1.3 Potential mechanisms underlying ROS inhibition by SGLT-2i’s
Several factors contribute to the anti-oxidative effect of SGLT-2i’s.
SGLT-2i’s revert upregulation of NOXs and inhibit oxidative stress in
the macro- and micro- vascular system (Ganbaatar et al. , 2020;
Kuno et al. , 2020). In diabetic mice, EMPA reduced NOX2
expression at messenger RNA (mRNA) levels in aortic endothelium
(Ganbaatar et al. , 2020). Correspondingly, EMPA also suppressed
the increase of NOX2 and NOX4 in renal tissue of rats with acute kidney
injury (Kuno et al. , 2020). An in vitro study showed that
EMPA exerted a similar inhibitory capacity like GKT136901, a specific
inhibitor for NOX1 and NOX4, on ROS generation in HCAECs undergoing
enhanced stretch. Combination of EMPA and GKT136901 did not further
reduce the stretch-induced ROS production, suggesting that the
anti-oxidative effect of EMPA is mediated via NOXs (Li et al. ,
2021). Furthermore, EMPA prevented hyperglycaemia-induced mitochondrial
disruption, thereby attenuating the overproduction of cytosolic ROS and
mitochondrial ROS (mtROS) in ECs isolated from mice and humans (Juniet al. , 2021; Zhou et al. , 2018). This mechanism is
further supported by the fact that induction of mitochondrial fission
abrogated the inhibitory effects of EMPA on mtROS in mice CMECs (Zhouet al. , 2018).
Another potential mechanism that might explain the antioxidant effects
of SGLT2i’s is the direct inhibition of the sodium-hydrogen exchanger
(NHE) by SGLT2i’s, firstly discovered in CMs (Baartscheer et al. ,
2017; Uthman et al. , 2018). A recent study from our laboratory
showed that 10 µM cariporide blocked the increase of oxidative stress in
HCAECs undergoing enhanced cyclic stretch, and this effect of cariporide
on ROS production was not further enhanced when combined with EMPA.
These data indirectly suggest the involvement of NHE in the
anti-oxidative capacity of EMPA in ECs (Li et al. , 2021). For the
first time, Uthman et al directly proved that EMPA inhibited ROS
production in ECs via NHE inhibition: EMPA treatment lowered the NHE
activity and Na+ concentration in human ECs triggered
by TNF-α (measured with SNARF-AM and SBFI-AM fluorescence probes
respectively), and also mitigated the increased ROS production. The
combination of EMPA and cariporide did not demonstrate additional ROS
reduction in cells, showing that EMPA reduced TNF-α induced ROS
production via NHE inhibition (Uthman et al. , 2022).
Yet, there is still an ongoing discussion regarding the role of NHE in
the inhibitory effect on ROS of SGLT-2i’s. In support of our finding,
Cappetta et al. previously reported NHE inhibition by DAPA in
“non-stimulated” human umbilical vein endothelial cells (HUVECs)
(Cappetta et al. , 2020). In contrast, using cardiac microvascular
ECs exposed to uremic serum, Juni et al. recently observed a stronger
ROS inhibitory capacity of 1 µM EMPA when compared to 10 µM cariporide
(63% vs 38%), indicating that part of the anti-oxidative effect of
EMPA might be unrelated to NHE inhibition (Juni et al. , 2021).
Chung et al. reported a neutral effect of EMPA (1-30 µM) on NHE activity
within isolated rat CMs (Chung et al. , 2020), which is in
contrast to the studies of Baartscheer et al., Uthman et al. and
Zuurbier et al. showing that 1 µM EMPA inhibited the NHE activity in
both isolated cardiac myocytes and isolated intact hearts of different
rodents (mice and rabbits) (Baartscheer et al. , 2017; Uthmanet al. , 2018; Zuurbier et al. , 2021). Diversities in the
employed methodology might explain the differences between these
studies.
The involvement of sodium glucose co-transporter 1/2 (SGLT-1/2) in the
anti-oxidative effect of SGLT-2i’s has been recently investigated.
Recent studies showed that high glucose and angiotensin II (Ang II)
increased the expression of SGLT-1 and -2 in porcine ECs, and that EMPA
showed an inhibitory effect on the induced SGLT-1/2 expression
(Khemais-Benkhiat et al. , 2020; Park et al. , 2021). At 24
h, sotagliflozin (a dual inhibitor for SGLT-1 and -2) and empagliflozin
abolished the Ang II-induced ROS production. Reduction of extracellular
glucose and Na+ concentrations significantly inhibited
the pro-oxidant reaction to Ang II, indicating the crucial role of
SGLT-1 and -2 in a glucose and sodium dependent ROS production (Parket al. , 2021). Intriguingly, the sustained oxidative stress
triggered by Ang II could also be alleviated by inhibition of NHE, NCX
and NOXs, further supporting the functional link between
NHE/Na+/Ca2+ pathway and ROS
production by NOXs within ECs (Park et al. , 2021). However,
expression of SGLT-2 in ECs is still a matter of debate, especially in
the case of human cells. Mancini et al. showed the absence of SGLT-2 at
mRNA level in HUVECs (Mancini et al. , 2018), corresponding with
the most recent study of Juni et al. using human CMECs (Juni et
al. , 2021). In contrast, using Western blot, Behnammanesh et al.
detected the presence of SGLT-2 in human ECs (Behnammanesh et
al. , 2019). Uthman et al. also reported a potential existence of SGLT-2
in human ECs at protein level with a commercially available antibody
(Uthman et al. , 2019). But this signaling for SGLT-2 protein
persisted after the target gene being silenced at mRNA level, and the
qPCR revealed no existence of SGLT-2 (Uthman et al. , 2019).