.

Inhibition of Na+-H+ exchange by N,N'-dicyclohexylcarbodiimide in isolated rat renal brush border membrane vesicles.

The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.

Lysine uptake by rat renal brush-border membrane vesicles.

Lysine uptake by isolated rat renal brush-border membrane vesicles occurs via a single saturable system plus a significant diffusion component with indication of a single energy of activation on an Arrhenius plot. Initial uptake is not sodium dependent, and intravesicular accumulation of lysine at longer time points is greatest in the absence of sodium. Accumulation levels differ in the presence or absence of NaCl or choline chloride and are specific for the cation used. Lysine uptake is membrane potential sensitive and inhibitable by cystine, dibasic amino acids, and cycloleucine. Heteroexchange diffusion of lysine with cystine and lysine with arginine occurs, but no heteroexchange occurs with cycloleucine, indicating that lysine shares a transport system with cystine and dibasic amino acids but not with cycloleucine.

Renal reabsorption and utilization of hydroxybutyrate and acetoacetate in starved rats.

Are there increases in the renal reabsorption (R) and utilization (Q) of ketoacids during starvation? R and Q of D-3-hydroxybutyrate and acetoacetate were measured in anesthetized normally fed (C) and 3-day starved (S) rats, using clearance and arteriovenous extraction methods. R was nearly complete in both groups at low filtered ketoacids loads (less than 2mumol/g X min). At higher filtered loads, R of the two ketoacids increased in proportion to their filtered loads (r2 greater than 0.95) but with significantly steeper slopes in the S than in the C group; fractional R of the two ketoacids were significantly higher in the S than in the C animals. Saturable and nonsaturable components for ketoacid reabsorption were evident, but only the nonsaturable component was significantly increased with starvation. Increased R of ketoacids during starvation was in the absence of changes in the initial rates of hydroxybutyrate influx into isolated rat renal brush-border membrane vesicles. Thus the rate-limiting step for ketoacid reabsorption through the nonsaturable pathway may not be located at the luminal membrane of the proximal cells. The increases in R of ketoacids in the S group were associated with simultaneous decreases in the net rates of renal utilization of the two ketoacids. Thus increased renal conservation of ketoacids is a transport and not a metabolic event. Overall, during complete starvation, both the increased R and the decreased renal Q of the ketoacids contribute to increase the availability of these metabolites to other organs.

In vitro phosphorylation of rat kidney proximal tubular brush border membranes.

The phosphorylation of rat renal brush border membrane protein was analyzed after incubation of cortical slices with 32P-orthophosphate and compared with the phosphorylation by gamma-32P-ATP of isolated brush border vesicles. Phosphate incorporation into brush border membranes isolated from slices was linearly related to the incubation time as well as to the specific activity of orthophosphate present during slice incubation. Incorporation of phosphate into proteins reached an equilibrium after about 60 min, whereas incorporation of phosphate into lipids increased continuously. In brush border membranes isolated from slices incubated with orthophosphate (32P), the addition of cAMP or theophylline produced a dephosphorylation of a 47,000-dalton protein; no increased phosphorylation was observed. In brush border membranes, phosphorylated with gamma-32P-ATP, cAMP and dibutyryl cAMP (dB-cAMP) produced an increase in phosphorylation but no dephosphorylation. Sodium-dependent phosphate transport in brush border membranes was not altered by an incubation of slices with cAMP or dB-cAMP. These observations suggest that the phosphorylation machinery of isolated rat renal brush border membranes does not correspond with the mechanisms leading to phosphate incorporation into brush border membrane proteins in the intact cell.

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.