Ultrastructural Pathology, 2013; 37(6): 433–439 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2013.833562
ORIGINAL ARTICLE
Ultrastructural Pathology of Rat Lung Injury Induced by Ischemic Acute Kidney Injury* Dongyu Zang, PhD1, Youzhi Shao, MD2,3,4, and Xiaoming Li, PhD3,4 Department of Thoracic Surgery, The Third Affiliated Hospital, 2Central Laboratory, Department of Histology and Embryology, College of Basic Medicine, and 4Laboratory of Renal Biology, Liaoning Medical University, Jinzhou, Liaoning, China
ABSTRACT Ischemic acute kidney injury (AKI) is a common complication during inpatient hospitalization, and often induces acute lung injury (ALI). A lot of studies have concentrated on the relevance between AKI and ALI, but the underlying mechanisms of AKI- associated ALI have remained unclear until now. One reason is that evidence of the ultrastructural pathology of AKI-associated ALI has been scarce and needed to be accumulated. The aims of present study are to observe ultrastructural changes, and to reveal leukocyte trafficking of ALI induced by ischemic AKI in rats. For this purpose light microscopy (LM) and electron microscopy (EM), as well as morphometric analysis, were employed in present study. LM observations revealed distinct regions of collapsed alveoli, hemorrhage in alveoli, and interstitial edema in AKI-induced ALI. EM examinations provided facts that alveolar epithelial cells, including type I and type II cells, were necrotic, and endothelia cells undergoing apoptosis as well as interstitial cells undergoing necroptosis were noted in AKI lungs. In addition, shrinkage and decreased or disappeared lamellar bodies were evident in alveolar type II cell of AKI rat lungs. Leukocyte numerical density on area (NA) in AKI lungs was significantly more than that in sham lungs. Based on the morphological criteria from EM examinations and morphometric analysis, a conclusion was that necrosis, including necroptosis, and apoptosis were involved in damaged lung induced by AKI. And inflammation also contributed to acute lung injury of rats with AKI. Keywords: Acute kidney injury, acute lung injury, apoptosis, necroptosis, necrosis, ultrastructure
microscopy, fewer of them were documented pathological features of AKI-associated ALI [3,4]. Particularly, with the new progression of alternative modalities of cell damage or death achieved in recent years a detailed study of the characteristics of ultrastructural changes of AKI-induced lung injury was needed. In addition, increased leukocyte trafficking as a hallmark of ALI has been introduced [5]. But if the trafficking was involved in AKI-induced lung injury, then a quantitative examination was needed. In order to examine ultrastructural pathology of AKI-associated lungs, the present study was designed to identify and characterize cell damage morphologies of AKI-associated lung using light microscopy (LM) and transmission electron microscopy (TEM) based on established rat models of AKI.
Ischemic acute kidney injury (AKI) occurs more often in various clinical settings, including shock, sepsis, kidney transplantation, and vascular surgery, but rarely in isolation [1]. It often causes acute lung injury (ALI) that becomes a significant cause of respiratory failure in the intensive care unit and carries a substantially higher mortality [2]. However, the underlying mechanisms of lung dysfunction in the setting of ischemic AKI remain unclear, even though there are a lot of clinical and laboratory studies describing the relevance between AKI and ALI. One reason is that few data have been available about morphological changes of AKI-induced lung injury. While previous studies have examined the morphology of pulmonary or extrapulmonary ALI using light microscopy in combination with electron
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*The authors are indebted to Yuling Liu and Yanqin Li for their electron microscopic assistance. Received 17 May 2013; Revised 29 July 2013; Accepted 7 August 2013; Published online 11 October 2013 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. E-mail: [email protected]
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434 D. Zang et al. Moreover, morphometric analysis was employed under the electron microscope for leukocyte trafficking in this kind lung injury.
Wistar male rats (n = 15), weighing 150–200 g, 2–3 months old were used for this study. All experimental procedures were reviewed and approved by the Liaoning Medical University Animal Care Committee. Rats were housed under pathogen-free conditions, acclimated, and maintained on a standard pellet diet for at least 1 week before surgical procedures.
