Brain Death Is Associated With Endoplasmic Reticulum Stress and Apoptosis in Rat Liver S. Caoa,b, T. Wanga,b, B. Yanb, Y. Lub, Y. Zhaoa, and S. Zhanga,b,* a
Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China; and bKey Laboratory of Hepatobiliary and Pancreatic Surgery & Digestive Organ Transplantation of Henan Province, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
ABSTRACT Cell death pathways initiated by stress on the endoplasmic reticulum (ER) have been implicated in a variety of common diseases, such as ischemia/reperfusion injury, diabetes, heart disease, and neurodegenerative disorders. However, the contribution of ER stress to apoptosis and liver injury after brain death is not known. In the present study, we found that brain death induces a variety of signature ER stress markers, including ER stresse specific X boxebinding protein 1 and up-regulation of glucose-regulated protein 78. Furthermore, brain death causes up-regulation of C/EBP homologous protein and caspase12. Consistent with this, terminal deoxynucleotidyl transferaseemediated 20 -deoxyuridine 50 -triphosphate nick-end labeling assay and transmission electron microscopy confirmed apoptosis in the liver after brain death. Taken together, the present study provides strong evidence supporting the presence and importance of ER stress and response in mediating brain deatheinduced apoptosis and liver injury.
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HE ENDOPLASMIC RETICULUM (ER) is the first compartment of the synthesis and folding of secreted, membrane-bound, and some organelle-targeted proteins. Optimum protein folding within the ER lumen is facilitated by adenosine triphosphate, Ca2þ, and an oxidizing environment [1]. Perturbations in cellular energy levels, mutations within proteins, or Ca2þ concentration can cause the accumulation and/or aggregation of unfolded proteins, a condition termed ER stress [2,3]. Cellular accumulation of mis-folded proteins triggers an integrated cellular reaction called unfolded protein response (UPR) to overcome the deleterious effects of ER stress. The UPR is regulated by the 3 ER transmembrane proteins: protein kinaseelike ER-resident kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) [4,5]. Glucose-regulated protein 78 (Grp78), an ER chaperone, is associated with the intraluminal regions of all 3 ER stress receptors in an inactive state. With conditions of ER stress, Grp78 is dissociated, and activation of the 3 ER stress receptors triggers UPR, which is a prosurvival response aimed at restoring normal ER function. However, when the UPR cannot be controlled, the organelle elicits apoptotic signals, leading to cellular dysfunction, death, and disease [6,7].
Liver transplantation has evolved from an experimental operation to a standard treatment today for end-stage disease. The majority of the donor livers are cadaveric, harvested from brain dead (BD) subjects. However, BD is known to induce cellular apoptosis and organ dysfunction [8,9]. The risk of kidney and liver graft dysfunction is higher in grafts from BD donors than in grafts from living donors [10,11]. Previous studies have shown inferior graft survival rates for the heart, lung, and kidneys from BD donors compared with those from living donors [12e14]. Increased levels of apoptosis mediators in the donor are associated with a poor graft dysfunction [15]. Thus, BD is believed to be an important antigen-independent risk factor that may damage organs before transplantation. This study was supported by the National Natural Science Foundation of China (grant 81171849). *Address correspondence to Shuijun Zhang, Key Laboratory of Hepatobiliary and Pancreatic Surgery & Digestive Organ Transplantation of Henan Province, the First Affiliated Hospital of Zhengzhou University, No. 1, East Jian She Road, Zhengzhou, 450052, Henan Province, China. E-mail: shuijunzhangzzu@gmail. com
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.04.016
Transplantation Proceedings, 46, 3297e3302 (2014)
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Because there have been no studies reporting the role of hepatic ER stress in organ dysfunction after BD, the aim of the present study was to determine whether induction of UPR during BD activates the ER stress death pathway under conditions that simulate transplantation of livers from BD donors. MATERIALS AND METHODS Animals and Ethics Statement Sixty adult male 300- to 350-g Sprague Dawley rats were used for all donor experiments. Rats were housed in standard cages with 5 animals per cage at 22 C, in relative humidity of 40% to 60%, with a lightedark cycle of 12/12 hours. Food and water were not restricted. The study was approved by the Ethics Committee for Animal Experimentation and Well-being at our hospital. All animal care and surgical procedures were performed in accordance with the guidelines of the animal care and use committee at Zhengzhou University. All efforts were made to minimize animal suffering and to reduce the number of animal used.
BD Model The donor rats were premedicated with atropine (0.25 mg) intraperitoneally. General anesthesia was induced with 1% pentobarbital (0.4e0.6 mL/100 g) intraperitoneally, and BD was produced by creating intracranial hypertension [16]. The scalp and the epicranial muscles and periosteum were excised, and a 2-mm hole was drilled with a 12-gauge needle through the skull 4 mm lateral to the sagittal suture. A No. 3 Fogarty catheter balloon (Fogarty Arterial Embolectomy Catheter, 3F, Edwards Lifesciences Co., Irvine, Calif, United States) was inserted epidurally into the burr hole and gradually inflated in increments of 20 mL of saline solution every 5 minutes to a final volume of w2400 mL. The saphenous artery was catheterized and connected to pressure transducers for continuous recording of artery pressure. Blood pressure >80 mm Hg was considered normotensive. Catheters were placed in the tail veins for fluid administration. BD was confirmed by flat-line electroencephalography, physical signs of apnea, and absence of brain stem reflexes [17]. Sham-operated rats undergoing the same surgical procedures, but without induction of BD, served as living-donor controls. Animals were killed at the indicated time after BD to obtain blood and liver samples for further analyses.
