Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

A fluorescence method for measurement of glucose transport in kidney cells. Diabetes Technol Ther 2011 Jul;13(7):743-51

Date

04/23/2011

Pubmed ID

21510766

Pubmed Central ID

PMC3118926

DOI

10.1089/dia.2011.0041

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.

CONCLUSION: Glucose transport in cultured kidney cells can be measured with the fluorescence method described in this study.

Author List

Blodgett AB, Kothinti RK, Kamyshko I, Petering DH, Kumar S, Tabatabai NM

Author

Suresh Kumar PhD Associate Professor in the Pathology department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

4-Chloro-7-nitrobenzofurazan
Animals
Biological Transport
COS Cells
Cell Line
Cells, Cultured
Cytochalasin B
Deoxyglucose
Fluorescent Dyes
Glucose
Glucose Transport Proteins, Facilitative
Humans
Kidney
Kidney Tubules, Proximal
Methylglucosides
Mice
Microscopy, Fluorescence
Phlorhizin
Sodium
Sodium-Glucose Transporter 1
Sodium-Glucose Transporter 2
Spectrometry, Fluorescence
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a