**2. Redox-Sensitive Signaling in Kidney Diseases**

ROS at low levels act as secondary messengers, activating signaling pathways and cellular enzymes and regulating several cellular processes such as cell proliferation, survival, and growth [13]. Among the plethora of ROS, hydrogen peroxide (H2O2) and nitric oxide ( •NO) are the central redox signaling agents [13–16]. These ROS induce oxidative posttranslational modifications (Ox-PTMs) in proteins that contain redox-sensitive amino acid residues, regulating their structure, localization, and function [17,18]. These redox-sensitive amino acids are arginine (Arg), cysteine (Cys), histidine (His), lysine (Lys), methionine (Met), proline (Pro), threonine (Thr), and tyrosine (Tyr) [19,20]. However, Cys and Met are most prone to be attacked by ROS. These amino acids contain oxidizable sulfur groups on the side chains, of which the oxidation states depend on the redox microenvironment changes [13]. Thus, the protein function is regulated by the oxidate states of these amino acids [21,22].

Cys residues perform structural functions, such as the assembly of iron-sulfur (Fe-S) groups, heme prosthetic groups, and zinc finger motifs and are essential in the active sites of enzymes. The sulfur in Cys is a large, polarizable, electron-rich atom. These characteristics give it high reactivity and the ability to adopt multiple oxidation states. In addition, pKa influences the formation of the nucleophilic thiolate anion (S−). Because the surrounding milieu influences pKa, the thiol (SH) protonation form of Cys depends on the cellular microenvironment. For example, at physiological pH (pH 7.3), the pKa of Cys is 8.3, which means that Cys is in a protonated and less reactive state [23]. However, if the cellular microenvironment becomes alkaline (pH < 7.8), Cys can adopt a deprotonated state S−. Note that positively charged amino acids adjacent to Cys can substantially decrease the pKa of SH. Furthermore, the presence of other contiguous positive charges, such as the positive partial charge of a dipole, can also reduce the pKa of Cys, making them more reactive [24]. On the other hand, since Cys oxidation reactions are predominantly bimolecular nucleophilic substitution (SN2) reactions in the protonated form, steric hindrance can prevent Cys oxidation [25]. In this way, in the steric hindrance, the surrounding amino acids and redox microenvironment will determine whether a Cys will be reactive to undergo redox modification, dictating selectivity for SH modification. Therefore, there is a wide range of Ox-PTMs that also depend on the present electrophiles. For example, H2O2 oxidizes SH, with an oxidate state of −2, in an oxidative microenvironment, producing sulfenic acid (R–SOH) with an oxidate state of 0 [26]. R-SOH can react with proximal SH groups to form disulfide bonds (S–S), with a −1 oxidate state or s-glutathionylation (R-SSG) by reacting with glutathione (GSH) (Figure 1) [27,28]. This reaction is reversed by glutaredoxin (Grx). However, under OS, R-SOH forms sulfinic acid (R–SO2H) with a +2 oxidate state or sulfonic acid (R–SO3H) with a +4 oxidate state. The latter is not enzymatically reversible [29]. R–SOH can also be condensed with another R–SOH to form thiosulfinate [R–S(O)–S–R']. R–S(O)–S–R', in turn, reacts with amine or amide groups to form sulfenamide (R–SN– R') [30]. Additionally, Cys can suffer from other modifications such as S-nitrosylation and persulfonation. Regarding S-nitrosylation, it comprises the •NO covalent link to the thyil radical (R–S•) to form S-nitrosothiol (R–SNO), [31] and in persulfonation, the hydrogen sulfide (H2S) reacts with R–S• to form persulfide (R–S–SH) [20].

