Oxidative Stress and Ischemia/Reperfusion Injury in Kidney Transplantation: Focus on Ferroptosis, Mitophagy and New Antioxidants

Although there has been technical and pharmacological progress in kidney transplant medicine, some patients may experience acute post-transplant complications. Among the mechanisms involved in these conditions, ischemia/reperfusion (I/R) injury may have a primary pathophysiological role since it is one of the leading causes of delayed graft function (DGF), a slow recovery of the renal function with the need for dialysis (generally during the first week after transplantation). DGF has a significant social and economic impact as it is associated with prolonged hospitalization and the development of severe complications (including acute rejection). During I/R injury, oxidative stress plays a major role activating several pathways including ferroptosis, an iron-driven cell death characterized by iron accumulation and excessive lipid peroxidation, and mitophagy, a selective degradation of damaged mitochondria by autophagy. Ferroptosis may contribute to the renal damage, while mitophagy can have a protective role by reducing the release of reactive oxygen species from dysfunctional mitochondria. Deep comprehension of both pathways may offer the possibility of identifying new early diagnostic noninvasive biomarkers of DGF and introducing new clinically employable pharmacological strategies. In this review we summarize all relevant knowledge in this field and discuss current antioxidant pharmacological strategies that could represent, in the next future, potential treatments for I/R injury.


Introduction
Kidney transplantation represents the most cost-effective modality of renal replacement therapy for patients with irreversible chronic kidney failure (end-stage renal disease, stage 5 chronic kidney disease) [1]. However, despite continuous technical and pharmaceutical progress in transplant medicine, some patients develop early acute post-transplant complications and experience a slow recovery of the renal function with the need for dialysis (generally during the first week after transplantation). This clinical condition, namely delayed graft function (DGF), has a significant social and economic impact as it is associated with prolonged hospitalization [2], poly-pharmacological approaches (particularly in the presence of concomitant acute allograft rejection) [3], and shorter graft survival [4].
The risk of DGF is higher in specific organ transplant programs using kidneys from non-heart-beating, elderly, multimorbid (e.g., diabetes, hypertension) donors, recipients with a previous allograft failure and/or allosensitized, and organs damaged by acute kidney injury and prolonged cold ischemia time [5,6].

Ferroptosis: Role in Kidney Allograft I/R Injury
Ferroptosis is a form of regulated cell death driven by iron accumulation, lipid peroxidation and subsequent plasma membrane rupture [27]. It is mainly characterized by: a nucleus lacking chromatin condensation, mitochondria with reduced volume and cristae, significant cell enlargement and plasma membrane rupture [28,29].
In the context of renal I/R, the iron accumulation, through the Fenton reaction, may generate a large amount of ROS (also increased by the concomitant mitochondrial dysfunction and NOX family activity) that can severely enhance intra-cellular oxidative stress and lipid peroxidation (Figure 1).
Oxidative stress, then, plays a key role in organ damage after I/R by activating ferroptosis, an iron-driven cell death characterized by iron accumulation, excessive ROS and lipid peroxidation products and mitophagy, the selective degradation of damaged mitochondria by autophagy.

Ferroptosis: Role in Kidney Allograft I/R Injury
Ferroptosis is a form of regulated cell death driven by iron accumulation, lipid peroxidation and subsequent plasma membrane rupture [27]. It is mainly characterized by: a nucleus lacking chromatin condensation, mitochondria with reduced volume and cristae, significant cell enlargement and plasma membrane rupture [28,29].
In the context of renal I/R, the iron accumulation, through the Fenton reaction, may generate a large amount of ROS (also increased by the concomitant mitochondrial dysfunction and NOX family activity) that can severely enhance intra-cellular oxidative stress and lipid peroxidation ( Figure 1).

