Ultrastructural Pathology, 2014; 38(3): 217–223 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2014.895788
ORIGINAL ARTICLE
Hepatocyte Necroptosis Induced by Ischemic Acute Kidney Injury in Rats Cuifen Bao, PhD1, Youzhi Shao, MD2,3, and Xiaoming Li, PhD2,3 Central Laboratory, 2Laboratory of Renal Biology, and 3Department of Histology and Embryology, Liaoning Medical University, Jinzhou, Liaoning, China
ABSTRACT While ischemic acute kidney injury (IAKI) is known often to cause hepatic injury, little is known about necroptosis involved in the hepatic injury. The purposes of this study were to identify necroptosis involvement and observe morphological changes of hepatocytes in hepatic injury induced by IAKI in rats. Based on successfully established IAKI rat models, enzyme-linked immunosorbent assay illustrated a significant higher level of tumor necrosis factor a in serums of IAKI animals. Tumor necrosis factor receptor a (TNFRa) and receptor interacting protein kinase 3 (RIPk3) showed significant higher expressions in immunoblot analyses and positive hepatocytes of RIPk3 immunohistochemical staining were also evident in livers of IAKI rats. In addition, light microscopy revealed necrotic lesions that contain hepatocytes ongoing necroptosis besides necrotic cells in IAKI livers. Electron microscopy revealed at least three types of necrotic hepatocytes, they were edema necrosis, vacuolization necrosis, and necroptosis. Hepatocytes undergoing necroptosis had both necrosis and apoptosis morphological characteristics, they were necrosis cytoplasm and apoptosis-like nucleus. Among cellular organelles of hepatocyte with necrosis, membranous structures, such as cell membrane, endoplasmic reticular system, and mitochondria were more vulnerable to the stress of IAKI and deformed nucleuses varied in shape and lytic or pyknotic chromatin appearances were noted under insults of IAKI. In conclusion, hepatocyte undergoing necroptosis, RIPk3-mediated necroptosis partly contributes to hepatic necrosis induced by IAKI. Both membranous structures and nucleuses of hepatocyte were vulnerable to ischemic acute kidney injury. Keywords: Acute kidney injury, hepatocyte, necrosis, RIPk3-mediated necroptosis, ultrastructure
INTRODUCTION
Unfortunately the exact mechanisms of hepatic dysfunction in the setting of ischemic AKI remain unclear until now. One reason is that there is limited pathological data available about AKI-associated ALI. Morphological criteria provided by previous studies come from only observations by light microscopy suggesting a role for necrosis. Necroptosis, as a new functional type cell death as well as a type necrosis has been introduced in recent years, and a crucial role for receptor interacting protein kinase (RIPk) activation, including RIPk1 or RIPk3, has been demonstrated during necroptosis and become a biomarker, in particular RIPk3, for identifying cells undergoing necroptosis [4]. Despite a lot of studies
Acute kidney injury (AKI) widely viewed as one frequent complication with severe implications for the critically ill patients [1]. AKI can mostly be caused by renal ischemia, called ischemic acute kidney injury (IAKI) that occurs in various clinical settings including shock, sepsis, renal transplantation, and vascular surgery. IAKI, however, rarely occurs in isolation. It has become apparent that clinically, much of the high patient mortality can be attributed to progression to remote organ failures. Among remote organ failures AKI-induced acute liver injury (ALI) often becomes a death cause of AKI patients [2,3].
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Received 8 December 2013; Revised 5 February 2014; Accepted 7 February 2014; Published online 28 March 2014 Correspondence: Dr. Xiaoming Li, Department of Histology and Embryology, Liaoning Medical University, No. 40, Section 3 Songpo Road, Linghe District, Jinzhou, Liaoning 121001, PR China. Fax: +86 416 4673425. E-mail: [email protected]
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218 C. Bao et al. describing the role of necrosis in IAKI-injured liver, two issues remain. First, there is a need to reveal if necroptosis is involved in hepatic dysfunction induced by IAKI. Second, exhibiting detail morphological, including ultrastructural changes of hepatocyte necrosis after IAKI is required. For these purposes, the effects of IAKI on hepatic necrosis were examined by enzyme-linked immunosorbent assay (ELISA) for tumor necrosis factor a (TNF-a), RIPk3 and tumor necrosis factor receptor a (TNFRa) immunoblot analyses and RIPk3 immunohistochemical staining as well as both light and electron microscopy.
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MATERIALS AND METHODS Rat model of IAKI This study used Wistar male rats (n = 19), weighing 150–200 g, 2–3 months old. All animals were acclimated and maintained on a standard pellet diet 1 week before the initiation of experiments. The animal protocol was reviewed and approved by the Liaoning Medical University Animal Care and Research Committee, and rats were housed under pathogenfree condition at least 1 week before surgical procedures. Surgical procedures were performed using strict sterile techniques under anesthesia with pentobarbital sodium (50 mg/kg ip). All animals were underwent a midline laparotomy with isolation of bilateral renal pedicles. For rats (n = 9) assigned to experimental ischemic AKI (IAKI), a non-traumatic microvascular clamp was applied across both renal pedicles for 60 min. After the allotted ischemia time, 60 min, the clamps were gently removed and animals were administered 2 mL of sterile saline intra-peritoneally, and incision closed with 4-0 silk suture. There were four IAKI animals, as only RIPk3 analysis controls, received necrostatin-1 injections (1.65 mg/kg) before incision closed. Sham rats (n = 5) received same surgical procedures only without placement of the vascular clamps. The rats (n = 5) assigned to bilateral nephrectomy (BNx) as renal dysfunction controls underwent similar procedures except that both renal pedicles were ligated with a 5–0 silk suture and kidneys were removed. At 24 h, following the experimental procedure, the rats were euthanized under anesthesia, and blood and liver tissues, were collected for analysis of both renal function and hepatic dysfunction and morphological examinations.
operation. Plasma concentrations of creatinine (Cr) and blood urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured by colorimetric methods using commercially available kits.
