Riboflavin transport by isolated perfused rabbit renal proximal tubules

Rabbit renal proximal tubular transport of riboflavin(RF) was examined by using the in vitro isolated tubule perfusiontechnique. We found that proximal tubules actively reabsorbed(J_lb) and secreted (J_bl)RF. At 0.1 µM RF concentration, J_bl wassignificantly higher than J_lb, resulting in anet secretion. This net secretion of RF was decreased at 0.01 µM RFconcentration and increased at 1 µM RF concentration. BothJ_lb and J_bl wereinhibited by lowering temperature or by adding iodoacetate, a metabolicinhibitor, and lumichrome, an RF analog, suggesting the involvement ofcarrier-mediated transport mechanisms. J_bl wasinhibited by probenecid, an anion transport inhibitor, and bypara-aminohippuric acid, an organic anion, suggesting therelevance of RF secretion to renal organic anion transport.J_bl was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca²⁺/calmodulin-dependent pathway on RFsecretion. Finally, we found that addition of chlorpromazine, aphenothiazine derivative, inhibited both J_lb andJ_bl, raising the concern about the nutritionalstatus in patients receiving such a type of medication.

     

Mechanism of organic anion transport across the apical membrane of choroid plexus

The mechanism and membrane localization of choroid plexus (CP) organic anion transport were determined in apical (or brush border) membrane vesicles isolated from bovine choroid plexus and in intact CP tissue from cow and rat. Brush border membrane vesicles were enriched in Na(+),K(+)-ATPase (20-fold; an apical marker in CP) and demonstrated specific, sodium-coupled transport of proline, glucose, and glutarate. Vesicular uptake of the anionic herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) was markedly stimulated by an inward sodium gradient but only in the presence of glutarate, indicating the presence of apical dicarboxylate/organic anion exchange. Consistent with this interpretation, an imposed outward glutarate gradient stimulated 2,4-D uptake in the absence of sodium. Under both conditions, uptake was dramatically slowed and overshoot was abolished by probenecid. Likewise, apical accumulation of 2,4-D by intact bovine choroid plexus tissue in vitro was stimulated by external glutarate in the presence of sodium. Glutarate stimulation was abolished by 5 mM LiCl. Identical findings were obtained using rat CP tissue, which showed both sodium/glutarate-stimulated 2,4-D (tissue/medium (T/M) approximately 8) and p-aminohippurate (T/M = 2) transport. Finally, since the renal exchanger (rROAT1) has been cloned in rat kidney, a rROAT1-green fluorescent protein construct was used to analyze exchanger distribution directly in transiently transfected rat CP. As predicted by the functional studies, the fluorescently tagged transporter was seen in apical but not basolateral membranes of the CP.     

Proteomic profiling of the mitochondrial inner membrane of rat renal proximal convoluted tubules

The proximal convoluted tubule is the primary site of renal fluid, electrolyte, and nutrient reabsorption, processes that consume large amounts of adenosine‐5′‐triphosphate. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to the onset of metabolic acidosis. To extend this analysis, a proteomic workflow was developed to characterize the proteome of the mitochondrial inner membrane of the rat renal proximal convoluted tubule. Separation by LC coupled with analysis by MS/MS (LC‐MS/MS) confidently identified 206 proteins in the combined samples. Further proteomic analysis identified 14 peptides that contain an N‐?‐acetyl‐lysine, seven of which are novel sites. This study provides the first proteomic profile of the mitochondrial inner membrane proteome of this segment of the rat renal nephron. The MS data have been deposited in the ProteomeXchange with the identifier PXD000121.     

Current-voltage relations of sodium-coupled sugar transport across the apical membrane ofNecturus small intestine

Summary The current-voltage (I–V) relations of the rheogenic Na-sugar cotransport mechanism at the apical membrane ofNecturus small intestine were determined from the relations between the electrical potential difference across the apical membrane, ^mc , and that across the entire epithelium, ^ms , when the latter was varied over the range ±200 mV, (i) under steady conditions in the presence of galactose and (ii) after the current across the apical membrane carried by the cotransporter,I _SNa ^m , is blocked by the addition of phloridzin to the mucosal solution.I _SNa ^m was found to be strongly dependent upon ^mc over the range –50 mV < ^mc <E _SNa ^m whereE _SNa ^m is the zero current or reversal potential. Over the range of values of ^mc encountered under physiological conditions the cotransporter may be modeled as a conductance in series with an electromotive force so thatI _SNa ^m =g _SNa ^m (E _SNa ^m – ^mc ) whereg _SNa ^m is the contribution of this mechanism to the conductance of the apical membrane and is near constant. In several instancesI _SNa ^m saturated at large hyperpolarizing or depolarizing values of ^mc .The values ofE _SNa ^m determined in the presence of 1, 5, and 15mm galactose strongly suggest that if the Na-galactose cotransporters are kinetically homogeneous, the stoichiometry of this coupled process is unity.Finally, the shapes of the observedI–V relations are consistent with the predictions of a simple kinetic model which conforms with current notions regarding the mechanico-kinetic properties of this cotransport process.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphate transport across the basolateral membrane of chick kidney proximal tubule cells

