A Fluorescent Glucose Transport Assay for Screening SGLT2 Inhibitors in Endogenous SGLT2-Expressing HK-2 Cells

The sodium-dependent glucose transporters 2(SGLT2)plays important role in renal reabsorption of urinal glucose back to plasma for maintaining glucose homeostasis.The approval of SGLT2 inhibitors for treatment of type 2 diabetes highlights the SGLT2 as a feasible and promising drug target in recent years.Current methods for screening SGLT2 inhibitors are complex,expensive and labor intensive.Particularly,these methods cannot directly measure nonradioactive glucose uptake in endogenous SGLT2-expressing kidney cells.In present work,human kidney cells,HK-2,was incubated with a fluorescent D-glucose derivant 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose(2-NBDG)and the fluorescent intensity of 2-NBDG was employed to measure the amount of glucose uptake into the cells.By optimizing the passages of HK-2 cells,2-NBDG concentration and incubation time,and by measuring glucose uptake treated by Dapagliflozin,a clinical drug of SGLT2 inhibitors,we successfully developed a new assay for measuring glucose uptake through SGLT2.The nonradioactive microplate and microscope-based high-throughput screening assay for measuring glucose can be a new method for screening of SGLT2 inhibitors and implied for other cell assays for glucose measurement extensively.

An optimised sequential extraction scheme for the evaluation of vanadium mobility in soils

Reviewing the current state of knowledge about sequential extraction applied for soil vanadium （V） fractionation, we identified an urgent requirement of an sequential extraction （SE） specified for V. Namely, almost all previous SE extracted only 8.4%-48% of total V in soils （excluding residue）. Thus, we proposed an eight-step SE for V fractionation in soils according to the knowledge gained from literature and our own dissolution experiments with model minerals. After extracting the mobilisable and adsorbed V with de-ionised water and 5 mmol/L phosphate, I moVL pyrophosphate was applied to gather organic matter bound V which minimised the artefact dissolving AI and Fe （hydr）oxides occurred when using HNO3-H202 for extraction. Extraction with 0.4 mol/L NH2OH. HCI was highly selective toward manganese oxides. Fractionation of different crystalline A1 and Fe （hydr）oxides associated V with 1 mol/L HCI, 0.2 mol/L oxalate buffer and 4 mol/L HCI at 95℃especially improved the extractability of V incorporated with crystalline phase associated V. The suitability of our new SE scheme was confirmed by its higher selectivity against the target phases and higher extraction efficiencies （55%-77% of total V） with model minerals and 6 soils of different properties than previous SE.