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 evaluate the specificity for the contraluminal sulfate transport system the inhibitory potency of phenol- and sulfonphthaleins, of sulfamoyl-compounds (diuretics) as well as diphenylamine-2-carboxylates (Cl- channel blockers) on the 35SO4(2-) influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1) Phenolsulfonphthalein (phenol-red) inhibited with an app. Ki-value of 1.7 mmol/l, while analogs which had additional Br-atoms in position 3 and/or 5, i.e. bromphenol-blue, bromcresol-purple and bromcresol-green, inhibited with an apparent Ki of 0.1 and 0.5 mmol/l respectively. 2) Phenolphthalein and tetrabromphenolphthalein did not inhibit, while the disulfonate dyes bromsulfalein, fuchsin acid and indigocarmine inhibited with a Ki between approximately equal to 1 and 3 mmol/l. The highest inhibitory potency in this class of compounds was seen with orange G (app. Ki 0.07 mmol/l). The monosulfonate dyes tested, fluorescein-sulfonate and orange I inhibited moderately with an app. Ki of approximately equal to mmol/l. 3) The 3-sulfamoyl compounds inhibited to a varying degree, when they had a neighbouring -NH-group (furylmethylamino-group), i.e. in position 6 to the COOH or SO3H-group, or when they had a phenoxy-group in position 4. 4) 4-sulfamoylbenzoate and the related compounds probenecid, acetazolamide and hydrochlorothiazide inhibited with an app. Ki between 4 and 7 mmol/l. 5) All diphenylamine-2-carboxylate analogs inhibited with an app. Ki between 3 and 5 mmol/l, even when the -NH-group was replaced by an = O-group or the benzene ring was replaced by a pyrimidine ring, but not when it was replaced by a thiophen ring.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contraluminal sulfate transport in the proximal tubule of the rat kidney

In order to study the specificity of the contraluminal sulfate transport system the inhibitory potency of salicylate analogs (5 mmol/l each) on the 35SO4(2-) influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 2-hydroxybenzoate (salicylate), per se, did not inhibit contraluminal 35SO4(2-) influx. The same holds when an additional NH2-group was introduced in position 4 or 5, or when an additional Cl-group was introduced in position 4. When an additional Cl- or NO2-group was introduced in position 5 a moderate inhibition was seen (app. Ki approximately equal to 4 mmol/l). However, introduction of 2 Cl- or 2NO2-groups in position 3 and 5 creates compounds with strong inhibitory potency (app. Ki approximately equal to 0.5 mmol/l). 2-hydroxy-3,5-iodobenzoate inhibited too, but with a smaller inhibitory potency (app. Ki approximately equal to 2.3 mmol/l). 2-hydroxybenzoate analogs, which have a carboxy- or sulfo-group in position 5, exerted strong inhibition, those with a acetyl- or butyryl-group exerted moderate inhibition. 1-Naphthol-2-carboxylate did not inhibit, while 1-naphthol-4-sulfamoyl-2-carboxylate did. Amongst the dihydroxybenzoates, 2,3- and 2,5-dihydroxybenzoate did not inhibit contraluminal 35SO4(2-) influx, while 2,4- and 2,6-dihydroxybenzoate did. The data indicate that a hydroxy-group in ortho-position and an electro-negative group in the meta-position to the carboxyl group and paraposition to the hydroxy-group are essential for interaction with the contraluminal sulfate transport system. The ability of 2,6-dihydroxybenzoate to inhibit might be explained by its ability to undergo mesomeric conformation.     

