Acute kidney injury (AKI) is one of the common complications in the immediate postoperative period of liver transplantation and associated with significant morbidity and mortality. Over 30% to 50% recipients have experienced postoperative AKI.1 A wide array of factors are responsible for the prevalence of AKI. Hypotension has been considered to be a crucial independent risk factor of renal ischemia-reperfusion (I/R) injury during perioperative period of liver transplantation.2 Several pharmacologic agents with the potential to reduce I/R injury have been characterized so far,3,4 but none has yet been translated into clinical application.
Although the mechanisms underlying I/R injury are complex, both apoptosis and autophagy play vital roles in the progression of cell death induced by I/R.5,6 Although the comprehensive picture depicting apoptotic regulation has been well explored, the mechanism of autophagy is still unclear. Accumulating evidence reveals that the crosstalk between autophagy and apoptosis is considered to be the turning point of cell fate, and several pathways have been delineated to provide mechanistic insight into this connection.7,8 In the network, some regulators, such as Bcl-2 family members and PI3K/Akt signaling proteins in the process of apoptosis, have been found to modulate autophagy simultaneously.9,10 P53 is a crucial transcription factor which mediates the cell cycle arrest associated with I/R injury.11 Intriguingly, several recent literatures have highlighted the functional connection of p53 between autophagy and apoptosis.12
The activation of autophagy by p53 is partly attributed to the inhibition of the negative regulator of autophagy (mammalian target of rapamycin) and the stimulation of AMP kinase or damage-regulated autophagy modulator (DRAM).13 In several settings (in vitro and in vivo), pharmacological or genetic inhibition of p53-mediated autophagy contributed to apoptosis in response to diverse stresses, pointing to p53 appears to be a significant component in the network controlling apoptosis and autophagy.14,15 Also, the activation of p53 was speculated to be therapeutically desirable for I/R treatment.
As a novel antioxidant, hydrogen possesses antioxidative property by selectively neutralizing cytotoxic reactive oxygen species, such as •OH and ONOO.16,17 Ohsawa et al18 demonstrated the protective effect of hydrogen against cerebral I/R injury via attenuating the production of cytotoxic reactive oxygen species. In addition, hydrogen could also suppress diseases induced by inflammation, such as acute peritonitis, intestinal injury, and so on.19,20 However, the therapeutic mechanism underlying its protective effects in organ transplantation has not been completely elucidated.
Herein, the present study was aimed to explore the protective effect of hydrogen-rich saline (HRS) on AKI of rats after orthotropic liver transplantation (OLT) and to investigate the potential role of p53-mediated autophagy in hydrogen-offered protection.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats weighing 220 ± 10 g were obtained from the Laboratory Animal Center, The People's Liberation Army Military Academy of Medical Sciences, Beijing, China. All animal care and experimental procedures were strictly under obligations of Institutional Animal Care and Use Committee of Tianjin First Center Hospital. All efforts were made to minimize suffering. Acclimatized to the environment for 5 days, rats were randomly assigned into 4 groups: sham-operated (sham), OLT, OLT rats injected with 6 mL/kg HRS through infrahepatic vena cava 5 minutes before ischemia (HS), and OLT rats pretreated with 6 mL/kg HRS and chloroquine (60 mg/kg) intraperitoneally just before ischemia (CQ).
Materials and Reagents
Chloroquine (Sigma, St. Louis, MO) was diluted in phosphate-buffered saline (PBS). Malondialdehyde (MDA) and superoxide dismutase (SOD) detection kits were purchased from Jiancheng Biotech (Jiangsu, China). The primers of caspase-3 and cytochrome c (cyt c) were purchased from Augct Biotech (Beijing, China). The antibodies against p53, p-p53, DRAM, Beclin1, microtubule-associated protein light 3-II (LC3-II), p62, caspase-3, cyt c, glyceraldehyde-3-phosphate dehydrogenase, and tubulin were purchased from Cell Signaling Technology (Beverly, MA). Lysosome-associated membrane protein-2 (LAMP-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Hydrogen was dissolved in normal saline under the pressure of 0.5 MPa for 7 hours to reach the saturation by using the apparatus (Hydrovita Biotechnology Co, Beijing, China). Then, the saturated HRS was stored under atmospheric pressure at 4°C and then sterilized with γ radiation. The concentration of HRS was 0.6 mmol/L measured by gas chromatography. Hydrogen-rich saline would be prepared every week to maintain the saturated concentration.
Establishment of Rat OLT Models
The OLT model was performed as described by Kamada et al.21 Donor liver was harvested in a standardized procedure, which included dissecting the liver from the ligaments and vessels, cannulating the common bile duct with a 22G stent, retracting liver lobes in situ to minimize the harvest-dependent injury. Then, the graft was flushed with 4°C solution under a pressure of 10 cm H2O before implantation. After removing from the original liver, the OLT operation was performed in the hepatectomized recipient rat by the following steps: anastomosing the suprahepatic vena cava with a running 7/0 polyproplene suture, inserting cuffs into the related vessels to reestablish the blood flow and connecting the bile duct with the splint technique. During the operation, the portal vein was clamped for 25 to 30 minutes. The rats were allowed to drink glucose water without any food. All the samples were collected 6 hours after reperfusion.
The Assay of Mean Arterial Pressure
The right carotid artery was catheterized with a polyethylene catheter (outer diameter, 0.96 mm; inner diameter, 0.66 mm) to monitor mean arterial pressure (MAP),22 the recording time points of which were as follows: before the OLT operation, before vascular clamped, the anhepatic phase (1 minute, 15 minutes), after portal vein unclamped (reperfusion, 1 minute, 15 minutes), before operation finished, and after operation for 10 minutes. The carotid artery was filled with heparinized saline (30 U/mL) to prevent the clot formation.
The tissues were embedded in paraffin after fixation in 10% phosphate-buffered formalin for 12 hours. Then they were sectioned at 4-μm thick and stained with hematoxylin-eosin before the examination under light microscopy (Olympus BX51, Tokyo, Japan). Each sample was examined by the same pathologist in a blinded study. Morphological changes were scored from 0 to 4 to assess the degree of renal damage as described by Jablonski et al23 (Table 1).
Determination of Renal Function
The renal function of rats after OLT was evaluated using the parameters of blood urea nitrogen (BUN) and serum creatinine (Cr), which were measured using an automated clinical chemistry analyzer (AU5400; Beckman Coulter).
Assessment of Oxidative Stress Parameters
Each sample from different groups was homogenized and centrifuged for detecting the level of MDA and SOD by commercially available kits. The concentration of MDA was measured by the thiobarbituric acid method. The SOD activity was measured using the nitroblue tetrazolium method.
Deoxynucleotide Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling Assay
To identify the apoptotic nuclei quantitatively, the terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) was performed. Briefly, tissue sections were deparaffinized and rehydrated by heating at 65°C, followed by incubation in protease K (10 μg/mL) for 30 minutes at room temperature. After washing in PBS twice, the sections were exposed to the TUNEL reaction mixture for 1 hour at 37°C in a humidified atmosphere without light. The apoptotic cells stained with 4′,6-diamidino-2-phenylindole were examined by the fluorescence microscopy. The apoptotic index was calculated in a total of 10 representative fields (400× magnification) from each tissue section.
Transmission Electron Microscopy
Kidney samples from each group were carefully immersed in the fixative solution (2.5% glutaraldehyde) overnight. Then, the tissues of approximately 1 mm3 were washed with PBS for 3 times, fixed in 10% buffered glutaraldehyde and 1% osmic acid, dehydrated, embedded, and finally sliced. The ultrastructure of autophagosome was observed under a transmission electron microscope by the same histologist who was unaware of the group of each sample.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from renal tissue using RNAeasy kit (Qiagen, Hilden, Germany). Also, 4 μg was reverse-transcribed into complementary (c)DNA by using the first-strand cDNA synthesis kit (Takara, Japan). The thermal cycling conditions were as follows: denaturation for 1 minute at 95°C, 40 cycles of 5 seconds at 95°C, 30 seconds at 58°C, and 30 seconds at 72°C, and dissolve curve analysis for 15 seconds at 95°C, 1 minute at 60°C, and 15 seconds at 95°C. The primer sequences used to amplify the desired cDNA were shown in Table 2. The polymerase chain reaction products were normalized to β-actin levels.
Western Blot Analysis
Kidney tissues were rapidly lysed in 200 μL of extraction protein buffer. After homogenization on ice for 30 minutes, the supernatant was centrifuged at 12 000g for 10 minutes, and the content would be calculated with Bradford assay. Then total protein were boiled in water for 5 minutes to denature the protein. Equal amounts of protein were loaded on 10% sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and transferred to the polyvinylidene difluoride membrane. After being blocked in 5% (w/v) skimmed milk powder-Tris–buffered saline with 0.1% Tween 20 for 1 hour at 37°C, the membranes were probed with antibodies against p53, p-p53, DRAM, Beclin1, LC3-II, p62, LAMP-2, caspase-3, cyt c, glyceraldehyde-3-phosphate dehydrogenase and tubulin overnight at 4°C, respectively. Subsequently, the membranes were incubated with secondary peroxidase-conjugated antibodies at room temperature for 2 hours. Blots were quantified by Image-Pro Plus Software. The glyceraldehyde-3-phosphate dehydrogenase and tubulin were taken as endogenous control, and the densitometry values were used for normalizing the expression of different proteins.
Histologic damage scores were expressed as median ± interquartile range and the statistical significance was calculated by Kruskale-Wallis H test. Other data were expressed as mean ± SD and analyzed by 1-way analysis of variance. A P value less than 0.05 was considered to be statistically significant. All statistical tests were performed with SPSS20.0.
The Levels of Rat MAP During OLT
According to Table 3, it shows a significantly lower value of MAP during the anhepatic phase. After the blood flow was reestablished, the MAP gradually recovered to the normal value. The results above indicated that blood reflux disorders including the severe hypotension during the anhepatic phase might be partly responsible for the AKI after OLT.
Effect of HRS Pretreatment on Renal Function of Rats Induced by OLT
To investigate the effect of HRS on renal damage after OLT, we checked the level of Cr and BUN in serum (Figures 1A, B). Orthotropic liver transplantation dramatically elevated the Cr and BUN levels compared with the sham-treated rats (P < 0.05). The increase in Cr and BUN values were reduced by administering HRS (P < 0.05). Pretreatment with chloroquine aggravated the renal damage compared with the HS group (P < 0.05). These findings suggest that HRS could improve the renal function of rats after OLT.
Effect of HRS on Renal MDA and SOD in OLT Rats
To uncover the potential antioxidative property of HRS, the presence of oxidative stress in rats was evaluated. Our results showed that the activity of SOD was drastically decreased with the elevated level of MDA after OLT compared with sham group. Pretreatment with HRS markedly suppressed the production of MDA accompanied by the increase of SOD activity in the OLT rats, exhibiting significant antioxidative activity. However, chloroquine counteracted the effect of HRS against oxidative stress compared with the HS group (P < 0.05) (Figures 1C, D).
HRS Reduced the Histopathologic Damage of Rats Induced by OLT
Sections obtained at 6 hours after reperfusion were evaluated for histopathologic analysis. Compared with the sham group, the histologic damage scores of the other 3 groups were higher (P < 0.05). Cellular vacuolization, irregularities in cytoplasm of epithelial cells, and loss of brush borders were observed in OLT group with a dramatically higher Jablonski score (P < 0.05). Moderate cytoplasmic vacuolation and lower tubular debris were seen in the HS group with the reduced damage score (P < 0.05). However, chloroquine counteracted the protective effect of HRS (Figures 2A, C).
Ultrastructural Changes of Autophagic Vacuoles in Renal Cells After OLT
Figure 2B shows the ultrastructural changes of renal tubular cells after OLT. The sham group shows the normal nuclei, mitochondria, and endoplasmic reticulum. Orthotropic liver transplantation induced the accumulation of double membrane structures containing undigested cytoplasmic organelles and proteins, which are known as autophagosomes and autolysosomes. Contrasted to OLT group, HS group showed a higher extent of autophagic phenomenon. Fewer autophagosomes were found after the pretreatment of chloroquine.
