title: A fluorescence method for measurement of glucose transport in kidney cells.
abstract: BACKGROUND
  Diabetes may alter renal glucose reabsorption by sodium (Na(+))-dependent glucose transporters (SGLTs). Radiolabeled substrates are commonly used for in vitro measurements of SGLT activity in kidney cells. We optimized a method to measure glucose uptake using a fluorescent substrate, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG).
  
  
  METHODS
  Uptake buffers for 2-NBDG were the same as for (14)C-labeled α-methyl-d-glucopyranoside ([(14)C]AMG). Cell lysis buffer was optimized for fluorescence of 2-NBDG and Hoechst DNA stain. Uptake was performed on cultures of primary mouse kidney cells (PMKCs), the LLC-PK(1) proximal tubule cell line, or COS-7 cells transiently overexpressing mouse SGLT1 or SGLT2 by incubating cells at 37°C in buffer containing 50-200 μM 2-NBDG. Microscopy was performed to visualize uptake in intact cells, while a fluorescence microplate reader was used to measure intracellular concentration of 2-NBDG ([2-NBDG](i)) in cell homogenates.
  
  
  RESULTS
  Fluorescent cells were observed in cultures of PMKCs and LLC-PK(1) cells exposed to 2-NBDG in the presence or absence of Na(+). In LLC-PK(1) cells, 2-NBDG transport in the presence of Na(+) had a maximum rate of 0.05 nmol/min/μg of DNA. In these cells, Na(+)-independent uptake of 2-NBDG was blocked with the GLUT inhibitor, cytochalasin B. The Na(+)-dependent uptake of 2-NBDG decreased in response to co-exposure to the SGLT substrate, AMG, and it could be blocked with the SGLT inhibitor, phlorizin. Immunocytochemistry showed overexpression of SGLT1 and SGLT2 in COS-7 cells, in which, in the presence of Na(+), [2-NBDG](i) was fivefold higher than in controls.
  
  
  CONCLUSIONS
  Glucose transport in cultured kidney cells can be measured with the fluorescence method described in this study.
authors.affiliation: Kidney Disease Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Amy B
authors.initials: AB
authors.lastname: Blodgett
authors.forename: Rajendra K
authors.initials: RK
authors.lastname: Kothinti
authors.forename: Ivan
authors.initials: I
authors.lastname: Kamyshko
authors.forename: David H
authors.initials: DH
authors.lastname: Petering
authors.forename: Suresh
authors.initials: S
authors.lastname: Kumar
authors.forename: Niloofar M
authors.initials: NM
authors.lastname: Tabatabai
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003571:Cytochalasin B / Q000494:pharmacology; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D005947:Glucose / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative / Q000037:antagonists & inhibitors; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008757:Methylglucosides / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D010695:Phlorhizin / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051297:Sodium-Glucose Transporter 2 / Q000235:genetics / Q000378:metabolism; D000077203:Sodium-Glucose Transporter 2 Inhibitors; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D051246:Glucose Transport Proteins, Facilitative; D008757:Methylglucosides; C495675:Slc5a1 protein, mouse; C490299:Slc5a2 protein, mouse; D051269:Sodium-Glucose Transporter 1; D051297:Sodium-Glucose Transporter 2; D000077203:Sodium-Glucose Transporter 2 Inhibitors; D003571:Cytochalasin B; C027020:methylglucoside; D003847:Deoxyglucose; D012964:Sodium; D010695:Phlorhizin; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose