Defective insulin-stimulated equilibrative nucleoside transporter-2 activity and altered subcellular transporter distribution drive the loss of adenosine homeostasis in diabetic kidney disease progression

Abstract

Aim: Progression of diabetic nephropathy (DN) is linked to the dysregulated increase of adenosine and altered signaling properties. A major contribution to the maintenance of physiological extracellular adenosine levels relies on cellular uptake activity through plasma membrane nucleoside transporters. Because kidney cells are responsive to insulin, this study aims to determine how DN affects insulin regulation of the equilibrative nucleoside transporter-2 (ENT2).

Methods: Human Podocytes and rat glomeruli were used to study ENT2 regulation. The effects of diabetes and insulin on ENT2 mediated transport activity were determined measuring the fraction of total adenosine uptake in sodium-free medium which is inhibitable by hypoxanthine. Alterations in ENT2 subcellular distribution were assessed in the kidney of people affected with DN and diabetic rats. The consequences of impaired ENT2 activity on the kidney were evaluated using dipyridamole in an animal model.

Results: Insulin upregulates ENT2 uptake activity by increasing the V_max, thus counteracting decreased adenosine uptake due to high d-glucose and achieving extracellular adenosine homeostasis. Insulin promoted ENT2 translocation to the plasma membrane dependent on PI3-kinase/Akt signaling and actin cytoskeleton integrity. However, in diabetic rats, the insulin-mediated induction of ENT2 activity was lost. Additionally, reduced Akt activation in response to insulin correlated with decreased ENT2 distribution at the plasma membrane. Kidney tissues from diabetic rats and human DN biopsies showed ENT2 redistribution to an intracellular pattern, evidencing dysfunctional adenosine uptake. Through ENT inhibition, we evidenced increased proteinuria and induced alpha-smooth muscle actin as a result of profibrotic activation of cells in the kidney.

Conclusion: Deficient insulin regulation of ENT2 activity contributes to chronically high adenosine levels and glomerular alterations that underline diabetic kidney disease progression.

Keywords: Adenosine; Diabetic nephropathy; Equilibrative nucleoside transporters; Insulin.