S. Klöss

Sodium dependence of the amino acid transport in the proximal convolution of the rat kidney

SummaryWith the technique of stop flow microperfusion with simultaneous capillary microperfusion the zero net flux transtubular concentration differences (Δc) of labelled amino acids which are equivalent to their active transport rates were measured. Alll-amino acids tested (phenylalanine, histidine, aminobicycloheptane-carboxylic acid, aminoisobutyric acid; lysine, ornithine, arginine; aspartic acid; proline and glycine) showed a considerable Δc, i.e. active transport rate. When, however, the ambient sodium was replaced by choline the Δc values dropped to zero. An analysis of the Na+ dependence of the ornithine transport revealed that the sodium-dependence is of the mixed type, i.e. thatKmdecreased andVmax increased with increasing Na+ concentration to the same extent.In contrast to other biological systems no mutual interaction between the Na+-dependentd-glucose andl-histidine transport could be observed.Incidental to these studies it was observed that the active transport rate ofd-histidine was in the range of 40% of that of thel-isomer while ford-phenylalanine it was only in the range of 10% of the active transport of thel-isomer. Furthermore it was found that thel-aspartic acid transport was already saturated at a luminall-aspartic acid concentration of 0.05 mmol/l while that ofl-phenylalanine was not saturated even at a luminal concentration of 9 mmol/l.

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

SummaryUsing 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).

Specificity and sodium dependence of the active sugar tansport in the proximal convolution of the rat kidney

SummaryWith the technique of stop flow microperfusion with simultaneous capillary perfusion, the zero net flux transtubular concentration difference (Δc) of labelled sugars was measured.The following sequence of Δc values, which are a measure for the active transtubular transport rate, were evaluated:d-glucose ≅β methyl-d-glycoside >α-methyl-d-glycoside >d-galactose >3-O-methyl-glucose >d-allose. When 10−4 M phlorrhizin was given in the luminal perfusate the Δcs dropped to zero (±8%). Δc-values in the same range i.e. indicating no active transport, were found for:l-glucose,d-mannose, 2-deoxy-d-glucose,d-fructose,d-glucosamine, 6-deoxy-d-galactose (=d-fucose),d-ribose and the reference polyalcohold-mannitol. Inhibition of thed-galactose δc was achieved by 15 mmol/l of the following sugars: α-methyl-d-glycoside ≅d-glucose ≅ 6-deoxy-d-glucose >3-O-methyl-d-glucose an no significant inhibition byd-xylose andd-mannose. Against Δc of α-methyl-d-glucose the following inhibitory potency was observed:d-glucose >6-deoxy-d-glucose >3-O-methyl-d-glucose ≅d-galactose >d-xylose and no inhibition byd-mannose.When the ambient sodium was replaced by choline, the Δc values of all actively transported sugars dropped toward zero. An analysis of the Na+ dependence of the α-methyl-d-glycoside transport revealed that the sodium dependence is of the affinity type i.e. that onlyKmincreased with increasing Na+ concentration whileVmax remained almost constant.From these data one can conclude: 1. The Crane specificity, i.e. that only the α-position of the OH-group on carbon atom 2 is essential, which was found for the intestinal hexose transport holds for the rat proximal kidney tubule, too. 2. The hexose transport system in the rat works only when Na+-ions are present. The sodium ions augment the affinity of the hexose transport system for the hexoses.

Coupling between proximal tubular transport processes

SummaryThe rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (α-methyl-d-glycoside), H+ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (Δc) of these substances. In the case of calcium the electrochemical potential differenceΔc +zFciΔϕ/RT) was the criterion employed. The rate of isotonic Na+-absorption (JNa) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4′isothiocyanatostilbene 2,2′-disulfonic acid) was observed in rats.Ouabain (1 mM) applied peritubularly incompletely inhibited JNa (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, α-methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H+ (glycodiazine) transport and PAH transport was only slightly affected.When SITS (1 mM) was applied from both sides of the cell it inhibited H+ (glycodiazine) transport by 72% and reduced JNa by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H+ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied.When the HCO3− buffer or physically related buffers were omitted from the perfusate the absorption of Na+ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected.The data are consistent with the hypothesis that the active transport processes of histidine, α-methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na+-Ca2+ countertransport located at the contraluminal cell side. The residual Na+ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H+ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO3− from the cell and secondary to that, the luminal H+-Na+ exchange and consequently the Na+ reabsorption. In the absence of HCO3− buffer in the perfusates the luminal H+-Na+ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onPi reabsorption observed under these conditions might be explained by possible variations in intracellular pH.

Secretion and contraluminal uptake of dicarboxylic acids in the proximal convolution of rat kidney

The transport of dicarboxylic acids in the proximal convolution was investigated by measuring: a) the zero net flux transtubular concentration difference ofdl-methyl-succinate, b) its 2-s influx from the interstitium into tubular cells, and c) its 3.5-s efflux from the tubular lumen. With the first method a luminal concentration exceeding the peritubular concentration was observed, thus indicating a net active transtubular secretion of this slowly metabolized substance.All transport steps, luminal and contraluminal, as well as the overall transport, were Na+-dependent and inhibited by lithium (apparentKi ≈ 1.8 mmol/l). The overall transport of methylsuccinate, as well as the contraluminal influx into proximal tubular cells, could be inhibited by paraaminohippurate and H2-DIDS with an apparentKi of ≈ 1.8 mmol/l, by taurocholate with an apparentKi ≈ 3.` mmol/l and by pyruvate with an apparentKi ≈ 5 mmol/l, but not by sulfate, thiosulfate,l-lactate, oxalate and urate. As judged from the inhibition of contraluminal methylsuccinate influx by 48 dicarboxylic acids (aliphatic and aromatic), a specificity pattern was observed similar to that of inhibition of luminal efflux of 2-oxoglutarate [22]: a preference of dicarboxylates in the transconfiguration with a chain length of 4–5 carbons; little change in the inhibitory potency with CH3−, OH−, SH−and O=, but strong reduction with a NH3+ in the 2 position; only a small reduction of inhibitory potency with 2,3 disubstituted SH and OH analogs; preference of the dicarboxylic benzene in the 1,4 position and of the diacetyl benzene in the 1,2 position. The data indicate a Na+-dependent dicarboxylic transport system at the contraluminal cell side of the proximal tubule which is very similar to the luminal transport system for dicarboxylic acids.

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 Ki 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-α-methylation does not change this behaviour. N-α-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-α-benzoylation of L-lysine is ineffective. N-α-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 α- and β-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 α-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 α-NH3+is eliminated by N-acetylation the affinity is further augmented.

Phosphate transport in the proximal convolution of the rat kidney. I. Tubular heterogeneity, effect of parathyroid hormone in acute and chronic parathyroidectomized animals and effect of phosphate diet.

SummaryThe standing droplet method was applied in combination with microperfusion of the peritubular blood capillaries to determine the build up of transtubular concentration differences of phosphate (Pi) in proximal convoluted tubules.As revealed in experiments with chronic parathyroidectomized (PTX) rats, the time dependent decrease of the intraluminal Pi concentration, or increase of transtubular Pi concentration difference (

Active sulfate reabsorption in the proximal convolution of the rat kidney: Specificity, Na+ and HCO 3 − dependence

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