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 sulfate esters and sulfonate compounds on the 35SO4(2-) influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1. From 10 sulfate monoesters tested 9 inhibited contraluminal sulfate influx with an app. Ki between 0.6 and 6 mmol/l; the two sulfate diesters tested, however, did not. 2. Out of 8 aliphatic sulfonate compounds only three, having a NH- or OH-group in a suitable position, exerted a moderate inhibition (app. Ki ca. 2-6 mmol/l). 3. Amongst 14 benzene sulfonates tested only 2 compounds (5-nitrobenzene-sulfonate and 2-hydroxy-5-nitrobenzenesulfonate) inhibited with a Ki less than 5 mmol/l. 4. Out of 10 naphthalene sulfonates tested 8 inhibited with a Ki less than 5; the highest inhibition was seen with the NH-containing 8-anilinonaphthalene-1-sulfonate (ANS), but no inhibition with 2 compounds containing an amino group. 5. From the polycyclic sulfonates pyrene-3-sulfonate and anthracene-1-sulfonate inhibited with a Ki of approximately 2 mmol/l, while no inhibition was seen with anthracene-2-sulfonate. 6. Out of 4 amino-sulfonates tested benzene-1-amino-sulfonate and a similar benzyl-analog inhibited with a Ki of 1 mmol/l and smaller; cyclohexyl-1-amino-sulfonate (cyclamate), however, inhibited only slightly (app. Ki of 6 mmol/l). The data indicate that sulfate monoesters are well accepted by the contraluminal sulfate transport system. The affinity of sulfonate compounds to this system depends on neighbouring OH-groups --NH-groups, meta-positioned electronegative groups or a hydrophobic moiety in an appropriate position.     

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

Using the stop-flow peritubular capillary microperfusion method contraluminal transport of corticosteroids was investigated (a) by determining the inhibitory potency (apparent K _i values) of these compounds against p-aminohippurate (PAH), dicarboxylate (succinate) and sulphate transport and (b) by measuring the transport rate of radiolabelled corticosteroids and its inhibition by probenecid. Progesterone did not inhibit contraluminal PAH influx but its 17- and 6-hydroxy derivatives inhibited with an app. K_i of 0.36 mmol/l. Introduction of an OH group in position 21 of progesterone, to yield 11-deoxycorticosterone, augments the inhibitory potency considerably (app. K _{i, PAH} of 0.07 mmol/l). Acetylation of the OH-group in position 21 of 11deoxycorticosterone, introduction of an additional hydroxy group in position 17 to yield 11-deoxycortisol or in position 11 to yield corticosterone brings the app. K _{i, PAH} back again into the range of 0.2–0.4 mmol/l. Acetylation of corticosterone or introduction of a third OH group to yield cortisol does not change the inhibitory potency, but, omission of the 21-OH group or addition of an OH group in the 6 position reduces or abolishes it. Cortisol and its derivatives prednisolone, dexamethasone and cortisone exert similar inhibitory potencies (app. K _{i, PAH} 0.12–0.27 mmol/l). But again, omission of the 21-OH group in cortisone or addition of a 6-OH group reduces or even abolishes the inhibitory potency against PAH transport. The interaction of corticosterone was not changed when 11, 18-epoxy ring (aldosterone) was formed. On the other hand, the interaction was considerably augmented if the 11-hydroxy group was changed to an oxo group in 11-dehydrocorticosterone (app. K _{i, PAH} 0.02 mmol/l). When the A ring of corticosterone is saturated and reduced to 3, 11-tetrahydrocorticosterone the inhibitory potency is not changed very much. But if more than four OH or oxo groups are on the pregnane skeleton or if the OH in position 21 is missing, the inhibitory potency decreases drastically (app. K_{i, PAH} 0.7–1.7 mmol/l). Introduction of a 21-ester sulphate into corticosterone, cortisol and cortisone does not change app. K _{i, PAH} very much. Glucuronidation, however, reduces it (app. K_{i, PAH} 1.2 mmol/l). None of the tested corticosteroids interacts, in concentrations applicable, with dicarboxylate transport and only the sulphate esters interact with sulphate transport.Radiolabelled cortisol, d-aldosterone, 11-dehydrocorticosterone, and corticosterone are rapidly transported into proximal tubular cells. With the latter three compounds no sign of saturation and no transport inhibition with probenecid could be seen. Only with cortisol was a shift toward saturation observed. In addition, cortisol transport could be inhibited by probenecid. The data indicate that corticosteroids interact with the contraluminal renal PAH transporter, whereby hydroxylation in position 21 augments, and hydroxylation in the 6 or 3, 17 position reduces interaction. However, as tested so far, simple diffusion seems to prevail when corticosteroids cross the cell membrane. Sulphation makes corticosteroids also a substrate for the sulphate transporter.     