Figure 1.
Schematic representation of the mechanisms of ferroptosis and mitophagy in renal ischemia/reperfusion (I/R) injury. During I/R several pathways contribute to ferroptosis: (i) the overproduction of ROS by NADPH oxidase (NOX), nitric oxide synthase (NOS), xanthine oxidoreductase (XOR) and mitochondria promotes lipid peroxidation and plasmatic membrane rupture; (ii) the reduction in glutathione (GSH) content inhibits glutathione peroxidase 4 (GPX4) activity and its protective action against membrane lipid peroxidation; (iii) I/R can indirectly induce ferritinophagy which causes the degradation of intracellular ferritin, and the increment of intracellular labile iron pool. Mitophagy is activated in I/R through both ubiquitin-dependent and ubiquitin-independent mechanisms and seems to have a protective role in I/R injury by reducing the release of reactive oxygen species from dysfunctional mitochondria. In physiological conditions, PINK1 is imported into mitochondria where it is cleaved by the intramembrane serine protease presenilin associated rhomboid-like (PARL) and ultimately degraded. When mitochondria are damaged, and lose their membrane potential, PINK1 accumulates on the mitochondrial outer membrane (MOM) and recruits Parkin. Parkin ubiquitinates several mitochondrial substrates such as voltage-dependent anion-selective channel protein (VDAC) and dynamin-1-like protein (DRP1). These ubiquitinated proteins can recruit mitophagy receptors (such as optineurin, p62) that link mitochondria to autophagosomes through interacting with LC3. This causes an autophagic Figure 1. Schematic representation of the mechanisms of ferroptosis and mitophagy in renal ischemia/reperfusion (I/R) injury. During I/R several pathways contribute to ferroptosis: (i) the overproduction of ROS by NADPH oxidase (NOX), nitric oxide synthase (NOS), xanthine oxidoreductase (XOR) and mitochondria promotes lipid peroxidation and plasmatic membrane rupture; (ii) the reduction in glutathione (GSH) content inhibits glutathione peroxidase 4 (GPX4) activity and its protective action against membrane lipid peroxidation; (iii) I/R can indirectly induce ferritinophagy which causes the degradation of intracellular ferritin, and the increment of intracellular labile iron pool. Mitophagy is activated in I/R through both ubiquitin-dependent and ubiquitinindependent mechanisms and seems to have a protective role in I/R injury by reducing the release of reactive oxygen species from dysfunctional mitochondria. In physiological conditions, PINK1 is imported into mitochondria where it is cleaved by the intramembrane serine protease presenilin associated rhomboid-like (PARL) and ultimately degraded. When mitochondria are damaged, and lose their membrane potential, PINK1 accumulates on the mitochondrial outer membrane (MOM) and recruits Parkin. Parkin ubiquitinates several mitochondrial substrates such as voltage-dependent anion-selective channel protein (VDAC) and dynamin-1-like protein (DRP1). These ubiquitinated proteins can recruit mitophagy receptors (such as optineurin, p62) that link mitochondria to autophagosomes through interacting with LC3. This causes an autophagic engulfment of the organelle necessary for its degradation. The ubiquitin-independent mechanism is regulated by mitophagy receptors that localize on MOM, such as BCL2 interacting protein 3 (BNIP3), BNIP3-like (BNIP3L/NIX), and FUN14 domain containing 1 (FUNDC1). These proteins bridge mitochondria to autophagosome by directly interacting with LC3. Two pathways may trigger ferroptosis: the extrinsic and the intrinsic pathway [27]. The extrinsic pathway is initiated through the inhibition of the cystine/glutamate exchanger of the membrane, namely the XC system, that mediates the entry of cystine into the cells, which is used to synthesize glutathione (GSH) [30], a cofactor used by glutathione peroxidase 4 (GPX4) to eliminate lipid peroxides in the cell membranes. Therefore, inhibition of the XC system indirectly reduces the activity of GPX4 with consequent accumulation of lethal lipid peroxides and induction of ferroptosis. Several agents such as erastin, sulfasalazine, and sorafenib, by blocking the XC system, are able to elicit ferroptosis through this mechanism.
The intrinsic pathway is mainly induced by drugs or small-molecule inhibitors such as RSL3, ML162, ML210, FIN56 and FINO2 which can directly or indirectly inhibit GPX4 activity [31]. Additionally, the molecules regulating iron uptake, storage, and utilization (such as ferritin, transferrin, and lactotransferrin) can influence ferroptosis by increasing levels of labile iron (free-iron source that was relatively accessible for Fenton reaction) in the cell [32]. Transferrin and lactotransferrin are proteins responsible for iron transport that, binding to their receptors, mediate the import of Fe into the cytoplasm. Ferritin is the intracellular iron-storage protein that can be degraded by lysosomes in a process termed ferritinophagy and increases free iron levels thus promoting ferroptosis [33] (Figure 1).
Recent studies have reported that ferroptosis may be involved in the pathophysiological pathway associated with the I/R injury [29,38].
Su et al. [39] demonstrated that pannexin 1, a membrane channel involved in regulating ATP release as a DAMP molecule able to activate apoptosis or autophagy signaling in oxidative condition [40,41], may activate ferroptosis in a mouse model of renal I/R injury [39]. Knockout of the panx1 gene in mice subjected to I/R is associated with a lower increment of serum creatinine and decreased tubular cell death together with decreased lipid peroxidation compared with wild-type mice. This protective effect seemed mediated by the inactivation of the MAPK/ERK pathway and the up-regulation of the antioxidant gene heme oxygenase-1 (HO-1).
The anti-ferroptosis protective effects may also be exerted by the activity of the Augmenter of Liver Regeneration (ALR), a sulfhydryl oxidase enzyme localized in the intermembrane space of mitochondria. This enzyme participates in the "disulfide relay system" that mediates the import of proteins to the intermembrane space [42] and has anti-apoptotic and anti-oxidative properties. ALR expression was significantly increased in ischemic rats and the administration of recombinant human ALR, by enhancing the proliferation of renal tubular cells and attenuating tubular cell apoptosis, effectively reduced tubular injury and ameliorated the impairment of renal function [43,44].
The protective role of ALR in ferroptosis could also be mediated by a reduction of ROS levels via its interaction with the GSH-GPX4 system [45] and by promoting the clearance of damaged mitochondria (a mechanism called mitophagy) [46]. Therefore, ALR activation may represent a possible future prevention therapeutic strategy for I/R-induced allograft injury.

