Henrik Jessen

Taurine and β-alanine transport in an established human kidney cell line derived from the proximal tubule

The transport mechanisms of taurine and beta-alanine by an immortalized human embryonic kidney epithelial cell line (IHKE) were examined. The uptake of these beta-amino acids was characterized by two Na(+)-dependent transport components, whereas an inwardly directed H(+)-gradient only stimulated amino acid influx to a small extent and in the absence of sodium. Competition experiments revealed that taurine and beta-alanine drastically reduced the uptake of one another by the high-affinity Na(+)-dependent transport system. However, some alpha-amino acids could also compete with the beta-amino acids, but with a low affinity. Examinations of the effect of different anions on the Na(+)-dependent uptake of taurine at a low amino acid concentration (240 nM) revealed a specific requirement for Cl-, whereas Cl- had no measurable effect at a higher concentration (1.0 mM) of taurine. In addition, activation of taurine transport as a function of Na+ and Cl- concentration indicated a probable coupling ratio of 3 Na+/1 Cl-/1 taurine for the high-affinity carrier. Finally, cellular regulation of taurine transport was indicated by the finding that pretreatment with taurine containing media decreased the activity of the taurine transporter(s).

A comparative study on the uptake of α-aminoisobutyric acid by normal and immortalized human embryonic kidney cells from proximal tubule

We investigated whether an immortalized human kidney epithelial cell line (IHKE), compared with normal embryonic cells (NHKE), can be used as a representative system with which to characterize the transport of neutral amino acids in the proximal tubule of the human kidney. The IHKE cell line, immortalized by treatment with NiSO4, exhibited microvilli and enzyme markers specific for highly specialized tubule cells. The Na(+)-dependent uptake of alpha-aminoisobutyric acid (AIB) by IHKE and NHKE cells occurred by means of a single transport system with identical half-saturation constants, but the capacity for uptake was higher in the immortalized cells. Proton-dependent influx of AIB was also mediated by a single transport component with similar uptake characteristics in both types of cells. Imposition of an H(+)-gradient to a Na(+)-gradient reduced the sodium dependent uptake of AIB with the exception of short incubation time (1 min), where addition of a proton gradient produced a marked increase in the Na(+)-dependent influx of AIB in NHKE but not in IHKE cells. Competition experiments revealed that the Na(+)-dependent uptake at 50 microM AIB was reduced by neutral alpha-amino acids in the two cell lines. L-Glutamate, L-aspartate, L-arginine and the beta-amino acid taurine had no effect. Only in the IHKE cell line did addition of 5 mM L-lysine produce a slight inhibition. Except for L-proline all of the neutral and acidic amino acids tested reduced the H(+)-dependent uptake of AIB in the IHKE cell line. By contrast, addition of L-aspartate did not influence the transport of AIB in NHKE cells. L-Arginine, but not L-lysine decreased the influx in both cell lines. We conclude that the IHKE cell line has retained the capability to accumulate AIB by transport protein(s) similar to those present for neutral alpha-amino acids in NHKE cells.

Renal transport of taurine in luminal membrane vesicles from rabbit proximal tubule

The uptake of taurine by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule was examined. In pars convoluta, the transport of taurine was characterized by two Na(+)-dependent (Km1 = 0.086 mM, Km2 = 5.41 mM) systems, and one Na(+)-independent (Km = 2.87 mM) system, which in the presence of an inwardly directed H(+)-gradient was able to drive the transport of taurine into these vesicles. By contrast, in luminal membrane vesicles from pars recta, the transport of taurine occurred via a dual transport system (Km1 = 0.012 mM, Km2 = 5.62 mM), which was strictly dependent on Na+. At acidic pH with or without a H(+)-gradient, the Na(+)-dependent flux of taurine was drastically reduced. In both kind of vesicles, competition experiments only showed inhibition of the Na(+)-dependent high-affinity taurine transporter in the presence of beta-alanine, whereas there was no significant inhibition with alpha-amino acids, indicating a beta-amino acid specific transport system. Addition of beta-alanine, L-alanine, L-proline and glycine, but not L-serine reduced the H(+)-dependent uptake of taurine to approx. 50%. Moreover, only the Na(+)-dependent high-affinity transport systems in both segments specifically required Cl-. Investigation of the stoichiometry indicated 1.8 Na+: 1 Cl-: 1 taurine (high affinity), 1 Na+: 1 taurine (low affinity) and 1 H+: 1 taurine in pars convoluta. In pars recta, the data showed 1.8 Na+: 1 Cl-: 1 taurine (high affinity) and 1 Na+: 1 taurine (low affinity).

Demonstration of H+‐ and Na+‐coupled co‐transport of beta‐alanine by luminal membrane vesicles of rabbit proximal tubule.