Lungs collected from AKI and sham rats for morphological observations were cut into pieces and fixed in formalin (10% phosphate buffered, pH = 7.4) for paraffin section and stained by H&E. The pieces fixed by 2.5% glutaraldehyde were for electron microscopy. After 48-h fixation and 2-h postfixing by 1% osmium tetraoxide, the pieces for electron microscopy were washed in Na cacodylate buffer, 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 TEM with a JEOL 1200EX electron microscope.
Surgical Procedures for Ischemic AKI Rat Models
Morphometric Analysis for Leukocyte Infiltration
All 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 = 5) assigned to ischemic AKI, a nontraumatic microvascular clamp was applied across both renal pedicles for 60 min. After the allotted ischemia time, the clamps were gently removed, animals were administered 2 mL of sterile saline intraperitoneatlly, and the incisions were closed with 4–0 silk suture. Sham rats (n = 5) underwent the same surgical procedures but 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, kidneys, and lungs were collected for analysis.
In order to optimize sampling efficiency [6], one ultrathin section from each rat of various groups was selected randomly. All the sections for morphometric analysis were recorded in the electron microscope at a magnification of 800. The micrographs were enlarged to a total magnification of 2400. Numerical density on area (NA) of leukocytes, i.e., the number of leukocytes relative to lung tissue area (reference area), measured as N/mm2, was determined by point counting from a coherent square lattice. The distance between the lines of the lattice was 2.4 cm, corresponding to 10 mm on the electron micrograph. The formula NA = N/mm2 = N/p 10 mm2 was used, then multiplied with 1 mm2. The number per 1 mm2 lung tissue area was estimated.
MATERIALS AND METHODS Animal Care
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Morphological Examinations of Lung Injury
Renal Examinations Blood samples were obtained via tail vein at 24 h after renal ischemia, bilateral nephrectomy, and sham operation. Blood concentration of creatinine (Cr) and blood urea nitrogen (BUN) were determined by colorimetric methods using commercially available kits. Kidneys from sham and AKI animals were fixed in formalin (10% phosphate buffered, pH = 7.4). After fixation, the renal tissues were rinsed then embedded in paraffin. Paraffin sections were cut and stained by hematoxylin and eosin for light microscopy.
Statistical Analysis Data are presented as mean values SE. Differences at the 95% confidence level were considered significant. Statistical analysis was performed using analysis of variance (ANOVA) and t tests between the sham group and each AKI group.
RESULTS Renal Examinations Renal ischemia led to a significant rise in serum Cr and blood BUN relative to sham-operated animals. And bilateral nephrectomy resulted in a significant elevation in serum Cr and BUN levels related to sham group animals (Figure 1). Ischemic AKI and BNx animals have nearly same renal dysfunction. Ultrastructural Pathology
Ultrastructural Pathology of Rat Lung Injury 435
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FIGURE 1. Serum creatinine and bun of various group animals. Serum creatinine (A) and blood urea nitrogen (B) were determined in all groups. Both AKI and BNx led to a significant rise in serum creatinine and BUN relative to sham animals.
FIGURE 2. Light micrographs of various group lungs, 400. A photo of sham animal lung. (A) Normal histology of lung could be observed. (B) Distinct regions of alveolar collapse were illustrated in AKI-associated lungs. (C) Alveolar hemorrhage and debris in alveolar lumen could be seen in AKI-induced lung.
Pathologically, AKI illustrated typical tubular necrosis, particularly in out medullar regions. Tubular cells were often disrupted and cellular debris appeared in the tubular lumen.
LM Observations of Lung Injury With low-magnification light microscopy, sham group lungs showed a normal histology (Figure 2A). Distinct regions of alveolar collapse of varied size were evident in lungs after ischemic AKI (Figure 2B). And focal alveolar hemorrhage, cellular debris in alveolar lumen could be seen in AKI-associated lungs. Leukocyte infiltration was seen in septum of alveoli (Figure 2C).
FIGURE 3. An endothelial cell undergoing apoptosis was featured by its pyknotic nucleuses in AKI-induced lung, and basement membrane was denudede (arrows). 6500.
TEM Observations of Lung Injury Endothelial cells undergoing apoptosis were featured by their pyknotic nuclei (Figure 3) and sometimes pyknotic chromatin was marginated (Figure 4). Alveolar type I cells often had a pathological appearance in the thin portion composing a part of the blood–air barrier. Figure 5 shows a thin portion of a sham alveolar type I cell. Swelling cytoplasm of the thin portion was evident in lungs with AKI (Figure 6). The thin portion became a vacuole-like structure due to swollen cytoplasm. Disrupted cell !