Mitophagy: Another Player in Kidney Allograft I/R Injury
Damaged or dysfunctional mitochondria harm the cell by producing a large amount of ROS and releasing pro-apoptotic factors. Thus, timely removal of these organelles is critical to cellular homeostasis and viability [47].
Mitophagy is the mechanism of selective degradation of damaged mitochondria via autophagy [48] that is executed by a ubiquitin-dependent and ubiquitin-independent pathway. The former is regulated by the PTEN-induced putative kinase 1 (PINK1)-Parkin pathway. PINK1 is a mitochondrial serine/threonine kinase and Parkin is a cytosolic ubiquitin E3 ligase. In physiological conditions, PINK1 is imported into mitochondria where it is cleaved by the intramembrane serine protease presenilin associated rhomboid-Antioxidants 2022, 11, 769 5 of 17 like (PARL) and ultimately degraded [49]. When mitochondria are damaged and lose their membrane potential, the import of PINK1 is hindered leading to an accumulation of this kinase at the mitochondrial outer membrane (MOM). Subsequently, PINK1 recruits Parkin and activates its ligase activity [50]. Parkin ubiquitinates several mitochondrial substrates such as Mitofusin 2 (Mfn2), voltage-dependent anion-selective channel protein (VDAC), and dynamin-1-like protein (DRP1). These ubiquitinated proteins can recruit mitophagy receptors (such as optineurin, p62, NBR1) that link mitochondria to autophagosomes through interacting with LC3. This causes an autophagic engulfment of the organelle necessary for its degradation [49,51].
Mitophagy is also regulated by proteins that participate in mechanisms of fusion and fission of these organelles. Fusion results in a single mitochondrion being formed from previously independent structures [55], generating networks with continuous membranes and matrix lumen [56]. Fission produces one or more daughter organelles and, in the case of reduced mitochondrial membrane potential, segregates this organelle for elimination by autophagy [56].
The coordination of fission/fusion and mitophagy seems to be mediated by FUNDC1. In physiological conditions, this receptor anchors dynamin-related GTPases optic atrophy 1 (OPA1) toward the inner surface of the MOM. In response to mitochondrial stress, the disassembly of the FUNDC1-OPA1 complex and the recruitment of Drp1 promote mitochondrial fission and mitophagy [57].
This complex and fascinating multifactorial autophagic mechanism may play a protective role in allografts undergoing I/R injury.
The protective effects of mitophagy on kidney undergoing I/R injury were observed after ischemic preconditioning [64], a short period of non-lethal ischemia-reperfusion that protect solid organ against subsequent extended I/R injury [65]. The up-regulation of mitophagy via the PINK1-Parkin pathway improved mitochondrial function, minimized ROS production and enhanced cell survival [64].
All these findings suggest that mitophagy, preserving mitochondrial quality and tubular cell survival, could represent a valuable protective mechanism against I/R injury that should be promoted by pharmacological interventions.