Characterization of the phosphate transport system across the basolateral membrane of renal proximal tubule has been attempted using isolated proximal tubule cells prepared from chicks. The Pi efflux system is independent of Na+ ions and is not influenced by the nature of the chief anion present in the bathing medium. Pi efflux is not sensitive to DIDS and it is concluded that a generalized anion transporter of band III type is not the chief agent for facilitating Pi exit from the cell across the basolateral membrane. Inhibition of efflux by vanadate is evidence for a specific carrier protein in the membrane. The carrier probably possesses thiol group(s) that are essential for activity. The carrier may effect electroneutral transport of Pi possibly in exchange for OH- ions. The activity of the transport process is not stimulated by depleting the cells of phosphate or inhibited by rearing the chicks on a vitamin D-deficient diet. The system is unlikely to be of great importance for the expression of various regulatory mechanisms that act on the kidney to control the excretion of Pi. The activity declines as the chicks mature however.     

pH-stat experiments in proximal renal tubules

Summary The pH-stat technique has been used to measure H⁺ fluxes in gastric mucosa and urinary bladder in vitro while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1n HC1-filled microelectrodes to inject OH^– or H⁺ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H⁺-ion conductance). These ratios were reduced after luminal 10^–4 m acetazolamide, during injection of OH^–, but they increased during injection of H⁺. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H⁺ fluxes per cm² epithelium. The relation betweenJ _H ⁺ and pH gradient showed saturation kinetics of H fluxes, both during OH^– and H⁺ injection. This kinetic behavior is compatible with inhibition ofJ _H by luminal H⁺. It is also compatible with dependence on Na⁺ and H⁺ gradients of a saturable Na/H exchanger. H⁺-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H⁺ flow is mediated by a membrane component, most likely the Na⁺–H⁺ exchanger.     

Phosphate transport after acute changes in total NAD content in renal proximal tubules

Suspensions of proximal tubules were obtained by collagenase digestion of rat renal cortex followed by centrifugation on a percoll gradient. NAD content in tubules incubated at 37 degrees C was decreased by 40-60% compared with tubules incubated at 4 degrees C. This change occurred within 30 min and was maintained for up to 2 hr. Inhibitors of NAD hydrolysing enzymes prevented the depletion of cellular NAD at 37 degrees C. Acute changes in proximal tubule NAD content at 37 degrees C were not accompanied by changes in phosphate uptake by brush border membrane vesicles subsequently prepared from the same tubules. In contrast, incubation of tubules with parathyroid hormone (10(-6) M) produced the expected inhibition (20%) of brush border membrane transport of phosphate. One implication of these findings is that acute changes in total NAD content of proximal tubules at 37 degrees C may not influence the phosphate transport system in the renal brush border membrane. Other interpretations are discussed.     

The role of facilitated diffusion in glucose transport across the apical membrane of enterocytes

In chronic experiments on Wistar rats, glucose and galactose absorption in the isolated loop of the small intestine considerably decreased in presence of both phloridzine am phloritine (inhibitors of the glucose transporters SGLT1 and GLUT2). The load of the isolated loop with glucose or galactose solutions scarcely influenced the absorption of 2-deoxi-D-glucose (substrate for GLUT2). According to the immunocytochemical analysis by means of confocal microscopy, after the load of the isolated loop with glucose (75 mM) the labels to GLUT2 and proteinkinase C (PKC betalI) were concentrated mainly in the apical part of the enterocytes, whereas after the load with the Ringer solution--in the basal part of the enterocytes. It was shown on the mathematical model that the part of the facilitated diffusion in the total glucose absorption was considerably lesser in comparison with the active transport mediated by SGLT1. Thus the findings support the hypothesis about a recruitment of the transporter GLUT2 into the apical membrane of the enterocytes and its involvement in glucose transfer across this membrane. However, under natural conditions, the active transport is the main mechanism of glucose absorption, whereas the facilitated diffusion plays a certain role only at high carbohydrate loads.     

