M.Iqbal Sheikh

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).

Mechanisms of uptake of ketone bodies by luminal-membrane vesicles.

The energetics and location of renal transport of acetoacetate, beta-hydroxybutyrate, alpha-hydroxybutyrate and gamma-hydroxybutyrate by luminal-membrane vesicles from either whole cortex or pars convoluta or pars recta of rabbit proximal tubule were studied. Addition of either acetoacetate or beta-hydroxybutyrate or its analogues to dye-membrane-vesicle suspensions in the presence of Na+ gradient (extravesicular greater than intravesicular) resulted in absorbance changes indicative of depolarizing event(s). Valinomycin enhanced the Na+-dependent uptake of monocarboxylic acids, provided a K+ gradient (intravesicular greater than extravesicular) was present. By contrast, Na+-dependent uptake of these compounds was nearly abolished by ionophores that permit Na+ to pass through the luminal-membrane via another channel, either electrogenically (e.g. gramicidin D) or electroneutrally (e.g. nigericin). These results established that the Na+-dependent transport of ketone bodies and analogues by luminal-membrane vesicles is an electrogenic process. Eadie-Hofstee analysis of saturation kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for these compounds. Tubular localization of the transport systems was studied by the use of vesicles derived from pars convoluta and pars recta. In pars recta uptake of all these compounds was mediated by means of a single high affinity common transport system. Uptake of these compounds by vesicles from pars convoluta was carried out via a relatively low affinity but common transport system. The physiological importance of the transport systems is discussed.

Effect of vanadate on the renal accumulation of p-aminohippurate in the rabbit kidney tubules in vitro

Abstract The possible involvement of Na + -K + -ATPase on renal organic anion transport systems was investigated by examining the effect of vanadate and ouabain on the uptake of p -aminohippurate (PAH) by rabbit kidney cortical slices. Addition of increasing concentration of vanadate (0.01–0.1 mM) reduced Na + -K + transport and renal steady-state aerobic accumulation of PAH to 80–50 per cent of the control value, without any significant decrease in tissue concentration of ATP. The rapid Na + -independent component of PAH transport was unaffected by the presence of vanadate (0.1 mM) or ouabain (0.2 mM), whereas the slowly equilibrating component, which makes the largest contribution to the final accumulation of PAH was ca . 50 per cent inhibited, by vanadate and almost abolished by ouabain. The anaerobic uptake of PAH was unaffected by a high concentration of vanadate (1 mM), suggesting that vanadate does not competitively inhibit the transport of organic anions. On the basis of the above-mentioned findings, it is suggested that PAH transport is probably linked in a direct manner to the function of Na + -K + -ATPase.

Characteristics of uptake of short chain fatty acids by luminal membrane vesicles from rabbit kidney

The mechanisms of renal transport of short chain fatty acids by luminal membrane vesicles prepared from pars convoluta or pars recta of rabbit proximal tubule were studied by a Millipore filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Both luminal membrane vesicle preparations take up propionate and butyrate by strictly Na+-dependent transport systems, although with different characteristics. The uptake of short chain fatty acids by membrane vesicles from the pars convoluta was insensitive to changes in membrane potential, which is indicative of electroneutral transport of these compounds. Furthermore, kinetic studies showed that the Na+-dependent, but electrically silent transport of propionate is saturable (Km = 10.9 +/- 1.1 mM and Vmax = 3.6 +/- 0.2 nmol/mg protein per 20 s) and is unaffected by the presence of L- and D-lactate, indicating that these monocarboxylic acids did not share the same common transport system. In the luminal membrane vesicles from the pars recta, the uptake of propionate and butyrate was mediated by an Na+-dependent electrogenic transport process, since addition of the organic compounds to these vesicle/dye suspensions depolarized the membrane vesicles and the renal uptake of propionate and butyrate was enhanced by K+ diffusion potential induced by valinomycin. Competition experiments revealed that in contrast to the transport of propionate by vesicles from the pars convoluta, the Na+-dependent electrogenic transport of short chain fatty acids in vesicles from the pars recta occurred via the same transport system that is responsible for the reabsorption of L- and D-lactate in this region of rabbit kidney proximal tubule.

Nature of Na+-independent stimulation of renal transport of p-aminohippurate by exogenous metabolites

The relationship between p-aminohippurate (PAH) and Na+ transport in rabbit kidney cortical slices was examined under optimal metabolic conditions. Addition of lactate, pyruvate and acetate to the incubation medium stimulated PAH transport and accumulation, but had no effect on active Na+ efflux from the slices. Conversely, small concentration of F-, in the presence of acetate, decreased PAH transport and accumulation, but had no effect on Na+ efflux. These observations constitute evidence that, in addition to Na+ cotransport [M. I. Sheikh and J. V. Møller Biochem. J. 208, 243 (1982)], PAH uptake is metabolically stimulated by a Na+-independent pathway. This could occur either by a direct metabolic effect on the PAH carrier, or by exchange with intracellular anions that are generated in the presence of exogenous substrate. In support of the latter possibility the PAH carrier is demonstrated to function as an anion exchanger of PAH and fumurate after preloading of the slices with fumurate under anaerobic conditions.