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

In order to study the specificities of the contraluminal anion transport systems, the inhibitory potency of substituted benzene analogs on influx of [³H]PAH, [¹⁴C]succinate, and [³⁵S]sulfate from the interstitium into cortical tubular cells has been determined in situ: (1) Contraluminal [³H]PAH influx is moderately inhibited by benzene-carboxylate and benzene-sulfonate, and strongly by benzene-dicarboxylates,-disulfonates and carboxy-benzene-sulfonates, if the substituents are located at positions 1 and 3 or 1 and 4. The affinity of the PAH transporter to polysubstituted benzoates increases with increasing hydrophobicity, decreasing electron density at the carboxyl group and decreasing pK_a. Similar dependencies are observed for phenols. Benzaldehydes which do not carry an ionic negative charge are accepted by the PAH-transporter, if they possess a second partially charged aldehyde or NO₂-group. (2) Contraluminal [¹⁴C]succinate influx is inhibited by benzene 1,3- or 1,4-dicarboxylates,-disulfonates and 1,3-or 1,4-carboxybenzene-sulfonates. Monosubstituted benzoates do not interact with the dicarboxylate transporter, but NO₂-polysubstituted benzoates do. Phenol itself and 2-substituted phenol interact weakly possibly due to oligomer formation. (3) The contraluminal sulfate transporter interacts only with compounds which show a negative group accumulation such as 3,5-dinitro- or 3,5-dichloro-substituted salicylates. The data are consistent with three separate anion transport systems in the contraluminal membrane: The PAH transporter interacts with hydrophobic molecules carrying one or two negative charges (–COO^–, –SO ₃ ^– ) or two or more than two partial negative charges (–OH, –CHO, –SO₂NH₂, –NO₂). The dicarboxylate transporter requires two electronegative ionic charges (–COO^–, –SO ₃ ^– ) at 5–9 Å distance or one ionic and several partial charges (–Cl, –NO₂) at a favourable distance. The sulfate transporter interacts with molecules which have neighbouring electronegative charge accumulation.     

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

In order to evaluate the specificity of the renal contraluminal PAH transport system for amino acids, oligopeptides and their conjugates, the inhibitory potency of these substances against contraluminal [³H] PAH influx has been determined. For this, inhibition of ³H-PAH flux from the interstitium into cortical tubular cells of the rat kidney in situ has been measured. Apparent K_i values were evaluated by a computer program assuming competitive inhibition. Unconjugated amino acids (glycine, cysteine, alanine, leucine, phenylalanine, tyrosine, aspartate, glutamate, arginine, ornithine and lysine) do not inhibit [³H] PAH influx. The very hydrophobic tryptophan, however, does. N--methylation does not change this behaviour. N--acetylation does not evoke interaction with the PAH transporter when it occurs with glycine, cysteine (to yield mercapturic acid), arginine, ornithine and lysine. However, it renders alanine, leucine, phenylalanine, tryptophan, L-aspartate moderately, and L-glutamate strongly, inhibitory. The acetylated D-isomers of alanine, leucine and phenylalanine exert a higher inhibitory potency compared with the respective L-isomers. N--benzoylation of L-lysine is ineffective. N--benzoylation, however, evokes interaction with the PAH transporter, when it occurs with ornithine < arginine < histidine < glycine = leucine < alanine = phenylalanine = aspartate = glutamate. Dipeptides interact with the PAH transporter according to their hydrophobicity (Nozaki scale down to 0.9, Fauchère scale up to 1.0). N-acetylation does not change this behaviour. Hydrophobicity also renders oligopeptides, as angiotensin II, inhibitory against PAH transport. Similarly the anionic angiotensin I converting enzyme inhibitors Captopril, Enalapril and Ramipril inhibit contraluminal PAH influx. The same holds for the Amanita phalloides peptides - and -amanitin, phalloin, phallacidin and Tyr5-carboxymethyl antamanide. Conjugation with L-glutathione renders only strongly hydrophobic xenobiotics inhibitory against PAH transport: S-(4-azidophenacyl)-= S-(4-chlorophenacyl)-< S-(1,2,2-trichlorovinyl)-< S-(1,2,3,4,4-pentachlorobuta-dienyl)- < S-(n-decyl)-. Processing to the L-cysteine conjugate augments the inhibitory potency and additional N-acetylation of the -amino group augments it even more. Thus, the above mentioned conjugation, which creates hydrophobic molecules with a negative ionic charge, renders it reactive with the PAH transporter. If a remaining positive change at the -NH ₃ ⁺ is eliminated by N-acetylation the affinity is further augmented.     