Western Blot Analyses Protein expression of Grp78, X boxebinding protein 1 (XBP-1), C/EBP homologous protein (CHOP), and caspase-12 were examined by using Western blot analysis [18]. The frozen tissue samples were solubilized in radioimmunoprecipitation assay buffer on ice by using a homogenizer. All lysates were determined by using the Bradford protein assay with bovine serum albumin as a standard protein. Proteins from 30-mg samples were separated by electrophoresis on a sodium dodecyl sulfateepolyacrylamide gel (12% gel). The proteins were transferred to polyvinylidene fluoride membranes. After a 1-hour incubation in blocking solution (5% non-fat milk in 20 mM Tris/HCl, 150 mM NaCl, 0.1% Tween-20; TBS-T), the membranes were blotted with primary antibodies against Grp78 (1:1000, Cell Signaling Technology, Danvers, Mass, United States), XBP-1 (1:1000, Santa Cruz Technology, Santa Cruz, Calif, United States), CHOP (1:1000, Santa Cruz Technology), caspase-12 (1:1000, Santa Cruz Technology), or b-actin (1:1000, Beijing Biosynthesis) overnight at 4 C. After extensive rinsing with TBS-T
CAO, WANG, YAN ET AL buffer, the blots were incubated with horseradish peroxidasee conjugated antirabbit secondary antibodies (Beijing Biosynthesis). Specific bands were detected by using an enhanced chemiluminescence system and captured on radiograph film. Western blot analyses were performed in at least 3 independent experiments. The amount of protein was quantified by using Quantity One software (Bio-Rad Laboratories, Hercules, Calif, United States) and was expressed as the ratio relative to b-actin protein.
Real-time Polymerase Chain Reaction Expression of Grp78, XBP-1, CHOP, and caspase-12 was determined by using real-time polymerase chain reaction (PCR) with the Applied Biosystems 7500 Real-Time PCR System [19]. Total ribonucleic acid (RNA) was isolated from liver samples with TRIzol reagent (Invitrogen Corporation). Purity of RNA was determined spectrophotometrically by optical density 260/280-nm ratios. RNA 2 mg was used as a template for the synthesis of complementary DNA (cDNA) using the PrimeScript RTreagent Kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instruction. Primer pairs used for amplification of target genes were designed by using Prime 5.0 software and specificity checked using the National Center for Biotechnology Information Basic Local Alignment Search Tool. The sequences of primers were: Grp78, 50 -tgttccgctctaccatgaaac-30 (forward) and 50 -ctacagcctcatctgggttga-30 (reverse); XBP-1, 50 tgaatgccctggttactgaag-30 (forward) and 50 -agggtccaacttgtccagaat-30 (reverse); CHOP, 50 -cctgtcctcagatgaaattgg-30 (forward) and 50 accactctgtttccgtttcct-30 (reverse); and caspase-12, 50 -aaagggatagccactgctgat-30 (forward) and 50 -gagagccactcttgcctacct-30 (reverse). Quantitative PCR was performed in a 2-step real-time PCR by using SYBR Premix EX Taq with 2 mL of cDNA and 4-mM primers in a total reaction volume of 20 mL. The cycling conditions were as follows: the cDNA was denatured at 95 C for 30 seconds, followed by 40 amplification cycles (3 seconds at 95 C and 30 seconds at 60 C). The amplification and data acquisition were run on the real-time PCR system (ABI Prism 7500). Fluorescence signals from the amplified products were quantitatively assessed by using 7500 Fast System SDS Software. All samples were normalized to expression of b-actin. PCR products were subsequently analyzed by using agarose gel electrophoresis to check for correct products.
Immunohistochemistry Studies Briefly, 4-mm sections were cut from liver specimens and placed on slides [19]. Sections were deparaffinized by using xylene and rehydrated in graded ethanol concentrations. After washing in water, antigen retrieval was performed by using 0.01 M of citrate buffer (pH 6.0) for 10 minutes with the microwave irradiation on high setting. To inhibit endogenous peroxidase activity, sections were immersed in 3% H2O2 for 30 minutes. Sections were blocked with 30% goat serum for 1 hour. Sections were blotted with primary antibodies against Grp78 (1:400, Beijing Biosynthesis), XBP-1 (1:400, Beijing Biosynthesis), CHOP (1:400, Beijing Biosynthesis), and caspase-12 (1:400, Beijing Biosynthesis) overnight at 4 C without coverslips. After washing in phosphate-buffered saline (PBS), sections were incubated with secondary antibodies for 1 hour. Sections were washed in PBS and visualized with diaminobenzidine counterstained with hematoxylin. Slides were dehydrated and covered. Images were captured by using a fluorescence microscope (Olympus research inverted system microscope IX71, original magnification 400). Negative control sections consisted of saline instead of primary antibodies using the same methods.
BD AND ENDOPLASMIC RETICULUM STRESS AND APOPTOSIS IN RAT LIVERS
Terminal Deoxynucleotidyl TransferaseeMediated 20 -Deoxyuridine 50 -Triphosphate Nick-End Labeling Assay Sections at 4-mm thickness cut from paraffin-embedded rat livers were placed onto slides and dried at 65 C for 30 minutes [20]. The sections were dewaxed in xylene and rehydrated through a graded series of ethanol and double distilled water, followed by antigen retrieval in citrate buffer for microwave irradiation. Sections were blocked in Tris-HCl, 0.1 M pH 7.5, containing 3% bovine serum albumin and 20% normal bovine serum for 30 minutes at 20 C, followed by terminal deoxynucleotidyl transferaseemediated 20 -deoxyuridine 50 -triphosphate nick-end labeling (TUNEL) assay for 1 hour at 37 C (using an In Situ Cell Death Detection Kit, Fluorescein, Roche). Sections were counterstained by using 40 ,6-diamidino-2-phenylindole stain (Beyotime), and fluorescence was viewed in a fluorescence microscope (Olympus research inverted system microscope IX71, original magnification 400). Positive control sections underwent the same treatment but were incubated with DNase I for 10 minutes at 20 C before the labeling procedures. For negative controls, sections were incubated with label solution (without terminal transferase) instead of TUNEL reaction mixture. The results were expressed as the percentage of TUNEL-labeled cells per total cells in each experimental group.
