Pyrazinoate transport in the isolated perfused rabbit proximal tubule

The bidirectional tubular transport of pyrazinoate (PZA) was studied in the isolated perfused proximal S₂ segment of rabbit kidney. PZA reabsorption was a mechanism of large capacity, temperature-dependent and requiring a normal Na⁺/K⁺-ATPase activity. PZA reabsorption was reversibly decreased when lactate was added to the perfusate, indicating that it might occur through the sodium-lactate cotransport. The addition of PAH to the bath had a slight stimulatory effect on PZA reabsorption, suggesting a component of anion exchange in the overall PZA reabsorption. However, SITS added to either the perfusate or the bathing medium induced a non-significant decrease in PZA reabsorption, confirming the minor part of an anion exchange mechanism in this reabsorptive process. PZA reabsorption was not affected by the establishment of a bath-to-lumen H⁺ gradient, and was only moderately decreased after carbonic anhydrase inhibition by ethoxyzolamide, in opposition to what is known for the reabsorbed anion salicylate. The secretory transport of PZA was saturable and also dependent on a normal Na⁺/K⁺-ATPase activity. It is concluded that PZA is bidirectionally transported by facilitated mechanisms in the rabbit proximal S₂ segment, one major reabsorptive mechanism appearing to be a sodium-anion cotransport, which might be the sodium-lactate reabsorbing mechanism.     

Transport ofl-cystine in isolated perfused proximal straight tubules

Unidirectional fluxes ofl-³⁵S-cystine and intracellular³⁵S activity were measured in isolated perfused segments of rabbit proximal straight tubule. The absorptive (lumen-to-both) flux ofl-³⁵S-cysteine showed a tendency toward saturation within the concentration limits imposed by the low solubility of cystine (0.3 mmol·l^–1). In contrast, for the bath-to-lumen fluxes, there was a linear relation between the bathing solution concentration ofl-³⁵S-cystine and the rate of³⁵S appearance in the lumen. Nonlinear fitting of both sets of unidirectional flux data gave a maximal cystine transport rate (J _max) of 1.45±0.27 (SEM) pmol min^–1 mm^–1, a Michaelis constant (K _m) of 0.20±0.07 mmol·l^–1, and an apparent permeability coefficient of 0.27±0.11 pmol min^–1 mm^–1 (mmol·l^–1)^–1 (approximately 0.06 m/s). The³⁵S concentration in the cell exceeded that in the lumen by almost 60-fold during the lumen-to-bath flux, and exceeded the bathing solution concentration by 4.7-fold during the bath-to-lumen flux. Thus cystine was accumulated by the cells across either membrane, but over 77% of the intracellular activity was in the form of cysteine. Although the presence of luminall-lysine or cycloleucine inhibited the absorptive flux of cystine, neither amino acid affected the bath-to-lumen flux.Some of the work described here was presented as an abstract at the 8th International Congress of Nephrology, Athens, Greece, 1981     

Characteristics of phosphate transport in isolated proximal tubule.

The characteristics of inorganic phosphate transport in isolated perfused proximal tubules of the rabbit were examined using radioisotopic techniques. When tubules were perfused with an ultrafiltrate of rabbit serum, the mean lumen-to-bath flux of phosphate in the convoluted segment was 6.60 +/- 1.41 (SE) pmol/mm-min with a simultaneous back-to-lumen flux of 0.45 +/- 0.08. In the straight portion of the proximal tubule, the lumen-to-bath flux was significantly lower (P less than 0.01) at 2.22 +/- 0.48 pmol/min-min with a bath-to-lumen flux of 0.31 +/- 0.05. The lumen-to-bath flux was not affected by increases in the intraluminal phosphate concentration from 2.00 +/- 0.19 to 3.12 +/- 0.34 mM or by the isohydric replacement of bicarbonate in the ambient fluids with chloride. However, phosphate absorption was completely inhibited by ouabain 10(-5) M in the bath. These data indicate that phosphate absorption in these segments occurs by a mechanism other than independent diffusion and is saturated at phosphate concentrations characteristic of normal glomerular filtrate. There is no evidence for significant phosphate transport from bath to lumen.     