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

Summary We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (V _bl) in rabbit proximal convoluted tubule (PCT. Sl segment). The present study is aimed at understanding the origin of the membrane repolarization following the intial effect of addition of luminal cotransported solutes. Luminal addition of 10–15mMl-alanine produced a rapid and highly significant depolarization ofV _bl (20.3±1.1 mV,n=15) which was transient and associated with an increase in the fractional K⁺ conductance of the basolateral membrane (t _K) from 8 to 29% (P<0.01,n=6). Despite the significant increase int _K, the repolarization was only slightly reduced by the presence of basolateral Ba²⁺ (2mM,n=6) or quinine (0.5 mM,n=5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4isothiocyamostilbene-2,2-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pH _i ) determined with the fluorescent dye 2, 7-bis-2-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF), averaged 7.39±0.02 in control solution (n=9) and increased to 7.50±0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16±0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K⁺ conductance but also through a cellular acidification reducing the basolateral HCO ₃ ^– exit through the electrogenic Na-3(HCO₃) cotransport mechanism.     

Sodium-dependent phosphate transport across the apical membrane of alveolar epithelium in caprine mammary gland

NaPi IIb cotransporter is expressed in various tissues including mammary glands of mice. The physiological role of NaPi IIb in lactating mammary glands is still unclear. Therefore, it was the aim of the study to detect and to localize NaPi IIb protein in lactating goat mammary glands by Western analysis and immunohistochemistry. Furthermore, Na(+)-dependent P(i) uptake into apical membrane vesicles isolated from goat milk was determined using rapid filtration technique. NaPi IIb protein could specifically be detected in the apical membranes of lactating alveolar epithelial cells. Na(+)-dependent P(i) uptake into apical membrane vesicles could be measured, which was inhibited by phosphonoformic acid. The kinetic parameters were V(max) with 0.9 nmol/mg protein/10 s and K(m) with 0.22 mmol/L for P(i) affinity, K(m) value for Na(+) affinity 11 mmol/L. Stoichiometry of this mammary gland Na(+)/P(i) transport across the apical membranes seemed to be 1:1 P(i):Na(+) without cooperativity in P(i) and Na(+) binding as assessed by Scatchard and Hill plots. These features of Na(+)/P(i) transport suggest that it could be mediated by NaPi IIb. The quantitative role of this P(i) transport which is directed from the alveolar lumen into the epithelial cell of goat mammary gland will be the topic of further investigations.     

Physico-chemical mechanisms of organic acid transport in the apical membrane cells of the proximal kidney tubules in rats. I. Kinetic transport parameters and effect of the lipid phasic state on transport

The kinetics of the transport of 3H-para-aminohippuric acid (PAH) and the influence of the temperature on the initial rate of transport were studied on the vesicles of a purified fraction of the apical membrane isolated from cells of kidney proximal tubules. The PAH transport is accomplished owing to the facilitate diffusion mechanism. The apparent Michaelis constant at 36 degrees C was equal to 7.0 + 1.0 mM, the maximum rate was 15 nmol/min on 1 mg of protein, the inhibition constant for the PAH transport by probenecid being 0.5 mM. At 22 degrees C the apparent Michaelis constant was drastically increased. When the temperature dependence of the initial rate of PAH transport into vesicles was replotted in the form of the Arrhenius plot, there was a turning-point of the line at 28-30 degrees C. The same turning-point is shown on the Arrhenius plot for temperature dependence of alkaline phosphatase activity (a marker enzyme for the apical membrane). The electron paramagnetic resonance spectra analysis of 5-doxylstearate-labeled apical membrane preparation reveals a thermotropic transition near 21-29 degrees C. It is concluded that the function of the carrier and the activity of alkaline phosphatase depend on the phasic state of membrane lipids; the normal function of membrane proteins is possible under the liquid-crystalline state of the lipid bilayer.     

Glucose transport and metabolism in rat renal proximal tubules: multicomponent effects of insulin

