Oxidative stress as a potential target in

acute kidney injury

Anamaria Magdalena Tomsa1,*, Alexandru Leonard Alexa2,
Monica Lia Junie3,*, Andreea Liana Rachisan1 and Lorena Ciumarnean4
1
Department of Pediatrics II, University of Medicine and Pharmacy of Cluj-Napoca, Cluj-Napoca,
Romania
2
Department of Anesthesia and Intensive Care I, University of Medicine and Pharmacy of
Cluj-Napoca, Cluj-Napoca, Romania
3
Department of Microbiology, University of Medicine and Pharmacy of Cluj-Napoca,
Cluj-Napoca, Romania
4
Department of Internal Medicine IV, University of Medicine and Pharmacy of Cluj-Napoca,
Cluj-Napoca, Romania
* These authors contributed equally to this work.

ABSTRACT
Background: Acute kidney injury (AKI) is a major problem for health systems
being directly related to short and long-term morbidity and mortality. In the last
years, the incidence of AKI has been increasing. AKI and chronic kidney
disease (CKD) are closely interconnected, with a growing rate of CKD linked to
repeated and severe episodes of AKI. AKI and CKD can occur also secondary to
imbalanced oxidative stress (OS) reactions, inflammation, and apoptosis.
The kidney is particularly sensitive to OS. OS is known as a crucial pathogenetic
factor in cellular damage, with a direct role in initiation, development, and
progression of AKI. The aim of this review is to focus on the pathogenetic role of
OS in AKI in order to gain a better understanding. We exposed the potential
relationships between OS and the perturbation of renal function and we also
presented the redox-dependent factors that can contribute to early kidney injury.
In the last decades, promising advances have been made in understanding the
pathophysiology of AKI and its consequences, but more studies are needed in order
Submitted 11 July 2019 to develop new therapies that can address OS and oxidative damage in early
Accepted 16 October 2019
stages of AKI.
Published 13 November 2019
Methods: We searched PubMed for relevant articles published up to May 2019.
Corresponding author
Andreea Liana Rachisan,
In this review we incorporated data from different types of studies, including
[email protected] observational and experimental, both in vivo and in vitro, studies that provided
Academic editor information about OS in the pathophysiology of AKI.
Teresa Seccia Results: The results show that OS plays a major key role in the initiation and
Additional Information and development of AKI, providing the chance to find new targets that can be
Declarations can be found on therapeutically addressed.
page 14 Discussion: Acute kidney injury represents a major health issue that is still not fully
DOI 10.7717/peerj.8046 understood. Research in this area still provides new useful data that can help obtain
Copyright a better management of the patient. OS represents a major focus point in many
2019 Tomsa et al. studies, and a better understanding of its implications in AKI might offer the chance
Distributed under to fight new therapeutic strategies.
Creative Commons CC-BY 4.0

How to cite this article Tomsa AM, Alexa AL, Junie ML, Rachisan AL, Ciumarnean L. 2019. Oxidative stress as a potential target in acute
kidney injury. PeerJ 7:e8046 DOI 10.7717/peerj.8046
 Subjects Anatomy and Physiology, Internal Medicine, Nephrology
Keywords Acute kidney injury, Oxidative stress, Physiopathology, Reactive oxygen species,
Mithocondria

INTRODUCTION
Acute kidney injury (AKI) represents a syndrome in which the renal function deteriorates
due to a sudden drop in the glomerular filtration rate. Traditionally, it has been viewed
in an anatomical context (prerenal, intrarenal, and postrenal), while at the same time
it has been considered to rarely transition to chronic kidney disease (CKD) (Chawla
et al., 2014). Nowadays, the emphasis has been put on an improved understanding of
AKI and its pathophysiological mechanisms, as it is considered a major risk factor for
developing CKD. The fact that each episode of AKI significantly influences the outcome
and accelerates the development of CKD, while a patient who suffers from CKD is
more predisposed to suffer new episodes of AKI, has led to the idea that these entities,
previously considered independent from each other, actually represent an interconnected
syndrome (Chawla et al., 2014; He et al., 2017).
Up until recently, the real incidence of AKI was not known due to the fact that there was
no consensus on what AKI actually represents. The lack of a standard definition for
AKI led to different approaches to these patients, with each medical center using its own
definition for AKI (Mehta & Chertow, 2003). In 2004 the first standard definition was
created, based on the RIFLE criteria (Risk, Injury, Failure, Loss, End-stage kidney disease)
by the Second International Consensus Conference of the Acute Dialysis Quality Initiative
(ADQI) Group (Bellomo et al., 2004). Nowadays, the reported incidence shows that
this syndrome represents a major public health problem, with about one in four
hospitalized patients worldwide (Palevsky et al., 2013). The mortality rates vary by age, with
23.9% in adults and 13.8% in pediatric patients (Susantitaphong et al., 2013). The severity of
this syndrome in the affected patients is considered to be an independent risk factor for
their outcome and mortality (Ostermann & Chang, 2007; Bagshaw et al., 2008).
Emerging evidence highlights that AKI represents a major risk factor for developing
CKD independent from the renal function recovery in the affected patients, even in those
who seemed to have completely recover after an episode of AKI (Lameire et al., 2013;
Wald et al., 2009; Basile et al., 2016). Recent experimental results showed that the
transition from AKI to CKD may be caused by a maladaptive cellular response or by a
reparative response that has been misdirected or maladaptive (Basile et al., 2016; Agarwal
et al., 2016; Takaori et al., 2016). Also, oxidative stress (OS) plays a crucial role in both
AKI and CKD, as it has been considered a central aggravating factor (Tanaka, Tanaka &
Nagaku, 2014; Ruiz et al., 2013). Excessive accumulation of reactive oxygen species
(ROS) determines the activation of adaptive gene programs, which experimental mouse
models suggest they offer insufficient protection (Nezu et al., 2017).
This review aims to summarize the current knowledge about the role that OS plays in
the pathophysiology of AKI, in order to gain a better understanding of this ubiquitous
pathology. New biomarkers and new therapeutic strategies are needed in order to address
this syndrome; therefore, a detailed knowledge about its underlying mechanisms is