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membrane was noted (Figure 7), and necrotic thin portion with denudation of the basement membrane was also evident (Figure 3). Alveolar type II cells in sham animal lungs showed normal structures including lamellar bodies (Figure 8). Shrinkage or disappeared lamellar bodies were evident in some alveolar type II cells of AKI animal lungs. In addition, some alveolar type II cells illustrated swelling cytoplasm containing degenerative cellular organelles and disappeared lamellar bodies (Figure 9). In the late stage of necrotic cell
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FIGURE 4. An endothelial cell undergoing apoptosis was featured by nucleus with pyknotic and marginated chromatin. 6500.
FIGURE 7. Disrupted cell membrane were found in alveolar type I cell (arrows). 5000.
FIGURE 5. Thin portion of alveolar type I cell in a sham animal lung showed normal histology (arrows). 10,000. FIGURE 8. Alveolar type II cells in sham animal lungs showed normal structures, including lamellar bodies. 4000.
FIGURE 6. Thin portion of an alveolar type I cell showing swollen cytoplasm containing multivesicles. 10,000.
death, these alveolar type II cells with AKI contained disruptive membranous structures and membranous debris in swelling cytoplasm (Figure 10). Figure 11 shows normal histology of lung interstitium in a sham animal. However, focal edema was evident in the alveolar septum in AKI lungs. Moreover, interstitial cells undergoing necroptosis in AKI-induced lung injury were identified by their
FIGURE 9. Alveolar type II cells illustrated swelling cytoplasm containing degenerative cellular organelles and disappeared lamellar bodies in swelling cytoplasm were also observed. 4000.
Ultrastructural Pathology
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Ultrastructural Pathology of Rat Lung Injury 437
FIGURE 10. Another alveolar type II cell in the late stage of necrosis illustrated swollen cytoplasm containing disrupted membranous structures and membranous debris. 4000.
FIGURE 11. A photo showing normal histology of lung interstitium in a sham animal. 2500.
FIGURE 13. Another interstitial cell undergoing necroptosis was seen in alveolar septum of AKI-induced lung injury. 8000.
FIGURE 14. Leukocyte trafficking in the interstitial space in alveolar septum was easy to observe in AKI-induced ALI. 2500.
pyknotic nucleuses and pale (swelling) cytoplasm (Figures 12,13). Leukocyte trafficking in the interstitial space was easily observed (Figure 14).
Morphometric Analysis for Leukocyte Trafficking As observed in Figure 15, the leukocyte number per 1 mm2 lung tissue in AKI-induced lungs was significantly more than that in sham lungs (p50.05)
DISCUSSION FIGURE 12. An interstitial cell undergoing necroptosis was noted in alveolar septum of AKI-induced lung injury. 8000.
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First we successfully established the animal models of ischemic AKI, 60 min ischemia and 24 h reperfusion. Both renal dysfunction and renal pathology were evident from results of blood biochemical analysis and light microscopy observations. Such an ischemic
Ultrastruct Pathol Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
438 D. Zang et al.
FIGURE 15. Morphometric analysis of leukocyte trafficking in various the lung of various groups. The number of leukocyte per 1 mm2 of lung tissue in AKI animals was significantly more than that in sham animals (p5.05).