Effects of HRS Pretreatment on Renal Apoptosis After OLT
The TUNEL-positive cells stained brown in the nuclei indicate apoptosis. As shown in Figures 3A and B, The TUNEL-positive cells appeared very few in the sham group but were greatly increased in OLT group. Pretreatment with HRS significantly reduced the renal apoptosis in OLT rats (P < 0.05), whereas the number of apoptotic cells in CQ group and in OLT group were closer (P > 0.05).
In addition to TUNEL staining, the quantification of the apoptotic proteins, including cyt c and caspase-3 were determined by quantitative real-time polymerase chain reaction and Western blot. The results showed that the expression of caspase-3 and cyt c were low in the sham group, whereas the OLT group has the higher expression of caspase-3 and cyt c. Pretreatment with HRS decreased the elevated apoptotic gene expression in OLT rats. However, chloroquine inhibited the effect of HRS (P < 0.05) (Figures 3C, D). Consistently, similar results were found by Western blot, indicating that HRS could attenuate apoptosis (Figures 3E, F).
HRS Treatment Increased the Expression of Autophagy-Related Proteins in Renal Cells
To investigate whether autophagy was associated in the salutary effects of HRS, we assessed the expression of proteins related to autophagy using Western blot. Figures 4A and E show that LC-3II and Beclin-1 in OLT group were concurrently increased in contrast to the sham group. Also, HRS treatment significantly elevated the expression of LC-3II and Beclin-1 compared with the OLT group. Additionally, the expressions of LC-3II and Beclin-1 were downregulated by chloroquine administration. Reciprocally, HRS preconditioning did impede the level of p62 and LAMP-2 as compared with those in the sham and OLT groups (Figures 4B, F). These results supported the role of autophagy in HRS therapeutic effect in AKI after liver transplantation.
HRS Treatment Triggered the p53-DRAM Activation in Renal Cells
Considering the identification of p53 as the switch between autophagy and apoptosis, we focused our attention at the phosphorylation of p53 and its direct target DRAM. Figures 4C and G show that the p-p53 level in OLT group was increased compared with the sham group. The HRS treatment significantly elevated the expression of p-p53 compared with the OLT group. Moreover, DRAM activation was observed consistent with the change in p53, indicating that p53-DRAM activation induced the autophagy. However, the expressions of p-p53 and DRAM were not downregulated by chloroquine administration (P > 0.05)(Figure 4C, D, G, H), which confirmed the upstream position of those 2 proteins in autophagy signaling pathway.
Liver transplantation is an effective treatment for patients with the final stage liver disease,24 whereas AKI is considered to be a frequent postoperative complication and influences patient survival severely.25 In our investigation, we chose the OLT model of rats to explore the protective effects and the mechanism of HRS in this pathology. Compared with the simple hepatic I/R model, OLT model can simulate most procedures of clinical liver transplantation including intraoperative blood reflux disorder, which might partly contribute to AKI after liver transplantation. Our results showed that reperfusion would induce severe renal dysfunction characterized by the aggravated renal damage parameters and histopathology change.
Hydrogen is the most lightest and abundant element in nature. It has ranked the forefront of medical gas research as an important physiological protective factor with antioxidation, anti-inflammation, and anti-apoptosis.26-28 Accumulated evidences have shown that hydrogen is able to reach all cellular compartments because of its small size and can act as a scavenger no matter if it is administered via gas inhalation or HRS consumption.29,30 Up to now, the effect of hydrogen on AKI after liver transplantation has not been well characterized. Findings from our present study showed the potential therapeutic value of HRS in AKI by suppressing apoptosis and the activating p53-mediated autophagy.
Several studies have verified that apoptosis plays a prominent role in ischemia-induced renal dysfunction.31,32 The excessive attack of oxygen free radical and the weakening of antioxidant defense systems are known to be responsible from apoptosis.33 As in the case of many other organs, the kidney is also vulnerable to oxidative stress resulting from I/R injury.34 In the present study, we disclosed that HRS alleviated I/R-induced apoptosis as well as oxidative damage by upregulating the SOD activity with the reduction in MDA content.