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 mandatory. At this moment, there is little data about this subject in the literature, even
though OS represents a major focus point in many diseases. In this review we detailed the
main sources of OS in AKI, their mechanisms of action and their effect on different
cellular structures. Furthermore, we included the main antioxidants that can be found in
the renal tissues and data about OS biomarkers. We address this review to nephrology
residents and specialists, and to whomever wants to gain a more in-depth view in this
particular subject.

SURVEY METHODOLOGY
We completed an electronic literature search in the PubMed database and we included
pertinent articles published up to May 2019, that provided information about OS and its
role in the pathophysiology of AKI. In our search we used the following terms: “AKI,”
“acute kidney failure,” “acute kidney insufficiency” in combination with “OS,” “ROS” or
“reactive nitrogen species (RNS).” In order to write this review, we comprised data
from numerous types of studies, including observational and experimental studies, both
in vitro and in vivo, including randomized controlled ones. For the purpose of this review
we used overall selected papers.

Oxidative stress overview

Oxidative stress reprexidants, ROS and/or RNS which surpasses the endogenous
antioxidant capacity, due to various causes. Defined for the first time in 1985 by Stahland
and Sies, OS has recently been intensely studied as a regulatory element in various diseases.
A free radical (FR) is defined as a species that presents a single or more unpaired
electrons, and which exists autonomously (Halliwell, 2006). FRs are considered essential in
biological evolution as they play a major role in several biochemical reactions (Singh et al.,
2019).
Up to this moment, OS has been proved to represent a pathogenetic factor in
cardiovascular diseases, neurodegenerative diseases, cancer, aging, and many others
(Valko et al., 2007; Singh et al., 2019). OS also represents a major factor in the
development of kidney damage (Himmelfarb et al., 2004), therefore it might play a
crucial role in therapeutic intervention when targeted. Also, OS presents damaging
effects on biomolecules such as DNA, RNA, proteins, lipids, enzymes, etc. The changes
that reactive species (RS) make upon these molecules could be highlighted and used as
biomarkers for OS (Singh et al., 2019).
Are reactive species important in maintaining homeostasis?
It is considered that RS are important for maintaining homeostasis when they are present
in low concentrations, in certain compartments, at a certain moment. ROS and RNS are
responsible for normal redox signaling, and therefore they represent a promotor for
cell survival, growth, and proliferation (Ratliff et al., 2016).
In the kidney, the blood flow and the glomerular filtration rate are maintained at a
constant level by renal autoregulation, despite all the physiological changes or pathological
states that may take place in the body. Two key mechanisms are responsible for the renal
autoregulation: the myogenic response and tubuloglomerular feedback. Studies show

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 that both vasoreactivity and the myogenic response are influenced by the presence of RS
such as nitric oxide (NO) and superoxide (O−2 ) (Carlström, Wilcox & Arendshorst, 2015;
Just, 1997; Loutzenhiser et al., 2006; Navar et al., 2008).
Nitric oxide is a vasodilator factor generated by endothelial NO synthase (eNOS),
and it defends the renal cells (endothelial and mesangial cells) from fibrosis and apoptosis
by inducing the expression of antioxidative genes and promoting a physiological renal
hemodynamics (Ratliff et al., 2016). It is well known that NO is a major factor in
modulating the myogenic response (Carlström, Wilcox & Arendshorst, 2015), with
numerous studies showing that inhibition of NOS decreases renal blood flow and increases
the renal vascular resistance in both pressor dosages (Baylis & Qiu, 1996; Majid,
Williams & Navar, 1993) as well as in dosages that have no impact on the blood pressure
(Carlström, Wilcox & Arendshorst, 2015; Deng & Baylis, 1993). Also, the infusion of
NO donors (sodium nitroprusside) and NO precursors (L-arginine) seems to counteract
renal vasoconstriction (Kiyomoto et al., 1992; Kumagai et al., 1994).
Intracellularly, low levels of NO may inhibit cytochrome-c oxidase, altering the generation
of ROS in the mitochondria (Brown, 1995). When ROS produced by mitochondria
increase moderately, they stabilize hypoxia-inducible factor (HIF) in endothelial cells, and
stimulate the nuclear factor erythroid 2–related factor 2 (Nrf2) (Piantadosi & Suliman,
2006; Guzy et al., 2005). Both HIF and Nrf2 protect the renal tissues against OS, therefore
ROS may exhibit renoprotective effects in certain concentrations (Ratliff et al., 2016;
Nangaku & Eckardt, 2007). Nrf2 represents the main regulator of the response to OS
(Ruiz et al., 2013).
Interestingly, one study that focused on the effects of endogenous O−2 showed that it
becomes a crucial factor in maintaining a physiological tone in the renal vasculature when
it is induced by vascular NADPH oxidase (NOX) (Haque & Majid, 2004). This further
demonstrates that RS might play a crucial role in maintaining homeostasis.