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Transmission Electron Microscopy Liver fragments of w1 mm3 were fixed in 2.5% glutaraldehyde solution for 24 hours at 4 C. After fixation, samples were washed in PBS and then immersed in 1% osmic acid. The liver fragments were then dehydrated in an ethanol series and embedded in epoxy resin. Ultrathin sections were screened by using light microscopy. Samples were stained with uranyl acetate and lead citrate and examined under a JEM-1400 (transmission electron microscopy).
Statistical Analysis All values are presented as mean SEM. Statistical analysis was conducted by using one-way analysis of variance or the Student t test. P values .05 were considered significant.
RESULTS Initiation ER Stress After BD
In response to ER stress, there was marked up-regulation of signature ER stress markers, such as Grp78 and XBP-1, as measured by using Western blot analyses. Grp78 protein expression was increased in the liver of rats after BD compared with sham-operated rats with a maximum of 1.3 0.2 vs 0.4 0.1 at 6 hours, respectively (P < .05) (Fig 1A).
Fig 1. Initiation of endoplasmic reticulum (ER) stress after brain death (BD). (A) Glucose-regulated protein 78 (Grp78) and X boxe binding protein 1 (XBP-1) in rat liver at different times after BD as detected by using Western blot analysis. b-actin was used as the control. P < .05 for sham versus BD. (B) Immunostaining of Grp78 and XBP-1 in the rat liver after BD. (C) Grp78 and XBP-1 messenger ribonucleic acid levels in the rat liver analyzed by using quantitative polymerase chain reaction. Grp78 and XBP-1, P < .05 for sham versus BD.
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Consistent with the increase in Grp78, there was an increase in XBP-1 expression after BD at 6 hours. The localized expression of Grp78 and XBP-1 was investigated by using immunohistochemistry in both the BD and the control groups. The number of Grp78- and XBP1epositive cells increased after BD to 110 4 and 78 5, respectively (P < .05) (Fig 1B), with an accumulation of staining within cell cytoplasm. To determine whether the increased levels of Grp78 and XBP-1 protein were due to increased transcription or other regulatory mechanisms, we evaluated the levels of Grp78 and XBP-1 messenger ribonucleic acid (mRNA). According to real-time PCR, Grp78 and XBP-1 mRNA levels were increased in the BD group compared with the control group (P < .05). Grp78 mRNA expression increased after BD and peaked at 2 hours of BD (Fig 1C). XBP-1 mRNA levels increased and peaked at 4 hours of BD. Activation of Pro-apoptotic CHOP and Caspase-12 Hours After BD
Real-time PCR and immunohistochemistry analysis showed that the expression of CHOP and caspase-12 gradually increased from 0 to 6 hours after BD compared with the
CAO, WANG, YAN ET AL
sham operation control group (P < .05). CHOP mRNA expression increased 2-fold at 4 hours after BD compared with sham operation controls (Fig 2C). The caspase-12 mRNA level increased 2-fold at 6 hours after BD compared with sham operation controls. Immunohistochemistry staining showed increased CHOP and caspase-12 in the cell cytoplasm in the rat liver compared with controls (Fig 2B). We also analyzed the activation of CHOP and caspase-12 by using Western blot analysis. The ER stresseinducible leucine zipper-containing transcription factor CHOP gradually accumulated from 0 to 6 hours after BD, indicating that BD triggers an ER stress response in favor of proapoptotic factors (Fig 2A). Similarly, the specific ER stresse associated caspase-12 activation was in concordance with the highest values of CHOP pro-apoptotic protein levels. Ultrastructural examination of liver sections from the sham-operated rats showed no abnormalities. Liver cells were arranged in order, nuclear membranes were intact, and the nuclei were full and clear. With the passage of time after BD, the characteristic apoptotic features of hepatocytes (eg, chromatin condensation and clumping, appearance of cytoplasmic vacuoles) were noted but not in the sham-
Fig 2. Activation of pro-apoptotic C/EBP homologous protein (CHOP) and caspase-12 after brain death (BD). (A) CHOP and caspase12 in rat liver at various times after BD as detected by using Western blot analysis. b-actin was used as the control. P < .05 for sham versus BD. (B) Immunostaining of CHOP and caspase-12 in the rat liver after BD. (C) Glucose-regulated protein 78 and X boxebinding protein 1 messenger ribonucleic acid levels in the rat liver analyzed by using quantitative polymerase chain reaction. CHOP and caspase-12, P < .05 for sham versus BD. (D) Transmission electron microscopy changes in the sham group and in the BD group 6 hours after BD.
BD AND ENDOPLASMIC RETICULUM STRESS AND APOPTOSIS IN RAT LIVERS
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Fig 3. Effect of brain death (BD) on apoptosis. Terminal deoxynucleotidyl transferaseemediated 20 -deoxyuridine 50 -triphosphate nickend labeling assay showed positive nuclear staining by fluorescent antibodies for DNA fragmentation in apoptotic cells in representative liver sections from rats from the BD group and the sham group (original magnification 400), *P < .05 for sham versus BD.
