Renalase as a Novel Biomarker for Evaluating the Severity of Hepatic Ischemia-Reperfusion Injury

Hepatic ischemia-reperfusion (I/R) injury is a serious complication in clinical practice. However, no efficient biomarkers are available for the evaluation of the severity of I/R injury. Recently, renalase has been reported to be implicated in the I/R injury of various organs. This protein is secreted into the blood in response to increased oxidative stress. To investigate the responsiveness of renalase to oxidative stress, we examined the changes of renalase in cell and mouse models. We observed a significant increase of renalase expression in HepG2 cells in a time- and dose-dependent manner when treated with H2O2. Renalase expression also increased significantly in liver tissues that underwent the hepatic I/R process. The increased renalase levels could be efficiently suppressed by antioxidants in vitro and in vivo. Furthermore, serum renalase levels were significantly increased in the mouse models and also efficiently suppressed by antioxidants treatment. The variation trends are consistent between renalase and liver enzymes in the mouse models. In conclusion, renalase is highly sensitive and responsive to oxidative stress in vitro and in vivo. Moreover, renalase can be detected in the blood. These properties make renalase a highly promising biomarker for the evaluation of the severity of hepatic I/R injury.


Introduction
Ischemia is ceasing of blood supply, causing a shortage of oxygen; reperfusion is restoring of blood supply after ischemia. The ischemia-reperfusion (I/R) process occurs in many clinically important events, including hepatic resectional surgery, transplantation, trauma, and hemorrhagic shock [1][2][3]. Hepatic I/R injury is an inevitable complication causing severe cellular death, tissues damage, and liver dysfunction, which increases the mortality [4][5][6]. Reactive oxygen species (ROS), which are the main toxicants in oxidative stress, play critical roles in hepatic I/R injury [7][8][9][10].
Vascular endothelial cells, hepatic sinusoidal endothelial cells, Kupffer cells, or polymorphonuclear leukocytes are activated by the I/R process and produce a large amount of ROS in oxidative stress process, including superoxide, hydrogen peroxide (H 2 O 2 ), hydroxyl radicals, and nitric oxide. This ROS production is the main factor responsible for damaging liver parenchymal cells, increasing vascular permeability, and inducing inflammatory cell infiltration [2,[11][12][13][14][15]. H 2 O 2 is the most abundant and stable ROS. It leads to oxidative stress and is implicated in a variety of inflammatory diseases [16,17]. H 2 O 2 can be converted into hydroxyl radicals, which are extremely reactive and more toxic than other ROS [8,9]. In this context, H 2 O 2 is the perfect agent to establish an oxidative stress model in cells and also to simulate I/R injury in vitro to some extent.
It has been proposed that I/R injury is an intricate process in which the oxidant/antioxidant balance is changed in favor of the oxidants [18]. Treatment with superoxide dismutase (SOD) and catalase (CAT) reduces the ROS levels, as SOD rapidly dismutases the superoxide (O 2 − ) to H 2 O 2 , and CAT scavenges H 2 O 2 to produce water, which reduces the severity of oxidative stress [5]. Beside SOD and CAT, a variety of compounds and biomaterials has been developed as potential therapeutic agents for I/R injury [6,17]. However, it still lacks efficient, specific, and sensitive biomarkers for the accurate evaluation of the severity of oxidative stress in hepatic I/R injury [19].
Renalase, a ubiquitous flavin adenine dinucleotide-containing amino oxidase, has been implicated in the process of I/R injury [20]. Since first being identified in 2005, renalase has been reported to be synthesized in various organs, including kidney, heart, liver, and adipose tissues [21]. Renalase is secreted into the blood in response to increased oxidative stress [22][23][24]. The elevated renalase level under stress conditions makes renalase a potential biomarker for the evaluation of the severity of organ I/R injury.
In the present study, we demonstrated that renalase is a sensitive ROS-responsive gene in hepatocytes. In hepatic I/R injury mouse models, renalase was augmented in liver and blood. Moreover, the augmentation of renalase can be ameliorated by antioxidants pretreating, which can reduce the severity of oxidative stress, in vitro and in vivo. These findings provide evidence that renalase can serve as an efficient and sensitive biomarker for the early warning or evaluation of the severity of hepatic I/R injury. and 100 U/mL penicillin-100 g/mL streptomycin (Gibco, Carlsbad, CA, USA) and kept in a humidified atmosphere at 37 ∘ C with 5% CO 2 in an incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA).

In Vivo Hepatic I/R Model.
The in vivo hepatic I/R model was performed as previously described [3,16,17,25]. Male C57BL/6 mice, aged 8-12 weeks, were purchased from Beijing University (Beijing, China) and maintained on a chow diet in a 12 h light/12 h dark environment at 25 ∘ C in the Animal Care Facility of Tongji Medical College. Surgical procedures on mice were performed under sterile conditions by administration of pentobarbital sodium (50 mg/kg) by an intraperitoneal injection. One hour before the pentobarbital sodium anesthesia, the I/R+SOD+CAT group was intraperitoneally injected with 300 KU/kg SOD and 60 mg/kg CAT, whereas the sham and I/R mice groups were given physiological saline as the solvent by the same method. Laparotomy was performed by vertically opening 2.5-3 cm in the anterior part of the abdomen of the anesthetized mice. After identifying the portal triad and biliary tree, the main trunk of the hepatic artery and portal vein, except for the vasculatures to the right lower lobe, was clamped with a vascular clip to achieve ischemic injury to approximately 70% of the liver. After 1 h of ischemia, reperfusion was achieved by releasing the vascular clip. No vascular clamp was done for the sham group of mice. Then, the incision was closed with silk suture. Six hours after reperfusion, hepatic lobes underwent I/R and the corresponding hepatic lobes from the mice of the sham group were removed and used for further assays. Histological evaluations (H&E staining and IHC of cleaved caspase-3) were performed to quantify the degree of liver injury. Confocal immunofluorescence imaging of frozen sections was performed to detect the renalase levels. Western blotting and real-time qPCR were performed to detect the protein levels and mRNA expression of renalase in liver tissue. Blood was taken by eyeball extirpating and then centrifuged for serum separation, and the serum was used for detection of levels of the renalase and liver enzymes.

Western Blot Analysis.
As previously described [26][27][28], total cells and tissues were lysed using RIPA lysis buffer, and the protein concentration was determined with a BCA protein assay kit (Pierce Company, Rockford, IL, USA). Protein extracts were used for SDS-PAGE (Invitrogen, Carlsbad, CA, USA), and the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), which was blocked with 5% nonfat milk in TBS for 3 h and incubated with various primary antibodies overnight at 4 ∘ C. After incubation with HRP-conjugated secondary antibodies (diluted 1 : 5000) for 1 h at room temperature, the membranes were treated with ECL reagents (170-5061, Bio-Rad, Hercules, CA, USA) prior to visualization using a ChemiDoc MP imaging analysis system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The specific protein expression levels were normalized totubulin on the same nitrocellulose membrane. The following primary antibodies and dilutions were used: anti-renalase (GTX89570, diluted 1 : 1000) was purchased from GeneTex (Irvine, CA, USA); anti--tubulin (sc-9104, diluted 1 : 2000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.5.
Real-Time RT-RCR. As previously described [29,30], total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 2 g of total RNA was reversely transcripted using an RNA PCR Kit (Takara Biotechnology, Otsu, Japan), and the resulting cDNA was used as a PCR template. The mRNA levels were determined by real-time qPCR with an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions, and -tubulin was used as endogenous control.
The experiment was performed in triplicate. The relative gene expression levels were calculated using the comparative C T method applying the formula 2 −ΔΔC T . The primer sequences for real-time qPCR are listed in Table 1.

Confocal Immunofluorescence.
Fresh ischemia-reperfusion hepatic lobes were collected and rinsed in saline to remove remaining blood. Tissues were cut into 6-m thick sections with a freezing slicing microtome. The sections were then immersed and fixed in 4% paraformaldehyde at room temperature for one-half hour. Afterwards, the sections were incubated with 5% bovine serum albumin (BSA) and immunolabeled with the indicated antibody (renalase GTX89570, GeneTex) at 4 ∘ C overnight. After washing, the sections were incubated with Alexa Fluor 594 goat antirabbit IgG (R37117, Invitrogen) for 1 h. After washing, 4 ,6diamidino-2-phenylindole (DAPI) was added to stain the cell nuclei on ice. Tissue fluorescence was imaged on a confocal microscope (Alsi, Nikon). For the quantitative expression of renalase, the density of fluorescence was analyzed by the ImageJ 1.44p software.

Liver Histology and IHC.
Formalin-fixed liver specimens were embedded in paraffin blocks and cut into 5 m sections. The sections were then stained with hematoxylin and eosin for histology. For IHC, the sections were deparaffinized and rehydrated with ethanol and xylene and were heated to 95-98 ∘ C for 20 min in 10 mM citrate buffer, pH 6.0. After blocking with PBS containing 10% goat serum for 1 h at room temperature, the sections were incubated with a primary antibody 1 : 200 cleaved caspase-3 overnight at 4 ∘ C. After incubation with secondary antibodies, the sections were mounted and evaluated with an Olympus microscope.

Detection of Liver Enzymes in Serum.
Alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in serum were determined using automatic biochemical analyzer (TBA-40FR, Toshiba, Tokyo, Japan). Related detection kits were purchased from MedicalSystem Biotechnology Co., Ningbo, China. 2.10. Statistical Analysis. As described in previous studies [31,32], the statistical analysis was performed with the Statistical Package for Social Sciences (SPSS version 13.0; IBM Analytics, Chicago, IL, USA). All the data were expressed as mean ± SD (standard deviation, SD) and the difference was analyzed by a one-way ANOVA test. Statistical analysis was performed using Student's t-test for paired data. The difference was considered statistically significant for < 0.05.  Figure 1(a)). The maximal expression of renalase was detected for a concentration of 500 M. Renalase expression decreased slightly when incubated with a concentration of 1000 M compared with 500 M (Figure 1(a)).  mRNA expression of renalase (Figure 1(b)). The maximal expression of renalase was measured for an incubation time of 12 h. Renalase expression decreased slightly when incubated for 24 h (Figure 1(b)). This indicates that the cells may not tolerate the long-time incubation of H 2 O 2 . Further, the protein levels of renalase in HepG2 cells that were incubated with 500 M H 2 O 2 for either 6 h or 12 h were measured and found to be larger for the longer incubation ( < 0.01, Figures  1(c) and 1(d)).

Increased Renalase Expression and Histologic Damage in Hepatic I/R Injury Mouse Models and the Suppression to the Increased Renalase Expression by Antioxidants.
In hepatic I/R injury mouse models, the renalase mRNA expression in livers was significantly augmented ( < 0.01, Figure 3(a)), and the intraperitoneal preinjection of SOD and CAT decreased this augmentation significantly ( < 0.01, Figure 3(a)). The renalase protein levels in livers of the mouse models were significantly increased ( < 0.01, Figure 3(c)) and could also be suppressed by the intraperitoneal preinjection of SOD and CAT ( < 0.01, Figure 3(c)). This result was confirmed by Western blot (Figures 3(b) and 3(c)) and confocal immunofluorescence imaging (Figure 4(b)). The histological evaluation revealed that I/R caused hydropic degeneration and necrosis of hepatic tissue and cells. A significant improvement was observed in I/R+SOD+CAT group (Figure 4(a)). Consistent with the observed hydropic degeneration and necrosis during I/R, hepatic apoptosis was increased as seen by the brown intracellular staining of cleaved caspase-3 in most hepatocytes. A significant reduction of cleaved caspase-3 staining was observed in the I/R+SOD+CAT group (Figure 4(a)).

Increase of the Serum Levels of Renalase and Liver Enzymes in Hepatic I/R Injury Mouse Models and the Suppression to the Increased Renalase and Liver Enzymes by
Antioxidants. The liver enzymes (ALT, AST, GGT, ALP, and LDH) and renalase levels were measured in the serum of the hepatic I/R injury mouse models. The renalase levels were found to be significantly augmented in the hepatic I/R process ( < 0.01). The levels of ALT ( < 0.01), AST ( < 0.01), ALP ( < 0.05), and LDH ( < 0.01) elevated significantly in the hepatic I/R group compared to the sham group, whereas GGT increased slightly in the hepatic I/R group and with no statistical significance to the sham group ( > 0.05).
The increase of ALT, AST, LDH, and renalase levels could be effectively suppressed (renalase, ALT, and LDH: < 0.01; AST: < 0.05) by the intraperitoneal preinjection of SOD and CAT. ALP and GGT could also be suppressed by antioxidants, but with no statistical significance ( > 0.05) to the I/R group ( Figure 5).

Discussion
Hepatic I/R injury may occur in hepatic resections, liver transplantations, traumas, and vascular surgeries [33][34][35]. It is a serious complication in clinical practice. The redox balance in the process of hepatic I/R injury leads to accumulation of ROS, which presents the disease mechanism most commonly invoked in hepatic I/R injury [33]. Suppressing ROS-burst in oxidative stress is an efficient way to alleviate hepatic I/R injury. However, early warning, evaluation of the severity, or therapeutic effect of oxidative stress, which is even more important in clinical practice of hepatic I/R injury management, is a remaining challenge. This is due to the lack of efficient, specific, and sensitive biomarkers for the accurate evaluation of the severity of oxidative stress [19]. Biochemical markers used to evaluate the severity of oxidative stress include malondialdehyde (MDA) [36,37], ascorbic acid (AA)/dehydroascorbic acid (DHA) [38,39], and a series of inflammatory, proinflammatory, or anti-inflammatory biomarkers [40][41][42]. However, these markers face various limitations. MDA is a marker for lipid peroxidation, which usually proceeds in the I/R injury of the extremities [43]. A close relationship between MDA and cardiac necrosis markers has been reported [36], but whether the MDA level can reflect the severity of oxidative stress or I/R injury of other organs remains unclear. The determination of AA and DHA levels is challenging because of the unstable nature of these compounds [39]. Inflammatory, proinflammatory, and anti-inflammatory markers cover a wide range of cytokines. However, these cytokines are not specific for evaluating oxidative stress. Therefore, an efficient, specific, and sensitive biomarker for evaluating oxidative stress is still urgently needed.
The human renalase gene is located on chromosome 10 at q23.33 and encodes the conserved protein renalase consisting of 342 amino acids. Renalase was reported to efficiently degrade dopamine and epinephrine in the blood [21], and it has been verified that it is the only enzyme involved in the catecholamine metabolism that can be secreted into the blood cycle hitherto [44]. Previous studies indicated that renalase is multifunctional and closely related to oxidative stress conditions such as stroke, heart transplantation, or acute kidney injury [45][46][47][48]. Considering its tight relationship with oxidative stress, renalase is thought to play a role, or at least responsive, in the process of hepatic I/R injury. In particular, H 2 O 2 is considered as the most abundant and stable ROS. H 2 O 2 is a mild oxidant but is converted into hydroxyl radicals, which are extremely reactive and more toxic than other ROS [49,50]. Therefore, to investigate the responsiveness of renalase in hepatic cells under the condition of oxidative stress, H 2 O 2 was used to mimic the ROSburst in oxidative stress. For the cell model in vitro, the common hepatocellular carcinoma cell line HepG2 was chosen. Renalase expression in HepG2 cells showed a positive correlation with H 2 O 2 concentration and incubation time. These results suggest that renalase is responsive to the ROS-burst or oxidative stress in hepatic cells, and it is sensitive to the degree or severity and duration of oxidative stress.
