Shock, Publish Ahead of Print DOI : 10.1097/SHK.0000000000001006
Role of M2 macrophages in sepsis-induced acute kidney injury Authors: Li Xing1, Mu Genhua1, Song Chunmei2, Zhou Liangliang1, He Lei1, Jin Qin1 Lu Zhongqian1 Li Xing and Mu Genhua contributed equally to the work and should be considered co-first authors. Author's Institutions: 1. Department of ICU, Yancheng City No.1 People’s Hospital, yancheng 224005,China 2. Nursing College of Nantong University, nantong 226000,China Corresponding Author and addres: Lu Zhongqian, MD, PhD, Department of ICU, Yancheng City No.1 People’s Hospital, Yue He Road 16, Yancheng, 224005, China; Tel: 86-0515-66696618; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Running Head : Role of M2 macrophages
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Abstract Background: Sepsis is a major cause of acute kidney injury (AKI), with high rates of morbidity and mortality. M2 macrophages have been shown to play important roles in the secretion of antiinflammatory and tissue repair mediators. In this study, we investigate the role of M2 macrophages in sepsis-induced AKI by depleting these cells in vivo through the systemic administration of liposomal clodronate (LC). Methods: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham surgery. Biochemical and histological renal damage was assessed. Macrophage infiltration and M2 macrophage depletion were assessed by immunohistochemistry. RT-PCR was used to investigate the expression of the inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1) and found in inflammatory zone 1 (FIZZ1) mRNAs. Western blots were performed to assay the tissue levels of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α). Results: M2 macrophages were obviously detected 72 h after sepsis-induced AKI. Kidney injury was more severe, renal function was decreased, and blood creatinine and blood urea nitrogen (BUN) levels were higher following M2 macrophage depletion. M2 macrophage depletion significantly inhibited the proliferation of tubular cells. M2 macrophage depletion also downregulated IL-10 expression and increased TNF-α secretion during sepsis-induced AKI. Conclusions: M2 macrophages attenuate sepsis-induced AKI, presumably by upregulating IL-10 expression and suppressing TNF-α secretion. Keywords: M2 macrophages, sepsis, acute kidney injury, repair, liposomal clodronate, cytokines
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Introduction Acute kidney injury (AKI) is a serious problem that affects more than 20% of critically ill patients. The presence of AKI is an independent risk factor for mortality in septic patients (1). Although AKI develops in patients with a variety of clinical disorders, sepsis is a major cause that accounts for more than 50% of AKI cases in critically ill patients (2). Despite significant advances in the support of kidney function, the mortality rate of septic patients is still high (approximately 15–30%) partially due to our poor understanding of the mechanisms of AKI (3). Animal models of sepsis have been developed and have revealed that the pathogenesis of AKI is caused by a complex interaction between vascular endothelial cell dysfunction, subsequent inflammation and tubular cell damage (4). Twenty-four hours after injury, the remaining renal tubular cells undergo mitosis due to the function of many cell growth factors and the chemotactic factors; then, cell differentiation and migration along the basal membrane of the tubular duct occur to repair the missing cells (5-6). The capacity of kidney to regenerate functional tissue after an episode of acute injury is a major determinant of outcomes of patients with AKI. No specific therapy improves the rate or effectiveness of the repair process after acute renal injury Macrophages, the most common type of leukocyte involved in the renal injury process, play different roles in different stages of injury (7). Because of differences in the immune microenvironment, the macrophage divides into several phenotypes and functional subclasses that exhibit different functions. In the early stage of sepsis, the macrophage undergoes M1 differentiation, resulting in the production of inflammatory mediators and AKI. In the last stage, the M1 macrophage transforms into an M2 macrophage, generates immunosuppression, and accelerates tissue repair by promoting the repair and regeneration of the renal tubular epithelium (8). M1 macrophages upregulate the expression of pro-inflammatory mediators, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α), and increase their production of reactive oxygen and nitrogen
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
species (9). In contrast, anti-inflammatory M2 macrophages upregulate the expression of arginase-1 (Arg1), scavenger and mannose receptors, and the intracellular protein found in inflammatory zone 1 (FIZZ1) (10-11). iNOS expression has been used as a marker of M1 responses. Arg1 and FIZZ1 are classical inducers of M2 gene expression. Liposomal clodronate (LC) is a bisphosphonate encapsulated by liposome that is known to induce macrophage depletion in vivo (12). Upon administration, this drug is rapidly taken up by macrophages and free bisphosphonate is released intracellularly through the action of lysosomal phospholipases and induces rapid suicidal apoptosis of macrophages. Several studies have reported the specificity of intravenously administered LC in depleting macrophages but not neutrophils or lymphocytes (13). In the present study, we hypothesized that M2 macrophages exhibit protective effects on sepsisinduced AKI. We induced polymicrobial abdominal sepsis by performing a cecal ligation and puncture (CLP) operation on wild type (Sprague-Dawley, SD) rats and investigated the mechanisms of sepsisinduced AKI to test this hypothesis. M2 