Contraluminal bicarbonate transport in the proximal tubule of the rat kidney

In order to measure the contraluminal bicarbonate flux in situ we applied the stopped flow capillary microperfusion technique and measured the influx of¹⁴C-bicarbonate buffer into cortical tubular cells at pH 8. It was found that the influx in percent of the starting concentration is larger at 20 mmol/l bicarbonate than at 1 mmol/l, indicating a sigmoidal type influx curve. At 20 mmol/l bicarbonate the influx was inhibited by 44%, when Na⁺ was replaced by choline. Replacement of gluconate by chloride or sulfate did not change H¹⁴CO ₃ ^– influx. At this bicarbonate concentration, influx is inhibited by 10 mmol/l 4,4-diisothiocyanato-2,2-stilbenedisulfonate (DIDS) (22%), 5 mmol/l of the carbonic anhydrase blocker ethoxyzolamide (40%) as well as by 5 mmol/l of the arginine reagent 4-nitrophenylglyoxal (31%). At 1 mmol/l bicarbonate starting concentration, bicarbonate influx was inhibited when chloride in the perfusate was present or when sulphate was added. Replacement of sodium by choline did not change bicarbonate influx. Addition of DIDS and 8-anilino-naphthalene-1-sulfonate (5 mmol/l each) inhibited 1 mmol/l bicarbonate influx 39 and 49%, respectively. The para-aminohippurate transport blocker dipropylsulfamoyl-benzoate (probenecid), the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the SH group blocker 2-(3-hydroxymercuri-2-methoxypropyl)-carbamoyl-phenoxyacetate (mersalyl), and formate did not inhibit bicarbonate influx, at 20 and at 1 mmol/l H¹⁴CO ₃ ^– starting concentration. The data are compatible with the assumption of 1. a contraluminal (HCO ₃ ^– )₃/Na⁺ cotransporter inhibitable by DIDS, carbonic anhydrase inhibitors and 4-nitrophenylglyoxal, 2. a HCO ₃ ^– /anion exchange system, which accepts sulfate and chloride and is inhibitable by the anion exchange blockers DIDS and 8-anilino-naphthalene-1-sulfonate, and 3. a HCO ₃ ^– influx component which could not be influenced by Na⁺, Cl^–, nor by the inhibitors applied.     

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

Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent Ki values) of cyclic nucleotides and prostanoids against contraluminal p-aminohippurate (PAH), dicarboxylate and sulphate transport was evaluated. Conversely the contraluminal transport rate of labelled cAMP, cGMP, prostaglandin E2, and prostaglandin D2 was measured and the inhibition by different substrates was tested. Cyclic AMP and its 8-bromo and dibutyryl analogues inhibited contraluminal PAH transport with an app. Ki,PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. Ki,PAH values of cGMP and its analogues are with 0.27, 0.04 and 0.05 mmol/l, considerably lower. None of the cyclic nucleotides tested interacted with contraluminal dicarboxylate, sulphate and N1-methylnicotinamide transport. ATP, ADP, AMP, adenosine and adenine as well as GTP, GDP, GMP, guanosine and guanine did not inhibit PAH transport while most of the phosphodiesterase inhibitors tested did. Time-dependent contraluminal uptake of [3H]cAMP and [3H]cGMP was measured at different starting concentrations and showed facilitated diffusion kinetics with the following parameters for cAMP: Km = 1.5 mmol/l, Jmax = 0.34 pmol S-1 cm-1, r (extracellular/intracellular amount at steady state) = 0.91; for cGMP: Km = 0.29 mmol/l, Jmax = 0.31 pmol S-1 cm-1, r = 0.55. Comparison of app. Ki,cGMP with app. Ki,PAH of ten substrates gave a linear relation with a ratio of 1.83 +/- 0.5. All prostanoids applied inhibited the contraluminal PAH transport; the prostaglandins E1, F1 alpha, A1, B1, E2, F2 alpha, D2, A2 and B2 with an app. Ki,PAH between 0.08 and 0.18 mmol/l. The app. Ki of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1 alpha (0.22 mmol/l) and Iloprost (0.17 mmol/l) as well as that of leukotrienes B4 (0.2 mmol/l) was in the same range, while the app. Ki,PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1 alpha (0.77 mmol/l) and 2,3-dinor-6-keto-PGF1 alpha (0.57 mmol/l) as well as that of thromboxane B2 (0.36 mmol/l) was somewhat higher. None of these prostanoids inhibited contraluminal dicarboxylate transport and only PGB1, E2 and D2 inhibited contraluminal sulphate transport (app. Ki,SO4(2-) 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed Km = 0.61 mmol/l and Jmax of 4.26 pmol S-1 cm-1. It was inhibited by probenecid, sulphate and indomethacin. Contraluminal influx of PGD2, however, was only inhibited by probenecid. The data indicate that cyclic nucleotides as well as prostanoids are transported by the contraluminal PAH transporter. For prostaglandin E2 a significant uptake through the sulphate transporter occurs in addition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ

In order to study contraluminal hexose transport, concentration and time-dependent influx of³H-2-deoxy-d-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (K _m 1.3±0.2 mmol/l,J _max 0.67±0.16 pmol/s · cm) plus an additional diffusion term (withP=6·10^–8 cm²/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-d-glucose of 10.6. Since the extracellular to intracellular free water space as estimated from morphological data was 12, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-d-glucose influx, indicating that it is Na⁺-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-d-glucose influx could be revealed: strong inhibition byd-glucose, methyl--d-glucoside,d-mannose, 3-O-methyl-d-glucose, 2-deoxy-d-galactose, methyl--d-galactoside and 6-deoxy-d-glucose, moderate inhibition byd-galactose,l-glucose,l-mannose andd-fructose, no or borderline inhibition by methyl -d-glucoside, 2-deoxy-methyl--d-galactoside, 1-thio--d-glucose, 1-thio--d-galactose, 5-thio--d-glucose, myo-inositol and mannitol. The contraluminal 2-deoxy-d-glucose influx was also inhibited by phloretin, chlormerodrin and preperfusion with cytochalasin B. Starvation as well as streptozotocin diabetes has no influence on contraluminal 2-deoxy-d-glucose transport. Thus, in contrast to the luminal hexose transport system the contraluminal system is Na⁺-independent, does not require on OH-group at C-atom 2, acceptsl-glucose and fructose, but not an -methyl group at C-atom 1.     

Contraluminal transport of organic cations in the proximal tubule of the rat kidney

In order to study the quantitative structure/activity relationship of organic cation transport across the contraluminal side of the proximal renal tubule cell, the stopped-flow capillary microperfusion method was applied and the inhibitory potency (apparent K _i values) of different homologous series of substrates against N ¹-[³H]methylnicotinamide (NMeN⁺) transport was evaluated. Aniline and its ring- or N-substituted analogues as well as the aminonaphthalines do not interact with the contraluminal NMeN⁺ transporter except for the quaternary trimethylphenylammonium and pararosaniline, which bear a permanent positive charge, and for 1,8-bis-(dimethylamino)naphthaline, which forms an intramolecular hydrogen bond. If, however, one or more than one methylene group is interposed between the benzene ring and the amino group, the compounds interact with the contraluminal NMeN⁺ transporter in proportion to their hydrophobicity parameter, i.e. the octanol/water partition coefficient (log octanol). The catecholamines and other hydroxyl-substituted phenylethyl analogues also follow this rule. In addition, the N-heterocyclic pyridine, quinoline, isoquinoline and acridine analogues also interact with the contraluminal NMeN⁺ transporter, when their pK _a values are higher than 5.0, and, an inverse correlation between pK _a and log K _{i, NMeN} was observed. An exception to this rule are those hydroxy compounds of pyridine, quinoline and isoquinoline that show tautomerism. These compounds slightly inhibit NMeN⁺ transport despite low pK _a values. The quaternary nitrogen compounds of aniline and the N-heterocyclic analogues, as far as tested, all interact with the contraluminal NMeN⁺ transporter in relation to their hydrophobicity. The data indicate that the contraluminal NMeN⁺ transporter interacts with N-compounds according to their hydrophobicity and/or according to their basicity (affinity to protons). The reason for deviation of the aniline analogues and the OH-tautomeric heterocyclic N-compounds from this behaviour is discussed.     