operated group. In additional, rough ER was obviously dilated and swollen (Fig 2D). As presented in Fig 3, the number of TUNEL-positive cells was significantly increased in the BD group compared with the sham-operated group. In the sham group, the percentage of apoptotic cells was 2.0% 0.2%. With the passing of time after BD, the expression increased and peaked at 6 hours (8.5% 1.0%). DISCUSSION
BD has definite effects on hemodynamic stability, immunologic changes, and hormone regulation in donor organs [21e24]. It induces liver injury, which progresses even in cases in which the hemodynamic variables are stable [16]. The liver injury is characterized by liver cell dysfunction, in which apoptosis plays a role in addition to necrosis. This is accompanied by increased expression of the cytokines interleukin-6, interleukin-10, and tumor necrosis factor a in the liver, and the elevated levels of these cytokines in the serum after BD [8,12,25]. Proinflammatory cytokines can induce ER stress in the liver by cAMP responsive elementbinding protein (hepatocyte specific), which mediates an acute phase response in the liver. Grp78, an ER molecular chaperone, is a key regulator of ER stress transducers [26]. Grp78 binds and inhibits all 3 ER stress sensors (PERK, ATF6, and IRE1) in the absence of ER stress. Grp78 protein levels increase under the conditions of increased ER stress. In previous studies, we found that the expression of Grp78 was increased in livers obtained after BD compared with those from living donors. Studies have shown that Grp78 is antiapoptotic and plays critical cytoprotective roles in a wide variety of cancer cells by activation of UPR signaling to ameliorate misfolded protein aggregation in the ER [27]. Grp78 induction can
serve as a general indicator that ER is under stress and that the UPR is triggered. XBP-1, a critical transcription factor regulating UPR, leads to generation of ER stress and is one of the master regulators of ER-folding capacity. The first 2 pathways are initiated by the ER, PERK, and IRE1. XBP-1 up-regulates gene expression of ER chaperones. During ER stress, IRE1 is switched off earlier than PERK. With stimulation of the 3 ER stress sensors, IRE1 plays a key role in controlling the switch between adaptive responses and initiation of the apoptosis program [28]. Taken together, BD results in ER stress and activation of UPR. However, if the UPR cannot be controlled, the protective signaling switches to a pro-apoptotic response. Previous studies have also demonstrated that apoptosis in the liver occurs by activation of the cell surface death receptore mediated pathway and the mitochondrial pathway. The present results demonstrate that BD activates the expression of pro-apoptotic CHOP and caspase-12. The results showed significantly increased expression of CHOP with activation of caspase-12 after BD. We found that mRNA and protein levels of caspase-12 were significantly increased after BD. This may be evidence of the caspase-12einduced pro-apoptotic pathway of the ER stress/UPRemediating hepatic cell apoptosis after BD. Caspase-12edeficient cells have been shown to be resistant to ER stress inducers, reflecting the importance of caspase-12 in the apoptosis process [29]. The CHOP pathway has been shown to be involved in ER stress/UPReinduced apoptosis. We found a significant increase in CHOP expression in the liver after BD. The CHOP gene is transcriptionally induced by ER stress, and up-regulation of this gene is a marker of ER stress. In addition, there is increasing evidence that CHOP plays an important role in ER stresseinduced apoptosis. CHOP knockout cells have been found to be resistant to ER stresseinduced apoptosis [30]. The increased number of
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apoptotic cells stained by TUNEL and transmission electron microscopy methods is consistent with this result. Apoptotic morphologic changes, including nuclear fragmentation, chromatin condensation, swelling of the ER lumen, and dissociation of ribosomes, have been observed with the use of electron microscopy. In addition, the liver apoptosis index was significantly increased in the BD group compared with the sham-operated group. In conclusion, the present results indicate that increased ER stress responses are associated with apoptosis in the liver of rats subjected to BD compared with that in the sham group. Further studies using techniques such as ER stress inhibition may shed more light on the role of ER stress in BD-mediated physiological dysfunction.
REFERENCES [1] Gaut JR, Hendershot LM. The modification and assembly of proteins in the endoplasmic reticulum. Curr Opin Cell Biol 1993;5: 589e95. [2] Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007;8: 519e29. [3] Gardner BM, Pincus D, Gotthardt K, et al. Endoplasmic reticulum stress sensing in the unfolded protein response. Cold Spring Harb Perspect Biol 2013;5:a13169. [4] Lee K, Tirasophon W, Shen X, et al. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev 2002;16:452e66. [5] Ma K, Vattem KM, Wek RC. Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. J Biol Chem 2002;277:18728e35. [6] Roussel BD, Kruppa AJ, Miranda E, et al. Endoplasmic reticulum dysfunction in neurological disease. Lancet Neurol 2013;12: 105e18. [7] Shirakawa J, Togashi Y, Sakamoto E, et al. Glucokinase activation ameliorates ER stress-induced apoptosis in pancreatic beta-cells. Diabetes 2013;62:3448e58. [8] Van Der Hoeven JA, Moshage H, Schuurs T, Nijboer M, Van Schilfgaarde R, Ploeg RJ. Brain death induces apoptosis in donor liver of the rat. Transplantation 2003;76:1150e4. [9] Van Der Hoeven JA, Molema G, Ter HG, et al. Relationship between duration of brain death and hemodynamic (in) stability on progressive dysfunction and increased immunologic activation of donor kidneys. Kidney Int 2003;64:1874e82. [10] Weiss S, Kotsch K, Francuski M, et al. Brain death activates donor organs and is associated with a worse I/R injury after liver transplantation. Am J Transplant 2007;7:1584e93. [11] Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med 2000;342:605e12.