**Figure 1.** Reactive oxygen species (ROS) induce modifications in cysteine (Cys) and methionine (Met) residues. (**A**) ROS oxidize Cys residues in its thiol form, named thiol (SH) protein, forming (1) intermolecular bonds, (2) reversed by thioredoxin (Trx), or (3) S-glutathionylation (R–SSG). R–SSG can be reversed by (4) the glutaredoxin (Grx) enzyme, acquiring a sulfenic (R–SOH) form. Moreover, (5) R–SOH is formed through oxidation by H2O2 from SH. If oxidation continues, R–SOH forms (6) sulfinic (R–SO2H) or (7) sulfonic (R–SO3H); the last is irreversible. Furthermore, R–SOH can condense with another R-SOH to form (8) disulfide bonds (R–S–S–R') or (9) thiosulfinate [R–S(O)–S-R]. The latter can react with amide groups (–NH2), forming (10) sulfenamide (R–SN–R ). At the alkaline cellular microenvironment (pH > 7.8), R-SH proteins are deprotonated, forming (11) thiyl proteins (–S•). The latter can react with hydrogen sulfide (H2S) to form (12) persulfide (RSSH) by persulfination (R–S–S–H). –S• can also react with nitrosothiol (•NO), forming S-nitrosothiol (RSNO). (**B**) Met residues are oxidized to form (1) methionine sulfoxide [MetO (R–SOCH3)], reversed by (2) methionine sulfoxide reductase (Msr). However, if oxidation persists, MetO is further oxidized by H2O2 to (3) methionine sulfone [MetO2 (RSO2CH3)], which is irreversible. H2O2: hydrogen peroxide; HS•: hydrosulfide radical. Created with BioRender.com.

On the other hand, Met residues are oxidated by H2O2, generating methionine sulfoxide [MetO (R–SOCH3)] [32]. This oxidation is reversed by methionine sulfoxide reductase (Msr). However, if OS persists, MetO is transformed into methionine sulfone [MetO2 (RSO2CH3)], an enzymatically irreversible product [33].

The alkalinization of the mitochondrial matrix is due to pumping protons from the matrix into the intermembrane space (IMS). It has a tremendous impact on the potential reactivity of Cys, because alkalinization induces Cys protein residues to exist in an ionized state, which gives them a higher reactivity for ROS oxidation. Thus, mitochondria harbor a unique environment that promotes Cys modification.

As mentioned, there are Ox-PTMs classified as enzymatically reversible and irreversible. Ox-PTMs are reversible by thioredoxin (Trx), peroxiredoxin (Prx), Grx, glutathione peroxidase (GPx), Msr isoform A (MsrA), or the GSH [34,35]. Since reversible Ox-PTMs have enzymatic systems that remove oxidations, they are considered part of cellular signaling processes [34]. In contrast, irreversible ROS modifications are not considered part of cell signaling, because they do not have enzymatic reduction mechanisms [18].

Regarding cysteine persulfidation, it is a reversible Ox-PTM modification of SH to RSSH, which can be formed by reacting with H2S, more precisely HS−, and oxidized protein thiols, reaction between inorganic polysulfides and protein thiolates, and radical reaction by other reactive sulfur species [24]. Persulfidation regulates fine tune protein function, localization and interaction in cells [36]. It also modulates biological processes, including autophagy, cellular metabolism, inflammation, cell cycle, and cell death under physiological and pathological contexts [37]. It has been shown that the Cys' architecture and spatial arrangement determine if the residue can be persulfonated and the effect on protein function. Allosteric impediments protect Cys residues from being oxidized under SOH, preventing permanent damage and preserving protein function. Likewise, in a reduced environment, this Ox-PTM is reversed [38]. Additionally, persulfonation depends on ROS levels in the cells. The latter is supported, because the transient ROS increase might augment oxidized Cys forms (SOH and S–S), highly reactive to H2S [39]. Thus, the reversibility of this Ox-PTM relies on Cys residues and the cellular microenvironment.

Ox-PTMs inactivate numerous proteins that contain Cys groups, such as protein tyrosine phosphatases (PTPs). The inactivation of PTPs prevents the deactivation of protein tyrosine kinases (PTKs), maintaining the activation of cell signaling pathways, such as mitogen-activated protein kinases (MAPK), promoting cell proliferation [40]. In addition, ROS such as H2O2 have also been shown to promote Cys oxidation and the formation of S–S in proteins such as growth factor receptors (GFRs), inducing their activation and cell growth. Since oO-PTMs regulate cellular functions involving cell proliferation, growth, migration, differentiation, and death, the concentration of ROS must be balanced to maintain cell homeostasis and prevent kidney diseases [13].

