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).