CAO, WANG, YAN ET AL [12] Wilhelm MJ, Pratschke J, Beato F, et al. Activation of the heart by donor brain death accelerates acute rejection after transplantation. Circulation 2000;102:2426e33. [13] Zweers N, Petersen AH, Van Der Hoeven JA, et al. Donor brain death aggravates chronic rejection after lung transplantation in rats. Transplantation 2004;78:1251e8. [14] Pratschke J, Wilhelm MJ, Laskowski I, et al. Influence of donor brain death on chronic rejection of renal transplants in rats. J Am Soc Nephrol 2001;12:2474e81. [15] Daemen MA, van’t Veer C, Denecker G, et al. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest 1999;104:541e9. [16] Van Der Hoeven JA, Ter HG, Molema G, et al. Effects of brain death and hemodynamic status on function and immunologic activation of the potential donor liver in the rat. Ann Surg 2000;232: 804e13. [17] Pratschke J, Wilhelm MJ, Kusaka M, et al. A model of gradual onset brain death for transplant-associated studies in rats. Transplantation 2000;69:427e30. [18] Marderstein EL, Bucher B, Guo Z, et al. Protection of rat hepatocytes from apoptosis by inhibition of c-Jun N-terminal kinase. Surgery 2003;134:280e4. [19] Wu CT, Sheu ML, Tsai KS, et al. Salubrinal, an eIF2alpha dephosphorylation inhibitor, enhances cisplatin-induced oxidative stress and nephrotoxicity in a mouse model. Free Radic Biol Med 2011;51:671e80. [20] Komoike Y, Inamura H, Matsuoka M. Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells. Arch Toxicol 2012;86:37e44. [21] Mascia L, Mastromauro I, Viberti S, et al. Management to optimize organ procurement in brain dead donors. Minerva Anestesiol 2009;75:125e33. [22] Herijgers P, Leunens V, Tjandra-Maga TB, et al. Changes in organ perfusion after brain death in the rat and its relation to circulating catecholamines. Transplantation 1996;62:330e5. [23] Gramm HJ, Meinhold H, Bickel U, et al. Acute endocrine failure after brain death? Transplantation 1992;54:851e7. [24] Chen EP, Bittner HB, Kendall SW, et al. Hormonal and hemodynamic changes in a validated animal model of brain death. Crit Care Med 1996;24:1352e9. [25] Zhang SJ, Shi JH, Tang Z, et al. Protective effects of glycine pretreatment on brain-death donor liver. Hepatobiliary Pancreat Dis Int 2005;4:37e40. [26] Lee AS. The ER chaperone and signaling regulator GRP78/ BiP as a monitor of endoplasmic reticulum stress. Methods 2005;35: 373e81. [27] Yoshida H, Matsui T, Hosokawa N, et al. A time-dependent phase shift in the mammalian unfolded protein response. Dev Cell 2003;4:265e71. [28] Gorman AM, Healy SJ, Jager R, et al. Stress management at the ER: regulators of ER stress-induced apoptosis. Pharmacol Ther 2012;134:306e16. [29] Saleh M, Mathison JC, Wolinski MK, et al. Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice. Nature 2006;440:1064e8. [30] Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis 2002;7:335e45.
Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China; and bKey Laboratory of Hepatobiliary and Pancreatic Surgery & Digestive Organ Transplantation of Henan Province, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
ABSTRACT Cell death pathways initiated by stress on the endoplasmic reticulum (ER) have been implicated in a variety of common diseases, such as ischemia/reperfusion injury, diabetes, heart disease, and neurodegenerative disorders. However, the contribution of ER stress to apoptosis and liver injury after brain death is not known. In the present study, we found that brain death induces a variety of signature ER stress markers, including ER stresse specific X boxebinding protein 1 and up-regulation of glucose-regulated protein 78. Furthermore, brain death causes up-regulation of C/EBP homologous protein and caspase12. Consistent with this, terminal deoxynucleotidyl transferaseemediated 20 -deoxyuridine 50 -triphosphate nick-end labeling assay and transmission electron microscopy confirmed apoptosis in the liver after brain death. Taken together, the present study provides strong evidence supporting the presence and importance of ER stress and response in mediating brain deatheinduced apoptosis and liver injury.
T
HE ENDOPLASMIC RETICULUM (ER) is the first compartment of the synthesis and folding of secreted, membrane-bound, and some organelle-targeted proteins. Optimum protein folding within the ER lumen is facilitated by adenosine triphosphate, Ca2þ, and an oxidizing environment [1]. Perturbations in cellular energy levels, mutations within proteins, or Ca2þ concentration can cause the accumulation and/or aggregation of unfolded proteins, a condition termed ER stress [2,3]. Cellular accumulation of mis-folded proteins triggers an integrated cellular reaction called unfolded protein response (UPR) to overcome the deleterious effects of ER stress. The UPR is regulated by the 3 ER transmembrane proteins: protein kinaseelike ER-resident kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) [4,5]. Glucose-regulated protein 78 (Grp78), an ER chaperone, is associated with the intraluminal regions of all 3 ER stress receptors in an inactive state. With conditions of ER stress, Grp78 is dissociated, and activation of the 3 ER stress receptors triggers UPR, which is a prosurvival response aimed at restoring normal ER function. However, when the UPR cannot be controlled, the organelle elicits apoptotic signals, leading to cellular dysfunction, death, and disease [6,7].
Liver transplantation has evolved from an experimental operation to a standard treatment today for end-stage disease. The majority of the donor livers are cadaveric, harvested from brain dead (BD) subjects. However, BD is known to induce cellular apoptosis and organ dysfunction [8,9]. The risk of kidney and liver graft dysfunction is higher in grafts from BD donors than in grafts from living donors [10,11]. Previous studies have shown inferior graft survival rates for the heart, lung, and kidneys from BD donors compared with those from living donors [12e14]. Increased levels of apoptosis mediators in the donor are associated with a poor graft dysfunction [15]. Thus, BD is believed to be an important antigen-independent risk factor that may damage organs before transplantation. This study was supported by the National Natural Science Foundation of China (grant 81171849). *Address correspondence to Shuijun Zhang, Key Laboratory of Hepatobiliary and Pancreatic Surgery & Digestive Organ Transplantation of Henan Province, the First Affiliated Hospital of Zhengzhou University, No. 1, East Jian She Road, Zhengzhou, 450052, Henan Province, China. E-mail: shuijunzhangzzu@gmail. com
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.04.016
Transplantation Proceedings, 46, 3297e3302 (2014)
3297
3298
Because there have been no studies reporting the role of hepatic ER stress in organ dysfunction after BD, the aim of the present study was to determine whether induction of UPR during BD activates the ER stress death pathway under conditions that simulate transplantation of livers from BD donors. MATERIALS AND METHODS Animals and Ethics Statement Sixty adult male 300- to 350-g Sprague Dawley rats were used for all donor experiments. Rats were housed in standard cages with 5 animals per cage at 22 C, in relative humidity of 40% to 60%, with a lightedark cycle of 12/12 hours. Food and water were not restricted. The study was approved by the Ethics Committee for Animal Experimentation and Well-being at our hospital. All animal care and surgical procedures were performed in accordance with the guidelines of the animal care and use committee at Zhengzhou University. All efforts were made to minimize animal suffering and to reduce the number of animal used.