macrophages afforded partial renal protection in rats with sepsis-induced AKI. This beneficial effect was accompanied by decreased expression of proinflammatory cytokines and increased anti-inflammatory cytokine expression, suggesting that the possible mechanism of M2 macrophages is to contribute to renal repair. Materials and Methods Ethics statement All rats (Male SD rats weighing 250-270 g) were housed in specific pathogen-free cages at 23±2°C and 60±10% humidity, with a 12-hour light/12-hour dark cycle and free access to food and water. All animal procedures were performed in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Septic animal models CLP was performed using a previously described method (9), with slight modifications. Briefly, a 4-0 silk ligature was placed 15 mm from the cecal tip after laparotomy under isoflurane anesthesia. The cecum was punctured twice with an 18-gauge needle and gently squeezed to express a small amount of fecal material before being returned to the central abdominal cavity. In sham-operated animals, the cecum was located but neither ligated nor punctured. The abdominal incision was closed in two layers with 6-0 nylon sutures. After surgery, animals were fluid resuscitated with 40 ml/kg of subcutaneously administered sterile saline and given free access to water but not food. During the surgical procedure, body temperature was maintained at approximately 37°C. Animals were sacrificed by cervical dislocation after 72 h, blood was collected, and kidney samples were harvested and stored at -80°C until further analysis. Liposome preparation and administration Liposomal clodronate (LC) and phosphate-buffered saline liposomes were prepared according to previously described methods (14). Rats received an intraperitoneal bolus of 0.12-0.2 ml/20-25 g body weight of clodronate-encapsulated liposomes or phosphate-buffered saline liposomes (Formumax, USA) 48 h prior to sepsis-induced AKI, as previously reported. Blood chemistry assay After blood collection, the concentrations of blood urea nitrogen (BUN) and serum creatinine (Scr) were immediately analyzed using a Roche Diagnostic analyzer (Roche, Indianapolis, IN). The Scr content was determined using a creatinine (serum) colorimetric assay kit (Cayman Chem, Ann Arbor, MI).
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Renal histology analysis Tissues were fixed with 10% formalin and embedded in paraffin. Four-micrometer sections were stained with periodic acid-Schiff (PAS) reagent. Histological changes in the cortex and the outer stripe of the outer medulla (OSOM) were assessed by quantitative measurements of tissue damage. As tubular damage was mainly vacuolization, the damage was defined as tubular vacuolar degeneration. The degree of kidney damage was estimated in 200X magnification images of more than 100 randomly selected tubules for each animal using the following criteria: 0, normal; 1, area of damage
Role of M2 macrophages in sepsis-induced acute kidney injury Authors: Li Xing1, Mu Genhua1, Song Chunmei2, Zhou Liangliang1, He Lei1, Jin Qin1 Lu Zhongqian1 Li Xing and Mu Genhua contributed equally to the work and should be considered co-first authors. Author's Institutions: 1. Department of ICU, Yancheng City No.1 People’s Hospital, yancheng 224005,China 2. Nursing College of Nantong University, nantong 226000,China Corresponding Author and addres: Lu Zhongqian, MD, PhD, Department of ICU, Yancheng City No.1 People’s Hospital, Yue He Road 16, Yancheng, 224005, China; Tel: 86-0515-66696618; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Running Head : Role of M2 macrophages
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Abstract Background: Sepsis is a major cause of acute kidney injury (AKI), with high rates of morbidity and mortality. M2 macrophages have been shown to play important roles in the secretion of antiinflammatory and tissue repair mediators. In this study, we investigate the role of M2 macrophages in sepsis-induced AKI by depleting these cells in vivo through the systemic administration of liposomal clodronate (LC). Methods: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham surgery. Biochemical and histological renal damage was assessed. Macrophage infiltration and M2 macrophage depletion were assessed by immunohistochemistry. RT-PCR was used to investigate the expression of the inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1) and found in inflammatory zone 1 (FIZZ1) mRNAs. Western blots were performed to assay the tissue levels of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α). Results: M2 macrophages were obviously detected 72 h after sepsis-induced AKI. Kidney injury was more severe, renal function was decreased, and blood creatinine and blood urea nitrogen (BUN) levels were higher following M2 macrophage depletion. M2 macrophage depletion significantly inhibited the proliferation of tubular cells. M2 macrophage depletion also downregulated IL-10 expression and increased TNF-α secretion during sepsis-induced AKI. Conclusions: M2 macrophages attenuate sepsis-induced AKI, presumably by upregulating IL-10 expression and suppressing TNF-α secretion. Keywords: M2 macrophages, sepsis, acute kidney injury, repair, liposomal clodronate, cytokines
