Effect of adenosine triphosphate in renal ischemic injury: involvement of NF-kappaB

Renal ischemic/reperfusion injury in vivo results in a significant increase of acute renal failure (ARF) and death. Nevertheless, there are many limitations in using in vivo models of renal ischemic injury to elucidate the detailed mechanisms of renal injury. Adenosine triphosphate (ATP), an extracellular signal, has been shown to be an important factor in regulation of epithelial cell function. Thus, the present study was performed to establish in vitro ischemic model using primary cultured rabbit renal proximal tubule cells (PTCs) and to examine the effect of ATP in this model. We established an in vitro model of ischemic injury, causing severe depletion of intracellular ATP by using the combination of a mitochondrial respiration inhibitor (antimycin A), non-metabolizable glucose analog (2-deoxyglucose), and calcium ionophore (A23187) in PTCs. Indeed, this ischemic injury significantly increased LDH release, a marker of structural damage, and ATP blocked ischemic injury-induced LDH release. 2-Methylthio-ATP and ATP-gamma-S (P2Y purinoceptor agonists) also blocked ischemic injury-induced LDH release, whereas AMP-CPP (P2X purinoceptor agonist) did not block it. In experiments to examine the relationship between ischemic injury and NF-kappaB activation, ischemic injury increased NF-kappaB translocation, DNA binding activity, and CAT activity. On the other hand, ATP, ATP-gamma-S, or 2-methylthio-ATP protected ischemic injury-induced NF-kappaB activation. These results suggest that the protective effect of ATP on ischemic injury is, in part, related to inhibition of NF-kappaB activation via P2Y receptor in PTCs.