Intracellular potassium activity in the rabbit proximal straight tubule

Double-barreled liquid ion-exchanger microelectrodes were used to measure basolateral membrane potential (VBL) and intracellular potassium activity (aiK) in superficial proximal straight tubules (sPST) of the rabbit perfused in vitro. The mean +/- SE (number of cells in parentheses) value of VBL was -37.8 +/- 2.49 (20) vM and aiK was 48.6 +/- 2.27 (20) mM. The calculated Nernst equilibrium potential (EK) across the basolateral membrane was -68 mV. Lowering both potassium concentration to 0.1 mM reversibly decreased both VBL and aiK to -12.2 +/- 1.21 (19) mV and 11.3 +/- 1.29 (19) mM, respectively. Bath ouabain (10(-5) resulted in similar changes. These results demonstrate that intracellular potassium is actively accumulated in sPST perfused in vitro and that accumulation results primarily from Na-K-ATPase activity in the basolateral membrane. During recovery from low K bath, the temporal relationship between VBL and aiK and the effects of ouabain and high K bath on recovery are used to demonstrate directly electrogenic pumping. Lowering bath pH to 6.7 (HCO-3 = 5 mM) and the presence of 0.5 mM BaCl2 in the bath resulted in a large and rapid depolarization of VBL with little or no change in aiK. These results suggest that the response of VBL to both maneuvers is caused by a decrease in potassium permeability of the basolateral membrane.     

Chloride transport in the renal proximal tubule

The renal proximal tubule is responsible for most of the renal sodium, chloride, and bicarbonate reabsorption. Micropuncture studies and electrophysiological techniques have furnished the bulk of our knowledge about the physiology of this tubular segment. As a consequence of the leakiness of this epithelium, paracellular ionic transport--in particular that of Cl(-)--is of considerable importance in this first part of the nephron. It was long accepted that proximal Cl(-) reabsorption proceeds solely paracellularly, but it is now known that transcellular Cl(-) transport also exists. Cl(-) channels and Cl(-)-coupled transporters are involved in transcellular Cl(-) transport. In the apical membrane, Cl(-)/anion (formate, oxalate and bicarbonate) exchangers represent the first step in transcellular Cl(-) reabsorption. A basolateral Cl(-)/HCO(3)(-) exchanger, involved in HCO(3)(-) reclamation, participates in the rise of intracellular Cl(-) activity above its equilibrium value, and thus also contributes to the creation of an outwardly directed electrochemical Cl(-) gradient across the cell membranes. This driving force favours Cl(-) diffusion from the cell to the lumen and to the interstitium. In the basolateral membrane, the main mechanism for transcellular Cl(-) reabsorption is a Cl(-) conductance, but a Na(+)-driven Cl(-)/HCO(3)(-) exchanger may also participate in Cl(-) reabsorption.     

Axial heterogeneity of sodium-bicarbonate cotransport in proximal straight tubule of rabbit kidney

Intracellular microelectrodes were used to investigate rheogenic Na⁺(HCO ₃ ^– ) _n cotransport in different segments of isolated proximal straight tubule (PST) of rabbit kidney. In the first portion (S2 segment) the peritubular cell membrane potentialV _b averaged –46.0, SE±1.3 mV (n=20), while in the terminal portion (S3 segment) it averaged –68.3, SE±2.5 mV (n=10). This difference may reflect different modes of anion permeation across the peritubular cell membrane. In S2 segments, sudden 101 reduction of bath HCO ₃ ^– concentration caused a fast transient cell depolarization, V _b=–45.8, SE±1.2 mV (n=33) as expected from the presence of Na⁺(HCO ₃ ^– ) _n contransport. As the puncture site moved further distally, V _b declined and gradually changed its time course by superposition of a slower secondary depolarization. In this region the transient cell depolarization could be recuperated by inhibiting the peritubular K⁺ conductance with Ba²⁺ (1 mmol/l). In S3 segments, however, the HCO ₃ ^– -dependent transient cell depolarization was completely lost both in the absence and presence of Ba²⁺. In addition, sudden reduction of bath Na⁺ concentration did not acidify the cell, as it did in the S2 segment. The data indicate that the expression of Na⁺(HCO ₃ ^– ) _n cotransport in the peritubular cell membrane gradually diminishes towards the end of the S2 segment and is lost in the S3 segment.     

