**1. Introduction: The Redox System**

In general terms, the reduction-oxidation (redox) system consists of the coupling and coordination of various electron gradients that are generated by serial reduction-oxidation enzymatic reactions. These reduction-oxidation reactions happen in fundamental biological processes in every cell [1] and lead to the production of radical oxidants or free radicals [2]. Free radicals are in fact oxygen or nitrogen metabolites that contain an unpaired electron and are thus partially reduced [3]. These free radicals have strong oxidizing capacity and can be classified into reactive oxygen species (ROS) and reactive nitrogen species (RNS) [4] (Table 1).

**Table 1.** Main known reactive oxygen species (ROS) and reactive nitrogen species (RNS).


**Citation:** Llorens-Cebrià, C.; Molina-Van den Bosch, M.; Vergara, A.; Jacobs-Cachá, C.; Soler, M.J. Antioxidant Roles of SGLT2 Inhibitors in the Kidney. *Biomolecules* **2022**, *12*, 143. https://doi.org/ 10.3390/biom12010143

Academic Editor: Liang-Jun Yan

Received: 16 December 2021 Accepted: 13 January 2022 Published: 16 January 2022

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The redox system is especially important during energy metabolism to generate adenosine triphosphate (ATP). The most important source of energy is glucose which is transformed into pyruvate via glycolysis. Pyruvate enters the tricarboxylic acid or Krebs cycle and reduces nicotinamide adenine dinucleotide (NAD) to NADH. Further, fatty acids (FA) can undergo β-oxidation to generate acetyl-CoA which then enters the Krebs cycle [5,6]. The NADH produced during the Krebs cycle is oxidized again in the mitochondrial respiratory chain [7]. The complexes that constitute the mitochondrial respiratory chain are NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V). These enzymes are able to oxidize NADH, starting the electron transport across the mitochondrial membrane and generating an electrochemical potential that allows complex V to generate ATP. Additionally, the electrons transported from complex I, through II and III, to complex IV are used to reduce oxygen to water [8,9]. ROS are generated as a byproduct of electron transfer. Therefore, mitochondria are considered the main source of ROS [10]. Approximately, 1–2% of the O2 consumed in the mitochondria is incompletely metabolized [11]. Here, the predominant origin of ROS is the respiratory chain (mainly complexes I and III), necessary for ATP synthesis. However, other mitochondrial enzymes have been reported to be sources of ROS, such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, or succinate dehydrogenase, which are a part of the Krebs cycle [12]. Additionally, p66shc, an adaptor protein with proapoptotic activity, is involved in the production of ROS [13]. In vitro and in vivo experiments have shown that this protein can generate ROS by oxidizing cytochrome C [14,15]. The NADPH oxidase (NOX) family of enzymes are another source of ROS. NOX are multiunit enzymes that use NADPH as an electron donor to reduce oxygen, leading to the generation of superoxide. There are seven members of the NOX family, NOX1–5 and DUOX1 and 2. NOX enzymes are expressed in different tissues and differ in the domains that constitute them, for example, DUOX1 and 2 have an additional peroxidase domain at their N-terminal end [16,17] (Figure 1).

**Figure 1.** Sources of reactive oxygen species. Reactive oxygen species (ROS) are generated at different sites within the cell. Respiratory chain complexes I and III (in blue) produce ROS as a byproduct of electron transfer in mitochondria. Several enzymes involved in the Krebs cycle are also reported as sources of ROS. p66shc (in purple), by oxidizing cytochrome c (Cyt C), is able to generate ROS within the mitochondria intermembrane space. The NADPH oxidase (NOX) family of enzymes (in brown) are another important source of ROS by reducing oxygen to generate superoxide. MIM: mitochondria inner membrane.

Regarding RNS, these comprise nitric oxide (NO) and its derivatives, such as peroxynitrite (ONOO−), dinitrogen trioxide (N2O3), dinitrogen tetraoxide (N2O4), nitrogen dioxide (NO2), or S-nitrosothiols (RSNO) [18,19] (Table 1). NO is produced from the metabolism of L-arginine [20]. The enzyme responsible for its synthesis is nitric oxide synthase (NOS) that converts L-arginine into L-citrulline, forming NO during the reaction. There are three forms of NOS: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). eNOS and nNOS are constitutively expressed while iNOS is only expressed under specific stimuli like infection or trauma. The different NOS isoforms are expressed in different types of cells in which NO is involved in signaling and regulation of several cellular functions [21]. In mitochondria, NO reacts with complex III of the respiratory chain, inhibiting the electron transfer and enhancing the production of O2 −. O2 − is highly reactive and may combine with NO, leading to the formation of peroxynitrite (ONOO−). ONOO− is a powerful RNS that can irreversibly inhibit electron transport, which is pathological [18,22–24].

Both ROS and RNS are necessary for normal cell function as they modulate cellsignaling pathways and cellular processes. However, overproduction of any of them is pathological since it can damage macromolecules like proteins, lipids, or DNA [25]. DNA damage can lead to defective complexes I and III, which may result in a shutdown of mitochondrial energy production [26] and further increase ROS levels [27,28]. This eventually ends up causing mitochondrial damage and apoptosis [29,30]. Therefore, to regulate the generated ROS, there is a network of antioxidant systems to alleviate this stress. Superoxide dismutase (SOD) is an enzyme that contributes to the elimination of some oxidants. According to its subcellular location, we can differentiate between SOD1 (in the cytosol and mitochondrial intermembrane space), SOD2 (in the mitochondrial matrix), and SOD3 (in the extracellular matrix) [31]. SOD enzymes convert the superoxide radical into hydrogen peroxide [32,33] that is finally detoxified by catalase or glutathione peroxidase [34,35]. Catalases decompose H2O2 into water and oxygen, and glutathione peroxidases reduce H2O2 to water [36]. Glutathione has also a role in the antioxidant system, as it can either react directly with ROS and RNS or act as a cofactor for various enzymes helping the cell to maintain its redox status [37]. Further, exogenous and synthetic antioxidants can also be effective to prevent oxidative stress [38,39].