Antioxidants and Ferroptosis/Mitophagy Regulators
Several pharmacological agents with anti-oxidant potentials have been proposed for the treatment of I/R injury, including those targeting the nuclear factor erythroid 2-related factor 2 (Nrf2), hydrogen sulfide (H 2 S), mitochondria-targeting antioxidants, drugs with anti-oxidant potential, and other specific ferroptosis and mitophagy regulators (Table 1). The nuclear factor erythroid 2-related factor 2 (Nrf2) is an inducible transcription factor that regulates the expression of antioxidant response elements [66] (Figure 2).
In physiological conditions Nrf2 binds to Kelch-like ECH-associated protein-1 (Keap1) in the cytoplasm and is degraded by the ubiquitin-proteasome pathway [67]. Under oxidative stress, Nrf2 escapes from degradation thanks to the inactivation of Keap1, forms dimers with a member of the small Maf proteins in nuclei, binds to anti-oxidant response elements, and activates transcription of the antioxidant genes [68].
In the course of renal I/R, the hyperactivation of Nrf2 by 1- In physiological conditions Nrf2 binds to Kelch-like ECH-associated protein-1 (Keap1) in the cytoplasm and is degraded by the ubiquitin-proteasome pathway [67]. Under oxidative stress, Nrf2 escapes from degradation thanks to the inactivation of Keap1, forms dimers with a member of the small Maf proteins in nuclei, binds to anti-oxidant response elements, and activates transcription of the antioxidant genes [68].
Nrf2 also regulates the expression of genes encoding for proteins mediating iron metabolism and is able to prevent the ferroptotic cascade, such as ferritin light and heavy chain (FTL/FTH1), ferroportin (SLC40A1) [70,71], GPX4, and HO-1, by which ferroptosis is inhibited and I/R-associated kidney injury alleviated [72,73].

