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

1Kidney 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). 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, poly-pharmacological approaches (particularly in the presence of concomitant acute allograft rejection), and shorter graft survival.

2The 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.

3Particularly, during ischemia, the significant reduction in oxygen supply and the consequent cellular switch from an aerobic to an anaerobic metabolism, may decrease the rate of ATP production and cause the accumulation of lactate (leading to acidosis). Consequently, Na+/K+ ATPases, Na+/H+ and Ca2+-ATPase pumps can become dysfunctional, and sodium, hydrogen and calcium accumulate in the cytoplasm with consequent hyper-osmolality, a rise in water transport across cell membranes, and cellular swelling.

4During reperfusion, the rapid increase of oxygen and pH normalization, may enhance cytosolic calcium concentration activating cysteine proteases (e.g., calpains, caspases) and triggering the apoptotic pathway. Moreover, calcium overload stimulates the opening of the mitochondrial permeability transition pores (mPTP) that allow the release of substances such as cytochrome C, succinate and mitochondrial DNA which can induce cell death through apoptosis and necrosis and act as danger/damage-associated molecular patterns (DAMP) promoting activation of both the innate and adaptive immunity. In addition, these mechanisms may result in progressive interstitial fibrosis.

5Furthermore, the overproduction of reactive oxygen species (ROS) following ischemia/reperfusion (I/R) may be induced by deregulation of numerous enzymes able to reduce molecular oxygen forming superoxide and/or hydrogen peroxide such as NADPH oxidase, nitric oxide synthase (NOS), the mitochondrial electron transport chain, and xanthine oxidoreductase (XOR).

6XOR is a complex molybdoflavoenzyme that controls the rate-limiting step of purine catabolism and exists in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO). XDH preferably uses NAD+ as an electron acceptor, while XO uses O2 as the terminal electron acceptor thereby exhibiting the ability to generate ROS. This enzyme converts hypoxanthine into xanthine generating superoxide (O2−) and hydrogen peroxide (H2O2) that play an important role in mediating the recruitment and/or activation of leukocytes that orchestrate the tissue damage.

7Additionally NADPH oxidases, multimeric complexes that generate superoxide or H2O2, composed of seven family members (NOX1–5, DUOX1–2), are implicated in the production of ROS following I/R. NOX enzymes use oxygen as final electron acceptors via NADPH, FAD, and heme groups. The DUOX enzymes predominately produce hydrogen peroxide along with NOX-4, while the remaining NOX isoenzymes largely produce superoxide. NOXs are constitutively inactive and require cell stimulation to translocate to the membrane and generate ROS. In I/R this enzymatic complex may be activated by several chemical mediators that are produced and released by cells, such as: hypoxia inhibitory factor-1α (HIF-1α), phospholipase A2, arachidonic acid, complement system, cytokines such as TNF-α and IL-1β from macrophages and mast cells.

8Another source of ROS is the uncoupled NOS that produces nitric oxide (NO) by the conversion of L-arginine to L-citrulline using NADPH as a reducing substrate and tetrahydrobiopterin (BH4) as a redox-sensitive cofactor. This enzyme, under hypoxic conditions may be converted to an O2-generating enzyme due to the reduced concentration of BH4, increasing the oxidative damage.

9Furthermore, mitochondria, organelles that generate most of the chemical energy needed to power the cell, contribute to ROS production through univalent reduction of O2 mainly by the leakage of electrons at complex I and α-ketoglutarate dehydrogenase.

10During ischemia, the alteration of mitochondrial structure, the high NADH/NAD+ ratio, and the accumulation of the citric acid cycle metabolite succinate exacerbate this process.

11Oxidative 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.

12Ferroptosis is a form of regulated cell death driven by iron accumulation, lipid peroxidation and subsequent plasma membrane rupture. It is mainly characterized by: a nucleus lacking chromatin condensation, mitochondria with reduced volume and cristae, significant cell enlargement and plasma membrane rupture.

13In 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).

14Two pathways may trigger ferroptosis: the extrinsic and the intrinsic pathway. 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), 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.

15The 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. 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. 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 (Figure 1).

16Contrarily enzymatic and non-enzymatic systems (CoQ10, vitamin E, ferrostatins, and liproxstatins), together with membrane repair systems, prevent lipid peroxidation and protect the cells from ferroptosis.

17Recent studies have reported that ferroptosis may be involved in the pathophysiological pathway associated with the I/R injury.

18Su et al. 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, may activate ferroptosis in a mouse model of renal I/R injury. 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).

19The 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 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.

20The protective role of ALR in ferroptosis could also be mediated by a reduction of ROS levels via its interaction with the GSH-GPX4 system and by promoting the clearance of damaged mitochondria (a mechanism called mitophagy).

21Therefore, ALR activation may represent a possible future prevention therapeutic strategy for I/R-induced allograft injury.

22Damaged 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.

23Mitophagy is the mechanism of selective degradation of damaged mitochondria via autophagy 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-like (PARL) and ultimately degraded. 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. 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.

24The 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 (Figure 1).

25Mitophagy 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, generating networks with continuous membranes and matrix lumen. Fission produces one or more daughter organelles and, in the case of reduced mitochondrial membrane potential, segregates this organelle for elimination by autophagy.

26The 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.

27This complex and fascinating multifactorial autophagic mechanism may play a protective role in allografts undergoing I/R injury.