H+ ion secretion in proximal tubule of low-CO2/HCO 3 − perfused isolated rat kidney

Acidification in proximal tubule of the isolated rat kidney, perfused in vitro, was studied by stopped-flow microperfusion techniques, using Sb microelectrodes to measure luminal pH. The kidney was perfused with mammalian Ringer's solution at pH 7.4 buffered by 20 mmol/l phosphate and containing 7.5 g/100 ml bovine albumin, equilibrated with air. Final urine pH was 6.88±0.5. Steady-state pH in proximal segments was 6.81±0.03 (n=80), and acidification half-time (t/2) 7.25±0.33 (80) s, giving a net secretory H⁺ ion flux of 0.51±0.05 nmol·cm^–2·s^–1. This flux was about 70% of in vivo (blood perfused kidneys). During luminal perfusion with solutions at pH 6.2, back-flux of H⁺ was 0.82 ±0.08 nmol·cm^–2·s^–1, with an alkalinizationt/2 of 6.33 ±0.34 (34) s. The difference between acidification and alkalinizationt/2 was not significant. This is compatible with a pump-leak system of H⁺ transport. The back flux of H from the lumen was markedly reduced in low Na⁺ perfused kidneys in the presence of 10^–4 mol/l amiloride in the lumen, indicating that this process is mediated by the luminal Na/H exchanger. Observations in the presence of high K levels suggest that it may have also a charged component. 10^–4 mol/l acetazolamide added to the kidney perfusate reduced acidification to 0.5% of control, and 10^–6 mol/l SITS to 25% of control. Thus, despite the lowpCO₂ (0.1–0.4 kPa, or 1–3 mm Hg), the CO₂/Hco ₃ ^– buffer system still plays an important role in tubular acidification in this preparation.     

Effects of ouabain and temperature on cell membrane potentials in isolated perfused straight proximal tubules of the mouse kidney

In isolated perfused segments of the mouse proximal tubule, the potential difference across the basolateral cell membrane (PDbl) was determined with conventional microelectrodes. Under control conditions with symmetrical solutions it amounted to –62±1 mV (n=118). The potential difference across the epithelium (PDte) was –1.7±0.1 mV (n=45). Transepithelial resistance amounted to 1.82±0.09 k cm (n=28), corresponding to 11.4±0.6 cm². Increasing bath potassium concentration from 5 to 20 mmol/l depolarized PDbl by +24±1 mV (n=103), and PDte by +1.6±0.1 mV (n=19). Thus, the basolateral cell membrane is preferably conductive to potassium. Rapid cooling of the bath perfusate from 38°C to 10°C led to a transient hyperpolarization of PDbl from –60±1 to –65±1 mV (n=21) within 40 s followed by gradual depolarization by +18±1% (n=14) within 5 min. The transepithelial resistance increased significantly from 1.78±0.11 k cm to 2.20±0.21 k cm (n=15). Rapid rewarming of the bath to 38°C caused a depolarization from –61±2 mV (n=17) to –43±2 mV (n=16) within 15 s followed by a repolarization to –59±2 mV (n=10) within 40 s. Ouabain invariably depolarized PDbl. During both, sustained cooling or application of ouabain, the sensitivity of PDbl to bath potassium concentration decreased in parallel to PDbl pointing to a gradual decrease of potassium conductance. Phlorizin hyperpolarized the cell membrane from –59±2 to –66±1 mV (n=13), virtually abolished the transient hyperpolarization under cooling, and significantly reduced the depolarization after rewarming from +17±2 mV (n=16) to +9±3 mV (n=9).The present data indicate that the contribution of peritubular potassium conductance to the cell membrane conductance decreases following inhibition of basolateral (Na⁺+K⁺)-ATPase. Apparently, cooling from 37° to 10°C does not only reduce (Na^–+K⁺)-ATPase activity but in addition luminal sodium uptake mechanisms such as the sodium glucose cotransporter. As a result, cooling leads to an initial hyperpolarization of the cell followed by depolarization only after some delay.Parts of this study have been presented at the 60th and 61th Meeting of the Deutsche Physiologische Gesellschaft, Dortmund 1984 and Berlin 1985     