1. The characteristics of renal transport of beta‐alanine by luminal membrane vesicles isolated from either the proximal convoluted part (pars convoluta) or the proximal straight part (pars recta) of rabbit proximal tubule were investigated. 2. In vesicles from pars convoluta two transport systems have been characterized: (1) a Na+‐dependent system with intermediate affinity (half‐saturation 2.7 mM), and (2) a Na+‐independent system, which in the presence of a H+ gradient (extravesicular greater than intravesicular) can drive the uphill transport of beta‐alanine into these vesicles. This is the first demonstration of H+‐beta‐alanine co‐transport across luminal membrane of rabbit kidney proximal convoluted tubule. 3. By contrast, in membrane vesicles from pars recta, transport of beta‐alanine was strictly dependent on Na+ and occurred via a dual transport system, namely a high‐affinity (half‐saturation 0.16 mM) and a low‐affinity system (half‐saturation 9.3 mM). 4. The demonstration of competition between the Na+‐gradient‐dependent uptake of beta‐alanine and taurine, without appreciable inhibition by alpha‐amino acids in vesicles from pars convoluta as well as from pars recta, strongly suggests that the luminal membrane of proximal tubule has transport systems for the reabsorption of beta‐amino acids which are distinct from alpha‐amino acid transport systems.

Na(+)‐ and H(+)‐gradient‐dependent transport of alpha‐aminoisobutyrate by luminal membrane vesicles from rabbit proximal tubule.

1. The characteristics of renal transport of alpha‐aminoisobutyrate (AIB) by luminal membrane vesicles isolated from either the proximal convoluted part (pars convoluta) or the proximal straight part (pars recta) of rabbit proximal tubule were investigated. 2. Transport of AIB in vesicles from pars convoluta was mediated by both Na(+)‐dependent and Na(+)‐independent systems, which in the presence of an inwardly directed H+ gradient can drive the uphill transport of AIB into these vesicles. 3. By contrast, in luminal membrane vesicles from pars recta, transient accumulation of AIB was only dependent on Na+. Lowering pH without a H+ gradient (pHi = pH0 = 5.5) completely abolished the Na(+)‐dependent transient accumulation of AIB in these vesicle preparations. 4. Attempts to determine the stoichiometry of both the Na(+)‐AIB and H(+)‐AIB transporters located in these two segments of proximal tubule suggested that one Na+ and one H+ ion may be involved in the transport of AIB. 5. Sodium‐dependent uptake of AIB in vesicles from pars convoluta was competitively inhibited by L‐serine and L‐phenylalanine, whereas the presence of L‐proline, L‐alanine and glycine had no significant effect. By contrast, the H(+)‐gradient‐dependent uptake of AIB was drastically reduced (30% of the control value) by L‐proline, L‐alanine and glycine, while L‐serine and L‐phenylalanine had no significant effect. 6. On the other hand, pars recta vesicles exhibited a different transport specificity. L‐Phenylalanine, L‐serine, L‐alanine and glycine, but not L‐proline competitively inhibited the uptake of AIB, providing evidence for the existence of a common transport system for AIB, L‐phenylalanine, L‐serine, L‐alanine and glycine in this segment of rabbit proximal tubule.

Uptake of neutral α- and β-amino acids by human proximal tubular cells

The transport characteristics of amino acids in primary cell cultures from the proximal tubule of human adults (AHKE cells) were examined, using α-aminoisobutyric acid (AIB) and β-alanine as representatives of α- and β-amino acids, respectively. The Na+-gradient dependent influx of AIB occurred by a single, saturable transport system, whereas the Na+-gradient dependent uptake data for β-alanine could be described in terms of two-independent transport components as well as one-transport one-leak model with identical kinetic constants for the high-affinity system. Competition experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, l-aspartic acid, and l-arginine. By contrast, the influx of β-alanine was only drastically reduced by β-amino acids, whereas the inhibition by neutral α-amino acids was relatively low. Nor did l-arginine and l-aspartic acid affect the uptake of β-alanine into AHKE cells. Comparison with the results obtained for normal (NHKE) and immortalized (IHKE) embryonic cells suggested an unaltered expression of the types of transport carriers for neutral α- and β-amino acids in the embryonic and AHKE cells. However, the uptake capacity of the above-mentioned transport proteins was relatively smaller in the embryonic kidney compared with the adult human kidney, which may explain, at least partly, the phenomenon of physiologic amino aciduria in neonates.

Mechanism of transport of L-alanine by luminal-membrane vesicles from pars recta of rabbit proximal tubule

The characteristics of renal transport of L‐alanine by luminal‐membrane vesicles from proximal straight tubules (pars recta) of rabbit kidney were investigated. The following picture emerges from transport studies. Two electrogenic and Na+ requiring systems confined to this region of the nephron exist for the transport of L‐alanine. In addition to Na+, the transport of L‐alanine was influenced by H+. However, H+ does not substitute for Na+, but instead potentiates the Na+ effect. Modification of histidyl residues of the intact luminal‐membrane vesicles by diethylpyrocarbonate (DEP), completely abolished the transient renal accumulation of L‐alanine. Substrate and Na+‐protection experiments suggest that histidyl residues may be at or close to the active site of the L‐alanine transporter in membrane vesicles from pars recta.

