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 proinflammatory mediators, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α), and increase their production of reactive oxygen and nitrogen 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 sepsis-induced 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 sepsis-induced 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
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-h light/12-h 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.
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
LC and phosphate-buffered saline liposomes were prepared according to previously described methods (13). Rats received an intraperitoneal bolus of 0.12 to 0.2 mL/20 to 25 g body weight of clodronate-encapsulated liposomes or phosphate-buffered saline liposomes (Formumax, Palo Alto, Calif) 48 h before 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, Ind). The Scr content was determined using a creatinine (serum) colorimetric assay kit (Cayman Chem, Ann Arbor, Mich).
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 200× magnification images of more than 100 randomly selected tubules for each animal using the following criteria: 0, normal; 1, area of damage less than 25% of tubules; 2, damage in 25% to 50% of tubules; 3, damage in 50% to 75% of tubules; and 4, 75% to 100% of tubules were affected. The histological analysis was performed by two pathologists graded sections in a blind fashion to avoid bias.
RNA was isolated from snap-frozen kidneys stored at 80°C using standard procedures (RNeasy, QIAGEN). RNA was treated with DNase1 (Invitrogen), and reverse transcription was performed using 0.5 μg of total RNA (iScript, Bio-Rad). PCR was performed on 1/20 of the RT product using the following primer pairs: iNOS forward 5′-GAATTCCCAGCTCATCCGGT-3′ and reverse, 5′-GGTGCCCATGTACCAACCGGT-3′; Arg-1 forward, 5′-CCGCAGCATTAAGGAAAGC-3′ and reverse, 5′-CCCGTGGTCTCTCACATTG-3′; FIZZ1 forward, 5′-CTATCCCTCCACTGTAACGAAG-3′ and reverse, 5′-AGTAGTCCAGTCAACGAGTAAG-3′; and β-actin forward, 5′-CAGTAACAGTCCGCCTAGAA-3′ and reverse, 5′-GATTACTGCTCTGGCTCCTA-3′. The cycling conditions were a melting temperature (Tm) of 55°C for 30 cycles, and PCR products were visualized with ethidium bromide staining after separation on 2% agarose gels and photographed.
Renal tissue sections were subjected to immunohistochemical staining for F4/80 and Fi67. For immunohistochemical staining, 3-mm renal sections were deparaffinized and rehydrated in a graded alcohol series. The sections were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity and then incubated in buffered normal horse serum to block nonspecific binding. Before immunochemistry, sections were subjected to antigen retrieval by immersion in 0.1 mol/L citrate buffer (pH 6.0) for 25 min, followed by heating in an electrical pressure cooker for 5 min. Sections were incubated with rabbit polyclonal anti-F4/80 (1:100; eBioscience, San Diego, Calif) and mouse monoclonal anti-CDFi67 (1:100; Abcam) primary antibodies overnight at 4°C. Control experiments omitted either the primary or secondary antibody. On the next day, sections were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rat anti-mouse secondary antibody (Beijing Zhongshan Biotechnology Co., Beijing, China) for 1 h at room temperature. Then, 3,3-diaminobenzidine tetrahydrochloride (DAB; Beijing Zhongshan Biotechnology Co., Beijing, China) was applied to the slides to develop a brown color. Counterstaining was performed with hematoxylin, and photomicrographs were captured with an Olympus camera.
Western blot analysis
Whole-cell extracts were prepared. Protein samples were resolved on 10% SDS-PAGE gels, transferred onto PVDF membranes, and blocked with 5% skim milk. Subsequently, the primary antibody incubation was performed overnight at 4°C, followed by an incubation with a HRP-conjugated secondary antibody conjugated for 1.5 h. Protein bands were detected using an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, Ill).
The results are presented as the means ± SEM. The Wilcoxon test was used to analyze nonparametric data. After the normal distribution of the data was assessed with the Kolmogorov–Smirnov test, statistical comparisons between experimental groups were evaluated using Student t test and one-way ANOVA with SPSS 20.0 software (SPSS Inc., Chicago, Ill); a P value <0.05 was considered significant.
M2 differentiation after sepsis-induced AKI
The expression of the Arg-1 and FIZZ1 mRNAs was increased in M2 macrophages. However, the expression of the iNOS mRNA expression was higher in M1 macrophages. We subjected rat renal tissue sections to real-time PCR analysis to determine the expression of markers of M1/M2 macrophages at different time points in rats submitted to CLP surgery (Fig. 1A). High levels of iNOS mRNA expression were detected 24 h postinjury, followed by a subsequent decrease (Fig. 1B). FIZZ1 (Fig. 1C) and Arg-1 (Fig. 1D) mRNA expression remained low in the sham-operated group. However, their levels significantly increased at 48 h. Rat peritoneal macrophages underwent M2 differentiation at 48 h, and the expression of M2 macrophage markers was obvious at 72 h after sepsis-induced AKI.