What reactive species are generated in AKI?

The main etiologies of AKI are linked to ischemia and hypoxia. Due to the decrease in
the renal blood flow, the cellular nutrient and oxygen uptake is limited, leading do the
development of inflammation and acute tubular necrosis (Basile, Anderson & Sutton,
2012). Renal ischemia and reperfusion represent major triggers of ROS with consequential
damage in renal function and tissue integrity (Deng & Baylis, 1993). In sepsis-induced
AKI there is an extensive immune response that is responsible for renal vasoconstriction,
endothelial injury, and localized hypoxia, which ultimately triggers the formation of
ROS (Andrades et al., 2011). Further, uremia is highly associated with an increase in
circulating levels of indole and carbonyl compounds, which are able to upregulate systemic
OS (Tanaka et al., 2017).
Some of the initial changes that are observed with AKI development are depletion
of ATP and changes in the structure of the mitochondria, leading to alterations
and dysfunction in the energetic metabolism (Kiyomoto et al., 1992; Tanaka et al., 2017;
Tracz et al., 2007). Each type of renal cell presents a different number of mitochondria,
which is considered to be one of the major sources of ROS (Duchen & Szabadkai, 2010),

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 therefore the production of ROS varies between the renal structures. Mitochondria is also
responsible for producing molecules that counteract the OS (Singh et al., 2019). In the
kidney, NOX and the mitochondrial respiratory chain are considered to be the main sources
of ROS production (Sureshbabu, Ryter & Choi, 2015).
One study evaluated the mitochondrial function, structure and redox state in rodents
with induced nephrotoxic and ischemic AKI via in vivo exogenous and endogenous
multi-photon imaging. The results showed changes in mitochondrial NADH levels and
proton motive force, and upregulated levels of mitochondrial O2, along with disjointed
mitochondria. They concluded that mitochondria represents a major source of ROS, and
that an alteration in mitochondrial function plays an important role in the early phase
of renal ischemia (Hall et al., 2013; Tanaka et al., 2017). In contrast, after gentamycin
exposure, the first alterations to appear were at lysosomal level in the renal epithelium,
along with anomalies in brush border cells. The mitochondrial dysfunction, with changes
in morphology of the mitochondria, alterations in NADH levels, RS and altered proton
motive force occurred later in the development of nephrotoxic AKI (Tanaka et al.,
2017; Deisseroth & Dounce, 1970). Mitochondria is, therefore, directly responsible for
increased levels of OS in AKI.
Despite the abundance of studies conducted on this topic, the exact mechanism through
which RS are generated in AKI remains unknown. However, OS might represent a
potential therapeutic target. Table 1 summarizes the main ROS found in AKI.
The production of the superoxide anion O2− is the result of the one-electron reduction of
oxygen in its molecular form (Noiri, Addabbo & Goligorsky, 2011). Superoxide can be
generated by a large variety of oxidase enzymes, and can also be generated inside the
mitochondria by components of the electron transport chain. O−2 is mainly transported
through anion channels, therefore the diffusion across different membranes is limited.
Superoxide anion is a rather selective FR, which is able to form a non-radical ROS (H2O2)
via dismutation. The reaction can take place spontaneously, but it can also be facilitated
by enzymatic catalysis (Ratliff et al., 2016). The presence of the superoxide anion
triggers a cascade of events, as its presence leads to the generation of other ROS. O−2 may
also be produced from xanthine by xanthine oxidase (XO), and from NADPH and NADH
by various oxidase enzymes which are induced by an inflammatory response (Deng &
Baylis, 1993). The most potent action of O−2 is represented by the scavenging of NO.
As the levels of superoxide anion increases, it is able to disrupt the iron-sulfur centers, and
it may react with catecholamines (Ratliff et al., 2016). Local ischemia and cytokines
generated in AKI induced by sepsis activate the endothelium of the renal vasculature and
recruit cells from the immune system that are able to generate O−2 via NOX (Kiyomoto
et al., 1992).
Hydrogen peroxide (H2O2) can be generated by dismutation but also by oxidases
which are able to directly reduce the molecular oxygen. H2O2 diffuses across different
biological membranes in a similar way to water, which makes it able to express its oxidative
properties in other cellular compartments and even in other cells. Peroxides are able to
react with different molecules containing iron, leading to generation of additional ROS.