In kidney physiology, 25% of mitochondrial Cys in proteins can suffer nitrosylation, and around 70% of these proteins depend on the activity of endothelial nitric oxide synthase (eNOS) [41]. These nitrosylations are protective in the kidneys. For instance, Zhou et al. [42] reported that in I/R, the denitrosylase enzyme aldo-keto reductase family 1 member A1 (AKR1A1) is associate with kidney damage. In contrast, its deletion is protective in this organ. The authors also found that S-nitroso-coenzyme A (CoA) reductase induces pyruvate kinase isoform M2 (PKM2) nitrosylation, inhibiting its activity. This nitrosylation favors the pentose phosphatase pathway instead of glycolysis. The latter reduces equivalents increase, attributed to ROS detoxification, which has a protector effect during I/R [42]. S-glutathionylation is also a protective Ox-PTM in renal mitochondria [43]. In the kidneys, the reduction of mitochondrial protein S-glutathionylation induced by folic acid is associated with acute kidney damage [44]. The latter has been demonstrated, because of glutathionylated proteins decrease and the levels of GSH and the activity of the enzymes involved in S-glutathionylation in the mitochondria 24 h (h) after treatment with folic acid [44]. In the cisplatin-induced AKI model, the reduction of Met is a protective Ox-PTM, since MsrA deficiency exacerbates cisplatin-induced damage by increasing mitochondrial susceptibility [45]. Furthermore, mice MsrA−/<sup>−</sup> are more sensible to kidney injury induced by I/R [46]. The deficiency of MsrA, cystathionine-β-synthase (CBS), and cystathionine-γ-lyase (CSE), which are enzymes involved in the transsulfuration pathway, decreases homocysteine and H2S [46]. Low levels of H2S have also been reported after unilateral ureteral obstruction (UUO) due to CBS and CSE decrease, inducing superoxide anion radical (O2 •−) and H2O2 production. The latter promotes the augment of oxidative damage markers such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) [47]. The MsrA deficiency in this model contributes to fibrosis development by increasing collagen deposition and augmenting fibrosis markers [48]. Therefore, MsrA regulates the Met metabolism and the production of H2S in the kidney [49].

Indeed, H2S regulates several kidney biological processes. CSE and CBS are expressed in the kidney, producing H2S that controls sodium reabsorption and glomerular filtration [50]. In addition, CSE is commonly expressed in endothelial and mesangial cells

and podocytes [51]. In kidney diseases, H2S ameliorates renal damage [52]. It has been shown that H2S attenuates kidney injury during I/R and diabetic kidney disease (DKD). In animal models of I/R, H2S donors administration before or after I/R ameliorates renal damage by decreasing OS, inflammation, and apoptosis [53]. Furthermore, in patients, it has been found that CSE messenger RNA (mRNA) expression positively correlates with glomerular filtration rate recovery after two weeks of kidney transplantation [54], suggesting a protective role of H2S. Supporting the latter, CBS and CSE levels and H2S production are low in animal models suffering I/R [53]. Regarding DKD, patients with type 2 diabetes show lowed H2S plasma levels than non-diabetic people [55]. In UUO, the low production of H2S is also due to the fact that CBS levels are decreased [56]. In this model, H2S decrease is related to fibrosis and inflammation development. Following this, in vitro, sodium hydrosulfide (NaHS) treatment reduces fibrosis and inflammation by inhibiting transforming growth factor-beta 1 (TGFβ1) [56]. Together, these data support the relevance of H2S signaling in kidney injury.

Cys persulfidation is an essential process in kidney physiology and disease, protecting against kidney injury [24]. Persulfidation in Cys 105 of mitochondrial glyceraldehyde 3-phosphate dehydrogenase (GAPDH) elevates its enzymatic activity [57]. In contrast, the persulfidation in Cys 156 or Cys 152 induces enzymatic deactivation [58]. Moreover, the persulfidation in Keap 1 Cys 150 promotes nuclear factor erythroid 2–related factor 2 (Nrf2) release and translocation to the nucleus, activating its genes targets [59]. This mechanism is critical for regulating redox homeostasis in the kidney. In renal pathologies, Nrf2 is deactivated, leading to OS increase [60,61]. In the kidney, persulfides regulate blood pressure and sodium reabsorption. In line with this, in tubular epithelial cells, the persulfidation of epidermal growth factor receptor (EGFR) (Cys 797 and 798) results in the loss of function of sodium/potassium-adenosine triphosphatase (Na+/K+-ATPase) [62]. Cys persulfide is produced by CSE and CSB, which are depleted in kidney diseases. However, a previous study showed that mice lacking CSE and CBS have significant levels of Cys persulfide proteins, which suggested the possibility of alternative processes [63–65]. Indeed, cysteinyl-transfer RNA (tRNA) synthetases (CARSs) act as cysteine persulfide synthases in vivo [66]. Moreover, CARSs are also involved in the regulation of mitochondrial bioenergetics (oxygen consumption and membrane potential) and dynamics (dynaminrelated protein 1 (Drp1)) [66]. Thus, this mechanism may be active in kidney diseases, which deserves investigation. The latter might suggest therapeutic targeting OS-induced mitochondrial dysfunction in renal pathologies.