Kinetics studies on the renal transport of probenecid in vitro.

1. The kinetic parameters of renal transport of probenecid have been assessed by studying the uptake of the drug in rabbit kidney tubules incubated in an electrolyte medium under various conditions. 2. The added compounds inhibited the uptake of probenecid both by kidney cortical slices and separated renal tubule preparations in the following order: p-aminohippurate less than phenol red less than bromophenol blue less than bromocresol green. A reversible competitive inhibitory effect of these organic anions on the renal accumulation of the drug was observed. 3. The Km for renal uptake of probenecid in separated tubules (0.04 mM) and the KI values calculated in this system for p-aminohippurate (0.5 mM), phenol red (0.09 mM), bromophenol blue (0.02 mM) and bromocresol green (0.015 mM) were found to be in good agreement with the corresponding KI value of probenecid and Km values of these compounds previously observed in various kidney tissue preparations. 4. On the basis of above mentioned findings, it is concluded that probenecid, p-aminohippurate and various phenolsulphonphthalein dyes are transported by the common renal organic anion transport system.

Demonstration of Na+-selective channels in the luminal-membrane vesicles isolated from pars recta of rabbit proximal tubule

Characteristics of 22Na+ fluxes through Na+ channels in luminal‐membrane vesicles isolated from either pars recta or pars convoluta of rabbit proximal tubule were studied. In NaCl‐loaded vesicles from pars recta, transient accumulation of 22Na+ is observed, which is inhibited by amiloride. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using either these membrane vesicles loaded with different anions, or an outwardly directed K+ gradient with a K+ ionophore valinomycin. The vesicles containing the channel show a cation selectivity with the order Li+ > Na+ > K+. The amiloride‐sensitive 22Na+ flux is dependent on intravesicular Ca2+. In NaCl‐loaded vesicles from pars convoluta, no overshoot for 22Na+ uptake is observed. Furthermore, addition of amiloride to the incubation medium did not influence the uptake of 22Na+ in these vesicle preparations. It is concluded that Na+ channels are only present in pars recta of rabbit proximal tubule.

The kinetic parameters of renal transport of p-aminohippuratein vitro

Abstract 1. 1. Influx and efflux of p- amino[ 3 H]hippurate in separated renal tubules, prepared from cortex of rabbit kidney, have been measured under a variety of conditions after preequilibration with unlabeled p-aminohippurate. 2. 2. p-Aminohippurate transport is characterized by a rapid and a slow component which are described in influx experiments by (A1 − A1exp (−s1t)) and (A2 − A2exp(−s2t)), respectively, where A1 and A2 are the equilibria accumulations, s1 and s2 are constants, and t is time. At 0.075 mM medium p-aminohippurate, A1 = 3.2, A2 = 7.8, s1 = 1.88 min−1, s2 = 0.056 min−1. The efflux of p-aminohippurate is also described by similar equations and constants. 3. 3. Preincubation with probenecid or 2,4-dinitrophenol resulted in a proportionate decrease of A1 and A2, whereas s1 and s2 were unaffected. 4. 4. At high medium concentrations of p-aminohippurate, A1 and A2 were also reduced to the same extent, while s1 and s2 remained unaltered. Influx of p-aminohippurate approached a saturation limit under these conditions. 5. 5. It is concluded that the rapid component represents active transport of p-aminohippurate, presumably occurring at the peritubular membrane. An analysis of the data shows that the decrease of A2 observed under various conditions may be explained as a secondary effect of the accompanying decrease of A1. It is proposed that the slow component is due to intracellular compartmentation of p-aminohippurate.

The mechanism of urate transport in rabbit kidney tubules in vitro

SummaryThe steady-state concentration of urate in slices and separated tubules of rabbit kidney cortex incubated in an electrolyte- and urate-containing medium has been examined under different conditions.1.The ratio between the concentration of urate in tubule water and medium (T/MUr) under aerobic conditions had a constant value of 0.4 in slices at medium concentrations of urate below 0.10 mM. However, renal accumulation of urate was evident at medium concentrations above 0.15 mM.2.Addition of probenecid (5 mM) resulted inT/MUr values for slices of 0.4 under aerobic conditions, irrespective of the medium concentration of urate.3.T/MUr values were close to unity during anaerobic incubation of slices and were not affected by the presence of probenecid in the medium under these conditions.4.The accumulation of urate in tubules was similar to that observed in slices. The uptake of urate approached a maximal value at a medium concentration of 5 mM.5.Low concentrations of probenecid, 2,4-dinitrophenol,p-aminohippurate, and hippurate competitively inhibited the uptake of urate in tubules at high medium concentrations of urate.6.It is concluded that tubular accumulation of urate is a consequence of tubular secretion, whereas extrusion of urate from tubules may be part of the reabsorptive process for urate in the functioning kidney.

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.