Fig 2: Kinetics of adenosine uptake mediated by ENTs are affected by D-glucose and insulin. Primary cultured podocytes were exposed to 5mM or 25mM D-glucose for 24 h and supplemented with 10nM insulin the last 30 minutes. Adenosine transport activity (0–500 µM adenosine, 60 seconds to 22 °C) mediated by ENT1 (A) and ENT2 (B) were measured. Data were adjusted to Michaelis-Menten equations without a lineal component. Graphs represent means ± S.E. n = 9.

Fig 3: ENT2-dependent uptake is increased by insulin receptor signaling through PI3K in rat glomeruli. ENT2-mediate uptake (10 µM adenosine, 60 seconds to 22 °C) was measured in purified glomeruli from healthy male rats exposed to 5mM or 25 mM D-glucose (D-Glc) for 24 h and supplemented with insulin the last 30 minutes. Signaling through insulin receptor and intracellular pathways were characterized by using AG1024 inhibitor of the tyrosine kinase activity of the receptor and LY294002 PI3-kinase inhibitor. Graphs represent the means ± S.D. *P < 0.05 versus 5 mM D-Glc; #P < 0.05 vs 25mM D-Glc n = 9.

Fig 4: D-glucose and insulin affect adenosine uptake activity mediated by ENT1 and ENT2 in rat glomeruli. (A) Total adenosine uptake (10 µM adenosine, 60 seconds, 22 °C) mediated by sodium-independent transporter systems was measured in purified glomeruli from healthy male rats exposed to 5mM or 25 mM D-glucose (D-Glc) for 24 h and supplemented with insulin the last 30 minutes. (B) The effect of D-glucose and insulin treatments on particular uptake activity mediated by ENT1 and ENT2 are shown. ENT1-mediated uptake is the fraction of total adenosine transport in sodium-free buffer sensitive to NBTI and ENT2-mediated uptake is the fraction of total transport inhibited by hypoxanthine. ENT1-mediated uptake was the fraction of total adenosine transport in sodium-free buffer sensitive to NBTI and ENT2-mediated uptake was the fraction of total transport inhibited by hypoxanthine. The graphs represent the means ± S.D. *P < 0.05 versus 5 mM D-Glc; #P < 0.05 vs 25mM D-Glc n = 9.

Fig 5: Protein levels of ENT2 are downregulated by inhibition of PI3K signaling pathway in podocytes. Primary cultured podocytes were exposed to insulin and LY294002 PI3-kinase inhibitor for 48 h. Representative western blot analyses of ENT1 (A) and ENT2 (B) are shown. For western blots the membrane of each gel was cut in pieces comprising proteins in the range of 45–70 kDa and 10–45 kDa. ENT1 or ENT2 were detected in the upper piece and ß-actin in the respective piece containing lower molecular weight proteins. Graphs represent the means ± S.D. *P < 0.05 versus 5 mM D-Glc; #P < 0.05 vs insulin; n = 5.