Contraluminal transport of organic cations in the proximal tubule of the rat kidney

In order to study the characteristics of contraluminal organic cation transport from the blood site into proximal tubular cells the stopped-flow capillary perfusion method was applied. The disappearance of N ¹-[³H]methylnicotinamide (NMeN⁺) and [³H]tetraethylammonium (TEA⁺) at different concentrations and contact times was measured and the following parameters evaluated: K _m,NMeN = 0.54 mmol/l, J _max,NMeN = 0.4 pmol s^–1 cm^–1; K _m,TEA = 0.16 mmol/l, J _max,TEA = 0.8 pmol s^–1 cm^–1. TEA⁺ inhibited NMeN⁺ transport and NMeN⁺ the uptake of TEA⁺. Thereby, the K _i values for inhibition correspond closely to the K _m values for uptake. Similar inhibitory potencies of ten organic cation against TEA⁺ and NMeN⁺ transport provide further evidence for a common transport system. Omission of HCO ₃ ^– , or Na⁺ and addition of K⁺ (with or without Ba²⁺) reduce NMeN⁺ transport, while omission of K⁺ (with or without valinomycin) or addition of thiocyanate has no effect. Since the manoeuvres that depolarize contraluminal electrical potential difference reduce NMeN⁺ transport, cell-negative electrical potential difference is suggested as a driving force for contraluminal organic cation transport from the interstitium into the cell. Furthermore, the inhibitory potency (app. K _i values) of homologous series of primary, secondary, tertiary and hydroxy amines as well as of mono- and bisquarternary ammonium compounds against NMeN⁺ transport was tested. The inhibitory potency increased in the sequence methyl < ethyl < propyl < butyl and primary < secondary < tertiary amines < quarternary ammonium compounds. With the amines a reversed correlation between K _i,NMeN and the octanol/water partition coefficient (log octanol) is seen. With quarternary ammonium compounds the inhibitory potency decreases with increasing molecular size: tetrabutyl- > tetrapentyl- > tetrahexyl- > tetraheptyl > tetraoctylammonium. Introducing two OH groups into triethylamine reduces the inhibitory potency while introduction of two OH groups into diethylamine or three OH groups into triethylamine abolishes the inhibitory potency as a result of reduced hydrophobicity. With choline (trimethylethanolamine) and its analogues the reversed correlation between K _i,NMeN and log octanol was also seen. Molecules with a similar hydrophobic moiety to those of the monoammonium compounds, but with two ammonium groups, showed only a small or no inhibitory potency against NMeN⁺ transport. The data indicate that (a) hydrophobic moieties are important for the interaction with the contraluminal organic cation transporter, and (b) the size of the molecule can be a limiting factor. The reduced or missing interaction of the bisquarternary compound might be caused either by the second charge and/or reduced hydrophobicity and/or too large size of a molecule.     

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

In order to study the characteristics of contraluminal para-aminohippurate transport into proximal tubular cells the stopped flow capillary perfusion method was applied. The disappearance of³H-paraaminohippurate from the capillary perfusate at different concentrations and contact times was measured and saturation type behaviour was found with aK _m of 0.08±0.01 (SE) mmol/l,J _max of 1.1±0.1 pmol·s^–1·cm^–1 andr, the final extracellular/intracellular distribution ratio of 0.93±0.03. Omission of Na⁺ from the capillary test perfusate caused a small reduction of contraluminal PAH uptake at small transport rates (0.1 mmol/l PAH in the test perfusate) but not at high transport rates (1.0 mmol/l PAH in the test perfusate). Change of K⁺ between 0 and 40 mmol/l and pH between 6.0 and 8.0 did not influence contraluminal PAH uptake. Isotonic replacement of chloride by gluconate, nitrate, sulfate, phosphate, methanesulfonate or increase in bicarbonate to 50 mmol/l did not influence PAH uptake at small transport rates. But isotonic sulfate and phosphate, as well as 50 mmol/l HCO ₃ ^– and 25 mmol/l Hepes in isotonic solutions reduced PAH uptake at high transport rates. Addition of 5 mmol/l Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, Cd²⁺ to isotonic Na⁺-gluconate solution did not influence PAH uptake except for Mg²⁺ and Mn²⁺ which inhibited uptake at small transport rates only. Preperfusion of the peritubular capillaries with rat serum, Na⁺ gluconate (Ca²⁺-+Mg²⁺-free), Na⁺ gluconate (Ca²⁺-+Mg²⁺-free) plus 10 mmol/l lactate or pyruvate or 0.1 mmol/l 2-oxoglutarate did not influence PAH uptake at small PAH transport rates, but inhibited at high transport rates. Preperfusion of the capillaries for 10 s with Na⁺-, Ca²⁺- and Mg²⁺-free solutions reduced PAH uptake in the presence of Na⁺ at both transport rates. A second 10 s preperfusion — after the first 10 s Na⁺-, Ca²⁺-, Mg²⁺-free preperfusion — with serum or solutions which contained Na⁺ and Ca²⁺ or Mg²⁺ restored the PAH fluxes to control values. The data are compatible with the hypothesis that contraluminal PAH uptake occurs by a saturable transport mechanism in exchange for other intracellular anions rather than in cotransport with Na⁺ ions. It was, however, not possible to identify the type of counteranions involved. The large effect of cation replacement on para-aminohippurate transport, which was reported in many previous studies with kidney slices, is not a direct effect on the para-aminohippurate transporter, but is rather caused indirectly via cell metabolism and/or changed ion gradients.     

