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 a-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 378C in buffer containing 50-200 mM 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

Novel application of complementary imaging techniques to examine in vivo glucose metabolism in the kidney

“…We observed the cellular uptake of 2-NBDG in the proximal tubule was similar from the apical and basolateral side of the cell, suggesting uptake by both SGLT and the glucose transporter (GLUT) as observed in studies in isolated cells (2,7,23,33).…”

“…In this study, we employed positron emission tomography (PET) and intravital multiphoton microscopy (MPM) to assess cortical and proximal tubule glucose tracer uptake, respectively, following experimental perturbations of kidney metabolism. Applying dynamic image acquisition PET with [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] fluoro-2-deoxyglucose ( 18 F-FDG) and tracer kinetic modeling, we found that an intracellular compartment in the cortex of the kidney could be distinguished from the blood and urine compartments in animals. Given emerging literature that the tumor suppressor protein p53 is an important regulator of cellular metabolism, we demonstrated that PET imaging was able to discern a threefold increase in cortical 18 F-FDG uptake following the pharmacological inhibition of p53 in animals.…”

Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene

“…Lipolysis and glucose uptake assays in 3T3-L1 cells Lipolysis and glucose uptake assays were performed as previously described in fully differentiated 3T3-L1 adipocytes (54,56). See the Supplemental Methods for additional details.…”

A Quantitative Approach to Screen for Nephrotoxic Compounds In Vitro

“…Time and dosedependent uptake of glucose was measured after incubation of cells with 100, 200, or 500 mM of the fluorescent substrate 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) over the course of 120 min in 96-well plates. 41 Competitive inhibition of SGLT activity was determined in transwell plates after incubation with 250 mM 2-NBDG for 1 h in the presence or absence of 1000 mM phlorizin (SGLT inhibitor) in Na+ and Na+ free buffers. Inhibition of Na+-independent, facilitative transporter activity (GLUT) was shown after pretreatment of HPTECs for 10 min with 50 mM cytochalasin B (SGLT inhibitor) followed by 1 h incubation with 250 mM 2-NBDG in Na+ and Na+ free buffers.…”