Macrophage infiltration during sepsis-induced renal injury
Renal cortical tissue sections were stained with a specific antibody for the rat macrophage marker F4/80 to assess the degree of macrophage infiltration during renal injury after the CLP procedure (Fig. 2). Sham-operated rats exhibited minimal interstitial macrophage staining. Rats that were subjected to CLP surgery exhibited significant macrophage accumulation after 72 h. The degree of sepsis-induced macrophage infiltration was significantly reduced in the presence of LC; thus, macrophages were effectively depleted in this model.
M2 macrophage depletion increased renal damage and dysfunction during sepsis-induced renal injury
Histological examinations (Fig. 3A) and tubular damage scoring (Fig. 3B) were performed on samples 72 h after sepsis-induced renal injury. Widespread damage was noted in the form of a dilated, flattened, and swollen epithelium and the loss of proximal tubular epithelial cells, along with luminal cast formation. The damage was more severe in the LC+CLP group than in the CLP group and the control (Ctl) group. The LC+CLP group exhibited a significant increase in BUN (Fig. 3C) and Scr (Fig. 3D) levels 72 h postinjury compared with the CLP and Ctl groups. Based on these data, M2 macrophages reduced renal tubular damage and renal dysfunction after sepsis-induced renal injury.
M2 macrophage depletion inhibited tubular cell proliferation
We subjected tissue sections to an immunohistochemical analysis to examine Ki67 expression in kidneys 72 h after sepsis-induced renal injury (Fig. 4). Ki67 was expressed at low levels in sham-operated animals. However, it was readily detected in the CLP group and the Ctl group compared with the LC+CLP group. Thus, M2 macrophage depletion inhibited tubular cell proliferation.
M2 macrophage depletion downregulated IL-10 expression
Because the highest total number of M2 macrophages and M1 macrophage depletion were observed on day 3, we subsequently studied IL-10 expression at this time point. Forty-eight hours before the onset of CLP, rats were treated with LC. Western blots of whole kidney homogenates showed a marked increase in the total IL-10 protein concentrations in the CLP group and the Ctl group compared with those in the LC+CLP group (Fig. 5).
M2 macrophage depletion increased TNF-α secretion during sepsis-induced AKI
We subsequently studied TNF-α expression at this time point. Forty-eight hours before the onset of CLP, rats were treated with LC. The LC+CLP group and the Ctl group exhibited a significant downregulation of TNF-α expression compared with the LC+CLP group (Fig. 6).
Sepsis is frequently implicated in the development and worsening of AKI. The incidence of AKI and the mortality rate in patients with AKI remain high, despite improvements in renal replacement techniques (1, 14). As key components of the innate immune response, macrophages play an important role in the initiation or progression of inflammatory disease (15). In recent years, polarized macrophages have attracted increasing attention, with the aim of altering disease outcomes (16, 17). Recent studies have reported a beneficial role for macrophages, depending on their phenotype and the location and nature of the injury. Inflammation is closely related to macrophage activation: M1 macrophages exert proinflammatory activities, whereas M2 macrophages are involved in resolving inflammation (18–20). According to the potential mechanisms, M2 macrophages play important roles in the secretion of anti-inflammatory mediators (e.g., IL-4 and IL-10) and tissue repair and remodeling in the course of viral myocarditis and acute nephritis (21, 22). However, few studies have examined the role of M2 macrophages in sepsis-induced AKI.
LC is a bisphosphonate encapsulated by liposomes that is known to induce macrophage depletion in vivo. We sought to determine which kind of macrophage, M1 or M2, was predominantly present in renal tissue sections from rats with sepsis at various time points. M2 macrophages were obviously detected at 72 h after sepsis-induced AKI, and M1 macrophages were significantly decreased. In this study, rats received intraperitoneal LC injection 48 h after sepsis-induced AKI. Immunohistochemistry revealed a significant decrease in macrophage infiltration at 72 h after sepsis-induced AKI. Furthermore, LC depleted M2 macrophages, but not M1 macrophages, in our study.
The histopathological changes in AKI vary, depending on the cause. More severe damage was noted in response to M2 macrophage depletion in our study, resulting in tubular dilation and necrosis and apoptotic cell death, suggesting that rats in which M2 macrophages had been depleted were more susceptible to sepsis-induced AKI. Furthermore, kidney injury (histopathological score) showed positive correlations with the increases in the Scr and BUN levels in the CLP-induced septic rats.