#### **3. Ox-PTMs Regulate Manganese Superoxide Dismutase (Mn-SOD) in Kidney Injury**

Mn-SOD is in the mitochondrial matrix, while copper/zinc-SOD (Cu/Zn-SOD) is in the space of the inner mitochondrial membrane (IMM) and IMS. These two enzymes catalyze the dismutation of O2 •− to H2O2 in the mitochondrial matrix and IMS, respectively [67]. This dismutation is crucial to avoid the O2 •−-induced ferric iron (Fe3+) to ferrous iron (Fe2+) reduction in Fe-S clusters of critical enzymes such as aconitase (Acn). The latter leads to the release of Fe2+ and the inactivation of these enzymes [68]. During Fenton/Haber–Weiss reaction, Fe2+ reacts with H2O2 to produce a highly reactive ROS, hydroxyl radical (•OH), so the regulation of O2 •− is essential to maintain mitochondrial homeostasis. Moreover, O2 •− overproduction, directly and indirectly, leads to the inactivation of Mn-SOD, promoting mitochondrial dysfunction. For instance, O2 •− can react with •NO to produce peroxynitrite (ONOO−), and ONOO− can induce Mn-SOD deactivation via nitration of Tyr34 residue in its active site [69].

Mn-SOD can also be S-glutathionylated in Cys 196, avoiding irreversible oxidation of SH. Renal I/R injury induces O2 •− and ONOO− production [70], increasing nitration levels of mitochondrial proteins such as Mn-SOD and cytochrome c (cyt c), inactivating them and inducing OS and mitochondrial dysfunction [71–74]. Likewise, in folic-acid-induced renal damage, Mn-SOD activity reduction in isolated mitochondria is related to decreased mitochondrial S-glutathionylation [44], making it more susceptible to nitration. Moreover, in human renal transplants and experimental rat models of chronic renal nephropathy, there are elevated levels of ONOO− [72].

In DKD, mitochondrial OS reduces Mn-SOD enzyme activity due to Tyr nitration of this enzyme. Interestingly, the use of resveratrol, a potent antioxidant, reduces OS and Tyr nitration of Mn-SOD, preserving Mn-SOD activity. Moreover, kidneys of mice treated with streptozotocin to induced diabetic nephropathy (DN) show nitration in Mn-SOD Tyr 34, which results in a decrease of Mn-SOD. However, antagonists of thromboxane A2 receptors reduce diabetes-induced renal injury, which is associated with Mn-SOS Tyr nitration reduction [75].

In models of hypertension-related kidney injury, where hypertension is induced by angiotensin II (Ang II), the production of O2 •− is promoted through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) [76]. In these models, Mn-SOD activity deactivation is also associated with Tyr nitration, inducing OS [77]. Moreover, spontaneously hypertensive rats treated with N(G)-nitro-L-argininemethyl ester (L-NAME), a potent nitric oxide synthase (NOS) inhibitor, reduce nitration of Mn-SOD, preserving its activity [78].

OS closely regulates aging-related kidney dysfunction, primarily by mitochondrial ROS (mtROS). A protein closely associated with aging is Klotho. This protein induces the activation of transcription factors such as forkhead box O (FoxO) that cause the expression of antioxidant enzymes such as Mn-SOD. Interestingly, Klotho−/<sup>−</sup> mouse models show high nitrotyrosine levels in Mn-SOD [79].