AKI model has been approved in the AKI research field [7,8]. ALI has an early exudative phase that is featured by interstitial and intra-alveolar edema, intra-alveolar hemorrhage, collapsed alveoli, and neutrophil accumulation. Actually, our studies have identified that AKI in rats causes lung edema, alveolar hemorrhage, and increased leukocyte trafficking, all of which are hallmarks of the ALI exudative phase. The most significant finding is that lung cell injury was induced by AKI in our models. To identify relevant models of cell damage morphologically involved in AKI-induced lung injury we performed ultrastructural analysis. A cell should be considered dead when any one of the following ultrastructural criteria is met: (1) the cell has lost the integrity of its plasma membrane; (2) the cell, including its nucleus, has undergone complete fragmentation into discrete bodies; (3) its corpse has been engulfed by an adjacent cell [9,10]. We identified alveolar cells as necrosis for their swollen cytoplasm and disrupted cell membrane. Necrosis has traditionally been considered an accidental cell death that is not subject to cellular regulations. Necrotic cell death is morphologically characterized by an increase in cell volume, swelling of organelles, and plasma membrane rupture, followed by leakage of intracellular contents. However, it has now been established that at least a part of necrotic cell death may be executed through a mechanism termed ‘‘necroptosis.’’ Necroptosis also shares some of same core regulators as apoptosis during upstream and downstream mechanisms [11,12]. Thus, necroptosis is morphologically characterized by its necrotic cytoplasm and apoptosis-like nucleus and as a type of necrosis has been accepted in public recommendations by Nomenclature Committee on Cell Death (NCCD) [9,10]. The most novel finding of this study is that necroptosis involved in lung injury is induced by ischemic AKI. Interstitial cells undergoing necroptosis are illustrated in Figures 12 and 13. Besides necrosis and apoptosis, endothelial cells undergoing apoptosis that contributes to increased pulmonary vascular permeability
were observed in septum capillaries in this study [13]. So we suggest that necrosis, including necroptosis, a new alternative type of necrosis, and apoptosis were involved in lung injury induced by AKI. In the current study a thin portion necrotic of type I cells, particularly swelling cytoplasm, as well as edema of interstitial space caused increased thickness of the blood–air barrier. In the analysis of intracellular surfactant, lamellar bodies are seen as the morphological equivalent of the intracellular surfactant pool. This approach has been used in various animal models [14,15]. Ultrastructural analysis in this study showed that type II cell necrosis and decreased shrinkage or disappeared lamellar bodies were involved in AKI-associated ALI. Thus, we suggest that surfactant dysfunction due to alveolar type II cell injuries, for example, decreased, shrunken, or disappeared lamellar bodies in type II cells, even with cellular necrosis, was involved in early ALI induced by AKI. Morphologically, both increased thickness of the blood–air barrier and alveolar collapse were direct reasons of respiratory failure in exudative phase of AKI-induced ALI. Experiments in rodents have shown that kidney ischemia–reperfusion facilitates lung inflammation and leukocyte trafficking plays a complex and important role in mediating the pulmonary inflammatory response and dysfunction during ischemic AKI [8,16]. As illustrated in Figure 14, lungs with AKI had significant increased leukocyte trafficking and the number of leukocyte per 1mm2 lung tissue was significantly increased relative to sham rat lungs. So a conclusion is that increased leukocyte trafficking was involved in the early phase of AKI-associated ALI. In summary, based on morphological criteria necrosis, including necroptosis as the principal cell damage, and apoptosis were involved in ALI induced by AKI. And leukocyte infiltration was evident in the early phase of AKI-associated ALI.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a grant from Natural Science Foundation of Liaoning Province, grant number 201202135.
REFERENCES 1. Metnitz PG, Krenn CG, Steltzer H, et al. Effect of acute renal failure requiring renal replacement therapy on outcome in critically ill patients. Crit Care Med 2002;30: 2051–8. Ultrastructural Pathology
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Ultrastructural Pathology of Rat Lung Injury 439 2. Faubel S. Acute kidney injury and multiple organ dysfunction syndrome. Minerva Urol Nefrol 2009;61: 171–88. 3. Anderson WR, Thielen K. Correlative study of adult respiratory distress syndrome by light,scanning,and transmission electron microscopy. Ultrastruct Pathol 1992;16: 615–28. 4. Menezes SLS, Bozza PT, Castro Faria Neto HC, et al. Pulmonary and extrapulmonary acute lung injury: inflammatory and ultrastructural analyses. J Appl Physiol 2005;98: 1777–83. 5. Bonventre JV, Zuk A. Ischemic acute renal failure: an inflammatory disease? Kidney Int 2004;66: 480–5. 6. Weibel ER, Hsia CC, Ochs M. How much is there really? Why stereology is essential in lung morphometry. J Appl Physiol 2007;102: 459–67. 7. Golab F, Kadkhodaee M, Zahmatkesh M, et al. Ischemic and non-ischemic acute kidney injury cause hepatic damage. Kidney Int 2009;75: 783–92. 8. Hassoun HT, Grigoryev DN, Lie ML, et al. Ischemic acute kidney injury induces a distant organ functional and genomic response distinguishable from bilateral nephrectomy. Am J Physiol Renal Physiol 2007;293: F30–40. 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.