In this review, we will focus on oxidative stress-mediated mechanisms of kidney tubular dysfunction. Further, the contribution of the sodium/glucose co-transporter type 2 (SGLT2) in diabetic kidney disease (DKD) onset and the antioxidant potential of the SGLT2 inhibitors will be discussed.

#### **2. Oxidative Stress-Induced Tubular Impairment**

The tubular system has a very precise and highly regulated transport capacity between the tubule lumen and the bloodstream. A broad range of tubular transporters contribute to ion, amino acid, glucose, and other solute reabsorption. Therefore, tubular cells require a sufficient energy supply to sustain the high rate of exchange within the two compartments. Tubular cells are the most energy-demanding cells of the body after cardiac cells [40]. Their metabolism is highly dependent on oxygen consumption to produce ATP by oxidative phosphorylation. The oxidation of a glucose molecule provides a high energy yield (30–32 ATP) and is used as a substrate in every cell. However, in the kidney, each section of the tubule has its own preferences in terms of energy substrates, including amino acids, ketone bodies, or FA. Strikingly, FAs are an important energy fuel in tubular cells, providing 106–129 ATP through β-oxidation within the mitochondria [41]. Proximal tubular cells (PTC), located at the first section of the tubular system (segments S1, S2, and S3), are particularly energy-consuming, as 80% of the biomolecules filtered by the glomerulus are reabsorbed by these cells [42,43]. In PTC, ATP generation relies mostly on oxidative phosphorylation of FA rather than of glucose. However, under pathological conditions, such as acute kidney injury (AKI), which is associated with hypoxia [44], PTCs undergo a metabolic shift towards glycolysis [45].

As mentioned above, mitochondrial metabolism is a source of ROS that directly modulates cell-signaling pathways, protein function through post-translational modifications, and influences cell survival, proliferation, and apoptosis [46]. However, increased ROS production results in oxidative stress that causes damage to lipids, proteins, and nucleic acids, eventually disrupting cellular homeostasis. Overall, elevated ROS levels can lead to mitochondrial dysfunction, bioenergetics defects, altered gene expression, and cell death. PTCs have a high mitochondrial density that makes them susceptible to ROS-induced cell damage [42]. In vitro experiments performed using the HK-2 cell line (human PTCs) demonstrated an increase of oxidative stress, mitochondrial destabilization, DNA damage, apoptosis, and cell senescence when treated with hydrogen peroxide (H2O2) [47]. Aragno et al. showed that rats subjected to unilateral ischemia/reperfusion (I/R) injury exhibit high hydrogen peroxide levels in the cytosol obtained from kidney homogenate and high levels of nitrite/nitrate in serum compared to control and sham-operated animals. Consequently, the proximal tubules showed dilation and cell debris and cast formation in the lumen. These effects were attenuated in rats supplemented with dehydroepiandrosterone (DHEA), a steroid with antioxidant properties [48]. Beyond the respiratory chain, there are other sources of oxidants in the kidney. NOX enzymes have been recognized as a primary source of ROS in the kidney, especially NOX4. NOX4-derived H2O2 mediates several cell functions, but excessive levels can induce inflammation, apoptosis, fibrosis, and cell damage. NOX4 has constitutive activity, hence, the amount of H2O2 depends on the expression level. As a result, it can be damaging or beneficial depending on its abundance at the given time [49]. Normally, the levels of ROS increase in some AKI and chronic kidney disease (CKD) models, secondary to the overexpression of NOX4 [50]. Upregulation of renal NOX4 plays an important role in several pathologies, like DKD, hypertensive nephropathy, and polycystic kidney disease by increasing ROS levels and mitochondrial damage [51–55]. It has been described that mitochondrial ROS and NOX-produced ROS can enhance damage and depolarization of the mitochondrial membrane potential [46,56,57]. The total absence of NOX4 has also been reported to be pathological since it results in fibrosis and oxidative stress [58]. This could be in part related to the fact that H2O2 generated by NOX4 enhances nuclear factor erythroid 2-related factor 2 (Nrf2) stability, a regulator of antioxidant activity [59].

Excessive ROS production can damage mitochondria [60]. Mitochondrial damage has been recognized as a main contributor to tubular necrosis and apoptosis. Tubular cell necrosis involves disruption of respiration complexes, loss of mitochondrial membrane potential, and mitochondrial membrane transition, while apoptosis is caused by mitochondrial outer membrane permeabilization and release of apoptogenic factors [61]. Mitochondrial damage secondary to necrosis or apoptosis is especially problematic for the tubular cells as it may compromise its normal function and ultimately lead to renal dysfunction. Despite the effects of oxidative stress in kidney impairment having been studied extensively [42,46], its complexity and interconnection with a broad range of metabolic and cellular pathways make it puzzling to decipher its full implications in the onset, progression, and development of renal pathology.