Immunoblot analyses of TNFRa and RIPk3 Only livers of sham and IAKI animals were available for TNFRa analysis. Livers of sham, IAKI, and IAKI with necrostatin-1 injection were available for RIPk3 analysis. All liver tissue was homogenized immediately by ultrasonication after isolation in RIPA solution containing inhibitors that inhibit enzyme degradation. Then liver tissue homogenates were centrifuged at 12,000 rpm for 20 min. Aliquots from tissue homogenates were assayed for protein measurement using the Bradford protein assay. Equal amounts of protein (50 mg) were then loaded in each well of 12% Tris–glycine gels (Bio-Rad, Hercules, CA). After electrophoresis for 90 min at 125 V of constant voltage, the gel was blotted onto a nitrocellulose membrane by electrophoretic transfer at 70 V of constant voltage for 1– 2 h. The membrane was then washed, blocked with blocking solution, and probed with cleaved TNFRa and RIPk3 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively. Immunoreactive bands were visualized by electrochemiluminescence.
RIPk3 immunohistochemical staining Immunohistochemistry (IHC) staining was performed on paraffin-embedded sections. RIPk3 (IHC) staining paraffin sections from sham, IAKI, and IAKI with necrostatin-1 livers were used. After paraffin removal in xylene, the sections were rehydrated and submitted to heat-steam treatment for 30 min in a 10-mM citric acid monohydrate solution. The endogenous peroxidase activity was quenched by incubating the specimen for 5 min with peroxidase Block. The specimens were then incubated with antibody of active antibody of active RIPk3 (Santa Cruz) overnight at 4 C followed by incubation with the labeled polymer for 30 min. Staining was completed by incubation with 3,3-diaminobenzidine (DAB+) substrate-chromogen, which results in a brown-colored precipitate at the antigen site.
ELISA for plasma tumor necrosis factor a Assessment of renal function and hepatic dysfunction Blood samples were collected via tail vein at 24 h after renal ischemia, bilateral nephrectomy, and sham
Plasma tumor necrosis factor a (TNF-a) from sham and IAKI animals were measured with rat-specific ELISA kits according to the manufacturer’s instructions (R&D Ltd., Minneapolis, MN). Ultrastructural Pathology
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Histological analysis of hepatic injury
Immunoblot analysis of TNFRa
Sham and IAKI livers collected for morphological observations were cut pieces, and fixed in formalin (10% phosphate buffered, pH = 7.4) for paraffin section and stained by H.E for light microscopy (LM). The pieces fixed partly by 2.5% glutaraldehyde were for transmission electron microscopy (TEM). After 48-h immersion fixation and 2-h post-fixation in 1% osmium tetraoxide the pieces for electron microscopy were washed in Na cacodylate buffer, and dehydrated in a series of graded alcohols, and passed through two changes of propylene oxide, then embedded in Epon 812. Semithin sections (1 mm) were cut and stained with toluidine blue and observed by light microscopy. Then ultrathin sections were cut and stained with lead citrate and uranyl acetate for transmission electron microscopy with a JEOL 1200EX electron microscope.
As shown in Figure 2, TNFRa was stronger expressed in IAKI livers than that in sham livers.
Immunoblot analysis of RIPk3 RIPk3 expressions were exhibited in Figure 3. IAKI liver expressed much stronger than either sham or IAKI + necrostatin-1 livers.
RIPk3 immunohistochemical staining Positive staining resulted in brown participates near to cell membrane. As illustration in Figure 4 sham livers exhibited negative staining in Figure 4(a) and positive staining in IAKI liver section (Figure 4b).
Statistical analysis A mean and SD was calculated for serum Cr, BUN, ALT, AST, and TNF-a of each group animals. Statistical analysis was performed using analysis of variance (ANOVA) and t tests between sham and IAKI group. The criterion for statistical significance was set at p5 0.05.
FIGURE 2. Representation of immunoblot analysis of TNFRa proteins in various group livers TNFRa protein expressions increase following IAKI treatment. Bands quantified relative to b-actin as internal loading control.
RESULTS Renal function and hepatic dysfunction Like bilateral nephrectomy renal ischemia led to a significant rise in serum Cr and blood BUN relative to sham-operated animals (Figure 1a and b). ALT and AST levels of animals subjected to AKI (ischemia or bilateral nephrectomy) were much higher than that of sham-operated animals (Figure 1c and d).
FIGURE 3. A representation of immunoblot analysis of RIPK3 in various group animal livers. Only IAKI animal liver illustrates significant stronger expression of RIPk3.
FIGURE 1. Effects of IAKI on renal and hepatic function of various group animals. Serum Cr (mg/100 mL; a), BUN (mg/100 mL; b), ALT (U/L; c), and AST (U/L; d) were determined in three group animals. IAKI and BNx led to a significant rise in serum Cr, BUN ALT, and AST, respectively, relative to sham animals (*p5 .05). !
