The Effect Of Intermedin (IMD) On Hypoxia/Reoxygenation-induced NRK-52E Apoptosis

Objective The hypoxia/reoxygenation(H/R) injury models of rat renal tubular epithelial cells (NRK-52E), which were transfected the eukaryotic expression vector of rat IMD, were established to simulate I/R injury in vivo, to investigate the effect and mechanism of intermedin (IMD) on hypoxia/reoxygenation(H/R)-induced rat renal tubular epithelial cells (NRK-52E) apoptosis.Methods (1) To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 4h and then with reoxygenation for 12h. The models were evaluated by detecting living cell count, cell viability and the activity of lactate dehydrogenase (LDH) in the culture medium. (2) NRK-52E cells were transfected by transfection complex comprising optimal proportion of pIRES2-EGFP/IMD plasmid and Fugene HD reagents. Transfection efficiency was tested by observation for EGFP made by fluorescent microscope and flow cytometery (FCM) after 48h. The cell groups with high transfection efficiency were screened for two weeks or so by incubating with medium containing G418(300μg/ml), and the positive cloning NRK-52E cells of stable expression IMD obtained. (3) NRK-52E cells were divided into 4 groups:control group, model group, primitive vector group and IMD vector group. The last three groups were exposed to H/R condition, and the apoptosis of NRK-52E cells were determined by flow cytometry, the serine-threonine kinase (Akt) activity were determined by enzyme linked immunosorbent assay, and the Caspase-3 activity were determined by colorimetry after H/R.Results (1) After hypoxia 4h and reoxygenation 12h, cell count and cell viability decreased significantly and the activity of LDH increased significantly; the model was established successfully. (2) NRK-52E cells was transfected with the optimal transfection proportion of pIRES2-EGFP/IMD to Fugene HD reagent as 3μg:12μl, transfection efficiency evaluated by fluorescence microscope and FCM showed that the transfection efficiency increased with the prolongation of time. The positive transfected cells after G418 screening were found clone-like proliferation on the third day, and the untransfected cells died in large bulk on the fifth day. Then the positive transfected cells increased gradually; and the positive cloning cells blending into slices were found on the fourteenth day. (3) Phosphorylated Akt:Compared with the control group, the level of Akt phosphorylation in last three groups significantly increased after H/R. Compared with model group and the empty vector group, Akt phosphorylation levels significantly increased in IMD group. There is no significant diffenrence between model group and the empty vector group. (4) Caspase-3 activity:Compared with the control group, the activity of Caspase-3 in last three groups significantly increased after H/R. Compared with model group and the empty vector group, the activity of Caspase-3 significantly reduced in IMD group. There is no significant diffenrence between model group and the empty vector group. (5) Apoptosis:Compared with the control group, the apoptosis of the cells in last three groups significantly increased after H/R. The apoptosis of the cells asignificantly reduced in IMD group, contrasted with model group and primitive vector group, respectively. There is no significant diffenrence between model group and the empty vector group.Conclusion IMD may against hypoxia/reoxygenation-induced NRK-52E apoptosis by activating Akt to decrease Caspase-3 activity.