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 Table 1 The main reactive species in AKI.
Superoxide anion O2− Induced by NOX, XO, and Maintains a physiological Generates other ROS In sepsis-induced
(Ratliff et al., 2016; other enzymes role in the renal (e.g., forms H2O2 via AKI, O2− is
Deng & Baylis, 1993; Generated by the vasculature dismutation) generated by
Kiyomoto et al., 1992; mitochondrial Scavenges NO immune cells
Basile, Anderson & respiratory chain Disrupts the iron-sulfur
Sutton, 2012) centers
Hydrogen peroxide H2O2 Generated by dismutation Diffuses across biological
(Ratliff et al., 2016; Generated by oxidases membranes to other
Dennis & Witting, from molecular oxygen cellular compartments
2017) and other cells
Reacts with
iron-containing
molecules, releasing
additional ROS
Hydroxyl radical HO− Generated by Fenton Lipid peroxidation, with
(Brown, 1995; reaction subsequent membrane
Bogdan, 2001; Dennis damage
& Witting, 2017) Generates additional
ROS
Hypochlorous acid HClO Generated by MPO in Lipid soluble molecule
(Ratliff et al., 2016; inflammatory cells Reacts with amines,
Kiyomoto et al., 1992) producing chloroamines
Peroxynitrite (Ratliff ONOO− Generated from nitric Major inhibitor of the Oxidizes or nitrates thiols
et al., 2016) oxide reacting with mitochondrial respiration Oxidizes and nitrates
superoxide anion chain fatty acid
Generated by heme Irreversibly disrupts the
peroxidase enzymes centers of iron-sulfur
from the nitrate
metabolism
Nitric oxide (Ratliff NO Generated by eNOS and Vasodilator and counteracts Soluble gas Mice deficient in
et al., 2016; eNOS renal vasoconstriction Larger levels of NO iNOS were
Carlström, Wilcox & Modulates the myogenic interact with bound iron, resistant to renal
Arendshorst, 2015; response and produce NO-derived IRI
Kiyomoto et al., 1992; Protects endothelial and RNS that can nitrosate
Kumagai et al., 1994; mesangial cells from thiols
Brown, 1995; Ling fibrosis and apoptosis
et al., 1999) Induces antioxidative
genes
Inhibits cyt-c oxidase and
alters the generation of
ROS in the mitochondria
Binds to guanylate cyclase
and regulates the
production of cGMP

When peroxides react with Fe2+ they generate the hydroxyl radical (HO−), known as the
Fenton reaction. When peroxide is metabolized by specific heme peroxidase in the
presence of other molecules such as chloride or nitrite, it can generate hypochlorous acid,
and nitrogen dioxide (Ratliff et al., 2016; Dennis & Witting, 2017).
The hydroxyl radical (HO−) may be generated by the Fenton reaction. This radical
alters and reacts with almost every cellular component, generating additional ROS

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 (Brown, 1995). It produces lipid peroxidation with subsequent membrane damage and
toxic compounds release, including aldehydes (Dennis & Witting, 2017).
Hypochlorous acid (HClO), a highly reactive lipid soluble molecule (Ratliff et al., 2016),
is generated by phagocyte myeloperoxidase in inflammatory cells when local ischemia is
present (Kiyomoto et al., 1992), and it may react with amines, producing chloroamines.
Peroxynitrite (ONOO−) is generated when superoxide anion reacts with NO.
The spontaneous decomposition of peroxynitrite generates nitrogen dioxide (NO2). Also,
NO2 is generated by heme peroxidase enzymes from the nitrate metabolism. These RS are
able to activate a variety of signaling mechanisms by oxidizing or nitrating thiols,
sometimes influencing processes that are also targeted by peroxide. When RNS react with
unsaturated fatty acids (FA), the result is oxidized and nitrated FA which present a
multitude of biological actions. ONOO− seems to be a major inhibitor of the mitochondrial
respiration chain, irreversibly disrupting the centers of iron-sulfur (Ratliff et al., 2016).
Nitric oxideNO represents a soluble gas that is able to bind to ferrous sites of heme. It is
generated by NOS enzymes, and in normal amounts it is a part of physiological signaling
processes: it binds to guanylate cyclase in order to regulate the production of cGMP,
and it binds to cytochrome c oxidase to regulate the mitochondrial respiration. While it is
mainly generated by regulating the activity of nNOS (NOS1) and eNOS (NOS3), an
inflammatory status is able to induce iNOS (NOS2) expression (iNOS—inducible NO
synthase; nNOS—neuronal NO synthase). This process leads to the generation of larger
quantities of NO which consequently modulates the inflammatory processes. Larger levels
of NO interact with bound iron, and produce NO-derived RNS that can nitrosate thiols.
When the concentration of NO equalizes the levels of local antioxidant enzymes, it
competes with them for the scavenging of O−2 , generating ONOO−(Ratliff et al., 2016;
Dennis & Witting, 2017). However, it has been shown that when using agents to inhibit the
global production of NO, including the NO production from constitutive eNOS, the
effect on renal ischemia-reperfusion injury (IRI) was not renoprotective (Yaqoob,
Edelstein & Schrier, 1996).
Another study showed that mice who were deficient in iNOS were resistant to renal IRI,
concluding that a constant release of NO from NOS2 could be a pathogenetic factor
(Ling et al., 1999). NOS2 is constitutively expressed in the renal tissue (Thomas et al., 2015),
underlining that NO presents a physiological role in kidney functioning (Araujo & Welch,
2006). These mechanisms may potentially contribute to the development of renal disease.
Carbonyls, nitrated lipids, and unsaturated aldehydes are just some of the toxic reactive
molecules that can be generated by ROS and RNS. These molecules are able to trigger
signaling processes, at low levels of OS. As the level of OS increases, these molecules
promote oxidative damage and cellular death (Ratliff et al., 2016; Dennis & Witting, 2017).
Out of all possible etiologies for AKI, two of the most common are IRI and sepsis
(Piantadosi & Suliman, 2006).