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FIGURE 4. Micrographs of livers with RIPk3 immunohistochemical stain. (a) A photograph of RIPK3 immunohistochemical staining of sham animal liver shows negative reaction. (b) A photograph of RIPK3 immunohistochemical staining of IAKI liver reveals positive reaction (brown color) near to cell membrane of hepatocytes (arrows). (c) A photograph of RIPK3 immunohistochemical staining of IAKI+necrostatin-1 animal liver shows negative reaction.
necrosis were swollen and contained lytic nuclei and liver cells undergoing necroptosis were swollen but contained pyknotic nuclei in the necrotic lesion (Figure 6c). Figure 6(d), a higher magnification image illustrated normal hepatic plates and sinusoid in sham livers.
FIGURE 5. ELISA of TNF-a in serum of various group animals. Both IAKI and IAKI+necrostatin animals have significant elevations of TNF-a levels in serum relative to sham animals (*p5.05).
Figure 4(c) represented IAKI + necrostatin-1 section also showed negative staining like sham section.
ELISA for TNF-a Figure 5 illustrates that both IAKI and IAKI + necrostatine-1 animals had significant higher levels of TNF-a in serum relative to sham rats (p50.05).
Histological examinations Light microscopic observations With lower magnification light microscopy necrotic lesions in IAKI-induced liver were seen by their pale staining, for an instance, a lesion near to the surface of liver was noted in Figure 6(a), however, sham rat livers illustrated normal histology, such as hepatic plate and sinusoid in order (Figure 6b). With higher magnification light microscopy the necrotic lesion illustrated hepatic plates in mess, eosinophilic and flocculent materials, fragmentations of pyknotic chromatin, and leucocytes. Hepatocytes undergoing pale
Electron microscopic observations The liver cell of sham animals had one or two nucleuses having nucleoli and also abundant endoplasmic reticulum-both smooth and rough and mitochondria. Three types of cell necrosis could be morphologically divided according to ultrastructural appearances in livers of IAKI animals, they were hepatocytes with edema or vacuolization necrosis and necroptosis. Hepatocytes with edema necrosis exhibited a deformed nucleus and swelling cytoplasm containing swollen mitochondria, dilated endoplasmic reticule and disruptive cell membrane (Figure 7a). In late stage of edema necrosis, the cell exhibited a cytoplasm with matrix of lower electron density containing a deformed nucleus and destroy debris of cellular organelles. Besides, in IAKI-associated livers, hepatocytes undergoing necroptosis were observed and they had edema cytoplasm and a pyknotic nucleus. Their cytoplasm matrix with a lower electron density, and membranous debris in cytoplasm and a nucleus with pyknotic and marginated chromatin showing the highest electron density were characteristic of necroptosis (Figure 7b). Hepatocytes with vacuolization necrosis were characteristics by multiple vacuoles in cytoplasm and dilated endoplasmic reticular system, these vacuoles were clear and varied in size and number, and usually appeared throughout the cytoplasm. Besides vacuoles and dilated endoplasmic reticule disrupted cell membrane was often observed (Figure 7c). Sometime, in particular near to necrosis lesion, a part of cytoplasm was replaced by non-structural, proteinaceous materials, and shrinkage nucleus with pyknotic chromatin was noted in the non-structural material (Figure 7d). Figure 8 showed a collection of nuclueses in various changes, deformed Ultrastructural Pathology
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FIGURE 6. Light microscopic images of each group livers. (a and b) 100 and (c and d) 600 H.E staining. In (a), a low magnification image of IAKI animal liver shows a necrotic lesion noted by its pale staining. In (b), sham liver shows normal liver histology. In (c), a higher magnification image of (b) (*) shows hepatic plates in disorder consisting of eosinophilic material, leukocytes, debris of chromatin or pyknotic nucleus, cell undergoing necroptosis (arrow) and hepatocytes with pale necrosis (arrow heads) in center of necrotic region. In (d), a higher magnification image of sham liver exhibits normal hepatic plates consisting of normal hepatocytes.
nucleuses with lytic or pyknotic chromatin in IAKI hepatocytes.
DISCUSSION First of all, rat IAKI models was successfully established in this study induced by 60-min renal ischemia that model have been approved in AKI research field [5]. In the current study, ischemic AKI lead to high level of serum Cr and BUN significantly relative to sham-operated animals and nearly same as that of BNx animals 24 h later. Rats subjected to IAKI and BNx rapidly developed acute hepatic dysfunction. Higher level of serum ALT and AST was observed in animals with IAKI significantly relative to sham rats. The first notable finding of this study is that rats subjected to ischemic AKI not only suffered from acute liver dysfunction but also developed morphologically necrotic lesions in varied sizes in livers. Recently, Park and Kadkhodaee provided a fact that focal necrotic lesions and individual hepatocyte necrosis were manifested at 24 h after ischemic renal injury [5–7]. The most significant finding was that we first observed individual hepatocyte undergoing necroptosis involving in necrosis lesions induced by IAKI under light microscope as well as electron !
