Ian Kippen

Transport of p-aminohippuric acid, uric acid and glucose in highly purified rabbit renal brush border membranes.

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na+ + K+)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%. Transport of D-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (EA = 11.3--37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 muM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.

Effect of drugs on urate binding to plasma proteins

The effect of various drugs on urate binding to plasma proteins was investigated in normal subjects. Whereas allopurinol, aspirin, phenylbutazone, probenecid, and sulphinpyrazone all significantly reduced plasma urate concentrations, only aspirin, phenylbutazone, and probenecid significantly impaired urate binding. Colchicine and indomethacin in the doses administered had no significant effect on plasma urate concentrations or binding. In the case of aspirin, urate binding was reduced to 25% of normal, and this effect was quickly abolished after cessation of therapy. Phenylbutazone reduced urate binding to 56% and probenecid to 46% of normal; this impairment was still detected four days after cessation of therapy. Drugs may impair urate binding by competition for plasma protein binding sites, with displacement of bound urate. Impairment of urate binding in vivo by administration of certain drugs may be relevant to the precipitation of acute gouty arthritis, to the formation of gouty tophi, and to the augmentation of uricosuria. Furthermore, the role of drugs must be seriously considered during all studies on urate binding in patients with gout.

Biochemical properties of anti-inflammatory drugs—XII: Inhibition of urate binding to human albumin by salicylate and phenylbutazone analogues and some novel anti-inflammatory drugs

Abstract We have previously reported that 0.2 mM phenylbutazone and salicylate inhibit the binding of urate in vitro to human albumin by over 50 per cent. This communication describes similar studies with over 60 other compounds. Only gentisate, per-fluorosalicylate, diiodosalicylate, salicylimide, Trimethazone, Diflumidone, the fenamic acids, 3-hydroxycinchophen and several acidic sulfonamides were as potent as phenyl-butazone or salicylate. A structure-action relationship was delineated for salicylate analogues. There was a consistent relationship between the ability of an acidic compound to displace albumin-bound urate and its relative avidity for the primary dansylamide (DNSA)-binding site(s) of the albumin. Some advantages of using DNSA, vis a vis other dyestufis, for measuring drug binding to albumin are discussed. Since some of the uricosuric action of certain drugs may depend on the displacement of urate from albumin or other proteins by the drugs, the assays in vitro for urate displacement which are described may facilitate pharmacological screening for potential uricosuric activity.

Effects of dibutyryl cyclic amp on the transport of α-methyl-d-glucoside and α-aminoisobutyric acid in separated tubules and brush border membranes from rabbit kidney

Abstract The effect of dibutyryl cyclic AMP on the transport of α-methyl- d -glucoside and α-aminoisobutyric acid in separated tubules and purified brush border membranes from rabbit kidney was investigated using a rapid filtration procedure. Dibutyryl cyclic AMP stimulated the uptake of α-methyl- d -glucoside and α-aminoisobutyric acid by separated renal tubules in agreement with prior studies utilizing renal slices (Rea, C. and Segal, S. (1973) Biochim. Biophys. Acta 311, 615–624; Weiss, I.W., Morgan, K. and Phang, J.M. (1972) J. Biol. Chem. 247, 760–764). However, in contrast to previous reports, no preincubation of the tissue with dibutyryl cyclic AMP was required for stimulation of transport to be manifest. Dibutyryl cyclic AMP stimulated oxygen consumption by separated tubules suggesting that stimulation of transport may occur by a linkage with renal oxidative metabolism. Dibutyryl cyclic AMP increased the uptake of α-aminoisobutyric acid into purified renal brush border membranes. However the uptakes of α-methyl- d -glucoside, proline, leucine and phosphate into brush border membranes were significantly inhibited.

Some Characteristics of Uric Acid Uptake by Separated Renal Tubules of the Rabbit

Uric acid is generally believed to be both secreted and reabsorbed by the mammalian renal proximal tubule. The degree to which reabsorption and secretion take place is highly species dependent. Uric acid uptake by slices of rabbit renal cortex has been used by previous investigators as a model of secretory transport of this compound. In 1961, Platts and Mudge (1) demonstrated that uptake of uric acid into rabbit kidney cortical slices is influenced by a variety of factors including hypoxia and the presence of various metabolic substrates and drugs. Berndt and Beechwood (2) subsequently showed that this uptake was pH dependent, and was influenced by the medium concentrations of sodium and potassium. Both groups of investigators obtained uric acid slice to medium ratios of approximately 2.5 indicating concentrative uptake of uric acid.