Nucleotide inhibition of phosphate transport in the renal proximal tubule

The observation that NAD inhibits sodium-dependent phosphate (P) uptake by the luminal brush border membrane (BBM) of the proximal tubule prompted us to examine the specificity and mechanism of this process. Addition of 10(-5) M NAD to the perfusate of isolated perfused rabbit proximal straight tubules inhibited lumen-to-bath P flux by approximately 50%. ADP-ribose had an identical effect, whereas nicotinamide had no effect. ADP and 5'-AMP (10(-5) M) also inhibited P flux. Na-dependent uptake of 32P by rabbit BBM vesicles was inhibited by 0.1-0.3 mM NAD, ADP-ribose, ADP, ATP, 5'-AMP, and GDP, which were preincubated with the vesicles for 30 min. The kinetics of inhibition showed an apparent increase in the Km for P but no change in Vmax. These findings are consistent with "competitive inhibition." The nucleotides inhibited P uptake even when BBM alkaline phosphatase was inhibited by 96% with 10 mM theophylline. Evidence of nonspecific phosphatase activity was present, since incubation of BBM with 0.1 mM solution of nucleotides for 30 min resulted in an elevation of free P in the medium of approximately 0.15-0.22 mM. Correction of 32P specific activity for this change resulted in values for Km and Vmax that were not significantly different from control. The "competitive inhibition" could thus be ascribed to an isotope-dilution effect. There was no evidence to suggest that NAD caused ADP-ribosylation of the luminal membrane. These studies indicate that adenine and guanine nucleotides do not inhibit P transport by a direct action on the luminal membrane of the proximal tubule but do inhibit lumen-to-bath P flux in isolated perfused proximal tubules at concentrations of 10(-5) M. Since there is no direct inhibitory effect of these compounds at the level of the BBM, it is possible that they inhibit P transport by altering some event subsequent to the transfer of P across the luminal membrane.     

Contraluminal sulfate transport in the proximal tubule of the rat kidney

In order to study the specificity for the contraluminal sulfate transport system the inhibitory potency of disulfonates, di-, tricarboxylates and sulfocarboxylates on the³⁵SO ₄ ^2– influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1) Methane- and ethane-disulfonate as well as benzene-1,3-disulfonate inhibit contraluminal³⁵SO ₄ ^2– influx (with an (app.K _i of <6 mmol/l), while benzene-1,2- and 1,4-disulfonate do not. 2) The inhibitory potency of 1,3-benzene disulfonate is slightly augmented by an additional NH₂ ^– or OH-group in position 4. However, OH-groups at position 4 and 5 or 4 and 6 abolish the inhibitory potency. 3) The naphthalene disulfonates tested inhibit only if they have an OH-group in ortho-position to one SO₃H group. 4) The stilbene disulfonates H₂DIDS and DNDS inhibit the contraluminal³⁵SO ₄ ^2– influx with high (app.K _i0.8 mmol/l), DADS with lower potency (app.K _i6 mmol/l). 5) Amongst the tested aliphatic di- and tricarboxylates inhibition was exerted by oxalate (app.K _i 1.1 mmol/l) and maleate (app.K _i 3.8 mmol/l), but not by malonate, hydroxymalonate and citrate. 6) Out of the tested benzenedicarboxylates only those inhibit which have the COO^–-groups directly on the ring in 1,2 and 1,3 position (app.K _i 4.0 and 2.7 mmol/l), but not in the 1,4 position. An additional OH-group in position 4 augments the inhibitory potency of 1,3 benzene-dicarboxylates (app.K _i 0.8 mmol/l), while an OH group on position 5 abolishes it. 7) The benzene tricarboxylates (BTC) inhibit in the sequence 1,2,3-BTC>1,3,5-BTC>1,2,4-BTC (app.K _i 0.9, 1.5 and 4.2 mmol/l, respectively). 8) The carboxy-benzene-sulfonates inhibit also in the 1,2 and 1,3 position only (app.K _i 6.7 and 5 mmol/l), but not in the 1,4 position. Addition of an –OH-group to the 3-carboxy-1-benzene-sulfonate forming 4-hydroxy-3-carboxy-1-benzene-sulfate augments the inhibitory potency drastically (app.K _i 0.32 mmol/l), while a NH₂ substitution at the same position leaves it unchanged (app.K _i 4.7 mmol/l). If, however, ethylamine instead of NH₂ is used as substituent, the inhibitory potency is almost as high as of 4-hydroxy-3-carboxy-1-benzene-sulfonate (app.K _i0.6 mmol/l). Amongst the dicarboxy-benzene-sulfonates, 3,4-carboxy-benzene-1-sulfonate inhibits (app.K _i ca. 2 mmol/l), while 3,5-carboxy-benzene-1-sulfonate does not. The data indicate that a strong interaction of substrate with the sulfate transporter is given, when two charged groups (COO^– and/or SO ₃ ^– ) are present in a distance equivalent to the meta-position on the benzene ring and an additional hydrogen bond forming OH- or –NH-group. Hydrogen bond forming groups and charged groups in other positions usually abolish the inhibitory potency.     