Ischemia-reperfusion injury induced AKI

Ischemia-reperfusion injury represents a major cause of AKI, and the main cause
of delayed renal graft function, and renal graft loss after kidney transplantation

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 (Bogdan, 2001). In IRI, the reperfusion phase is the crucial moment when the most IRI
damage might occur. The initial event that takes place immediately after reperfusion is a
sudden increase in superoxide anion production in the mitochondria which is released
inside the cell, and represents the main trigger for the pathology that follows reperfusion
(Zweier, Flaherty & Weisfeldt, 1987; Chouchani et al., 2016). In IRI, this mechanism plays
a crucial role in initiating AKI, but also in maintaining it (Nath & Norby, 2000). Also,
the early inflammatory response in IRI consists mainly of neutrophils, which generate
ROS, and are recruited by ROS (Friedewald & Rabb, 2004).
One study that focused on the pathophysiology of renal IRI showed that pericytes
express adhesion protein-1, a key protein that generates a local H2O2 gradient which
further stimulates the neutrophil infiltration (Tanaka et al., 2017), proving that RS are
directly involved in developing renal injury following ischemia.
Another study concluded that, following renal IRI, Nrf2-regulated cell defense genes
presented significantly high levels in the kidneys of wild-type mice but not in Nrf2-
knockout (−/−) mice (Leonard et al., 2006; Liu et al., 2009). The same study showed that the
loss of Nrf2 leads to an increase in severity of renal IRI, which also proves that OS plays a
crucial role in the pathogenesis and outcome of renal IRI.
Tracz et al. (2007) and Piantadosi & Suliman (2006) showed that mice who were heme
oxygenase-1 knockout (HO-1−/−) had increased sensitivity to renal IRI, and expressed a
higher inflammatory response and oxidative damage and an increase in mortality when
compared to wild-type mice.
Another study demonstrated that mice deficient in transient receptor potential
melastatin 2 (TRPM2) who were subjected to renal IRI showed resistance to OS and
apoptosis (Gao et al., 2014). TRPM2 is a nonselective cation channel (for calcium, sodium,
and potassium) activated by OS, ADP-ribose, tumor necrosis factor-a (TNF-a), and
intracellular calcium, all of which are considered to be increased in kidney ischemia.
Gao et al. showed that TRPM2-knockout mice are resistant to IRI, with a reduction in OS,
NOX activity, and apoptosis in the kidney. Also, they obtained similar results by
pharmacologically inhibiting TRPM2. The cationic channel was identified mainly in the
proximal tubule epithelial cells, and Gao et al. showed that its effects are attributable to
expression in parenchymal cells. Also, they showed that the activation of RAC1, a
constituent of the NOX complex, was promoted by TRPM2 following renal ischemia.
In contrast, inhibition of RAC1 in vivo reduced ischemic injury and OS. This further
shows that OS and ischemic injury are closely interrelated.

Sepsis-induced AKI
In intensive care units (ICU), sepsis represents the major cause of AKI (Uchino et al.,
2005). Of all the bacterial pathogens that induce AKI, Gram-negative bacteria represent a
major threat due to the composition of their outer membrane. One of the main
components of their outer membrane is represented by lipopolysaccharide (LPS),
an endotoxin considered to be an important trigger of the inflammatory response in sepsis.
These LPSs are recognized by Toll-like receptor (TLR), a transmembrane protein

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 expressed in renal tubules. Once the LPS bind to TLRs, the immune system is triggered,
and the acquired immunity develops an antigen-specific response (Medzhitov, 2001).
TLR4 binds specifically to the LPS ligand, and their binding triggers a cascade of events
that leads to OS and a pro-inflammatory response, with new cytokines and mediators
being further released. TNF-a and interleukin-1β (IL-1β) are just two of the newly
generated molecules which consequently promote the H2O2 formation, with oxidative
damage which further enhances the inflammatory response (Li & Engelhardt, 2006).
Many studies suggest that both sepsis and ischemia upregulate TLR4 (Wu et al., 2007;
Pulskens et al., 2008; El-Achkar et al., 2006). Hato et al. (2015) studied the effect of
endotoxin preconditioning and concluded that it confers renal epithelial protection in
vivo, by preventing peroxisomal damage, abolishing OS, and tubule injury. OS was
measured in both preconditioned and nonpreconditioned mice, with measurements
performed after 4 h from LPS inoculation intraperitoneally. High levels of OS were
determined in proximal tubules of NP wild-type mice, whereas in preconditioned
wild-type mice OS was absent. TLR4−/− mice showed no OS, which confirms that TLR4
plays a crucial role in LPS signaling pathway. Also, in preconditioned mice, KIM-1,
and NGAL, markers for tubular injury, were significantly reduced, suggesting renal
protection. This study proves that sepsis indeed upregulates TRL4, and leads to high levels
of OS in renal proximal tubules, with subsequent oxidative damage.
TLR9 identifies bacterial DNA with unmethylated CpG motifs (Tsuji et al., 2016), but
the TLR9 pathway is also activated by endogenous mitochondrial DNA (mtDNA) (Zhang,
Itagaki & Hauser, 2010). Tsuji et al. (Gao et al., 2014) studied the role of mtDNA via
TLR9 in septic AKI and showed that cytokine production, tubular mitochondrial disorders
(ATP depletion), and splenic apoptosis are inflicted by the circulating mtDNA released
in the early phase of sepsis. They used an animal model of sepsis in wild-type and TLR9−/−
mice, but they also injected mitochondrial debris intravenously. The results show that
TLR9−/− mice presented a lower bacteremia than wild-type mice, but with a higher level of
leucocyte migration. Also, TLR9−/− mice presented lower levels of IFN-γ and IL-12 than
wild-type mice, with attenuated production of superoxide in the proximal tubule cells.
When injected with mitochondrial debris containing a substantial amount of mtDNA,
wild-type mice had increased plasma levels of IL-12, but not of IFN-γ and TNF-a, while
the levels of IL-12 in TLR9−/− mice did not increase as much. This study further shows
that sepsis upregulates certain receptors that stimulate the production of RS and
oxidative damage.
While sepsis remains ubiquitous, aforementioned studies suggest that the cascade of
events that it triggers, including OS damage and inflammatory response, could be
counteracted at receptor level. More studies are needed in order to gain a better perspective
of how OS could be addressed in sepsis-induced AKI.