BD Model The donor rats were premedicated with atropine (0.25 mg) intraperitoneally. General anesthesia was induced with 1% pentobarbital (0.4e0.6 mL/100 g) intraperitoneally, and BD was produced by creating intracranial hypertension [16]. The scalp and the epicranial muscles and periosteum were excised, and a 2-mm hole was drilled with a 12-gauge needle through the skull 4 mm lateral to the sagittal suture. A No. 3 Fogarty catheter balloon (Fogarty Arterial Embolectomy Catheter, 3F, Edwards Lifesciences Co., Irvine, Calif, United States) was inserted epidurally into the burr hole and gradually inflated in increments of 20 mL of saline solution every 5 minutes to a final volume of w2400 mL. The saphenous artery was catheterized and connected to pressure transducers for continuous recording of artery pressure. Blood pressure >80 mm Hg was considered normotensive. Catheters were placed in the tail veins for fluid administration. BD was confirmed by flat-line electroencephalography, physical signs of apnea, and absence of brain stem reflexes [17]. Sham-operated rats undergoing the same surgical procedures, but without induction of BD, served as living-donor controls. Animals were killed at the indicated time after BD to obtain blood and liver samples for further analyses.
Western Blot Analyses Protein expression of Grp78, X boxebinding protein 1 (XBP-1), C/EBP homologous protein (CHOP), and caspase-12 were examined by using Western blot analysis [18]. The frozen tissue samples were solubilized in radioimmunoprecipitation assay buffer on ice by using a homogenizer. All lysates were determined by using the Bradford protein assay with bovine serum albumin as a standard protein. Proteins from 30-mg samples were separated by electrophoresis on a sodium dodecyl sulfateepolyacrylamide gel (12% gel). The proteins were transferred to polyvinylidene fluoride membranes. After a 1-hour incubation in blocking solution (5% non-fat milk in 20 mM Tris/HCl, 150 mM NaCl, 0.1% Tween-20; TBS-T), the membranes were blotted with primary antibodies against Grp78 (1:1000, Cell Signaling Technology, Danvers, Mass, United States), XBP-1 (1:1000, Santa Cruz Technology, Santa Cruz, Calif, United States), CHOP (1:1000, Santa Cruz Technology), caspase-12 (1:1000, Santa Cruz Technology), or b-actin (1:1000, Beijing Biosynthesis) overnight at 4 C. After extensive rinsing with TBS-T
CAO, WANG, YAN ET AL buffer, the blots were incubated with horseradish peroxidasee conjugated antirabbit secondary antibodies (Beijing Biosynthesis). Specific bands were detected by using an enhanced chemiluminescence system and captured on radiograph film. Western blot analyses were performed in at least 3 independent experiments. The amount of protein was quantified by using Quantity One software (Bio-Rad Laboratories, Hercules, Calif, United States) and was expressed as the ratio relative to b-actin protein.
Real-time Polymerase Chain Reaction Expression of Grp78, XBP-1, CHOP, and caspase-12 was determined by using real-time polymerase chain reaction (PCR) with the Applied Biosystems 7500 Real-Time PCR System [19]. Total ribonucleic acid (RNA) was isolated from liver samples with TRIzol reagent (Invitrogen Corporation). Purity of RNA was determined spectrophotometrically by optical density 260/280-nm ratios. RNA 2 mg was used as a template for the synthesis of complementary DNA (cDNA) using the PrimeScript RTreagent Kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instruction. Primer pairs used for amplification of target genes were designed by using Prime 5.0 software and specificity checked using the National Center for Biotechnology Information Basic Local Alignment Search Tool. The sequences of primers were: Grp78, 50 -tgttccgctctaccatgaaac-30 (forward) and 50 -ctacagcctcatctgggttga-30 (reverse); XBP-1, 50 tgaatgccctggttactgaag-30 (forward) and 50 -agggtccaacttgtccagaat-30 (reverse); CHOP, 50 -cctgtcctcagatgaaattgg-30 (forward) and 50 accactctgtttccgtttcct-30 (reverse); and caspase-12, 50 -aaagggatagccactgctgat-30 (forward) and 50 -gagagccactcttgcctacct-30 (reverse). Quantitative PCR was performed in a 2-step real-time PCR by using SYBR Premix EX Taq with 2 mL of cDNA and 4-mM primers in a total reaction volume of 20 mL. The cycling conditions were as follows: the cDNA was denatured at 95 C for 30 seconds, followed by 40 amplification cycles (3 seconds at 95 C and 30 seconds at 60 C). The amplification and data acquisition were run on the real-time PCR system (ABI Prism 7500). Fluorescence signals from the amplified products were quantitatively assessed by using 7500 Fast System SDS Software. All samples were normalized to expression of b-actin. PCR products were subsequently analyzed by using agarose gel electrophoresis to check for correct products.