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

In order to study the specificity for contraluminal para-aminohippurate (PAH) transport, the inhibitory potency of aliphatic dicarboxylates on 3H-PAH influx, as well as the inhibitory effect on 35SO4(2-)- and 3H-succinate influx, from the interstitium into cortical tubular cells in situ has been determined. The following was found: Testing a homologous series of dicarboxylates--ranging from the 2 C oxalate to the 10 C sebacate--PAH transport was inhibited by succinate (app. Ki 1.35 mmol/l), and all longer dicarboxylates, with high potency (app. Ki 0.05--0.35 mmol/l). Sulfate transport was inhibited only by oxalate (app. Ki 1.1 mmol/l), while dicarboxylate transport was inhibited by succinate, glutarate, adipate and pimelate with decreasing potency (app. Ki 0.04, 0.24, 0.91, 4.0 mmol/l, respectively). PAH transport was inhibited by succinate and glutarate with high potency (app. Ki 1.35 and 0.05 mmol/l), by the correspondent monomethylester to a lesser extent (app. Ki 1.7 and 0.74 mmol/l), but not by the dimethylester. On the other hand, the semialdehyde of succinate with a Ki-value of 1.2 mmol/l, had the same inhibitory potency as succinate itself, while the dialdehyde of glutarate (app. Ki 1.4 mmol/l) was much less potent as glutarate. Introduction of an oxo-, methyl- or sulfhydroxyl-group group onto the 2-position of succinate, or of an oxo-group onto the 2-position of glutarate moderately augmented the inhibitory potency against PAH-uptake. However, introduction of a 2-hydroxy group onto succinate or glutarate in the L-position reduced the inhibitory potency more than in the D-position. Introduction of two methyl-, sulfhydryl- or hydroxyl-groups in the 2-3 position of succinate reduced or abolished its inhibitory potency. The introduction of a 2-amino group onto succinate or glutarate abolished its effect on PAH transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contraluminal transport of small aliphatic carboxylates in the proximal tubule of the rat kidney in situ

In order to study the characteristic of contraluminal transport of hydrophylic small fatty acids the in situ stopped flow microperfusion technique [12] has been applied. By measuring with 4 s contact time the decrease in the contraluminal concentration of the respective radiolabelled substances the concentration dependence of the influx into the cortical cells was tested. The 4 s decrease in contraluminal concentration of chloroacetate,l-lactate,d-lactate, 3-hydroxybutyrate and acetoacetate was between 26% and 31%. For each substance the percent decrease was the same, no matter whether it was offered in a concentration of 0.1 or 10 mmol/l. Contraluminal disappearance of 0.1 mmol/ll-lactate was not influenced by 5 mmol/l H₂DIDS, probenecid, phloretin, mersalyl or cyanocinnamate, but it was significantly (37%) inhibited by 5-nitro-2-(phenyl-propyl-amino) benzoate, a blocker of the nonspecific anion channel. The percent decrease in propionate uptake was somewhat larger — between 36% and 39% — but again not different at 0.01, 0.1, 1.0 and 10 mmol/l. With pyruvate the contraluminal decrease was 20% at 0.1 mmol/l and 31% at 10 mmol/l. The percent disappearance of the aromatic pyrazinoate was 38% and 34% at 0.1 and 10 mmol/l and for nicotinate 42% and 22%, respectively. The disappearance of nicotinate (0.1 mmol/l) was significantly inhibited by 10 mmol/l pyrazinoate and paraaminohippurate (PAH). The data are in agreement with the hypothesis that the hydrophilic small fatty acids traverse the contraluminal cell side by simple diffusion, possibly via the unspecific anion channel [14], pyruvate via the dicarboxylic acid pathway in a cooperative manner and pyrazinoate, as well as nicotinate, via the PAH pathway.     