28Deficiency of BNIP3 or Pink1 and/or Parkin in rat models of renal I/R injury resulted in increment of ROS production, apoptosis, and tubulointerstitial inflammation. The same effects were obtained by the suppression of the mitophagic cascade by acting on proteins regulating fission (e.g., Drp1) or fusion (e.g., OPA1).

29The protective effects of mitophagy on kidney undergoing I/R injury were observed after ischemic preconditioning, a short period of non-lethal ischemia-reperfusion that protect solid organ against subsequent extended I/R injury. The up-regulation of mitophagy via the PINK1-Parkin pathway improved mitochondrial function, minimized ROS production and enhanced cell survival.

30All 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.

31Several 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 (H2S), mitochondria-targeting antioxidants, drugs with anti-oxidant potential, and other specific ferroptosis and mitophagy regulators (Table 1).

32The nuclear factor erythroid 2–related factor 2 (Nrf2) is an inducible transcription factor that regulates the expression of antioxidant response elements (Figure 2).

33In physiological conditions Nrf2 binds to Kelch-like ECH-associated protein-1 (Keap1) in the cytoplasm and is degraded by the ubiquitin-proteasome pathway. 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.

34In the course of renal I/R, the hyperactivation of Nrf2 by 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazolide (CDDO) in the initial phase of the ischemia process prevents the progression of ROS-mediated tubular damage by inducing the expression of genes involved in anti-oxidant response [NADPH: quinone acceptor oxidoreductase 1 (Nqo1), Sulfiredoxin-1 (Srxn1) and Thioredoxin Reductase 1 (Txnrd1)], glutathione metabolism [Glutamate-Cysteine Ligase Modifier Subunit (Gclm) and Glutathione S-Transferase Mu 1 (Gstm1)], and NADPH synthesis [Glucose-6-Phosphate Dehydrogenase (G6pd) and Phosphogluconate Dehydrogenase (Pgd)].

35Nrf2 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), GPX4, and HO-1, by which ferroptosis is inhibited and I/R-associated kidney injury alleviated.

36Contrarily, silencing Nrf2 in mice undergoing I/R injury, triggered worse renal function and elevated histological tubular damage, increased renal vascular permeability, oxidative stress, and apoptosis compared to wild-type mice.

37Hydrogen sulfide (H2S) 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.

38Several studies have reported the protective effect of soluble forms of H2S (such as sodium hydrosulfide or sodium sulfide) in animal models of I/R injury (Table 2).

39During renal I/R injury, the expression of the enzyme cystathionine gamma-lyase that catalyzes H2S formation is up-regulated and consequently, H2S production, as well as its plasmatic concentration, increased. 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 with positive functional and histological effects.

40Another protective mechanism mediated by H2S is based on its ability to induce hypometabolism (50% reduction in oxygen consumption and 60% in carbon dioxide output). The demand for O2 is reduced to such an extent that H2S-treated mice can survive in 5% O2 for over 6 h.

41In a mouse model of renal I/R injury, H2S administrated before the ischemic insult may preserve renal function, prevent apoptosis and limit the influx of leukocytes and granulocytes into the renal interstitium. Contrarily, a post-ischemic treatment with H2S may not exert any protective effects. These results demonstrated that the reduction in O2 demand during hypoxia prevents the activation of detrimental pathways associated with I/R.

42According 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.

43More recently Zhao et al. also found that a water-soluble H2S 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.

44These findings indicate that the H2S-producing system may play a critical role in the recovery from acute kidney injury and prevention of progression to chronic kidney disease.

45The commonly used antioxidants could be ineffective in limiting mitochondrial ROS production, due to their low penetrance to the mitochondria interior. To overcome these limitations, mitochondria-targeting anti-oxidants have been developed to provide their delivery to the mitochondrion interior. These molecules have been used in numerous pre-clinical and clinical studies (Table 3).

46MitoQ: a quinone comprises a lipophilic triphenylphosphonium (TPP) cation covalently linked by an aliphatic 10-carbon chain to an antioxidant ubiquinone moiety. The TPP lipophilic cation passes rapidly through biological membranes and its positive charge drives the extensive accumulation of these molecules into mitochondria where it acts as a chain-breaking anti-oxidant to prevent oxidative damage.

47In 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.

48Its ability to preserve mitochondrial integrity and function limits ferroptosis induced by loss of GPX4 or exposure to RSL3.

49Szeto-Schiller peptide SS-31 (also known as MTP-131, elamipretide, and bendavia) is a peptide agent that interacts with cardiolipin in the inner mitochondrial membrane and exerts strong anti-oxidant propriety.

50In 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. 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).

51In a recent Phase 2a prospective, multicenter, randomized, double-blind, placebo-controlled 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). 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.

52Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl or 4-hydroxy-tempo) is a stable piperidine nitroxide that scavenges superoxide anions and reduces the intracellular concentrations of Fe2+ and, hence, the formation of hydroxyl radicals via the Fenton or Haber-Weiss reactions.

53In 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).

54This 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).

55Mito-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. 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.

56XJB peptides are composed of 4-NH2-TEMPO, a stable nitroxide radical with anti-oxidant 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. 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.

57Dexmedetomidine is a highly selective and specific α2-adrenoreceptor agonist with a sedative effect.

58In 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.

59Edaravone (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.

60In the future, edaravone could be potentially employable in clinical organ preservation and transplantation.

61Besides 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. 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. However, additional studies (including clinical trials) should be undertaken to better address the clinical utility of these agents.

62There 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).