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10. Kroemer G, Galluzzi L, Vandenabeele P, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ 2009;16: 3–11. 11. Galluzzi L, Kroemer G. Necroptosis: a specialized pathway of programmed necrosis. Cell 2008;135: 1161–3. 12. Yuan J, Kroemer G. Alternative cell death mechanisms in development and beyond. Genes Dev 2010;24: 2592–602. 13. Hassoun HT, Lie ML, Grigoryev DN, et al. Kidney ischemia–reperfusion injury induces caspase-dependent pulmonary apoptosis. Am J Physiol Renal Physiol 2009;297: F125–37. 14. Fehrenbach A, Bube C, Hohlfeld JM, et al. Surfactant homeostasis is maintained in vivo during keratinocyte growth factor-induced rat lung type-II cell hyperplasia. Am J Respir Crit Care Med 2003;167: 1264–70. 15. Knudsen L, Waizy H, Fehrenbach H, et al. Ultrastructural changes of the intracellular surfactant pool in a rat model of lung transplantation-related events. Respir Res 2011;12: 79–89. 16. Deng J, Hu X, Yuen PS, et al. Alpha-melanocytestimulating hormone inhibits lung injury after renal ischemia/reperfusion. Am J Respir Crit Care Med 2004; 169: 749–56.
ORIGINAL ARTICLE
Ultrastructural Pathology of Rat Lung Injury Induced by Ischemic Acute Kidney Injury* Dongyu Zang, PhD1, Youzhi Shao, MD2,3,4, and Xiaoming Li, PhD3,4 Department of Thoracic Surgery, The Third Affiliated Hospital, 2Central Laboratory, Department of Histology and Embryology, College of Basic Medicine, and 4Laboratory of Renal Biology, Liaoning Medical University, Jinzhou, Liaoning, China
ABSTRACT Ischemic acute kidney injury (AKI) is a common complication during inpatient hospitalization, and often induces acute lung injury (ALI). A lot of studies have concentrated on the relevance between AKI and ALI, but the underlying mechanisms of AKI- associated ALI have remained unclear until now. One reason is that evidence of the ultrastructural pathology of AKI-associated ALI has been scarce and needed to be accumulated. The aims of present study are to observe ultrastructural changes, and to reveal leukocyte trafficking of ALI induced by ischemic AKI in rats. For this purpose light microscopy (LM) and electron microscopy (EM), as well as morphometric analysis, were employed in present study. LM observations revealed distinct regions of collapsed alveoli, hemorrhage in alveoli, and interstitial edema in AKI-induced ALI. EM examinations provided facts that alveolar epithelial cells, including type I and type II cells, were necrotic, and endothelia cells undergoing apoptosis as well as interstitial cells undergoing necroptosis were noted in AKI lungs. In addition, shrinkage and decreased or disappeared lamellar bodies were evident in alveolar type II cell of AKI rat lungs. Leukocyte numerical density on area (NA) in AKI lungs was significantly more than that in sham lungs. Based on the morphological criteria from EM examinations and morphometric analysis, a conclusion was that necrosis, including necroptosis, and apoptosis were involved in damaged lung induced by AKI. And inflammation also contributed to acute lung injury of rats with AKI. Keywords: Acute kidney injury, acute lung injury, apoptosis, necroptosis, necrosis, ultrastructure
microscopy, fewer of them were documented pathological features of AKI-associated ALI [3,4]. Particularly, with the new progression of alternative modalities of cell damage or death achieved in recent years a detailed study of the characteristics of ultrastructural changes of AKI-induced lung injury was needed. In addition, increased leukocyte trafficking as a hallmark of ALI has been introduced [5]. But if the trafficking was involved in AKI-induced lung injury, then a quantitative examination was needed. In order to examine ultrastructural pathology of AKI-associated lungs, the present study was designed to identify and characterize cell damage morphologies of AKI-associated lung using light microscopy (LM) and transmission electron microscopy (TEM) based on established rat models of AKI.