How do the kidneys combat oxidative stress?

Homeostasis is favored by balancing the production of both oxidants and antioxidants.
It has been proved that ROS play a physiological role in renal function, but when RS
are unregulated or upregulated with consequent local accumulation, they are able to

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 induce OS, irreversibly damaging DNA, RNA, lipids, proteins, causing organelle
dysfunctionality (Dennis & Witting, 2017). The oxidant production is usually
counterbalanced by formation of endogenous antioxidants which disrupt the damaging
effects of OS. High levels of ROS may overrun the antioxidant activity, promoting
dysfunction of the vasculature, triggering an inflammatory response, with consequent
cytotoxicity on renal tubule cells, mechanisms that are observed in the pathogenesis of AKI
(Ratliff et al., 2016).
Antioxidants represent the body’s system to combat the RS that are constantly released
inside the cell (Singh et al., 2019). They are molecules that intervene early in AKI
development, by scavenging RS. There are several antioxidant systems able to protect the
renal cells from OS (Ratliff et al., 2016). Exogenous antioxidants are mainly represented by
dietary and/or supplementation substances which downregulate OS, consequently
diminishing lipid peroxidation, and oxidative damage (Dennis & Witting, 2017). Table 2
contains the main antioxidants that can be found in the renal tissue.
Modulating AKI with the help of antioxidants represents a focus point for many human
and animal studies (Chatterjee, 2007), which have shown that nephrotoxicity and renal
ischemia induce an increased oxidative damage with low levels of local antioxidants
(Paller, Hoidal & Ferris, 1984; Baliga et al., 1999).
Superoxide dismutase (SOD) isoforms represent some of the major enzymes that fight
against OS, and they all can be found in the kidneys. They are localized in both the
extracellular space, and intracellular space (mitochondria, cytoplasm). SOD localization
varies by species, but their renal activity is comparable in human, mouse, sheep, etc.
(Marklund, 1984). SOD is a catalysator that helps the dismutation of O−2 into O2 and H2O2.
It is considered to be the first system to fight OS (Ratliff et al., 2016).
SOD1 (copper/zinc form of SOD) cannot be found inside the mitochondria, but is
commonly present in other intracellular spaces. It is responsible for 80% of the activity that
SOD has in the renal tissues (Matés, 2000), and for 1/3 of the SOD activity in the renal
vasculature, where it stops disruption of NO signaling (Carlström et al., 2010). SOD1
suppresses the clearing of NO by O2− inside the cellular compartments, maintaining the
production of H2O2 (Ratliff et al., 2016).
SOD2 (manganese form of SOD) is present in great quantities inside the mitochondria,
and it plays a key role in generating and releasing the peroxide from the mitochondria.
ONOO− is able to inactivate SOD2. Overexpression of this isoform, but not catalase,
ameliorates AKI induced by cisplatin in vitro (Davis, Nick & Agarwal, 2001), fact that
shows the importance of superoxide anion in AKI (Tanaka et al., 2017).
One study in which SOD2 and SOD1 were ablated in mice, showed that the pathological
phenotype was aggravated in SOD2-ablated mice, despite the fact that SOD1 is responsible
for most of the SOD activity (Schieber & Chandel, 2014). This data suggests that the
localization of RS is of great importance, indicating that mitochondria is crucial in the
initiation and evolution of AKI (Ratliff et al., 2016).
SOD3 is an extracellular copper/zinc SOD isoform responsible for protecting NO
from O−2 , and also for converting O−2 into H2O2 in the extracellular environment
(Ratliff et al., 2016).