Tubular cell damage of the kidney is another important mechanism in the pathogenesis of AKI. Ki67 is an antigen expressed in proliferating cells that is closely related to mitosis and is required for cell proliferation (23). The immunohistochemical analysis showed increased Ki67 expression in the kidneys of the CLP rat compared with those in the LC+CLP rat and revealed the protective effect of M2 macrophages on renal cells in sepsis-induced AKI. These findings were also consistent with a previous observation that M2 macrophages exerted a protective effect by promoting repair.
TNF-α is a potent proinflammatory mediator secreted by activated macrophages that exerts a variety of biological effects, such as cell differentiation, proliferation, and multiple proinflammatory effects. TNF-α plays a very important role in the early phase of sepsis by activating macrophages to differentiate into the M1 phenotype, thereby contributing to the progression of sepsis (24). In the present study, we observed increased levels of TNF-α in the kidneys of CLP rats, with more pronounced expression in the M2 macrophage-depleted rats. Based on these results, the protective effects of M2 macrophages on the repair of sepsis-induced AKI may be related to the reduction of TNF-α levels.
The potential renoprotective mechanism of IL-10 is likely complex, as IL-10 has multiple actions. IL-10 is a pluripotent cytokine produced by many activated immune cell types, including T helper (Th2) cells, B cells, macrophages, monocytes, and keratinocytes. IL-10 has been shown to suppress the production of proinflammatory mediators and to downregulate costimulatory molecules that are required for T-cell activation, inhibiting macrophage activation (25, 26). M2 macrophage depletion decreased IL-10 expression in our study, which was also increased in the CLP and Ctl groups. Thus, IL-10 secretion may have promoted tissue repair by M2 macrophages during the later stages of AKI in septic rats. The imbalance of proinflammatory and antiinflammatory cytokines may contribute to deterioration of sepsis and sepsis-induced AKI. Only two inflammatory cytokines were assessed in this study; therefore, analyses of additional cytokines and the balance between proinflammatory and anti-inflammatory cytokines may explain the mechanisms by which M2 macrophages modulate immune homeostasis in rats with sepsis-induced AKI in future studies.
With the advent of cell therapy, the repair of renal damage has been induced by injecting specific cytokines to stimulate the initial monocytes to enter the blood circulation. According to Heather M, an injection of IL-10-transduced macrophages into the renal artery of rats with nephrotoxic nephritis (NTN) reduced the levels of both biochemical and histological markers of glomerular inflammation (27). Our findings also allowed us to identify possible new links between the protective roles of M2 macrophages and IL-10 secretion. We hypothesized that IL-10 stimulated macrophage activation and recruitment via an autocrine positive feedback loop that also promoted IL-10 secretion. Although our findings are reliable, the related experiments have not yet been validated in human studies. The activation and regulation of M2 macrophages will be new regulatory targets for the treatment of sepsis-induced AKI in future studies.
Based on our findings, M2 macrophages play a protective role in sepsis-induced AKI, presumably by upregulating IL-10 expression and suppressing TNF-α secretion. Therefore, strategies that limit early macrophage infiltration or activation may represent a novel approach in the prevention or treatment of AKI in septic patients. However, the signaling pathway involved in the repair mechanism of M2 macrophages needs further investigation. We hope that our study will contribute to a better understanding of the complex events involved in renal sepsis-induced AKI, which is key for the development of more effective therapeutic strategies.
1. Schrier RW, Wang W. Acute renal failure and sepsis
. N Engl J Med
351: 159–169, 2004.
2. Silvester W, Bellomo R, Cole L. Epidemiology, management, and outcome of severe acute renal failure of critical illness in Australia. Crit Care Med
29: 1910–1915, 2001.
3. Wan L, Bagshaw SM, Langenberg C, Saotome T, May C, Bellomo R. Pathophysiology of septic acute kidney injury
: what do we really know? Crit Care Med
36 (4 Suppl.):S198–S203, 2008.
4. Jo SK, Sung SA, Cho WY, Go KJ, Kim HK. Macrophages contribute to the initiation of ischaemic acute renal failure in rats. Nephrol Dial Transplant
21: 1231–1239, 2006.
5. Benigni A, Morigi M, Remuzzi G. Kidney regeneration. Lancet
375: 1310–1317, 2010.
6. Humphreys BD, Valerius MT, Kobayashi A, Mugford JW, Soeung S, Duffield JS, McMahon AP, Bonventre JV. Intrinsic epithelial cells repair
the kidney after injury. Cell Stem Cell
2: 284–291, 2008.
7. Lee S, Huen S, Nishio H, Nishio S, Lee HK, Choi BS, Ruhrberg C, Cantley LG. Distinct macrophage phenotypes contribute to kidney injury and repair
. J Am Soc Nephrol
22: 317–326, 2011.
8. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest
122: 787–795, 2012.