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microscope. Necroptosis was featured by its necrotic cytoplasm (pale cytoplasm) containing apoptosis-like nucleuses (pyknotic chromatin nucleus). In addition, we found membranous destruction as common changes in various types of necrotic hepatocytes, including cell membrane, membranous organelles, for instances mitochondria, endoplasmic reticule were destructed. The loss of structural integrity of the plasma membrane is a hallmark of cell necrosis and represents the final endpoint at which a cell can no longer maintain its discrete identity from the environment [8,9]. Besides membranous destruction, nucleus insults, for instances mal-shape, mal-nucleolus, mass-chromatin and lysis or pyknotic chromatin and so on were also obviously observed in IAKIinduced hepatocyte injury under electron microscope. Though membranous destruction is universal indication of cell death, it does not imply the antecedent mechanisms leading to death. In contrast to the morphological definition of cell death mode biochemical definitions of cell death mode does imply biochemical explorations of dying cell. It has now been established that at least a part of necrotic cell death may be executed through a mechanism termed ‘‘necroptosis’’. Necroptosis may be activated upon stimulation by TNF-a, FasL, and TRAIL, and the very same ligands that can activate
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FIGURE 7. Transmission electron micrographs of hepatocyte necrosis in IAKI group animals. In (a), a hepatocyte undergoing edema (pale) necrosis in liver with IAKI shows swelling cytoplasm containing swollen mitochondria and membranous-like debris, and disrupted cell membrane (arrow). In (b), a hepatocyte undergoing necroptosis shows an edema cytoplasm and a nucleus with pyknotic and marginated chromatin. In (c), a hepatocyte undergoing vacuolization necrosis has multi-vacuoles, dilated endoplasmic reticule and disrupted cell membrane (arrow). In (d), a hepatocyte in late necrosis in the center of a necrotic lesion exhibits a deformed nucleus with pyknotic and marginated chromatin and a part of cytoplasm replaced by non-structural and proteinaceous material (*).
FIGURE 8. A series nucleus changes of necrotic hepatocytes in various group animals. (a) Normal nucleus was illustrated in sham liver. Deformed (b–d), nucleuses with lytic chromatin (e and f) and nucleuses with pyknotic chromatin (g and h) were noted in IAKI livers.
apoptosis in the death receptor pathway [10]. Necroptosis does also share some same core regulators as apoptosis does during upstream and downstream mechanism [11,12]. Thus, necroptosis is morphologically featured by its necrotic cytoplasm and apoptotic nucleus [13,14]. For these reasons,
necroptosis, a particular cell death routine was classified as a new type of necrosis [7,12,13]. Recently RIPk3, as homolog of RIPk1, a member of RIP kinase family, has been identified to be required for necroptosis. RIPk3 is recruited to RIPk1 to form a necrosis-inducing complex that is target of Ultrastructural Pathology
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Hepatocyte necroptosis necrostatine-1. The kinase activity of RIPk3 is essential for necroptosis execution. Such a term, RIPk3mediated necroptosis or RIPk3 dependent necrosis was lately introduced [4,13,14]. It is reasonable to employee RIPk3 as a biomarker of necroptosis, a programmed necrosis, to evaluate cell injury [4,14,15]. In this study, the data of ELISA for TNF-a in serums, TNFRa immunoblot analysis, IHC staining observations and even TEM examinations (Figure 8f) indicate RIPk3, as the determinant of hepatocyte necroptosis, possible in response to TNFRa family of deathinducing cytokines. Thus the most novel finding of this study is that necroptosis, RIPk3-mediated necroptosis as a type of necrosis was involved in hepatocyte injury induced by ischemic AKI. The time has come to switch from morphological to biochemical definitions of cell death modalities duo to the substantial progress in the biochemical exploration of cell dying. On the basis of the biochemical definitions of cell death modalities, parthanatos and necroptosis are defined by a series of precise, measurable biochemical features that are available for the possibilities of novel therapeutic targets for the deadly distant organ effects of AKI. Both PARP-1 and RIPk3 inhibitors may become viable candidates for a protective strategy in the treatment of AKIinduced hepatic injury patients In summary, we show in this study that functionally RIPk3-mediated necroptosis was involved in hepatic necrosis induced by ischemic AKI. Morphologically, membranous structures and nucleus of hepatocyte were predominantly vulnerable to the insults of IAKI.
DECLARATION OF INTEREST The authors are indebted to Dr Hongchao Ji for his technique assistance, and Ms. Yuling Liu and Ms. Yanqin Li for their electron microscopic assistance. PhD grand of LNMU (20101311); Natural Science Foundation of Liaoning Province (201202141).
