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

Abstract

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 microM and an EC(50) value of 14 microM. 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.

Figure 2

Adenosine and inosine pretreatment enhances cellular recovery after prolonged oxygen-glucose deprivation. Confluent NRK cultures were exposed to a 16-h-long combined oxygen-glucose deprivation (COGD) followed by an aerobic recovery period of the indicated length. Cellular viability was evaluated by the MTT (A) and Alamar Blue (AB) assays (B), and LDH activity (C) was measured in the supernatant. Adenosine was applied at 100 µM (ADE) and inosine at 300 µM (INO) preceding the hypoxic period and was present throughout the COGD. Controls (CTL) were exposed to hypoxia in complete culture medium and showed similar viability and LDH release to cells maintained under normoxic conditions. Data are shown as mean ± SEM values (n = 72). #P < 0.05 compared to CTL, *P < 0.05 compared to COGD.

Figure 1

Adenosine and inosine reduce NRK cell injury during prolonged oxygen-glucose deprivation. Confluent NRK cultures were subjected to combined oxygen-glucose deprivation (COGD) for the indicated lengths. Viability was determined after 4 h of recovery by the MTT assay (A) and LDH activity (B) was measured in the supernatant. Adenosine was applied at 100 µM (ADE) and inosine at 300 µM (INO) preceding the hypoxic period. Controls (CTL) were exposed to hypoxia in complete culture medium and showed similar viability and LDH release to cells maintained under normoxic conditions. Data are shown as mean ± SEM values (n = 72). #P < 0.05 compared to CTL, *P < 0.05 compared to COGD.

Figure 3

Dose-response curves for adenosine and inosine. Confluent NRK cultures were subjected to combined oxygen-glucose deprivation (COGD) for 16 h followed by a 4-h-long reoxygenation period. Adenosine (ADE) and inosine (INO) (1 µM-1 mM) were applied preceding the hypoxia induction and responses were calculated based on the viability values measured by the MTT assay. EC₅₀ values are shown in µM. Data are shown as mean± SEM (n = 9).

Figure 5

The cytoprotective effects of adenosine and inosine are partially mediated by activation of the A₃ adenosine receptor. Confluent NRK cultures were subjected to glucose deprivation (C, D) or combined oxygen-glucose deprivation (COGD, n = 96) for 16 h followed by a 4-h-long reoxygenation period (A, B, E, F). Adenosine (ADE, n = 36) and inosine (INO, n = 36) were applied at 30 µM (A, B) or at 100 and 300 µM respectively (n = 9 each) (E, F). A₃ adenosine receptor antagonist, MRS 1523 (A–D) and A₃ adenosine receptor agonist IB-MECA (E–F) were applied in the indicated concentrations (n = 9 or 18 each concentrations). Controls (CTL, n = 48) were exposed to hypoxia in complete culture medium. Viability was measured by the MTT assay (A, C, E) and LDH activities (B, D, F) were measured in the supernatant. Data are shown as mean ± SEM. #P < 0.05 compared to CTL, *P < 0.05 compared to ADE (A, B). +P < 0.05 compared to COGD (A, B), *P < 0.05 compared to COGD (C–F).

Figure 4

The effect of adenosine receptor antagonists on the cytoprotective effect of inosine. Confluent NRK cultures were subjected to combined oxygen-glucose deprivation (COGD, n = 96) for 16 h followed by a 4-h-long reoxygenation period. The A₁ adenosine receptor antagonist, CDPX (A, B), the A_2A adenosine receptor antagonist, CSC (C, D), the A_2B adenosine receptor antagonist, alloxazine (E, F), and the A₃ adenosine receptor antagonist, MRS 1523 (G, H) were applied in the indicated concentrations (n = 9) 30 min prior to the inosine pretreatment (INO, n = 36) and were present throughout the COGD period. Viability was measured by the MTT assay (A, C, E, G) and LDH activities (B, D, F, H) were measured in the supernatant. Controls (CTL, n = 48) were exposed to hypoxia in complete culture medium. Data are shown as mean ± SEM. #P < 0.05 compared to CTL, *P < 0.05 compared to INO, +P < 0.05 compared to COGD.

Figure 7

Adenosine kinase inhibition prevents the protective effects of adenosine and inosine. Confluent NRK cultures were subjected to combined oxygen-glucose deprivation (COGD, n = 48) for 16 h. Viability was measured after a 4-h-long recovery by the MTT assay (A, C), and cellular injury was determined by the LDH assay (B, D). Cells were pretreated with 100 µM adenosine (ADE, n = 9) (A, B) or 300 µM inosine (INO, n = 9) (C, D) 30 min prior to hypoxia induction. The adenosine kinase inhibitor (ABT 702, 30 µM) was applied 10 min prior to adenosine/inosine (n = 9, each) and was present throughout the COGD period. Controls (CTL, n = 48) were exposed to hypoxia in complete culture medium. Data are shown as mean ± SEM. #P < 0.05 compared to CTL, *P < 0.05 compared to ADE or INO, +P < 0.05 compared to COGD.

Figure 6

Adenosine deaminase blockade reduces the protective effect of adenosine. Confluent NRK cultures were subjected to combined oxygen-glucose deprivation (COGD, n = 48) for 16 h and viability was measured after a 4-h-long recovery by the MTT assay (A, C), and cellular injury was determined by the LDH assay (B, D). Cells were pretreated with 100 µM adenosine (ADE, n = 9) (A, B) or 300 µM inosine (INO, n = 9) (C, D) 30 min prior to hypoxia induction. The adenosine deaminase inhibitor (EHNA) was applied at 10 µM prior to adenosine/inosine (n = 9, each). Controls (CTL, n = 48) were exposed to hypoxia in complete culture medium. Data are shown as mean ± SEM. #P < 0.05 compared to CTL, *P < 0.05 compared to ADE or INO, +P < 0.05 compared to COGD.

Figure 8

Adenosine and inosine preserve ATP content of NRK cultures during combined oxygen-glucose deprivation. Confluent NRK cultures were subjected to combined oxygen-glucose deprivation (COGD, n = 18) for 16 h. Cells were pretreated with 300 µM adenosine (ADE), inosine (INO) or glucose (GLUC) 30 min prior to hypoxia induction (n = 18, each). The adenosine kinase inhibitor ABT 702 (30 µM) or the adenosine deaminase inhibitor EHNA (10 µM) were applied prior to adenosine/inosine and glucose addition and were present throughout the COGD period. Values represent ATP content (pmol) of 10⁵ NRK cells. Controls (CTL, n = 18) were exposed to hypoxia in complete culture medium and showed similar ATP content to cells maintained under normoxic conditions (not shown). Adenosine or inosine, applied during normoxia, failed to affect cellular ATP levels (not shown). Data are shown as mean ± SEM (n = 18). #P < 0.05 compared to CTL, *P < 0.05 compared to COGD, +P < 0.05 compared to ADE, §P < 0.05 compared to INO and †P < 0.05 compared to GLUC.

Figure 9

Scheme representing some of the proposed metabolic pathways for the conversion of adenosine and inosine into different substrates for the glycolytic pathway. The diagram also shows an alternative way to produce energy in the absence of glucose during hypoxic injury.