The function of autophagy in I/R injury is still controversial. From 1 perspective, autophagy can disassemble the cytoplasmic parts and intracellular organelles which are not necessary to eliminate damaged organelles and maintain the balance of protein synthesis.35 However, it has also been reported that uncontrolled autophagy can lead to the irreversible demise of cells.36 Our study explored the role of autophagy in regulating the process of AKI after OLT. According to the result of transmission electron microscopy, we found that the focal degradation of cytoplasmic areas was sequestered by the phagophore and its maturation into the autophagosome, which is the morphologic hallmark of autophagy. Molecularly, autophagosomes induced by PI3K are tightly regulated by autophagy-related genes (ATG), among which, the most well-known gene is ATG8, also called LC3 in mammalian cells. The cytosolic form of LC3 (LC3-I) is cleaved and lipidated to form LC3-II, which translocates rapidly onto the autophagosomal membrane. The LC3-II is a reliable marker to indicate the activation of autophagy.37 Beclin-1 is a unique autophagy-related protein and plays an important role in the recruitment of other autophagic proteins during the expansion of preautophagosomal membrane.38 The p62, also called sequestosome 1, can bind to LC3 and Atg8 family proteins directly and facilitate the selective degradation of autophagic cargo. Moreover, LAMP-2, abundant in late endosomes and lysosomes, serves as a critical determinant in autophagosome-lysosome fusion39 Compared with the sham group, LC3-II and Beclin-1 protein levels were upregulated with the reduced p62 and LAMP-2 expression in the renal tissues 6 hours after reperfusion. It suggested the impairment of autophagic flux of OLT-induced kidney which could be restored after HRS pretreatment.
Many stress pathways sequentially elicit autophagy and apoptosis, enabling cells to degrade organelles.40,41 It was already verified that autophagy and apoptosis might crosstalk with each other, and p53 appears to be a critical switch in the network.42 A more precise understanding of the relationship between autophagy and apoptosis in OLT-induced AKI is still required. Our initial analysis showed that p53 and DRAM expressions were both impeded in renal samples of rats after OLT. Pretreatment with HRS markedly provoked the phosphorylation of p53 and the transactivation of its target gene DRAM. It was noteworthy that the inhibition of autophagy by chloroquine did not effect the production of p53 and DRAM but could enhance the release of cyt c and caspase-3, confirming the upstream positions of p53 and DRAM in the signal pathway.
The schematic diagram illustrating the molecular mechanisms of HRS involved in the crosstalk between apoptosis and autophagy. The extracellular stress suppressed the phosphorylation of p53, which in turn facilitated the permeabilization of the mitochondria membrane characterized by cyt c and caspase-3 cleavage. Similarly, the downregulation of phosphorylated p53 blocked the autophagic flux through impeding the expression of DRAM. However, pretreatment with HRS induces renoprotection in the opposite manner (Figure 5). Subsequent research on inhibiting autophagy via chloroquine verified the antagonism between autophagy and apoptosis. It was the first time to report the molecular mechanism of HRS effect against AKI in rats induced by OLT. Similar research was just found in skeletal muscle of Sprague-Dawley rats.26 To better unravel the existence of molecular switches between these 2 cellular processes in the OLT rat models, numerous proteins need to be investigated.
This study has certain limitations. First, we used only 1 simple dose of the autophagy inhibitor (chloroquine), which was confirmed to be effective in our preliminary experiment. However, it is difficult for us to ensure that the doses we selected can completely block the generation and function of endogenous Beclin-1, and thus, the dose-dependent effect should be determined. Second, rat models have some limitations in mimicking human biological and physiological processes. Further research will focus on larger animal models.
In conclusion, our results indicate that HRS induces autophagy through activating the phosphorylation of p53, and autophagy activation exerts a renoprotective role via inhibiting the renal apoptosis during AKI after liver transplantation.
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