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REFERENCES 1. Elapavaluru S, Kellum JA. Why do patients die of acute kidney injury. Acta Clin Belg Suppl 2007;2: 326–31. 2. Faubel S. Acute kidney injury and multiple organ dysfunction syndrome. Minerva Urol Nefrol 2009;61: 171–88. 3. Yap SC, Lee HT. Acute kidney injury and extrarenal organ dysfunction: New concepts and experimental evidence. Anesthesiology 2012;116: 1139–48. 4. He S, Wang L, Miao L, et al. Receptor interacting protein kinase-3 determines cellular necrosis response to TNF-a. Cell 2009;137: 1100–11. 5. Golab F, Kadkhodaee M, Zahmatkesh M, et al. Ischemic and non-ischemic acute kidney injury cause hepatic damage. Kidney Int 2009;5: 783–92. 6. Park SW, Chen SW, Kim M, et al. Cytokines induce small intestine and liver injury after renal ischemia or nephrectomy. Lab Invest 2011;91: 63–84. 7. Kadkhodaee M, Golab F, Zahmatkesh M, et al. Effects of different periods of renal ischemia on liver as a remote organ. World J Gastroenterol 2009;15: 1113–18. 8. Kroemer G, Galluzzi L, Vandenabeele P, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 2009;16: 3–11. 9. Galluzzi L, Vitale I, Abrams JM, et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 2012;19: 107–20. 10. Cho YS, Chalia S, Moquin D, et al. Phosphorylation-driven assemble of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009;137: 1112–23. 11. Hitomi J, Christofferson DE, Ng A, et al. Identification of a molecular signaling network that regulates a cellular necrosis cell death pathway by a genome wide siRNA screen. Cell 2008;135: 1311–23. 12. Galluzzi L, Kroemer G. Necroptosis: A specialized pathway of programmed necrosis. Cell 2008;135: 1161–3. 13. Yuan J, Kroemer G. Alternative cell death mechanisms in development and beyond. Gene Dev 2010;24: 2592–602. 14. Price PM, Hodeify R. A possible mechanism of renal death after ischemia/reperfusion. Kidney Int 2012;81: 81–2. 15. Green DR, Oberst A, Dillon C, et al. RIPK-dependent necrosis and its regulation by caspases: A mystery in five acts. Mol Cell 2011;44: 9–16.
ORIGINAL ARTICLE
Hepatocyte Necroptosis Induced by Ischemic Acute Kidney Injury in Rats Cuifen Bao, PhD1, Youzhi Shao, MD2,3, and Xiaoming Li, PhD2,3 Central Laboratory, 2Laboratory of Renal Biology, and 3Department of Histology and Embryology, Liaoning Medical University, Jinzhou, Liaoning, China
ABSTRACT While ischemic acute kidney injury (IAKI) is known often to cause hepatic injury, little is known about necroptosis involved in the hepatic injury. The purposes of this study were to identify necroptosis involvement and observe morphological changes of hepatocytes in hepatic injury induced by IAKI in rats. Based on successfully established IAKI rat models, enzyme-linked immunosorbent assay illustrated a significant higher level of tumor necrosis factor a in serums of IAKI animals. Tumor necrosis factor receptor a (TNFRa) and receptor interacting protein kinase 3 (RIPk3) showed significant higher expressions in immunoblot analyses and positive hepatocytes of RIPk3 immunohistochemical staining were also evident in livers of IAKI rats. In addition, light microscopy revealed necrotic lesions that contain hepatocytes ongoing necroptosis besides necrotic cells in IAKI livers. Electron microscopy revealed at least three types of necrotic hepatocytes, they were edema necrosis, vacuolization necrosis, and necroptosis. Hepatocytes undergoing necroptosis had both necrosis and apoptosis morphological characteristics, they were necrosis cytoplasm and apoptosis-like nucleus. Among cellular organelles of hepatocyte with necrosis, membranous structures, such as cell membrane, endoplasmic reticular system, and mitochondria were more vulnerable to the stress of IAKI and deformed nucleuses varied in shape and lytic or pyknotic chromatin appearances were noted under insults of IAKI. In conclusion, hepatocyte undergoing necroptosis, RIPk3-mediated necroptosis partly contributes to hepatic necrosis induced by IAKI. Both membranous structures and nucleuses of hepatocyte were vulnerable to ischemic acute kidney injury. Keywords: Acute kidney injury, hepatocyte, necrosis, RIPk3-mediated necroptosis, ultrastructure
INTRODUCTION
Unfortunately the exact mechanisms of hepatic dysfunction in the setting of ischemic AKI remain unclear until now. One reason is that there is limited pathological data available about AKI-associated ALI. Morphological criteria provided by previous studies come from only observations by light microscopy suggesting a role for necrosis. Necroptosis, as a new functional type cell death as well as a type necrosis has been introduced in recent years, and a crucial role for receptor interacting protein kinase (RIPk) activation, including RIPk1 or RIPk3, has been demonstrated during necroptosis and become a biomarker, in particular RIPk3, for identifying cells undergoing necroptosis [4]. Despite a lot of studies
Acute kidney injury (AKI) widely viewed as one frequent complication with severe implications for the critically ill patients [1]. AKI can mostly be caused by renal ischemia, called ischemic acute kidney injury (IAKI) that occurs in various clinical settings including shock, sepsis, renal transplantation, and vascular surgery. IAKI, however, rarely occurs in isolation. It has become apparent that clinically, much of the high patient mortality can be attributed to progression to remote organ failures. Among remote organ failures AKI-induced acute liver injury (ALI) often becomes a death cause of AKI patients [2,3].
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Received 8 December 2013; Revised 5 February 2014; Accepted 7 February 2014; Published online 28 March 2014 Correspondence: Dr. Xiaoming Li, Department of Histology and Embryology, Liaoning Medical University, No. 40, Section 3 Songpo Road, Linghe District, Jinzhou, Liaoning 121001, PR China. Fax: +86 416 4673425. E-mail: [email protected]
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218 C. Bao et al. describing the role of necrosis in IAKI-injured liver, two issues remain. First, there is a need to reveal if necroptosis is involved in hepatic dysfunction induced by IAKI. Second, exhibiting detail morphological, including ultrastructural changes of hepatocyte necrosis after IAKI is required. For these purposes, the effects of IAKI on hepatic necrosis were examined by enzyme-linked immunosorbent assay (ELISA) for tumor necrosis factor a (TNF-a), RIPk3 and tumor necrosis factor receptor a (TNFRa) immunoblot analyses and RIPk3 immunohistochemical staining as well as both light and electron microscopy.
