4. Discussion
Pharmacological intervention targeting SGLT2 has recently attached great
concern as a novel therapeutic option for diabetes. Increasing evidences
have confirmed the extra renal protective effect of SGLT2 inhibitors
beyond the controlling of hyperglycemia.(Perkovic et al., 2019) However,
the mechanism through which SGLT2 inhibitors exert the renal protective
effect has been rarely reported. Here we find that the Empa can relieve
the tubular injury by alleviating the mitochondrial fission though an
AMPK-dependent pathway in diabetic mice. By activating AMPK, Empa can
remarkably rescues the dephosphorylation of DRP1S637 and inhibits the
mitochondria-translocation of DRP1, which eventually result in less
mitochondrial fission. The present study revealed the existence of a
glycemia independent mechanism of Empa that protects against
mitochondrial dysfunction and alleviates the tubular injury in diabetic
mice and HK2 cells.
DKD was traditionally considered as a
glomerular disease characterized
by mesangial expansion, reduction in capillary surface, and podocyte
loss which differ it from other glomerulonephritis.(Thomas et al., 2015)
However, increasing evidences have revealed the primary contribution of
tubular injury in the pathogenesis of DKD.(Bonventre, 2012; Tang & Lai,
2012) In the present study, we found the urinal biomarkers NAG and NGAL
which reflect the early tubular injury increased obviously at the
10th week without change in albuminuria in KK-Ay mice.
Since the albuminuria is an early clinical manifestation of DKD and
reflects glomerulopathy, our finding verifies the notion that diabetic
tubulopathy occurs earlier than
glomerular disease.
Recent clinical trials have confirmed the renal protective effect of
SGLT2-inhibitors(Mahaffey et al., 2018; Wanner, Inzucchi & Zinman,
2016). In present study, both the TUNEL and Western Blotting assay
consolidated that the Empa could alleviate apoptosis in the kidney of
KK-Ay mice. Recent animal and clinical studies have confirmed the
critical role of mitochondrial fission in the pathogenesis of
DKD.(Galvan et al., 2019; Jiang et al., 2019) However, the signaling
pathways by which hyperglycemia leads to mitochondrial fission are not
fully understood.(Wang et al., 2012) Here we found that the DRP1S637
decreased significantly and the mitochondria translocation of DRP1
increased remarkably in the kidney of KK-Ay mice. The DRP1
phosphorylation on serine-637 was previously reported to be involved in
Parkinson’s disease,(Dagda et al., 2011) myocardial ischemia-reperfusion
injury(Sharp et al., 2014) and regulation of ER morphology in stressed
pancreatic β-cells through its anti-fission effect.(Wikstrom et al.,
2013) Zhou et al. reported that the Empa could rescue the
dephosphorylation of DRP1S637 in cardiac microvascular endothelial cells
and alleviated the mitochondrial fission, but the intrinsic mechanism
remained unclear.(Zhou, Wang, Zhu, Hu, Chen & Ren, 2018)
To detect the exact mechanism through which the Empa impacts the DRP1
phosphorylation, we conducted a series of investigations. In present
study, we found an obviously decreased phosphorylation of AMPK and
DRP1S637 in KK-Ay mice kidney, and this change was reversed in the Empa
group. We presumed that the Empa may rescue the dephosphorylation of
DRP1S637 and alleviate the mitochondrial fission via an AMPK dependent
pathway. We also found an obvious increase of SP1 and PGAM5 expression
in KK-Ay mice kidney which were
reversed in Empa group. These findings remind us that some causative
relationship may exist among the AMPK, SP1 and PGAM5.
To verify our deduction, we performed in vitro experiments using HK2
cells. We found that the apoptosis increased significantly in HG treated
cells. Meanwhile, the increased mitochondrial fission and disrupted
mitochondrial membrane potential were detected in HK2 cells subjected to
HG. All these changes were alleviated in Empa treated cells. These
findings were consistent with the in vivo experiments.
Then we tested the phosphorylation of AMPK, the expression of SP1 and
PGAM5 in HK2 cells to testify our presumption. Here we found that the
AMPK activity decreased obviously in HG treated cells and the
expressions of SP1 and PGAM5 increased significantly. These changes were
rescued in Empa treated cells. Those above findings confirmed the
involvement of AMPK, SP1 and PGAM5 in the development of mitochondrial
fission and the Empa’s anti-fission effect. But if there is a causality
among these changes was unclear. So, we introduced the AMPK activator
and inhibitor to our study. We found that not only the AMPK activator
but also the Empa could increase the AMPK phosphorylation. Both the two
chemicals could decrease the SP1 and PGAM5 expressions and alleviate the
mitochondrial fission. However, the Compound C, an AMPK inhibitor
reversed the declining of SP1 and PGAM5 and blocked the anti-fission
effect in Empa treated cells. These findings suggested that the AMPK
located at the upstream of the AMPK/SP1/PGAM5 axis and played a central
role in the process of HG induced mitochondrial fission in HK2 cells.
To further illustrate how the SP1 and PGAM5 participate in the
development of the mitochondrial
fission, si-SP1 and si-PGAM5 were
introduced to our later investigations. We found that both the si-SP1
and si-PGAM5 could alleviated the HG induced mitochondrial fission, but
neither of them could activate the AMPK. Meanwhile, the Empa could not
only activate the AMPK but also decrease the SP1 and PGAM5 expression
and relive the HG induced mitochondrial fission in HK2 cells. These
phenomena consolidated our deduction that the AMPK located at the
upstream of the AMPK/SP1/PGAM5 axis and the
Empa could alleviated the
mitochondrial fission via an AMPK dependent pathway. Meanwhile, we found
that the si-SP1 could decrease the PGAM5 expression, but the si-PGAM5
had no effect on the SP1 protein. This result suggests that the SP1
could mediate the PGAM5 expression but it is not contrarily. Finally, we
verified the direct interaction between the SP1 and the PGAM5 by
luciferase reporter assay and chromatin immunoprecipitation (ChIP), and
our findings clearly showed that the SP1 could bind to the promotor of
PGAM5 at -539 nt and increased its expression.
In conclusion, the present study depicted an AMPK regulated and PGAM5
involved mitochondrial fission pathway in the development of diabetic
tubular disease (Figure 6), and we also revealed
a glycemia independent mechanism
of Empa that protects against the diabetic tubular injury.
Our finding could not only provide
the experimental and theoretical basis for the expansion of new
indications of Empa, but also revealed a new prospective therapeutic
target for DKD.