Ischemic acute kidney injury (AKI) occurs more often in various clinical settings, including shock, sepsis, kidney transplantation, and vascular surgery, but rarely in isolation [1]. It often causes acute lung injury (ALI) that becomes a significant cause of respiratory failure in the intensive care unit and carries a substantially higher mortality [2]. However, the underlying mechanisms of lung dysfunction in the setting of ischemic AKI remain unclear, even though there are a lot of clinical and laboratory studies describing the relevance between AKI and ALI. One reason is that few data have been available about morphological changes of AKI-induced lung injury. While previous studies have examined the morphology of pulmonary or extrapulmonary ALI using light microscopy in combination with electron
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3
*The authors are indebted to Yuling Liu and Yanqin Li for their electron microscopic assistance. Received 17 May 2013; Revised 29 July 2013; Accepted 7 August 2013; Published online 11 October 2013 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. E-mail: [email protected]
433
434 D. Zang et al. Moreover, morphometric analysis was employed under the electron microscope for leukocyte trafficking in this kind lung injury.
Wistar male rats (n = 15), weighing 150–200 g, 2–3 months old were used for this study. All experimental procedures were reviewed and approved by the Liaoning Medical University Animal Care Committee. Rats were housed under pathogen-free conditions, acclimated, and maintained on a standard pellet diet for at least 1 week before surgical procedures.
Lungs collected from AKI and sham rats for morphological observations were cut into pieces and fixed in formalin (10% phosphate buffered, pH = 7.4) for paraffin section and stained by H&E. The pieces fixed by 2.5% glutaraldehyde were for electron microscopy. After 48-h fixation and 2-h postfixing by 1% osmium tetraoxide, the pieces for electron microscopy were washed in Na cacodylate buffer, 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 TEM with a JEOL 1200EX electron microscope.
Surgical Procedures for Ischemic AKI Rat Models
Morphometric Analysis for Leukocyte Infiltration
All 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 = 5) assigned to ischemic AKI, a nontraumatic microvascular clamp was applied across both renal pedicles for 60 min. After the allotted ischemia time, the clamps were gently removed, animals were administered 2 mL of sterile saline intraperitoneatlly, and the incisions were closed with 4–0 silk suture. Sham rats (n = 5) underwent the same surgical procedures but 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, kidneys, and lungs were collected for analysis.
In order to optimize sampling efficiency [6], one ultrathin section from each rat of various groups was selected randomly. All the sections for morphometric analysis were recorded in the electron microscope at a magnification of 800. The micrographs were enlarged to a total magnification of 2400. Numerical density on area (NA) of leukocytes, i.e., the number of leukocytes relative to lung tissue area (reference area), measured as N/mm2, was determined by point counting from a coherent square lattice. The distance between the lines of the lattice was 2.4 cm, corresponding to 10 mm on the electron micrograph. The formula NA = N/mm2 = N/p 10 mm2 was used, then multiplied with 1 mm2. The number per 1 mm2 lung tissue area was estimated.
MATERIALS AND METHODS Animal Care
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Morphological Examinations of Lung Injury
Renal Examinations Blood samples were obtained via tail vein at 24 h after renal ischemia, bilateral nephrectomy, and sham operation. Blood concentration of creatinine (Cr) and blood urea nitrogen (BUN) were determined by colorimetric methods using commercially available kits. Kidneys from sham and AKI animals were fixed in formalin (10% phosphate buffered, pH = 7.4). After fixation, the renal tissues were rinsed then embedded in paraffin. Paraffin sections were cut and stained by hematoxylin and eosin for light microscopy.
Statistical Analysis Data are presented as mean values SE. Differences at the 95% confidence level were considered significant. Statistical analysis was performed using analysis of variance (ANOVA) and t tests between the sham group and each AKI group.
RESULTS Renal Examinations Renal ischemia led to a significant rise in serum Cr and blood BUN relative to sham-operated animals. And bilateral nephrectomy resulted in a significant elevation in serum Cr and BUN levels related to sham group animals (Figure 1). Ischemic AKI and BNx animals have nearly same renal dysfunction. Ultrastructural Pathology
Ultrastructural Pathology of Rat Lung Injury 435
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FIGURE 1. Serum creatinine and bun of various group animals. Serum creatinine (A) and blood urea nitrogen (B) were determined in all groups. Both AKI and BNx led to a significant rise in serum creatinine and BUN relative to sham animals.