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MATERIALS AND METHODS Rat model of IAKI This study used Wistar male rats (n = 19), weighing 150–200 g, 2–3 months old. All animals were acclimated and maintained on a standard pellet diet 1 week before the initiation of experiments. The animal protocol was reviewed and approved by the Liaoning Medical University Animal Care and Research Committee, and rats were housed under pathogenfree condition at least 1 week before surgical procedures. Surgical procedures were performed using strict sterile techniques under anesthesia with pentobarbital sodium (50 mg/kg ip). All animals were underwent a midline laparotomy with isolation of bilateral renal pedicles. For rats (n = 9) assigned to experimental ischemic AKI (IAKI), a non-traumatic microvascular clamp was applied across both renal pedicles for 60 min. After the allotted ischemia time, 60 min, the clamps were gently removed and animals were administered 2 mL of sterile saline intra-peritoneally, and incision closed with 4-0 silk suture. There were four IAKI animals, as only RIPk3 analysis controls, received necrostatin-1 injections (1.65 mg/kg) before incision closed. Sham rats (n = 5) received same surgical procedures only without placement of the vascular clamps. The rats (n = 5) assigned to bilateral nephrectomy (BNx) as renal dysfunction controls underwent similar procedures except that both renal pedicles were ligated with a 5–0 silk suture and kidneys were removed. At 24 h, following the experimental procedure, the rats were euthanized under anesthesia, and blood and liver tissues, were collected for analysis of both renal function and hepatic dysfunction and morphological examinations.
operation. Plasma concentrations of creatinine (Cr) and blood urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured by colorimetric methods using commercially available kits.
Immunoblot analyses of TNFRa and RIPk3 Only livers of sham and IAKI animals were available for TNFRa analysis. Livers of sham, IAKI, and IAKI with necrostatin-1 injection were available for RIPk3 analysis. All liver tissue was homogenized immediately by ultrasonication after isolation in RIPA solution containing inhibitors that inhibit enzyme degradation. Then liver tissue homogenates were centrifuged at 12,000 rpm for 20 min. Aliquots from tissue homogenates were assayed for protein measurement using the Bradford protein assay. Equal amounts of protein (50 mg) were then loaded in each well of 12% Tris–glycine gels (Bio-Rad, Hercules, CA). After electrophoresis for 90 min at 125 V of constant voltage, the gel was blotted onto a nitrocellulose membrane by electrophoretic transfer at 70 V of constant voltage for 1– 2 h. The membrane was then washed, blocked with blocking solution, and probed with cleaved TNFRa and RIPk3 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively. Immunoreactive bands were visualized by electrochemiluminescence.
RIPk3 immunohistochemical staining Immunohistochemistry (IHC) staining was performed on paraffin-embedded sections. RIPk3 (IHC) staining paraffin sections from sham, IAKI, and IAKI with necrostatin-1 livers were used. After paraffin removal in xylene, the sections were rehydrated and submitted to heat-steam treatment for 30 min in a 10-mM citric acid monohydrate solution. The endogenous peroxidase activity was quenched by incubating the specimen for 5 min with peroxidase Block. The specimens were then incubated with antibody of active antibody of active RIPk3 (Santa Cruz) overnight at 4 C followed by incubation with the labeled polymer for 30 min. Staining was completed by incubation with 3,3-diaminobenzidine (DAB+) substrate-chromogen, which results in a brown-colored precipitate at the antigen site.
ELISA for plasma tumor necrosis factor a Assessment of renal function and hepatic dysfunction Blood samples were collected via tail vein at 24 h after renal ischemia, bilateral nephrectomy, and sham
Plasma tumor necrosis factor a (TNF-a) from sham and IAKI animals were measured with rat-specific ELISA kits according to the manufacturer’s instructions (R&D Ltd., Minneapolis, MN). Ultrastructural Pathology
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Hepatocyte necroptosis
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Histological analysis of hepatic injury
Immunoblot analysis of TNFRa
Sham and IAKI livers collected for morphological observations were cut pieces, and fixed in formalin (10% phosphate buffered, pH = 7.4) for paraffin section and stained by H.E for light microscopy (LM). The pieces fixed partly by 2.5% glutaraldehyde were for transmission electron microscopy (TEM). After 48-h immersion fixation and 2-h post-fixation in 1% osmium tetraoxide the pieces for electron microscopy were washed in Na cacodylate buffer, and dehydrated in a series of graded alcohols, and passed through two changes of propylene oxide, then embedded in Epon 812. Semithin sections (1 mm) were cut and stained with toluidine blue and observed by light microscopy. Then ultrathin sections were cut and stained with lead citrate and uranyl acetate for transmission electron microscopy with a JEOL 1200EX electron microscope.
As shown in Figure 2, TNFRa was stronger expressed in IAKI livers than that in sham livers.
Immunoblot analysis of RIPk3 RIPk3 expressions were exhibited in Figure 3. IAKI liver expressed much stronger than either sham or IAKI + necrostatin-1 livers.
RIPk3 immunohistochemical staining Positive staining resulted in brown participates near to cell membrane. As illustration in Figure 4 sham livers exhibited negative staining in Figure 4(a) and positive staining in IAKI liver section (Figure 4b).
