Intermedin/adrenomedullin 2 protects against tubular cell hypoxia-reoxygenation injury in vitro by promoting cell proliferation and upregulating cyclin D1 expression.

AIM

Intermedin/adrenomedullin 2 (IMD/ADM2) is a newly discovered peptide closely related to adrenomedullin. We recently reported that IMD/ADM2 gene transfer could significantly reduce renal ischaemia/reperfusion injury. In this study, we evaluated the effect of IMD/ADM2 on cell proliferation and regeneration in a cultured rat renal tubular epithelial cell line (NRK-52E) of hypoxia-reoxygenation (H/R) injury.

METHODS

The H/R model in NRK-52E cells consisted of hypoxia for 1 h and reoxygenation for 2 h. IMD/ADM2 was overexpressed in NRK-52E cells using the vector pcDNA3.1-IMD. Enzyme-linked immunosorbent assays were used to measure the concentration of IMD/ADM2 in the culture medium, and real-time PCR and Western blotting were used to determine mRNA and protein levels. In addition, luciferase reporter assays and electrophoretic mobility-shift assays were performed to measure cyclin D1 promoter activity and transcription factor activity.

RESULTS

We found that IMD/ADM2 gene transfer markedly promoted cell viability and decreased lactate dehydrogenase (LDH) activity and cell apoptosis compared with that of H/R. IMD/ADM2 increased the phosphorylation of ERK and decreased the phosphorylation of JNK and P38. Furthermore, IMD/ADM2 promoted cell cycle progression with concomitant increases in the levels of cyclin D1 and cyclin E, and these effects were blocked by the inhibition of ERK, or the agonist JNK and P38. IMD/ADM2 also increased cyclin D1 promoter activity and AP-1 DNA-binding activity.

CONCLUSIONS

We demonstrated that IMD/ADM2 promotes renal cell proliferation and regeneration after renal H/R injury by upregulating cyclin D1 and that this upregulation seems to be mediated by the ERK, JNK, and P38 MAPK signalling pathways.