FIGURE 2. Light micrographs of various group lungs, 400. A photo of sham animal lung. (A) Normal histology of lung could be observed. (B) Distinct regions of alveolar collapse were illustrated in AKI-associated lungs. (C) Alveolar hemorrhage and debris in alveolar lumen could be seen in AKI-induced lung.
Pathologically, AKI illustrated typical tubular necrosis, particularly in out medullar regions. Tubular cells were often disrupted and cellular debris appeared in the tubular lumen.
LM Observations of Lung Injury With low-magnification light microscopy, sham group lungs showed a normal histology (Figure 2A). Distinct regions of alveolar collapse of varied size were evident in lungs after ischemic AKI (Figure 2B). And focal alveolar hemorrhage, cellular debris in alveolar lumen could be seen in AKI-associated lungs. Leukocyte infiltration was seen in septum of alveoli (Figure 2C).
FIGURE 3. An endothelial cell undergoing apoptosis was featured by its pyknotic nucleuses in AKI-induced lung, and basement membrane was denudede (arrows). 6500.
TEM Observations of Lung Injury Endothelial cells undergoing apoptosis were featured by their pyknotic nuclei (Figure 3) and sometimes pyknotic chromatin was marginated (Figure 4). Alveolar type I cells often had a pathological appearance in the thin portion composing a part of the blood–air barrier. Figure 5 shows a thin portion of a sham alveolar type I cell. Swelling cytoplasm of the thin portion was evident in lungs with AKI (Figure 6). The thin portion became a vacuole-like structure due to swollen cytoplasm. Disrupted cell !
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membrane was noted (Figure 7), and necrotic thin portion with denudation of the basement membrane was also evident (Figure 3). Alveolar type II cells in sham animal lungs showed normal structures including lamellar bodies (Figure 8). Shrinkage or disappeared lamellar bodies were evident in some alveolar type II cells of AKI animal lungs. In addition, some alveolar type II cells illustrated swelling cytoplasm containing degenerative cellular organelles and disappeared lamellar bodies (Figure 9). In the late stage of necrotic cell
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436 D. Zang et al.
FIGURE 4. An endothelial cell undergoing apoptosis was featured by nucleus with pyknotic and marginated chromatin. 6500.
FIGURE 7. Disrupted cell membrane were found in alveolar type I cell (arrows). 5000.
FIGURE 5. Thin portion of alveolar type I cell in a sham animal lung showed normal histology (arrows). 10,000. FIGURE 8. Alveolar type II cells in sham animal lungs showed normal structures, including lamellar bodies. 4000.
FIGURE 6. Thin portion of an alveolar type I cell showing swollen cytoplasm containing multivesicles. 10,000.
death, these alveolar type II cells with AKI contained disruptive membranous structures and membranous debris in swelling cytoplasm (Figure 10). Figure 11 shows normal histology of lung interstitium in a sham animal. However, focal edema was evident in the alveolar septum in AKI lungs. Moreover, interstitial cells undergoing necroptosis in AKI-induced lung injury were identified by their
FIGURE 9. Alveolar type II cells illustrated swelling cytoplasm containing degenerative cellular organelles and disappeared lamellar bodies in swelling cytoplasm were also observed. 4000.
Ultrastructural Pathology
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Ultrastructural Pathology of Rat Lung Injury 437
FIGURE 10. Another alveolar type II cell in the late stage of necrosis illustrated swollen cytoplasm containing disrupted membranous structures and membranous debris. 4000.
FIGURE 11. A photo showing normal histology of lung interstitium in a sham animal. 2500.
FIGURE 13. Another interstitial cell undergoing necroptosis was seen in alveolar septum of AKI-induced lung injury. 8000.
FIGURE 14. Leukocyte trafficking in the interstitial space in alveolar septum was easy to observe in AKI-induced ALI. 2500.
pyknotic nucleuses and pale (swelling) cytoplasm (Figures 12,13). Leukocyte trafficking in the interstitial space was easily observed (Figure 14).
Morphometric Analysis for Leukocyte Trafficking As observed in Figure 15, the leukocyte number per 1 mm2 lung tissue in AKI-induced lungs was significantly more than that in sham lungs (p50.05)
DISCUSSION FIGURE 12. An interstitial cell undergoing necroptosis was noted in alveolar septum of AKI-induced lung injury. 8000.