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Introduction Acute kidney injury (AKI) is a serious problem that affects more than 20% of critically ill patients. The presence of AKI is an independent risk factor for mortality in septic patients (1). Although AKI develops in patients with a variety of clinical disorders, sepsis is a major cause that accounts for more than 50% of AKI cases in critically ill patients (2). Despite significant advances in the support of kidney function, the mortality rate of septic patients is still high (approximately 15–30%) partially due to our poor understanding of the mechanisms of AKI (3). Animal models of sepsis have been developed and have revealed that the pathogenesis of AKI is caused by a complex interaction between vascular endothelial cell dysfunction, subsequent inflammation and tubular cell damage (4). Twenty-four hours after injury, the remaining renal tubular cells undergo mitosis due to the function of many cell growth factors and the chemotactic factors; then, cell differentiation and migration along the basal membrane of the tubular duct occur to repair the missing cells (5-6). The capacity of kidney to regenerate functional tissue after an episode of acute injury is a major determinant of outcomes of patients with AKI. No specific therapy improves the rate or effectiveness of the repair process after acute renal injury Macrophages, the most common type of leukocyte involved in the renal injury process, play different roles in different stages of injury (7). Because of differences in the immune microenvironment, the macrophage divides into several phenotypes and functional subclasses that exhibit different functions. In the early stage of sepsis, the macrophage undergoes M1 differentiation, resulting in the production of inflammatory mediators and AKI. In the last stage, the M1 macrophage transforms into an M2 macrophage, generates immunosuppression, and accelerates tissue repair by promoting the repair and regeneration of the renal tubular epithelium (8). M1 macrophages upregulate the expression of pro-inflammatory mediators, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α), and increase their production of reactive oxygen and nitrogen
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
species (9). In contrast, anti-inflammatory M2 macrophages upregulate the expression of arginase-1 (Arg1), scavenger and mannose receptors, and the intracellular protein found in inflammatory zone 1 (FIZZ1) (10-11). iNOS expression has been used as a marker of M1 responses. Arg1 and FIZZ1 are classical inducers of M2 gene expression. Liposomal clodronate (LC) is a bisphosphonate encapsulated by liposome that is known to induce macrophage depletion in vivo (12). Upon administration, this drug is rapidly taken up by macrophages and free bisphosphonate is released intracellularly through the action of lysosomal phospholipases and induces rapid suicidal apoptosis of macrophages. Several studies have reported the specificity of intravenously administered LC in depleting macrophages but not neutrophils or lymphocytes (13). In the present study, we hypothesized that M2 macrophages exhibit protective effects on sepsisinduced AKI. We induced polymicrobial abdominal sepsis by performing a cecal ligation and puncture (CLP) operation on wild type (Sprague-Dawley, SD) rats and investigated the mechanisms of sepsisinduced AKI to test this hypothesis. M2 macrophages afforded partial renal protection in rats with sepsis-induced AKI. This beneficial effect was accompanied by decreased expression of proinflammatory cytokines and increased anti-inflammatory cytokine expression, suggesting that the possible mechanism of M2 macrophages is to contribute to renal repair. Materials and Methods Ethics statement All rats (Male SD rats weighing 250-270 g) were housed in specific pathogen-free cages at 23±2°C and 60±10% humidity, with a 12-hour light/12-hour dark cycle and free access to food and water. All animal procedures were performed in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Septic animal models CLP was performed using a previously described method (9), with slight modifications. Briefly, a 4-0 silk ligature was placed 15 mm from the cecal tip after laparotomy under isoflurane anesthesia. The cecum was punctured twice with an 18-gauge needle and gently squeezed to express a small amount of fecal material before being returned to the central abdominal cavity. In sham-operated animals, the cecum was located but neither ligated nor punctured. The abdominal incision was closed in two layers with 6-0 nylon sutures. After surgery, animals were fluid resuscitated with 40 ml/kg of subcutaneously administered sterile saline and given free access to water but not food. During the surgical procedure, body temperature was maintained at approximately 37°C. Animals were sacrificed by cervical dislocation after 72 h, blood was collected, and kidney samples were harvested and stored at -80°C until further analysis. Liposome preparation and administration Liposomal clodronate (LC) and phosphate-buffered saline liposomes were prepared according to previously described methods (14). Rats received an intraperitoneal bolus of 0.12-0.2 ml/20-25 g body weight of clodronate-encapsulated liposomes or phosphate-buffered saline liposomes (Formumax, USA) 48 h prior to sepsis-induced AKI, as previously reported. Blood chemistry assay After blood collection, the concentrations of blood urea nitrogen (BUN) and serum creatinine (Scr) were immediately analyzed using a Roche Diagnostic analyzer (Roche, Indianapolis, IN). The Scr content was determined using a creatinine (serum) colorimetric assay kit (Cayman Chem, Ann Arbor, MI).
Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.
Renal histology analysis Tissues were fixed with 10% formalin and embedded in paraffin. Four-micrometer sections were stained with periodic acid-Schiff (PAS) reagent. Histological changes in the cortex and the outer stripe of the outer medulla (OSOM) were assessed by quantitative measurements of tissue damage. As tubular damage was mainly vacuolization, the damage was defined as tubular vacuolar degeneration. The degree of kidney damage was estimated in 200X magnification images of more than 100 randomly selected tubules for each animal using the following criteria: 0, normal; 1, area of damage