Statistical analysis A mean and SD was calculated for serum Cr, BUN, ALT, AST, and TNF-a of each group animals. Statistical analysis was performed using analysis of variance (ANOVA) and t tests between sham and IAKI group. The criterion for statistical significance was set at p5 0.05.
FIGURE 2. Representation of immunoblot analysis of TNFRa proteins in various group livers TNFRa protein expressions increase following IAKI treatment. Bands quantified relative to b-actin as internal loading control.
RESULTS Renal function and hepatic dysfunction Like bilateral nephrectomy renal ischemia led to a significant rise in serum Cr and blood BUN relative to sham-operated animals (Figure 1a and b). ALT and AST levels of animals subjected to AKI (ischemia or bilateral nephrectomy) were much higher than that of sham-operated animals (Figure 1c and d).
FIGURE 3. A representation of immunoblot analysis of RIPK3 in various group animal livers. Only IAKI animal liver illustrates significant stronger expression of RIPk3.
FIGURE 1. Effects of IAKI on renal and hepatic function of various group animals. Serum Cr (mg/100 mL; a), BUN (mg/100 mL; b), ALT (U/L; c), and AST (U/L; d) were determined in three group animals. IAKI and BNx led to a significant rise in serum Cr, BUN ALT, and AST, respectively, relative to sham animals (*p5 .05). !
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FIGURE 4. Micrographs of livers with RIPk3 immunohistochemical stain. (a) A photograph of RIPK3 immunohistochemical staining of sham animal liver shows negative reaction. (b) A photograph of RIPK3 immunohistochemical staining of IAKI liver reveals positive reaction (brown color) near to cell membrane of hepatocytes (arrows). (c) A photograph of RIPK3 immunohistochemical staining of IAKI+necrostatin-1 animal liver shows negative reaction.
necrosis were swollen and contained lytic nuclei and liver cells undergoing necroptosis were swollen but contained pyknotic nuclei in the necrotic lesion (Figure 6c). Figure 6(d), a higher magnification image illustrated normal hepatic plates and sinusoid in sham livers.
FIGURE 5. ELISA of TNF-a in serum of various group animals. Both IAKI and IAKI+necrostatin animals have significant elevations of TNF-a levels in serum relative to sham animals (*p5.05).
Figure 4(c) represented IAKI + necrostatin-1 section also showed negative staining like sham section.
ELISA for TNF-a Figure 5 illustrates that both IAKI and IAKI + necrostatine-1 animals had significant higher levels of TNF-a in serum relative to sham rats (p50.05).
Histological examinations Light microscopic observations With lower magnification light microscopy necrotic lesions in IAKI-induced liver were seen by their pale staining, for an instance, a lesion near to the surface of liver was noted in Figure 6(a), however, sham rat livers illustrated normal histology, such as hepatic plate and sinusoid in order (Figure 6b). With higher magnification light microscopy the necrotic lesion illustrated hepatic plates in mess, eosinophilic and flocculent materials, fragmentations of pyknotic chromatin, and leucocytes. Hepatocytes undergoing pale
Electron microscopic observations The liver cell of sham animals had one or two nucleuses having nucleoli and also abundant endoplasmic reticulum-both smooth and rough and mitochondria. Three types of cell necrosis could be morphologically divided according to ultrastructural appearances in livers of IAKI animals, they were hepatocytes with edema or vacuolization necrosis and necroptosis. Hepatocytes with edema necrosis exhibited a deformed nucleus and swelling cytoplasm containing swollen mitochondria, dilated endoplasmic reticule and disruptive cell membrane (Figure 7a). In late stage of edema necrosis, the cell exhibited a cytoplasm with matrix of lower electron density containing a deformed nucleus and destroy debris of cellular organelles. Besides, in IAKI-associated livers, hepatocytes undergoing necroptosis were observed and they had edema cytoplasm and a pyknotic nucleus. Their cytoplasm matrix with a lower electron density, and membranous debris in cytoplasm and a nucleus with pyknotic and marginated chromatin showing the highest electron density were characteristic of necroptosis (Figure 7b). Hepatocytes with vacuolization necrosis were characteristics by multiple vacuoles in cytoplasm and dilated endoplasmic reticular system, these vacuoles were clear and varied in size and number, and usually appeared throughout the cytoplasm. Besides vacuoles and dilated endoplasmic reticule disrupted cell membrane was often observed (Figure 7c). Sometime, in particular near to necrosis lesion, a part of cytoplasm was replaced by non-structural, proteinaceous materials, and shrinkage nucleus with pyknotic chromatin was noted in the non-structural material (Figure 7d). Figure 8 showed a collection of nuclueses in various changes, deformed Ultrastructural Pathology
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FIGURE 6. Light microscopic images of each group livers. (a and b) 100 and (c and d) 600 H.E staining. In (a), a low magnification image of IAKI animal liver shows a necrotic lesion noted by its pale staining. In (b), sham liver shows normal liver histology. In (c), a higher magnification image of (b) (*) shows hepatic plates in disorder consisting of eosinophilic material, leukocytes, debris of chromatin or pyknotic nucleus, cell undergoing necroptosis (arrow) and hepatocytes with pale necrosis (arrow heads) in center of necrotic region. In (d), a higher magnification image of sham liver exhibits normal hepatic plates consisting of normal hepatocytes.
nucleuses with lytic or pyknotic chromatin in IAKI hepatocytes.