Disturbance of the redox balance is characteristic for oxidative stress in the process of the hepatic I/R injury, which leads to ROS-burst and antioxidant consumption [33]. The most efficient way to ameliorate oxidative stress is replenishing ROS-scavenging antioxidants [17]. SOD is a common antioxidant, which is wildly distributed within mammalian organisms [51]. It is an important free radical scavenger, which has been clinically used in several countries. CAT is also an endogenous antioxidant, which can catalytically decompose H 2 O 2 [52,53], but has not been used in clinical practice. In this study, SOD and CAT were used for the preincubation of the cells. We observed that the SOD/CAT preincubation significantly ameliorated the elevation of the renalase expression in HepG2 cells. The application of antioxidants reduced the severity of oxidative stress, and the responsive elevation of the renalase expression was ameliorated accordingly. This result suggests that renalase is responsive to the therapeutic means for oxidative stress in hepatic cells. And the expression of renalase is closely related to the degree or severity of the oxidative stress. The hepatic I/R injury mouse model is the most commonly used animal model in various studies for hepatic I/R injury and oxidative stress in vivo. We used this model to investigate whether renalase is responsive to the hepatic I/R injury in vivo. Renalase expression was significantly increased in the livers of hepatic I/R injury mouse models. Intraperitoneal preinjection of SOD and CAT ameliorated the elevation of renalase in the livers significantly. Furthermore, a consistent high-and-low variation between the renalase expression in liver and the degree of liver damage, including tissue necrosis and cell apoptosis, has been observed. Liver enzymes are the most important evaluation indexes in the standard liver function test (LFT) panel. Serum levels of liver enzymes, including ALT, AST, GGT, ALP, and LDH, were increased by different degrees in the hepatic I/R injury mouse models. Consistently, the serum renalase levels of the hepatic I/R injury mouse models were significantly elevated. The elevations of serum levels of either the renalase or the liver enzymes could be significantly reduced by the intraperitoneal preinjection of SOD and CAT. These results suggest that renalase is responsive to hepatic I/R injury and therapeutic means. Its expression in liver tissue closely related to the severity of oxidative stress and the subsequent liver damage. Moreover, the serum renalase level can sensitively reflect the severity and therapeutic effect of oxidative stress in the process of hepatic I/R injury. Combining with the standard LFT panel, more accurate and specific evaluations of the hepatic I/R injury can be made.
In conclusion, to the best of our knowledge, this study firstly demonstrates the value and applicability of renalase as a promising biological marker for the evaluation of the severity of oxidative stress in hepatic I/R injury. We investigated the responsiveness of renalase to conditions related to oxidative stress in vitro and in vivo. Our results revealed that oxidative stress efficiently induces the elevation of renalase expression in vitro and in vivo. Furthermore, this elevated Oxidative Medicine and Cellular Longevity 9 renalase expression can be decreased by antioxidants. Its sensitive responsiveness to the severity of oxidative stress and convenient detection in the blood make renalase an ideal biomarker for the evaluation of the severity and therapeutic effect of hepatic I/R injury. In the future, clinical treatment decisions based on the detection of renalase activities in the blood may help to improve the clinical outcomes of hepatic resections, liver transplantations, traumas, or vascular surgeries. Nevertheless, the variation trend of serum renalase level in the hepatic I/R process under different conditions and time points has not been demonstrated in this study, which needs further investigations. Beside the I/R injury, there are various pathophysiological processes which are related to oxidative stress. Renalase may play roles in these processes, and the variation trend of renalase expression in these processes may be different from each other. Related researches may make renalase serve as a novel inspection item, which can assist the existing inspection items to make the evaluations of the diseases more accurate. Other potential applications of renalase in clinical practice require further explorations and investigations.

Disclosure
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.