Comparative study of the uptake of L-cysteine and L-cystine in the renal proximal tubule.

Summary1.The transport mechanisms of L-cysteine and L-cystine by luminal membrane vesicles isolated from either the proximal convoluted part (pars convoluta) or the proximal straight part (pars recta) of rabbit proximal tubuli were examined.2.The uptake of L-cysteine in pars convoluta is characterized by a single sodium-dependent transport system (Km = 0.58 mM), whereas the sodiumdependent influx of L-cysteine in pars recta proceeds via transport systems with different affinities (Km1 = 0.03 mM, Km2 = 5.84 mM). An H+-gradient enhanced the uptake of L-cysteine in pars recta and only in the presence of a Na+-gradient.3.The presence of a Na+ gradient stimulated the influx of L-cystine in both parts of the nephron, but to a lesser extent than for L-cysteine. In addition a considerable amount of binding of L-cystine to membrane vesicles was observed in both pars convoluta and pars recta.4.Stoichiometric studies indicated a coupling ratio of 1 Na+: 1 amino acid for the transport components involved in the uptake of L-cysteine and L-cystine along the proximal tubule.5.Competition experiments demonstrated that neutralα-amino acids inhibited the influx of L-cysteine in the proximal tubule. Basic and acidic amino acids had no effect on uptake of L-cysteine in pars convoluta, whereas a slight inhibitory effect of L-lysine and L-arginine was noted in pars recta.6.Both basic and neutral amino acids, but not L-glutamate inhibited the uptake of L-cystine in pars convoluta. This was in general also the case in pars recta. However, addition of L-proline did not influenced the uptake of L-cystine, and L-phenylalanine, L-asparagine, L-glutamine, L-leucine and L-methionine only inhibited at a high concentration (5 mM).

Adaptive regulation of taurine and β-alanine uptake in a human kidney cell line from the proximal tubule

1. The underlying mechanisms involved in the adaptive regulation of beta-amino acid uptake in the human proximal tubule were examined by use of an immortalized human embryonic kidney epithelial cell line (IHKE). 2. The results indicated that the adaptive response to maintain whole-body taurine homeostasis occurs predominantly via changes in the activity of the high-affinity taurine transport system by alterations in the uptake capacity and with an unaffected half-saturation constant. An adaptive response was not observed for the structurally related beta-alanine. 3. Only colchicine, which interferes with microtubule organization, was capable of blocking the response to alterations of taurine in cell medium, whereas inhibition of protein and nucleic acid synthesis by cycloheximide and actinomycin D, respectively, did not change the adaptive pattern. 4. Phorbol 12-myristate 13-acetate (PMA), mimicking the effects of diacylglycerol, induced inhibition of both beta-alanine and taurine uptake. By contrast, the Ca2(+)-ionophore A23187, mimicking the effects of IP3, only stimulated the uptake of taurine but not the influx of beta-alanine. However, the effect of PMA down-regulation and A23187 up-regulation was rapid and short-lived in contrast to the adaptive response, suggesting that the inositol phospholipid pathway involving diacetylglycerol and IP3 is less likely to be linked directly to the adaptive regulation, but rather plays a role in short-term regulation.

IGF-II receptors in luminal and basolateral membranes isolated from pars convoluta and pars recta of rabbit proximal tubule

The binding of 125I-labeled insulin-like growth factor-II (125I-IGF-II) to luminal and basolateral membrane vesicles isolated from pars convoluta and the straight part (pars recta) of rabbit proximal tubule was investigated. Analyses of the binding data by use of the general stoichiometric binding equation revealed, that in all preparations IGF-II was bound to one high-affinity binding site and other sites with lower affinities. The specificity of the high-affinity 125I-IGF-II binding to the membrane vesicles assessed by displacement by unlabeled IGF-II, IGF-I and insulin showed that IGF-I displaced 125I-IGF-II in the range 22.5-47.9 nM (IC50) whereas insulin did not effect 125I-IGF-II binding at all. beta-Galactosidase inhibited the 125I-IGF-II binding with half-maximal inhibition of 20-30 nM beta-galactosidase. D-Mannose 6-phosphate increased the binding of 125I-IGF-II and reversed the inhibitory effect of beta-galactosidase. Analyses of 125I-IGF-II binding curves in the presence of beta-galactosidase or D-mannose 6-phosphate demonstrated that none of these compounds changed the binding affinity of 125I-IGF-II for the membrane vesicles. The IGF-II/M6P receptor content in the luminal membranes was in the range 0.21-0.34 pmol IGF-II/M6P receptor per mg protein and very low compared to 2.27-2.86 pmol IGF-II/M6P receptor per mg protein in basolateral membranes.