9. Zhang XL, Guo YF, Song ZX, Zhou M. Vitamin D prevents podocyte injury via regulation of macrophage M1/M2 phenotype in diabetic nephropathy rats. Endocrinology
155: 4939–4950, 2014.
10. Meera G, Nair, Yurong Du, Jacqueline G. Alternatively activated macrophage-derived RELM-a is a negative regulator of type 2 inflammation in the lung. J Exp Med
206: 937–952, 2009.
11. Heo KS, Cushman HJ, Akaike M, Woo CH, Wang X, Qiu X, Fujiwara K, Abe J. ERK5 activation in macrophages promotes efferocytosis and inhibits atherosclerosis. Circulation
130: 180–191, 2014.
12. Van Rooijen N, Sanders A. Elimination, blocking, and activation of macrophages: three of a kind? J Leukoc Biol
62: 702–709, 1997.
13. Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods
174: 83–93, 1994.
14. Zarjou A, Agarwal A. Sepsis
and acute kidney injury
. J Am Soc Nephrol
22: 999–1006, 2011.
15. Scatizzi JC, Mavers M, Hutcheson J, Young B, Shi B, Pope RM, Ruderman EM, Samways DS, Corbett JA, Egan TM, et al. The CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages. Eur J Immunol
39: 820–825, 2009.
16. Zhang H, Wang X, Shen Z, Xu J, Qin J, Sun Y. Infiltration of diametrically polarized macrophages predicts overall survival of patients with gastric cancer after surgical resection. Gastric Cancer
18: 740–750, 2015.
17. Guoping Zheng, Menghua Ge, Guanguan Qiu, Qiang Shu, Jianguo Xu. Mesenchymal stromal cells affect disease outcomes via macrophage polarization. Stem Cells Int
18. Geng Y, Zhang L, Fu B, Zhang J, Hong Q, Hu J, Li D, Luo C, Cui S, Zhu F, et al. Mesenchymal stem cells ameliorate rhabdomyolysis-induced acute kidney injury
via the activation of M2 macrophages
. Stem Cell Res Ther
19. Lundie RJ, Webb LM, Marley AK, Phythian-Adams AT, Cook PC, Jackson-Jones LH, Brown S, Maizels RM, Boon L, O’Keeffe M, et al. A central role for hepatic conventional dendritic cells in supporting Th2 responses during helminth infection. Immunol Cell Biol
94: 400–410, 2016.
20. Gordon S, Plüddemann A, Martinez Estrada F. Macrophage heterogeneity in tissues: phenotypic diversity and functions. Immunol Rev
262: 35–36, 2014.
21. Wang Y, Wang YP, Zheng G, Lee VW, Ouyang L, Chang DH, Mahajan D, Coombs J, Wang YM, Alexander SI, et al. Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease. Kidney Int
72: 290–299, 2007.
22. Li K, Xu W, Guo Q, Jiang Z, Wang P, Yue Y, Xiong S. Differential macrophage polarization in male and female BALB/c mice infected with coxsackievirus B3 defines susceptibility to viral myocarditis. Circ Res
105: 353–354, 2009.
23. Berghoff AS1, Ilhan-Mutlu A, Wöhrer A, Hackl M, Widhalm G, Hainfellner JA, Dieckmann K, Melchardt T, Dome B, Heinzl H, et al. Prognostic significance of Ki67 proliferation index, HIF1 alpha index and microvascular density in patients with non-small cell lung cancer brain metastases. Strahlenther Onkol
190: 676–685, 2014.
24. Wu X, Xu W, Feng X, He Y, Liu X, Gao Y, Yang S, Shao Z, Yang C, Ye Z. TNF-a mediated inflammatory macrophage polarization contributes to the pathogenesis ofsteroid-induced osteonecrosis in mice. Int J Immunopathol Pharmacol
28: 351–361, 2015.
25. Mu W, Ouyang X, Agarwal A, Zhang L, Long DA, Cruz PE, Roncal CA, Glushakova OY, Chiodo VA, Atkinson MA, et al. IL-10 suppresses chemokines, inflammation, and fibrosis in a model of chronic renal disease. J Am Soc Nephrol
16: 3651–3660, 2005.
26. Ostmann A, Paust HJ, Panzer U, Wegscheid C, Kapffer S, Huber S, Flavell RA, Erhardt A, Tiegs G. Regulatory T cell derived IL-10 ameliorates crescentic GN. J Am Soc Nephrol
24: 930–942, 2013.
27. Lin SL, Li B, Rao S, Yeo EJ, Hudson TE, Nowlin BT, Pei H, Chen L, Zheng JJ, Carroll TJ, et al. Macrophage Wnt7b is critical for kidney repair
and regeneration. Proc Natl Acad Sci USA
107: 4194–4199, 2010.