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 Table 2 The main antioxidants fighting oxidative stress in AKI.
Superoxide dismutase SOD (Ratliff et al., 2016; Extracellular and Catalysator
Marklund, 1984; Matés, intracellular Generates O2 and H2O2 via dismutation
2000) (mitochondria, Considered the first system to fight oxidative stress
cytoplasm)
SOD1 (Ratliff et al., 2016; Cytoplasm Suppresses the clearing NO, Gene located on
Matés, 2000; Carlström maintaining the production chromosome 21 (21q22.1)
et al., 2010) (copper/ of H2O2
zinc isoform)
 dimer
SOD2 (Tanaka et al., Mitochondria (great Generates and releases Gene located on chromosome
2017; Davis, Nick & quantities) peroxide from the 6 (6q25.3)
Agarwal, 2001; Schieber mitochondria Overexpression ameliorates
& Chandel, 2014) Inactivated by ONOO− cisplatin-induced AKI in
(manganese isoform) vitro
 tetramer
SOD3 (Ratliff et al., 2016) Extracellular Protects NO from O2− Gene located on
(copper/zinc isoform) Converts O2− into H2O2 chromosome 4
 tetramer (4p15.3-p15.1)
Catalase (Ratliff et al., Contains heme groups Intracellular (mainly in in Converts H2O2 into O2 and H2O
2016; Deisseroth & with iron core the cytosol and in
Dounce, 1970; Hwang  tetramer peroxisomes)
et al., 2012; Vasko In cells with aerobic
et al., 2013) metabolism
Thioredoxin peroxidase Intracellular Converts H2O2 by oxidizing thioredoxin
systems (Ratliff et al.,
2016)
Glutathione and Intracellular (cytoplasm, Reduce H2O2
glutathione mitochondria, nucleus) Reduce lipid peroxides to lipid alcohols
peroxidase family  tripeptide and extracellular Regulate apoptosis vs. necrosis
(Araujo & Welch, (pulmonary surfactant) Modulate DNA synthesis and cellular division
2006; Dennis &
Witting, 2017; Oberley
et al., 2001; Fukai,
2009; Doi et al., 2004)
Edaravone (Tanaka Norphenazone MCI-186 Reduces ROS generation in the Improves kidney function in
et al., 2017; Doi et al., Synthetic medication renal tubular cells, in vitro rats with IRI, and in rats
2004; Satoh et al., Reduces lipid peroxidation, with nephrotoxicity
2003; Aksoy et al., in vivo
2015)
Vitamin C (Ratliff et al., Vitamin Reacts with oxidized forms of Shown to improve kidney
2016; Tanaka et al., Dietary/ enzymes and free radicals function in ischemia/
2017) Supplementation intake Cofactor for some enzymatic chemically/
reactions rhabdomyolysis-induced
AKI
Selenium (Matés, 2000; Trace element Participates in aerobic Deficiency linked to AKI
Vasko et al., 2013; respiration reducing FR Improves kidney function
Oberley et al., 2001; Upregulates antioxidants in toxic AKI
Fukai, 2009)
Sulforaphane (Hwang Isothiocyanate Improves kidney function
et al., 2012; Shokeir Dietary intake in renal IRI
et al., 2015)

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 Superoxide dismutase mimetic activity can be found in pharmacologic agents Tempol
and MnTMPyP which have proved to ameliorate both ischemia-induced AKI, and
sepsis-induced AKI (Chatterjee et al., 2000; Liang et al., 2009). Also, MnTMPyp (which is
both SOD and catalase mimetic) decreases SOD related to renal fibrosis after ischemic
AKI (Kim et al., 2009).
Catalase, an enzyme containing heme, is responsible for further converting H2O2
into O2 and H2O, without requiring additional cofactors (Ratliff et al., 2016). It is
ubiquitary in cells with aerobic metabolism, and high levels of catalase are found in renal
cells (Deisseroth & Dounce, 1970). Intracellular disposition shows that catalase is mainly
found in the cytosol and in peroxisomes (Ratliff et al., 2016). Its deficiency leads to
ROS accumulation inside the mitochondrial matrix, causing mitochondrial dysfunction
(Hwang et al., 2012). An experimental study of endotoxemia in mice showed that LPS can
downregulate the catalase activity, aggravating renal damage (Vasko et al., 2013).
Thioredoxin peroxidase systems also consume H2O2, while oxidizing thioredoxin.
The oxidized forms that result play a crucial role in several signaling mechanisms.
These peroxidases can be found in most cellular compartments (Ratliff et al., 2016).
Glutathione and glutathione peroxidase family are present in all cellular compartments
(including the nucleus, mitochondria, cytoplasm) (Araujo & Welch, 2006; Dennis &
Witting, 2017; Oberley et al., 2001). Glutathione is important for regulating the cellular
redox, while being a part of the controlling mechanisms responsible for the cellular
proliferation, DNA synthesis, and cellular apoptosis (Fukai, 2009). In the mitochondria,
glutathione regulates apoptosis and necrosis, while in the nucleus it modulates the cellular
division (Dennis & Witting, 2017).
Edaravone represents an approved stroke therapy in Japan. It has been studied in
multiple experimental models, and it has been shown that it reduces the generation of ROS
in vitro in the renal tubular cells, reduces lipid peroxidation in vivo (Tanaka et al.,
2017), improves kidney function in rats with IRI (Doi et al., 2004), and also in rats with
nephrotoxicity (Satoh et al., 2003). These results show that edaravone (norphenazone,
MCI-186) might be able to avoid preservation injury in kidney transplantation
(Tanaka et al., 2017).
Vitamin C is a well-known antioxidant that attenuates OS, inflammation, and
improves kidney function in AKI animal models (ischemia-induced, chemically-induced,
rhabdomyolysis-induced AKI) (Tanaka et al., 2017). Vitamin C plays an important role
for some enzymatic reactions as a cofactor, participating in several defense mechanisms.
Vitamin C redox is preserved by diverse components of redox systems in the cell, reacting
with oxidized forms of molecules (enzymes, FRs) (Ratliff et al., 2016).
Selenium (Se) represents a trace element that plays a role in cellular aerobic respiration
by reducing FRs (Aksoy et al., 2015). Selenium deficiency was linked to AKI in rodent
model (Iglesias et al., 2013). Also, several experimental studies showed improved kidney
function after Se supplementation in AKI induced by cisplatin (Matés, 2000), and by
gentamycin (Randjelovic et al., 2012). Selenium upregulated antioxidants in a pig model of
transplanted kidneys (IRI) (Třeška et al., 2002).

