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).