Brush Border Disaccharidases in Dog Kidney and Their Spatial Relationship to Glucose Transport Receptors
Total Page:16
File Type:pdf, Size:1020Kb
Brush Border Disaccharidases in Dog Kidney and Their Spatial Relationship to Glucose Transport Receptors Melvin Silverman J Clin Invest. 1973;52(10):2486-2494. https://doi.org/10.1172/JCI107439. Research Article The localization of disaccharidases in kidney has been studied by means of the multiple indicator dilution technique. A pulse injection of a solution containing Evans blue dye (plasma marker), creatinine (extracellular marker), and a 14C- labeled disaccharide (lactose, sucrose, maltose, and αα-trehalose), is made into the renal artery of an anesthetized dog, and the outflow curves are monitored simultaneously from renal venous and urine effluents. Lactose and sucrose have an extracellular distribution. Trehalose and maltose remain extracellular from the postglomerular circulation. About 75% of filtered tracer maltose or trehalose is extracted by the luminal surface of the nephron. Thin-layer chromatography of urine samples shows that all of the excreted 14C radiolabel is in the form of the injected disaccharide. Following the administration of phlorizin, all of the filtered radioactivity is recoverable in the urine, but chromatography of the urine samples now reveals that there is a significant excretion of [14C]glucose, approximating the amount previously extracted under control conditions (in the absence of phlorizin). It has been verified that no hydrolysis of maltose or trehalose to their constituent glucose subunits occurred during the transit of tracer between the point of injection (renal artery), and the point of filtration (glomerular basement membrane). Similarly, after addition of [14C]disaccharides to fresh urine there is no chromatographically recoverable [14C]glucose. It is concluded that there exist α-glucosidases […] Find the latest version: https://jci.me/107439/pdf Brush Border Disaccharidases in Dog Kidney and Their Spatial Relationship to Glucose Transport Receptors MELVIN SILVERMAN From the Department of Medicine, Division of Nephrology, Toronto General Hospital, Toronto M5G IL7, Ontario, Canada A B S T R A C T The localization of disaccharidases in kid- the proximal tubule and that these disaccharidases are ney has been studied by means of the multiple indicator located spatially superficial to the glucose transport dilution technique. A pulse injection of a solution con- receptors. taining Evans blue dye (plasma marker), creatinine (ex- marker), and a "C-labeled disaccharide (lac- tracellular INTRODUCTION tose, sucrose, maltose, and aa-trehalose), is made into the renal artery of an anesthetized dog, and the outflow The disaccharidases maltase and trehalase have been curves are monitored simultaneously from renal ve- demonstrated in membrane fractions prepared from rab- nous and urine effluents. Lactose and sucrose have an bit (2-4) and rat (5) kidney, but the functional sig- extracellular distribution. Trehalose and maltose remain nificance of these enzymes in the kidney remains un- extracellular from the postglomerular circulation. About known. 75% of filtered tracer maltose or trehalose is extracted It was suggested several years ago (6) that trehalase hy the luminal surface of the nephron. Thin-layer chro- may play a role in the active transport of glucose by matography of urine saml)les shows that all of the ex- the intestine as well as the kidney. In the intestine, there creted "C radiolabel is in the form of the injected is evidence that trehalase is located in the mucosal brush disaccharide. Following the administration of phlorizin, border, spatially superficial to the glucose transport all of the filtered radioactivity is recoverable in the urine, mechanism (7-9). This experimental finding, together but chromatography of the urine samples now reveals with other data summarized by Semenza and Rikova that there is a significant excretion of ["C]glucose, (10), argues against the participation of trehalase in approximating the amount previously extracted under the active transport of glucose. However, in more re- control conditions (in the absence of phlorizin). It has cent studies (11), using everted sacs of small intestine, been verified that no hydrolysis of maltose or trehalose new data have been obtained which imply the existence to their constituent glucose subunits occurred during of disaccharidases (maltase, trehalase, sucrase), internal the transit of tracer between the point of injection (renal (serosal) as well as external (mucosal) to the glucose artery), and the point of filtration (glomerular basement transport receptors. Also, papain digestion of kidney membrane). Similarly, after addition of ["4C]disaccha- brush border membranes (3) releases maltase into the rides to fresh urine there is