Immunohistochemistry Studies Briefly, 4-mm sections were cut from liver specimens and placed on slides [19]. Sections were deparaffinized by using xylene and rehydrated in graded ethanol concentrations. After washing in water, antigen retrieval was performed by using 0.01 M of citrate buffer (pH 6.0) for 10 minutes with the microwave irradiation on high setting. To inhibit endogenous peroxidase activity, sections were immersed in 3% H2O2 for 30 minutes. Sections were blocked with 30% goat serum for 1 hour. Sections were blotted with primary antibodies against Grp78 (1:400, Beijing Biosynthesis), XBP-1 (1:400, Beijing Biosynthesis), CHOP (1:400, Beijing Biosynthesis), and caspase-12 (1:400, Beijing Biosynthesis) overnight at 4 C without coverslips. After washing in phosphate-buffered saline (PBS), sections were incubated with secondary antibodies for 1 hour. Sections were washed in PBS and visualized with diaminobenzidine counterstained with hematoxylin. Slides were dehydrated and covered. Images were captured by using a fluorescence microscope (Olympus research inverted system microscope IX71, original magnification 400). Negative control sections consisted of saline instead of primary antibodies using the same methods.
BD AND ENDOPLASMIC RETICULUM STRESS AND APOPTOSIS IN RAT LIVERS
Terminal Deoxynucleotidyl TransferaseeMediated 20 -Deoxyuridine 50 -Triphosphate Nick-End Labeling Assay Sections at 4-mm thickness cut from paraffin-embedded rat livers were placed onto slides and dried at 65 C for 30 minutes [20]. The sections were dewaxed in xylene and rehydrated through a graded series of ethanol and double distilled water, followed by antigen retrieval in citrate buffer for microwave irradiation. Sections were blocked in Tris-HCl, 0.1 M pH 7.5, containing 3% bovine serum albumin and 20% normal bovine serum for 30 minutes at 20 C, followed by terminal deoxynucleotidyl transferaseemediated 20 -deoxyuridine 50 -triphosphate nick-end labeling (TUNEL) assay for 1 hour at 37 C (using an In Situ Cell Death Detection Kit, Fluorescein, Roche). Sections were counterstained by using 40 ,6-diamidino-2-phenylindole stain (Beyotime), and fluorescence was viewed in a fluorescence microscope (Olympus research inverted system microscope IX71, original magnification 400). Positive control sections underwent the same treatment but were incubated with DNase I for 10 minutes at 20 C before the labeling procedures. For negative controls, sections were incubated with label solution (without terminal transferase) instead of TUNEL reaction mixture. The results were expressed as the percentage of TUNEL-labeled cells per total cells in each experimental group.
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Transmission Electron Microscopy Liver fragments of w1 mm3 were fixed in 2.5% glutaraldehyde solution for 24 hours at 4 C. After fixation, samples were washed in PBS and then immersed in 1% osmic acid. The liver fragments were then dehydrated in an ethanol series and embedded in epoxy resin. Ultrathin sections were screened by using light microscopy. Samples were stained with uranyl acetate and lead citrate and examined under a JEM-1400 (transmission electron microscopy).
Statistical Analysis All values are presented as mean SEM. Statistical analysis was conducted by using one-way analysis of variance or the Student t test. P values .05 were considered significant.
RESULTS Initiation ER Stress After BD
In response to ER stress, there was marked up-regulation of signature ER stress markers, such as Grp78 and XBP-1, as measured by using Western blot analyses. Grp78 protein expression was increased in the liver of rats after BD compared with sham-operated rats with a maximum of 1.3 0.2 vs 0.4 0.1 at 6 hours, respectively (P < .05) (Fig 1A).
Fig 1. Initiation of endoplasmic reticulum (ER) stress after brain death (BD). (A) Glucose-regulated protein 78 (Grp78) and X boxe binding protein 1 (XBP-1) in rat liver at different times after BD as detected by using Western blot analysis. b-actin was used as the control. P < .05 for sham versus BD. (B) Immunostaining of Grp78 and XBP-1 in the rat liver after BD. (C) Grp78 and XBP-1 messenger ribonucleic acid levels in the rat liver analyzed by using quantitative polymerase chain reaction. Grp78 and XBP-1, P < .05 for sham versus BD.
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Consistent with the increase in Grp78, there was an increase in XBP-1 expression after BD at 6 hours. The localized expression of Grp78 and XBP-1 was investigated by using immunohistochemistry in both the BD and the control groups. The number of Grp78- and XBP1epositive cells increased after BD to 110 4 and 78 5, respectively (P < .05) (Fig 1B), with an accumulation of staining within cell cytoplasm. To determine whether the increased levels of Grp78 and XBP-1 protein were due to increased transcription or other regulatory mechanisms, we evaluated the levels of Grp78 and XBP-1 messenger ribonucleic acid (mRNA). According to real-time PCR, Grp78 and XBP-1 mRNA levels were increased in the BD group compared with the control group (P < .05). Grp78 mRNA expression increased after BD and peaked at 2 hours of BD (Fig 1C). XBP-1 mRNA levels increased and peaked at 4 hours of BD. Activation of Pro-apoptotic CHOP and Caspase-12 Hours After BD
Real-time PCR and immunohistochemistry analysis showed that the expression of CHOP and caspase-12 gradually increased from 0 to 6 hours after BD compared with the
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sham operation control group (P < .05). CHOP mRNA expression increased 2-fold at 4 hours after BD compared with sham operation controls (Fig 2C). The caspase-12 mRNA level increased 2-fold at 6 hours after BD compared with sham operation controls. Immunohistochemistry staining showed increased CHOP and caspase-12 in the cell cytoplasm in the rat liver compared with controls (Fig 2B). We also analyzed the activation of CHOP and caspase-12 by using Western blot analysis. The ER stresseinducible leucine zipper-containing transcription factor CHOP gradually accumulated from 0 to 6 hours after BD, indicating that BD triggers an ER stress response in favor of proapoptotic factors (Fig 2A). Similarly, the specific ER stresse associated caspase-12 activation was in concordance with the highest values of CHOP pro-apoptotic protein levels. Ultrastructural examination of liver sections from the sham-operated rats showed no abnormalities. Liver cells were arranged in order, nuclear membranes were intact, and the nuclei were full and clear. With the passage of time after BD, the characteristic apoptotic features of hepatocytes (eg, chromatin condensation and clumping, appearance of cytoplasmic vacuoles) were noted but not in the sham-
Fig 2. Activation of pro-apoptotic C/EBP homologous protein (CHOP) and caspase-12 after brain death (BD). (A) CHOP and caspase12 in rat liver at various times after BD as detected by using Western blot analysis. b-actin was used as the control. P < .05 for sham versus BD. (B) Immunostaining of CHOP and caspase-12 in the rat liver after BD. (C) Glucose-regulated protein 78 and X boxebinding protein 1 messenger ribonucleic acid levels in the rat liver analyzed by using quantitative polymerase chain reaction. CHOP and caspase-12, P < .05 for sham versus BD. (D) Transmission electron microscopy changes in the sham group and in the BD group 6 hours after BD.