Glucose transport and metabolism, and the effect of insulin thereon, was studied using suspensions of rat renal tubules enriched in the proximal component. [U-14C]Glucose oxidation is a saturable process (Km 3.1 +/- 0.2 mM; Vmax 14 +/- 0.2 mumole 14CO2 formed/g tissue protein per h). Glucose oxidation and [14C]lactate formation from glucose are inhibited in part by phlorizin and phloretin: the data suggest that the rate-limiting entry of glucose into the cell metabolic pool occurs by both the Na-glucose cotransport system (at the brush border) and the equilibrating, phloretin-sensitive system (at the basal-lateral membrane). Raising external glucose from 5 to 30 mM markedly increases aerobic and anaerobic lactate formation. Gluconeogenesis from lactate is not affected by variations of glucose concentrations. 24 h after streptozotocin administration, aerobic lactate formation is enhanced, as is the uptake of methyl alpha-D-glucoside by the tubules, while anaerobic glycolysis is depressed. Streptozotocin treatment (ST) increases both the Km and Vmax of glucose oxidation; gluconeogenesis and lactate oxidation are not affected. The effect of streptozotocin treatment on lactate formation are abolished by 1 mU/ml insulin. Streptozotocin treatment increases tissue hexokinase activity, decreases glucose-6-phosphatase, but has no significant effect on fructose-1,6-diphosphatase; phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase. The data demonstrate fast streptozotocin-induced changes in cellular enzymes of carbohydrate metabolism. The enhancing effect of streptozotocin on methyl alpha-glucoside uptake is transient: 8 days after administration of the agent, no significant difference from controls is found. It is concluded that under the given experimental conditions insulin enhances the equilibrating glucose entry by the phloretin-sensitive pathway at the basal-lateral membrane, and transiently inhibits the Na-glucose cotransport system.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

Chloride reabsorption by renal proximal tubules of necturus

Summary Movement of Cl from the lumen ofNecturus proximal tubule into the cells is mediated and dependent on the presence of luminal Na. Intracellular Cl activity was monitored with ion selective microelectrodes. In Cl Ringer's perfused kidneys, cell Cl activity was 24.5±1.1mm, 2 to 3 times higher than that predicted for passive distribution. When luminal NaCl was partially replaced by mannitol (capillaries perfused with Cl Ringer's) cell Cl decreased showing a sigmoidal dependence on luminal NaCl. Peritubular membrane potential was unaltered. Sulfate Ringer's perfusion of the kidneys washed out all cell Cl but did not alter peritubular membrane potential. Chloride did not enter the cell when the tubule lumen was perfused with 100mm KCl, LiCl, or tetramethylammonium Cl. Luminal perfusion of NaCl caused cell Cl to rise rapidly to the same value as the controls in the Cl Ringer's experiments. Perfusion of the tubule lumen with mixtures of NaCl and Na₂SO₄, while the capillaries contained sulfate Ringer's yielded a sigmoidal dependence of cell Cl on luminal NaCl activity. Chloride movement from the lumen into the proximal tubule cells required approximately equal concentrations of Na and Cl. Current clamp experiments indicated that intracellular chloride activity was insensitive to alterations in liminal membrane potential, suggesting that chloride entry was electrically neutral. The transcellular chloride flux was calculated to constitute about one half of the normal chloride reabsorption rate. We conclude that the cell Cl activity is primarily determined by the NaCl concentration in the tubule lumen and that Cl entry across the luminal membrane is mediated.     

Urate transport in erythrocytes: possible role of a transport membrane]

Urate transport seems dependant of ATP. So, we studied urate behaviour in relation with the isolated membrane: we observed a linkage between urate and a membrane protein of which molecular weight was estimated to twenty thousand. The exact role of this protein remains to be clarify.     

Electro-osmosis and the reabsorption of fluid in renal proximal tubules

The lateral intercellular spaces (LIS) are believed to be the final common pathway for fluid reabsorption from the renal proximal tubule. We postulate that electrogenic sodium pumps in the lateral membranes produce an electrical potential within the LIS, that the lateral membranes bear a net negative charge, and that fluid moves parallel to these membranes because of Helmholtz-type electro-osmosis, the field- induced movement of fluid adjacent to a charged surface. Our theoretical analysis indicates that the sodium pumps produce a longitudinal electric field of the order of 1 V/cm in the LIS. Our experimental measurements demonstrate that the electrophoretic mobility of rat renal basolateral membrane vesicles is 1 micron/s per V/cm, which is also the electro-osmotic fluid velocity in the LIS produced by a unit electric field. Thus, the fluid velocity in the LIS due to electro-osmosis should be of the order of 1 micron/s, which is sufficient to account for the observed reabsorption of fluid from renal proximal tubules. Several experimentally testable predictions emerge from our model. First, the pressure in the LIS need not increase when fluid is transported. Thus, the LIS of mammalian proximal tubules need not swell during fluid transport, a prediction consistent with the observations of Burg and Grantham (1971, Membranes and Ion Transport, pp. 49-77). Second, the reabsorption of fluid is predicted to cease when the lumen is clamped to a negative voltage. Our analysis predicts that a voltage of -15 mV will cause fluid to be secreted into the Necturus proximal tubule, a prediction consistent with the observations of Spring and Paganelli (1972, J. Gen. Physiol., 60:181).