ArticleDiabetes technology & therapeutics2011
A fluorescence method for measurement of glucose transport in kidney cells.
Article in Diabetes technology & therapeutics, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 49 papers, 1 of them a synthesis that pooled it.
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49 citing papers in PubMed, 1 synthesis or guideline pooled it, 83 citations in OpenAlex.
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
Abstract
backgroundDiabetes 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).
methodsUptake 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.
resultsFluorescent 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.
conclusionGlucose transport in cultured kidney cells can be measured with the fluorescence method described in this study.
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