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 3

Influence of renal ischemia on ENT expression.

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Influence of renal ischemia on ENT expression. (A) Ent1 and Ent2 transcr...
(A) Ent1 and Ent2 transcript levels in kidneys exposed to sham operation (Sham) or 30 minutes of ischemia, followed by reperfusion for the indicated time periods assessed by real-time RT-PCR relative to β-actin (n = 4 independent experiments). (B) Ent1 and Ent2 protein levels assessed by Western blotting (β-actin to control for loading conditions; 1 representative blot of 3 is shown). (C) Comparison of immunoreactivity for Ent1 and Ent2 in kidneys exposed to 30 minutes of renal ischemia and 2 hours of reperfusion or sham-operated controls (original magnification, ×400; fluorescent green: counterstaining for proximal tubules; fluorescent red [arrows]: staining for Ent1 or Ent2; 1 representative image of 3 is displayed). (D) Relative expression levels of Ent1 or Ent2 transcript in isolated tubules or glomeruli, relative to β-actin by real-time RT-PCR (n = 4 independent experiments). (E) ENT1 and ENT2 transcript levels in cultured HK-2 cells exposed for the indicated time periods to ambient hypoxia (1% oxygen) assessed by real-time RT-PCR relative to β-actin (n = 4 independent experiments). (F) ENT1 and ENT2 protein levels from HK-2 cells exposed to indicated time periods to ambient hypoxia (1% oxygen) and assessed by Western blot (β-actin to control for loading conditions; 1 representative blot of 3 is shown).