Contraluminal para-aminohippurate transport in the proximal tubule of the rat kidney

In order to study the specificity of the contraluminal para-aminohippurate (PAH) transport system, the inhibitory potency of monocarboxylates on the³H-PAH influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: if a homologous series of fatty acids with increasing chain length is tested, inhibition of contraluminal PAH influx is first seen with valerate (app.K _i 1.4 mmol/l), increasing up to nonanoate (app.K _i 0.06 mmol/l) and remaining in this range up to duodecanoate, the last compound of this series which is sufficiently water-soluble. Similarly, the inhibitory potency of aromatic monocarboxylates increases with increasing hydrophobicity. If the fatty acids are esterified, their inhibitory potency is lost. If they are transformed to the respective aldehydes their inhibitory potency is preserved at a reduced degree. Introduction of a hydrophobic methyl-, ethyl-, or propyl-group increases the inhibitory potency. A -, but not an -oxo-group augments the inhibitory potency of phenylpropionate analogs, an OH group diminishes it, and a NH₂ group abolishes it. Among phenyl-fatty acids an increase in affinity is observed from phenyl- < benzoylamine-< phenoxy- < benzoyl-acetate and-propionate. All monocarboxylate compounds, so far tested, do not inhibit contraluminal sulfate and Na⁺/succinate influx. The data indicate that the PAH transporter interacts with monocarboxylates and also with aldehydes which have a hydrophobic moiety. An additional oxo-group facilitates the interaction. Thus, the benzoyl compounds show the highest affinity observed.     

Contraluminal sulfate transport in the proximal tubule of the rat kidney

In order to study contraluminal sulfate transport the influx rate of³⁵SO ₄ ^2– from the interstitium into cortical tubular cells has been determined. Preloading of the rat with sulfate augmented contraluminal³⁵SO ₄ ^2– influx; preperfusion with sulfate-free solutions diminished it. The contraluminal³⁵SO ₄ ^2– influx in sulfate-loaded animals followed two parameter kinetics (K _m 1.4 mmol/l,J _max 1.2 pmol·s^–1·cm^–1). The contraluminal³⁵SO ₄ ^2– influx (starting concentration 10 mol/l) did not change when the K⁺ concentration was varied between 4 and 40 mmol/l and the Ca²⁺ concentration from zero to 3 mmol/l. Omission of Na⁺ from the perfusates augmented contraluminal³⁵SO ₄ ^2– influx markedly. The increase is larger at pH 6 than at pH 7.4. Changes of pH affect contraluminal³⁵SO ₄ ^2– influx only when the solutions are Na⁺- and K⁺-free. Under these conditions the³⁵SO ₄ ^2– influx decreased when the ambient pH was raised from pH 6.0 to pH 8.0. Thiosulfate, selenate, molybdate, oxalate, phosphate, arsenate, and bicarbonate exerted competitive inhibition, while formate, 2-oxoglutarate and paraaminohippurate showed a biphasic response: inhibition at 50 mmol/l, no inhibition at 150 mmol/l. Chloride and bicarbonate inhibited³⁵SO ₄ ^2– influx at 10 mol/l³⁵SO ₄ ^2– , but augmented sulfate influx at 5 mmol/l³⁵SO ₄ ^2– concentration in rats not preloaded with sulfate. The data indicate the presence of a contraluminal sulfate transport system which is shared by a variety of inorganic and organic anions. The biphasic behaviour of some anions suggests parallel pathways leading to a cis-inhibition at small and trans-stimulation at high anion concentrations. Na⁺ and H⁺ may be cotransported or interact with the transport system at a modifier site.