DISCUSSION First of all, rat IAKI models was successfully established in this study induced by 60-min renal ischemia that model have been approved in AKI research field [5]. In the current study, ischemic AKI lead to high level of serum Cr and BUN significantly relative to sham-operated animals and nearly same as that of BNx animals 24 h later. Rats subjected to IAKI and BNx rapidly developed acute hepatic dysfunction. Higher level of serum ALT and AST was observed in animals with IAKI significantly relative to sham rats. The first notable finding of this study is that rats subjected to ischemic AKI not only suffered from acute liver dysfunction but also developed morphologically necrotic lesions in varied sizes in livers. Recently, Park and Kadkhodaee provided a fact that focal necrotic lesions and individual hepatocyte necrosis were manifested at 24 h after ischemic renal injury [5–7]. The most significant finding was that we first observed individual hepatocyte undergoing necroptosis involving in necrosis lesions induced by IAKI under light microscope as well as electron !
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microscope. Necroptosis was featured by its necrotic cytoplasm (pale cytoplasm) containing apoptosis-like nucleuses (pyknotic chromatin nucleus). In addition, we found membranous destruction as common changes in various types of necrotic hepatocytes, including cell membrane, membranous organelles, for instances mitochondria, endoplasmic reticule were destructed. The loss of structural integrity of the plasma membrane is a hallmark of cell necrosis and represents the final endpoint at which a cell can no longer maintain its discrete identity from the environment [8,9]. Besides membranous destruction, nucleus insults, for instances mal-shape, mal-nucleolus, mass-chromatin and lysis or pyknotic chromatin and so on were also obviously observed in IAKIinduced hepatocyte injury under electron microscope. Though membranous destruction is universal indication of cell death, it does not imply the antecedent mechanisms leading to death. In contrast to the morphological definition of cell death mode biochemical definitions of cell death mode does imply biochemical explorations of dying cell. It has now been established that at least a part of necrotic cell death may be executed through a mechanism termed ‘‘necroptosis’’. Necroptosis may be activated upon stimulation by TNF-a, FasL, and TRAIL, and the very same ligands that can activate
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FIGURE 7. Transmission electron micrographs of hepatocyte necrosis in IAKI group animals. In (a), a hepatocyte undergoing edema (pale) necrosis in liver with IAKI shows swelling cytoplasm containing swollen mitochondria and membranous-like debris, and disrupted cell membrane (arrow). In (b), a hepatocyte undergoing necroptosis shows an edema cytoplasm and a nucleus with pyknotic and marginated chromatin. In (c), a hepatocyte undergoing vacuolization necrosis has multi-vacuoles, dilated endoplasmic reticule and disrupted cell membrane (arrow). In (d), a hepatocyte in late necrosis in the center of a necrotic lesion exhibits a deformed nucleus with pyknotic and marginated chromatin and a part of cytoplasm replaced by non-structural and proteinaceous material (*).
FIGURE 8. A series nucleus changes of necrotic hepatocytes in various group animals. (a) Normal nucleus was illustrated in sham liver. Deformed (b–d), nucleuses with lytic chromatin (e and f) and nucleuses with pyknotic chromatin (g and h) were noted in IAKI livers.
apoptosis in the death receptor pathway [10]. Necroptosis does also share some same core regulators as apoptosis does during upstream and downstream mechanism [11,12]. Thus, necroptosis is morphologically featured by its necrotic cytoplasm and apoptotic nucleus [13,14]. For these reasons,
necroptosis, a particular cell death routine was classified as a new type of necrosis [7,12,13]. Recently RIPk3, as homolog of RIPk1, a member of RIP kinase family, has been identified to be required for necroptosis. RIPk3 is recruited to RIPk1 to form a necrosis-inducing complex that is target of Ultrastructural Pathology
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Hepatocyte necroptosis necrostatine-1. The kinase activity of RIPk3 is essential for necroptosis execution. Such a term, RIPk3mediated necroptosis or RIPk3 dependent necrosis was lately introduced [4,13,14]. It is reasonable to employee RIPk3 as a biomarker of necroptosis, a programmed necrosis, to evaluate cell injury [4,14,15]. In this study, the data of ELISA for TNF-a in serums, TNFRa immunoblot analysis, IHC staining observations and even TEM examinations (Figure 8f) indicate RIPk3, as the determinant of hepatocyte necroptosis, possible in response to TNFRa family of deathinducing cytokines. Thus the most novel finding of this study is that necroptosis, RIPk3-mediated necroptosis as a type of necrosis was involved in hepatocyte injury induced by ischemic AKI. The time has come to switch from morphological to biochemical definitions of cell death modalities duo to the substantial progress in the biochemical exploration of cell dying. On the basis of the biochemical definitions of cell death modalities, parthanatos and necroptosis are defined by a series of precise, measurable biochemical features that are available for the possibilities of novel therapeutic targets for the deadly distant organ effects of AKI. Both PARP-1 and RIPk3 inhibitors may become viable candidates for a protective strategy in the treatment of AKIinduced hepatic injury patients In summary, we show in this study that functionally RIPk3-mediated necroptosis was involved in hepatic necrosis induced by ischemic AKI. Morphologically, membranous structures and nucleus of hepatocyte were predominantly vulnerable to the insults of IAKI.
DECLARATION OF INTEREST The authors are indebted to Dr Hongchao Ji for his technique assistance, and Ms. Yuling Liu and Ms. Yanqin Li for their electron microscopic assistance. PhD grand of LNMU (20101311); Natural Science Foundation of Liaoning Province (201202141).
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