Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice
Almut Grenz, … , Michail Sitkovsky, Holger K. Eltzschig
Almut Grenz, … , Michail Sitkovsky, Holger K. Eltzschig
Published January 24, 2012
Citation Information: J Clin Invest. 2012;122(2):693-710. https://doi.org/10.1172/JCI60214.
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Research Article

Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice

Abstract

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) — a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1–/– mice. Comprehensive examination of adenosine receptor–knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications.

Authors

Almut Grenz, Jessica D. Bauerle, Julee H. Dalton, Douglas Ridyard, Alexander Badulak, Eunyoung Tak, Eóin N. McNamee, Eric Clambey, Radu Moldovan, German Reyes, Jost Klawitter, Kelly Ambler, Kristann Magee, Uwe Christians, Kelley S. Brodsky, Katya Ravid, Doo-Sup Choi, Jiaming Wen, Dmitriy Lukashev, Michael R. Blackburn, Hartmut Osswald, Imogen R. Coe, Bernd Nürnberg, Volker H. Haase, Yang Xia, Michail Sitkovsky, Holger K. Eltzschig

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Figure 4

Influence of renal ischemia on ENT function and cellular localization of ENTs.

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Influence of renal ischemia on ENT function and cellular localization of...
(A) Model for measuring epithelial adenosine uptake. HK-2 cells were grown on inserts until they were fully confluent (approximately 7 days). Exogenous adenosine (5 μM adenosine containing 10 nCi of 8-14C-adenosine) was added to the basolateral or apical compartment, and dissipation of labeled adenosine was measured at indicated time points. (B) HK-2 cells were maintained under normoxic conditions or exposed to 24 or 48 hours of ambient hypoxia (1% oxygen). In subsets of experiments, dipyridamole (10 μM) was added to the supernatant. Adenosine measurements were performed in triplicate, and 1 representative experiment of 3 is shown. *P < 0.05 by t test compared with samples treated with 10 μM dipyridamole (Normoxia +DIP). (C) Basolateral adenosine dissipation. *P < 0.05 by t test compared with samples treated with 10 μM dipyridamole (Normoxia +DIP). (D) Either apical or basolateral surface proteins of confluent HK-2 cells grown on inserts were biotinylated. Immunoprecipitates were resolved by SDS-PAGE, and resultant Western blots were probed with streptavidin-HRP. Apical (ezrin) or basolateral (ATPase) surface markers were included as controls. A representative experiment of 2 is shown. (E) Quantification by densitometry (n = 2).