!
2013 Informa Healthcare USA, Inc.
First we successfully established the animal models of ischemic AKI, 60 min ischemia and 24 h reperfusion. Both renal dysfunction and renal pathology were evident from results of blood biochemical analysis and light microscopy observations. Such an ischemic
Ultrastruct Pathol Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
438 D. Zang et al.
FIGURE 15. Morphometric analysis of leukocyte trafficking in various the lung of various groups. The number of leukocyte per 1 mm2 of lung tissue in AKI animals was significantly more than that in sham animals (p5.05).
AKI model has been approved in the AKI research field [7,8]. ALI has an early exudative phase that is featured by interstitial and intra-alveolar edema, intra-alveolar hemorrhage, collapsed alveoli, and neutrophil accumulation. Actually, our studies have identified that AKI in rats causes lung edema, alveolar hemorrhage, and increased leukocyte trafficking, all of which are hallmarks of the ALI exudative phase. The most significant finding is that lung cell injury was induced by AKI in our models. To identify relevant models of cell damage morphologically involved in AKI-induced lung injury we performed ultrastructural analysis. A cell should be considered dead when any one of the following ultrastructural criteria is met: (1) the cell has lost the integrity of its plasma membrane; (2) the cell, including its nucleus, has undergone complete fragmentation into discrete bodies; (3) its corpse has been engulfed by an adjacent cell [9,10]. We identified alveolar cells as necrosis for their swollen cytoplasm and disrupted cell membrane. Necrosis has traditionally been considered an accidental cell death that is not subject to cellular regulations. Necrotic cell death is morphologically characterized by an increase in cell volume, swelling of organelles, and plasma membrane rupture, followed by leakage of intracellular contents. However, it has now been established that at least a part of necrotic cell death may be executed through a mechanism termed ‘‘necroptosis.’’ Necroptosis also shares some of same core regulators as apoptosis during upstream and downstream mechanisms [11,12]. Thus, necroptosis is morphologically characterized by its necrotic cytoplasm and apoptosis-like nucleus and as a type of necrosis has been accepted in public recommendations by Nomenclature Committee on Cell Death (NCCD) [9,10]. The most novel finding of this study is that necroptosis involved in lung injury is induced by ischemic AKI. Interstitial cells undergoing necroptosis are illustrated in Figures 12 and 13. Besides necrosis and apoptosis, endothelial cells undergoing apoptosis that contributes to increased pulmonary vascular permeability
were observed in septum capillaries in this study [13]. So we suggest that necrosis, including necroptosis, a new alternative type of necrosis, and apoptosis were involved in lung injury induced by AKI. In the current study a thin portion necrotic of type I cells, particularly swelling cytoplasm, as well as edema of interstitial space caused increased thickness of the blood–air barrier. In the analysis of intracellular surfactant, lamellar bodies are seen as the morphological equivalent of the intracellular surfactant pool. This approach has been used in various animal models [14,15]. Ultrastructural analysis in this study showed that type II cell necrosis and decreased shrinkage or disappeared lamellar bodies were involved in AKI-associated ALI. Thus, we suggest that surfactant dysfunction due to alveolar type II cell injuries, for example, decreased, shrunken, or disappeared lamellar bodies in type II cells, even with cellular necrosis, was involved in early ALI induced by AKI. Morphologically, both increased thickness of the blood–air barrier and alveolar collapse were direct reasons of respiratory failure in exudative phase of AKI-induced ALI. Experiments in rodents have shown that kidney ischemia–reperfusion facilitates lung inflammation and leukocyte trafficking plays a complex and important role in mediating the pulmonary inflammatory response and dysfunction during ischemic AKI [8,16]. As illustrated in Figure 14, lungs with AKI had significant increased leukocyte trafficking and the number of leukocyte per 1mm2 lung tissue was significantly increased relative to sham rat lungs. So a conclusion is that increased leukocyte trafficking was involved in the early phase of AKI-associated ALI. In summary, based on morphological criteria necrosis, including necroptosis as the principal cell damage, and apoptosis were involved in ALI induced by AKI. And leukocyte infiltration was evident in the early phase of AKI-associated ALI.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a grant from Natural Science Foundation of Liaoning Province, grant number 201202135.
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