Antioxidant Effects of Hydrogen Sulfide (H 2 S)
Hydrogen sulfide (H 2 S) is a membrane-permeable, gaseous mediator that inhibits oxidative damage through scavenging free radicals and ROS by increasing the level of GSH and thioredoxin, and the activation of Nrf2 signaling by inactivation of Keap1 [77,78].
Several studies have reported the protective effect of soluble forms of H 2 S (such as sodium hydrosulfide or sodium sulfide) in animal models of I/R injury [79][80][81][82][83][84] (Table 2). The H 2 S-induced reduction in metabolism before ischemia (PRE-TREATMENT/PREand POST-TREATMENT) protected against acute tubular necrosis, apoptosis, loss of mitochondrial integrity and mitochondrial swelling associated with I/R injury. The protection was less pronounced when H 2 S was administered after the hypoxic period (POST-TREATMENT) [82] Ischemic mice Mice received daily intraperitoneal administration of sodium hydrosulfide hydrate (NaHS; 500 µg/kg) beginning 2 days after ischemia until 8 days after surgery Exogenous supplement of H 2 S by NaHS after ischemia improved recovery of kidney function by accelerating tubular epithelial cell proliferation, suppressing interstitial cell proliferation and fibrosis. Furthermore, NaHS treatment reduced post-I/R oxidative stress by prevention of reduction of glutathione level [83] Ischemic mice Mice received GYY4137 (H 2 S donor) 50 mg/kg via intraperitoneal injection for 2 consecutive days before ischemia/reperfusion GYY4137 attenuated the deterioration of renal function and morphology by increasing the expression of anti-oxidant enzymes via activation of the Nrf2 pathway [84] During renal I/R injury, the expression of the enzyme cystathionine gamma-lyase that catalyzes H 2 S formation is up-regulated and consequently, H 2 S production, as well as its plasmatic concentration, increased [80]. This could represent a defensive mechanism of the kidney against I/R. In fact, the administration of exogenous NaHS (15 min before ischemia and 5 min before reperfusion) prevented the I/R-induced activation of caspase-3 as well as the decline in the expression of the apoptotic markers Bid and Bcl-2 [79] with positive functional and histological effects.
Another protective mechanism mediated by H 2 S is based on its ability to induce hypometabolism (50% reduction in oxygen consumption and 60% in carbon dioxide output) [85]. The demand for O 2 is reduced to such an extent that H 2 S-treated mice can survive in 5% O 2 for over 6 h [86].
In a mouse model of renal I/R injury, H 2 S administrated before the ischemic insult may preserve renal function, prevent apoptosis and limit the influx of leukocytes and granulocytes into the renal interstitium [82]. Contrarily, a post-ischemic treatment with H 2 S may not exert any protective effects. These results demonstrated that the reduction in O 2 demand during hypoxia prevents the activation of detrimental pathways associated with I/R [82].
According to these findings, Han et al. demonstrated, in an ischemic kidney mouse model, the capability of NaHS treatment to accelerate the regeneration of damaged tubular cells by activating anti-oxidant effects [83].
More recently Zhao et al. also found that a water-soluble H 2 S donor (GYY4137) was able to attenuate the deterioration of renal function and morphology in the renal I/R model by increasing the nuclear localization of Nrf2 [84].
These findings indicate that the H 2 S-producing system may play a critical role in the recovery from acute kidney injury and prevention of progression to chronic kidney disease.
In a mouse model of bilateral renal ischemia, followed by up to 24 h reperfusion, intra-venous administration of MitoQ 15 min prior to ischemia reduced the severity of I/R injury to the kidney by decreasing oxidative damage [88,89].
Its ability to preserve mitochondrial integrity and function limits ferroptosis induced by loss of GPX4 or exposure to RSL3 [105].
In a rat model of renal I/R injury, treatment with SS-31 protected mitochondrial structure and respiration during early reperfusion, accelerated recovery of ATP, reduced apoptosis and necrosis of tubular cells, and abrogated tubular dysfunction [93]. In addition, SS-31 seemed to be able to modulate the expression of members of the RAS system (an important regulator of kidney functions), in particular aminopeptidase A (APA) and Ang receptors (AT2R) [94].
In a recent Phase 2a prospective, multicenter, randomized, double-blind, placebocontrolled study Saad et al., assessed the safety, tolerability, and efficacy of IV administered elamipretide (clinical formulation of SS-31) for reduction of reperfusion injury in patients with severe atherosclerotic renal artery stenosis undergoing revascularization with percutaneous transluminal renal angioplasty (PTRA) [95]. Patients were treated before and during PTRA with elamipretide (0.05 mg/kg per hour intravenous infusion) or placebo. Compared to the placebo group, the patients who received elamipretide showed increased estimated GFR and a decline in systolic blood pressure after 3 months.
In a rat model of renal I/R injury, administration of tempol prior to and throughout reperfusion attenuated renal dysfunction at least partially through reduced renal activity of myeloperoxidase (MPO) and levels of malondialdehyde (MDA) [100].
This compound is currently under investigation in a clinical trial evaluating its ability to prevent many of the toxicities associated with cisplatin and radiation treatment (including the prevention of mucositis, nephrotoxicity, and ototoxicity) in head and neck cancer patients (NCT03480971).
Mito-TEMPO is a combination of the intracellular anti-oxidant piperidine nitroxide TEMPO (2,2,6,6-tetramethylpiperidin-1-yloxy) and the TPP cation which facilitates 1000-fold accumulation into the mitochondrial matrix and selectively targets mitochondrial ROS [110]. Administration of mito-TEMPO in rats after reperfusion and for 3 or 5 consecutive days after surgery restored the renal mtDNA level, mitochondrial mass, and ATP production with a consequently reduced inflammation and kidney injury [101].
XJB peptides are composed of 4-NH2-TEMPO, a stable nitroxide radical with antioxidant properties conjugated to a pentapeptide fragment from gramicidin S (Leu-d-Phe-ProVal-Orn), a natural membrane-active cyclopeptide antibiotic localized in the inner mitochondrial membrane [111]. The most studied of all the XJB peptides is XJB-5-131. Mice injected intraperitoneally with XJB-5-131 (10 mg/kg) 30 min prior to ischemia and for 3 consecutive days after surgery showed decreased kidney inflammation, regeneration and repair of injured renal tubular cells at least partially through the inhibition of I/R induced ferroptosis [102].