BD AND ENDOPLASMIC RETICULUM STRESS AND APOPTOSIS IN RAT LIVERS
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Fig 3. Effect of brain death (BD) on apoptosis. Terminal deoxynucleotidyl transferaseemediated 20 -deoxyuridine 50 -triphosphate nickend labeling assay showed positive nuclear staining by fluorescent antibodies for DNA fragmentation in apoptotic cells in representative liver sections from rats from the BD group and the sham group (original magnification 400), *P < .05 for sham versus BD.
operated group. In additional, rough ER was obviously dilated and swollen (Fig 2D). As presented in Fig 3, the number of TUNEL-positive cells was significantly increased in the BD group compared with the sham-operated group. In the sham group, the percentage of apoptotic cells was 2.0% 0.2%. With the passing of time after BD, the expression increased and peaked at 6 hours (8.5% 1.0%). DISCUSSION
BD has definite effects on hemodynamic stability, immunologic changes, and hormone regulation in donor organs [21e24]. It induces liver injury, which progresses even in cases in which the hemodynamic variables are stable [16]. The liver injury is characterized by liver cell dysfunction, in which apoptosis plays a role in addition to necrosis. This is accompanied by increased expression of the cytokines interleukin-6, interleukin-10, and tumor necrosis factor a in the liver, and the elevated levels of these cytokines in the serum after BD [8,12,25]. Proinflammatory cytokines can induce ER stress in the liver by cAMP responsive elementbinding protein (hepatocyte specific), which mediates an acute phase response in the liver. Grp78, an ER molecular chaperone, is a key regulator of ER stress transducers [26]. Grp78 binds and inhibits all 3 ER stress sensors (PERK, ATF6, and IRE1) in the absence of ER stress. Grp78 protein levels increase under the conditions of increased ER stress. In previous studies, we found that the expression of Grp78 was increased in livers obtained after BD compared with those from living donors. Studies have shown that Grp78 is antiapoptotic and plays critical cytoprotective roles in a wide variety of cancer cells by activation of UPR signaling to ameliorate misfolded protein aggregation in the ER [27]. Grp78 induction can
serve as a general indicator that ER is under stress and that the UPR is triggered. XBP-1, a critical transcription factor regulating UPR, leads to generation of ER stress and is one of the master regulators of ER-folding capacity. The first 2 pathways are initiated by the ER, PERK, and IRE1. XBP-1 up-regulates gene expression of ER chaperones. During ER stress, IRE1 is switched off earlier than PERK. With stimulation of the 3 ER stress sensors, IRE1 plays a key role in controlling the switch between adaptive responses and initiation of the apoptosis program [28]. Taken together, BD results in ER stress and activation of UPR. However, if the UPR cannot be controlled, the protective signaling switches to a pro-apoptotic response. Previous studies have also demonstrated that apoptosis in the liver occurs by activation of the cell surface death receptore mediated pathway and the mitochondrial pathway. The present results demonstrate that BD activates the expression of pro-apoptotic CHOP and caspase-12. The results showed significantly increased expression of CHOP with activation of caspase-12 after BD. We found that mRNA and protein levels of caspase-12 were significantly increased after BD. This may be evidence of the caspase-12einduced pro-apoptotic pathway of the ER stress/UPRemediating hepatic cell apoptosis after BD. Caspase-12edeficient cells have been shown to be resistant to ER stress inducers, reflecting the importance of caspase-12 in the apoptosis process [29]. The CHOP pathway has been shown to be involved in ER stress/UPReinduced apoptosis. We found a significant increase in CHOP expression in the liver after BD. The CHOP gene is transcriptionally induced by ER stress, and up-regulation of this gene is a marker of ER stress. In addition, there is increasing evidence that CHOP plays an important role in ER stresseinduced apoptosis. CHOP knockout cells have been found to be resistant to ER stresseinduced apoptosis [30]. The increased number of
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apoptotic cells stained by TUNEL and transmission electron microscopy methods is consistent with this result. Apoptotic morphologic changes, including nuclear fragmentation, chromatin condensation, swelling of the ER lumen, and dissociation of ribosomes, have been observed with the use of electron microscopy. In addition, the liver apoptosis index was significantly increased in the BD group compared with the sham-operated group. In conclusion, the present results indicate that increased ER stress responses are associated with apoptosis in the liver of rats subjected to BD compared with that in the sham group. Further studies using techniques such as ER stress inhibition may shed more light on the role of ER stress in BD-mediated physiological dysfunction.
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