3.4. The expression level of AMPK phosphorylation, SP1 and PGAM5 were recovered in Empa treated KK-Ay mice.
We found an obvious decrease of the AMPK phosphorylation and increased PGAM5 expression in KK-AY group and these changes were rescued in the Empa group (Fig. 2F, H). These phenomena remind us a possible connection between the AMPK and PGAM5, thus there may be a mediator correlated the AMPK activation to the PGAM5 expression. By searching the JASPAR online database, we selected the transcription factor SP1, which is predicted to be bound to the PGAM5 promoter region with high scores for analysis (Fig. 5A). Moreover, we found an opposite trend of change between the AMPK phosphorylation and the SP1 expression, thus the AMPK phosphorylation decreased and the SP1 expression increased in KK-AY group and all the changes were reversed in Empa group (Fig. 2F, H).
Lastly, we examined the changes of apoptosis and mitochondrial fission in the INS group, and we found that though the insulin administration could reverse the hyperglycemia in KK-Ay mice, no obvious alleviation of apoptosis and mitochondrial fission were founded. Meanwhile, no significant changes of SP1, PGAM5 and phosphorylated AMPK were detected. These findings reminded us that controlling hyperglycemia could not alleviate apoptosis and mitochondrial fission in diabetic kidney, thus the Empa’s anti-apoptosis and anti-fission effect was independent of its glycemia controlling function.
3.5. The increased apoptosis and mitochondrial fission in HG treated HK2 cell were alleviated in Empa exposed cells.
We next investigated the changes of apoptosis and mitochondrial dynamics in vitro by HK2 cells. According to the CCK8 assay (Supplemental Figure A), we selected the 8 umol/ml as the testing concentration of Empa in our in vitro study. Immunoblotting demonstrated notable increase of pro-apoptotic proteins such as BAX, Cleaved-CASPASE 3 in the HG group. Whereas the anti-apoptotic protein BCL-2 was down-regulated (Fig. 3A, E, F). The mito-CYT decreased significantly in the HG group which also proved the increase of apoptosis (Fig. 3C, F). However, all these effects were partially reversed by Empa administration. No significant difference of the BAX, Cleaved-CASPASE 3, mito-Cytc was found between the NG and MA group (Fig. 3A, C, E, F).
We next examined the changes of mitochondrial dynamics among the different groups in vitro. Decreased DRP1S637 expression was noted in HK2 cells subjected to HG, and no change of Drp1 was found (Fig. 3A, G, H). Meanwhile, we found an obvious increase of mito-Drp1 in cells subjected to HG (Figs 3C, H) and no notable change of Mfn2 was detected among the four groups (Fig. 3A, G). We also investigated the mitochondrial membrane potential (MMP) with JC-1 staining. We found that the HG treated cells presented with collapsed mitochondrial membrane potential, as evidenced by a higher green fluorescence and lower red fluorescence, then the red fluorescence was recovered in cells subjected to Empa (Fig. 3B). Lastly, we depicted the morphological change of mitochondria with Mito-Tracker Red imaged by laser scanning confocal microscopy, we found that the HG exposed cells showed small punctate and rounded mitochondria. However, mitochondria from Empa-treated cells recovered filamentous and tubular shape again (Fig. 3D).