Drugs with Antioxidant Properties
Dexmedetomidine is a highly selective and specific α2-adrenoreceptor agonist with a sedative effect.
In a rat model of I/R, dexmedetomidine, administered intraperitoneally at different dosages (from 10 to 100 ug/kg) at the starting of ischemia or reperfusion or after surgery, attenuated renal dysfunction, acute tubular necrosis and inflammatory response at least partially through increased renal p38 MAPK, anti-oxidant levels, and maintenance of autophagy [112][113][114][115].
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a potent scavenger of hydroxyl and peroxyl radicals. As recently reported in the literature, administration of edaravone (from 3 to 10 mg/kg) intravenously in a mouse model of I/R injury (by clamping of renal arteria) protected against kidney damage by reducing oxidative stress, inhibiting apoptosis, and improving mitochondrial injury through JAK/STAT signaling [116,117].
In the future, edaravone could be potentially employable in clinical organ preservation and transplantation.

Ferroptosis and Mitophagy Specific Agents:
Besides the aforementioned antioxidant agents that can have an indirect role on both ferroptosis and mitophagy, specific molecules have been proposed for the direct regulation of these two pathways, including ferrostatin-1 and liproxstatin, two specific inhibitors of ferroptosis that because of their reactivity as radical trapping antioxidants may allow to reduce the accumulation of lipid hydroperoxides [118]. Liproxstatin-1 was reported to be able to suppress ferroptosis in human renal proximal tubule epithelial cells, in Gpx4−/− kidney, and in an I/R-induced tissue injury models [37]. However, additional studies (including clinical trials) should be undertaken to better address the clinical utility of these agents.

Conclusions
There are no therapeutic strategies available in clinical practice to slow down the onset and development of the allograft damage induced by I/R injury. However, data obtained in vitro and in animal models suggest that modulation of ferroptosis and mitophagy could represent a future therapeutic tool to prevent or slow-down the progression of the allograft I/R injury. Moreover, some of the components of both biological mechanisms could be proposed as novel (and not invasive) early diagnostic biomarkers for I/R injury-induced allograft complications (mainly delayed graft function).
Author Contributions: S.G., V.V., F.S., V.C. and G.Z. searched the literature and wrote the manuscript. E.R., G.S.N. and G.S. contributed to the literature analysis and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.