no chromatographically re- supernate but leaves trehalase bound to the membrane coverable ['4C]glucose. matrix. It is concluded that there exist a-glucosidases with On the basis of these data, the suggestion that tre- maltase and trehalase activity along the brush border of halase plays a role in the intestinal and renal transport of glucose remains a viable hypothesis. This work was presented at the International Society of described (12, 13) how the mul- Mexico, and the We have previously Nephrology, October 1972, Mexico City, tiple indicator dilution technique can be adapted in dog Canadian Society for Clinical Investigation, January 1973, interactions Edmonton, Canada. A preliminary report of this work has kidney to differentiate between chemical been published in abstract form (1). occurring at the luminal (brush border) surface from Dr. Silverman is a scholar of the Medical Research events taking place at the antiluminal (basal and lateral) Council of Canada. membrane. The details of our method for distinguishing Received for publication 13 April 1973 and in reviscd form surfaces of the nephron have been 8 June 1973. between the opposing 2486 The Journal of Clinical Investigation Volume 52 October 1973 2486-2494 given in the previously mentioned publications (12, 13) Ill.), and 6.25 or 12.5 ,uCi/inl of the following radiolabeled and are described briefly in the Methods section below. test disaccharides: ['4C]sucrose and ["C]aa-trehalose, spe- The present study was undertaken to investigate the cific activity 360 mCi/mM (ICN Chemical and Radioiso- topes Div., Irvine, Calif.); [1-14C]lactose (14.9 mCi/mM); nature of the interaction between disaccharides, in par- and [U-14C]maltose (7 mCi/mM) (Amersham/Searle Corp., ticular trehalose and maltose, with the luminal and Arlington Heights, Ill.). 1 ml of this solution was removed antiluminal surfaces of the nephron and to determine the and saved for urine standards. T1824 (Evan blue dye), relationship (if any) to the glucose transport mechanism. (Matheson Coleman & Bell, Norwood, Ohio) was added to the remaining 3 ml to make a concentration of 2.5 mg/ml. METHODS The solution containing T1824 was used for injection and for preparing blood standards. The surgical and experimental procedures used in these Determinations of whole studies have been described in detail in previous publications arterial blood and plasma glucose (12-14). Briefly, a closed arterial injection of a mixture of concentrations were made at the beginning and end of each indicators (total volume of 0.35-0.45 ml) is made into the experimental run. (Glucostat, Worthington Biochemical left renal artery of an anesthetized Corp., Freehold, N. J.). Analytical determinations of T1824, mongrel dog, and timed creatinine, and radioactivity were made as previously de- serial samples are obtained simultaneously from left renal scribed (12). vein (30 samples in 15 or 30 s) and from right and left Blood pressure, blood pH, Po2 and Pco2 were determined ureters (30 urine samples in 5 or 10 min). Appropriate in- for each run. ternal standards are prepared in blood and urine collected At the end of each experiment, the kidney was prior to each experimental run. By analyzing the internal removed, weighed, and examined for any gross abnormality. standards along with the unknown samples in blood and All the chemical dilutions were performed using automatic urine the concentration of the injected indicators is deter- dilutors whose reproducibility is 0.6% (ADD System, Fisons mined. The data are expressed as fractional recovery (of the Scientific Apparatus Ltd., Loughborough, Leicestershire, total amount injected) per milliliter of whole blood, versus England) and the reproducibility of each experimental data time, for the renal venous effluent, and as fractional recovery point is better than 3%. versus time in the urine effluent. Recirculation corrections as Thin-layer chromatography was carried out on silica gel well as methods for calculation of blood flow, mean transit plates (Eastman Kodak, Rochester, N. Y.). Chromatography time and fractional tracer reabsorption from the urine have was performed on alcohol filtrates of samples from blood, been previously described (12, 13). urine, injection solutions, and unlabeled sugar standards of It is worth noting that the rationale for using our method glucose, trehalose, and maltose. For trehalose the solvent to differentiate luminal and antiluminal events is based upon was a mixture of n-butanol, ethanol, water, and ammonium having a valid reference marker for the extracellular space. hydroxide in a ratio of 45: 5: 49:1. The chromatograms In the case of the dog kidney it turns out that creatinine can were run for 3-4 h (10 cm migration of solvent front) and serve as such a marker. Then, if a test solute has a transit spots were visualized with