Cytoprotective effects of adenosine and inosine in an in vitro model of acute tubular necrosis

Background and purpose: We have established an in vitro model of acute tubular necrosis in rat kidney tubular cells, using combined oxygen-glucose deprivation (COGD) and screened a library of 1280 pharmacologically active compounds for cytoprotective effects. Experimental approach: We used in vitro cell-based, high throughput, screening, with cells subjected to COGD using hypoxia chambers, followed by re-oxygenation. The 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and the Alamar Blue assay measured mitochondrial respiration and the lactate dehydrogenase assay was used to indicate cell death. ATP levels were measured using a luminometric assay. Key results: Adenosine markedly reduced cellular injury, with maximal cytoprotective effect at 100 μM and an EC ₅₀ value of 14 μM. Inosine was also found to be cytoprotective. The selective A ₃ adenosine receptor antagonist MRS 1523 attenuated the protective effects of adenosine and inosine, while an A ₃ adenosine receptor agonist provided a partial protective effect. Adenosine deaminase inhibition attenuated the cytoprotective effect of adenosine but not of inosine during COGD. Inhibition of adenosine kinase reduced the protective effects of both adenosine and inosine during COGD. Pretreatment of the cells with adenosine or inosine markedly protected against the fall in cellular ATP content in the cells subjected to COGD. Conclusions and implications: The cytoprotection elicited by adenosine and inosine in a model of renal ischaemia involved both interactions with cell surface adenosine receptors on renal tubular epithelial cells and intracellular metabolism and conversion of adenosine to ATP.