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 Sulforaphane, a natural dietary isothiocyanate, has been shown to be renoprotective and
able to improve kidney function in an animal renal IRI model (Shokeir et al., 2015). In this
study, Shokeir et al. showed that ischemic preconditioning and sulforaphane both
enhanced gene expression (Nrf2, heme oxygenase-1 HO-1, NADPH-quinone
oxidoreductase1 NQO-1) and diminished the inflammatory (TNF-a, IL-1, ICAM-1) and
apoptotic markers (caspase-3), with better results for sulforaphane alone. Their combination
improved the antioxidant gene expression and attenuated the inflammatory genes.
Antioxidants play a leading role in fighting OS and preventing oxidative damage. Both
endogenous and exogenous (dietary/supplementation) seem to have a beneficial impact
on renal function, with renoprotective effects and good outcomes even on the suffering
kidneys.

Are there any biomarkers for detecting oxidative stress in early AKI?
The interest in defining biomarkers for acute illnesses is continuously increasing. Recent
studies focused on molecules that might increase specifically in OS. Some of them,
conducted in patients who suffered kidney damage induced by sepsis or by other critically
illnesses, highlighted the presence of plasmatic biomarkers from protein and lipid
oxidation that could be correlated with other markers for cytokines, pro-oxidative
mediators, and pro-inflammatory markers (Himmelfarb et al., 2004). Ischemia causes
alterations in DNA structure and leads to lipids peroxidation, leading to increased levels of
3-nitrotyrosine, a well-known biomarker for ROS and RNS (Noiri et al., 2001; Walker
et al., 2001).
One study showed that renal IRI causes increased urinary expression of thioredoxin1 (TRX),
making it a possible biomarker for OS (Piantadosi & Suliman, 2006; Kasuno et al., 2014).
Costa et al. (2018) conducted a study in septic shock patients admitted to the ICU and
determined the protein carbonyl concentration upon admission. The levels of protein
carbonyl were significantly higher in patients who developed septic AKI, and they were
also positively correlated with the SOFA score. Moreover, protein carbonyl concentration
was associated with development of septic AKI, and with mortality in these critical
patients. This study shows a novel molecule that could be used as a biomarker in AKI with
excellent reliability.
Recently, transfer RNA (tRNA) has been shown to be cleaved into half molecules under
OS, resulting tRNA-derived stress-induced fragments (tiRNAs) (Yamasaki et al., 2009).
Mishima et al. (2014) used a specific tRNA-specific modified nucleoside 1-methyladenosine
(m1A) antibody to show that changes in RNA structure occur much earlier than DNA
damage in OS exposure. Change in tRNA structure leads to further tRNA fragmentation,
reflecting early stages of OS damage. Authors suggest that detecting tRNA damage could be a
valuable tool for recognizing early organ damage and making a better medical decision.

CONCLUSIONS
Acute kidney injury, although common in clinical practice, is still poorly understood
from the pathophysiological point of view. This leads to addressing AKI with only
supportive therapies, instead of effective therapeutics to treat it. Research focuses on

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 understanding the molecular pathways that are present in renal injury, but the large
possible etiologies of AKI along with other comorbidities that patients might suffer
from, make it really problematic to draw a conclusion. Also, animal models used in the
experimental studies are more or less comparable to the human body from the
physiological and pathophysiological point of view. Recent data shows that OS plays a
crucial role in both initiating and further development of AKI, making it a possible target
for therapies. Oxidative damage leading to DNA and RNA alterations, peroxidation
of lipids, changes in the structure of proteins, represents an important cause of kidney
damage that must be addressed when attempting to treat AKI. ROS, RNS, FRs are closely
linked to AKI, and they must be very well understood in order to be targeted. Endogenous
antioxidants represent a first line against oxidative damage, and their downregulation
worsens the outcome in kidney injury. Antioxidant supplementation is intensely
studied, showing promising renoprotective effects. Moreover, research focuses on OS
biomarkers that can be detected in early phases of AKI, with apparently good results,
whether we talk about detecting novel molecules or using newly-constructed monoclonal
antibodies. Nonetheless, more studies are needed in order to establish new clinical
guidelines.
Oxidative stress in AKI, although accepted as a key component in the pathophysiology
of AKI, is yet to be understood as a target for novel therapeutics.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
The authors received no funding for this work.

Competing Interests
The authors declare that they have no competing interests.

Author Contributions
 Anamaria Magdalena Tomsa conceived and designed the experiments, prepared figures
and/or tables, approved the final draft.
 Alexandru Leonard Alexa conceived and designed the experiments, prepared figures
and/or tables, approved the final draft.
 Monica Lia Junie analyzed the data, authored or reviewed drafts of the paper, approved
the final draft.
 Andreea Liana Rachisan analyzed the data, authored or reviewed drafts of the paper,
approved the final draft, she participated in the correction of the whole manuscript.
 Lorena Ciumarnean analyzed the data, authored or reviewed drafts of the paper,
approved the final draft.

Data Availability
The following information was supplied regarding data availability:
This is a review article.

Tomsa et al. (2019), PeerJ, DOI 10.7717/peerj.8046 14/21

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