Transport of inorganic and organic substances in the renal proximal tubule

Summary The transport through the epithelial cell layer of the renal proximal tubule proceeds in principle by passive paracellular and active transcellular transport. The active transcellular transport is mostly secondary active. This means it proceeds coupled with the flux of Na⁺ ions, where-by the transcellular gradient of sodium, created by the (Na⁺+K⁺)-ATPase, located at the contraluminal cell side, provides the main driving force. Once in the cell the substances leave the other cell side by a Na⁺-independent, but carrier-mediated transport system. Using microperfusion and electrophysiological techniques as well as brush border membrane vesicle preparation the Na⁺-H⁺ countertransport and the Na⁺-cotransport of amino acids, phosphate, sulfate, thiosulfate, bile acids, aliphatic-aromatic monocarboxylic acids (lactate) and dicarboxylic acids was studied. Special emphasis will be given to the bidirectional transport of thiosulfate as well as to the specificity of the monocarboxylic acid and dicarboxylic acid transport system.     

Active Ca2+ reabsorption in the proximal tubule of the rat kidney

Summary Using the stop flow microperfusion technique with simultaneous capillary perfusion the rate of active Ca²⁺ reabsorption was evaluated by measuring the static head electrochemical potential difference as well as the permeability of the tubular wall for Ca²⁺ ions. Under control conditions the active Ca²⁺ transport was calculated to be 3.35×10^–13 mol/cm·s. It declined toward zero if the ambient Na⁺ was replaced by choline or lithium. Parallel experiments in the golden hamster showed that active Ca²⁺ transport, vanished completely if active Na⁺ transport was blocked by ouabain (1 mM). These data indicate that the active Ca²⁺ reabsorption from the proximal tubule depends on the active reabsorption of Na²⁺ presumably via a Na⁺–Ca²⁺ countertransport at the contraluminal cell membrane. The static head electrochemical potential difference of Ca²⁺ is the same in late and early proximal tubules. It is also not affected by the presence of acetazolamide (10^–4 M) by the absence of bicarbonate or glycodiazine buffer or by the absence or presence of phosphate (2 mM).     

Bidirectional active transport of thiosulfate in the proximal convolution of the rat kidney

Using the standing droplet method in the late proximal convolution and simultaneous microperfusion of the peritubular capillaries, the zero net flux transtubular concentration difference of thiosulfate at 45 s was determined, the latter being taken as a measure of active thiosulfate transport. Under control conditions, in the presence of Na⁺, near zero c values were observed. When 1 mmol/l carinamide or paraaminohippurate (PAH) were added to the perfusates significant reabsorptive c arose. However, when 7.5 mmol/l sulfate was added to the Na⁺-free secretory c values were observed. Tested under Na⁺-free conditions, the secretory c was not influenced by simultaneously present 5 mmol/l of SO ₄ ^2– but was diminished by 50 mmol/l SO ₄ ^2– . PAH (1 mmol/l), carinamide (0.2 mmol/l) and probenecid (1 mmol/l) decreased the secretory c by 48, 65 and 48%, respectively. The PAH secretion was not influenced, when thiosulfate or sulfate up to 50 mmol/l was added to both perfusates. Under Na⁺-free conditions the c of thiosulfate in early loops of the proximal convolution is higher than in late loops, while for PAH this pattern is reversed. Taken together with the previously published inhibition of sulfate reabsorption by thiosulfate the data indicate 1. thiosulfate is reabsorved by the Na⁺-dependent sulfate transport system and 2. thiosulfate is simultaneously secreted by a carinamide-, probenecid-and PAH-sensitive secretory system. The secretory system might also be shared by sulfate. The thiosulfate net flux is the result of the difference in the activity of the counteracting transporters, located at the luminal and contraluminal cell side. Is is possible that the higher activity of the transporter at one cell side leads to a reversal of the flux through the transporter at the other cell side.