Macrophages in Kidney Injury, Inflammation, and Fibrosis Qi Cao, David C. H. Harris and Yiping Wang

Physiology 30:183-194, 2015. doi:10.1152/physiol.00046.2014 You might find this additional info useful... This article cites 140 articles, 51 of which can be accessed free at: /content/30/3/183.full.html#ref-list-1 This article has been cited by 1 other HighWire hosted articles Transforming Medicine Through Physiology Gary Sieck Physiology, May , 2015; 30 (3): 173-174. [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: /content/30/3/183.full.html Additional material and information about Physiology can be found at: http://www.the-aps.org/publications/physiol

Physiology (formerly published as News in Physiological Science) publishes brief review articles on major physiological developments. It is published bimonthly in January, March, May, July, September, and November by the American Physiological Society, 9650 Rockville Pike, Bethesda, MD 20814-3991. Copyright © 2015 by the American Physiological Society. ISSN: 1548-9213, ESSN: 1548-9221. Visit our website at http://www.the-aps.org/.

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PHYSIOLOGY 30: 183–194, 2015; doi:10.1152/physiol.00046.2014

Macrophages in Kidney Injury, Inflammation, and Fibrosis Macrophages are found in normal kidney and in increased numbers in dis-

Qi Cao, David C. H. Harris, and Yiping Wang Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney, Sydney, New South Wales, Australia [email protected]

eased kidney, where they act as key players in renal injury, inflammation, and fibrosis. Macrophages are highly heterogeneous cells and exhibit distinct phenotypic and functional characteristics in response to various stimuli in the local microenvironment in different types of kidney disease. In kidney tissue necrosis and/or infection, damage- and/or pathogen-associated molecular patterns induce pro-inflammatory macrophages, which contribute to further tissue injury, inflammation, and subsequent fibrosis. Apoptotic cells and antiinflammatory factors in post-inflammatory tissues induced anti-inflammatory macrophages, which can mediate kidney repair and regeneration. This review summarizes the role of macrophages with different phenotypes in kidney injury, inflammation, and fibrosis in various acute and chronic kidney diseases. Understanding alterations of kidney microenvironment and the factors that Downloaded from on May 20, 2015

control the phenotype and functions of macrophages may offer an avenue for the development of new cellular and cytokine/growth factor-based therapies as alternative treatment options for patients with kidney disease.

Macrophage Heterogeneity and Phenotypes Macrophages belong to the family of mononuclear phagocytes and are considered to originate from a common myeloid progenitor in the bone marrow; however, recent studies have demonstrated that macrophages can be self-renewing embryo-derived populations referred to as tissue-resident macrophages (1, 42, 47). Macrophages perform a wide range of critical roles in homeostasis, surveillance, immune response, and tissue injury and repair (41, 44). Macrophages are present at an early time point of kidney development, and addition of CSF-1 improves development of branch tips and nephrons, suggesting a trophic role of macrophages in embryonic kidney development (102). They are an essential component of innate immunity and also generate adaptive immune responses by recruiting other immune cells such as lymphocytes. Macrophages are highly heterogeneous cells that are divided into subpopulations based on their distinct functions and anatomical location, including as examples Langerhans cells, Kupffer cells, microglial cells, and osteoclasts (101). These tissue-specific macrophage subpopulations can change their phenotype and function in response to local microenvironmental signals during tissue infection or injury (94). 1548-9213/15 ©2015 Int. Union Physiol. Sci./Am. Physiol. Soc.

The diversity of macrophage functions has led to several classification systems. Two well defined phenotypes are commonly referred to as classically activated macrophages (M1 macrophages), produced by exposure to LPS or IFN-␥, and alternatively activated macrophages (M2 macrophages), produced by Th2 cytokines such as IL-4 and IL-10 (Table 1) (43). The M1/M2 nomenclature mirrors the T helper 1 (Th1)-Th2 polarization of T cells. M1 macrophages produce a great amount and a great number of pro-inflammatory mediators and mediate antimicrobial defence and antitumour immunity. In contrast, M2 macrophages have anti-inflammatory functions and are involved in parasite containment, wound healing, and fibrosis (94, 114). The alternatively activated macrophages can be subdivided further into at least three subgroups: M2a macrophages induced by IL-4 and/or IL-13, M2b macrophages induced by immune complexes with LPS or IL-1␤, and M2c macrophages induced by IL-10, TGF-␤, or glucocorticoids (88). More recently, macrophages have been classified as classically activated macrophages (M1 macrophages), wound-healing macrophages (also known as M2a), and regulatory macrophages (also known as M2c) on the basis of their fundamental activation and function (92). However, these in vitro classifications of macrophages do not 183

REVIEWS Table 1. Macrophage activation states and functions Phenotype

Stimulation

Surface Markers

Secretion/Expression

Function

M1

IFN-␥, TNF-␣, LPS

MHC-II, CD86

M2a

IL-4, IL-13

Mannose receptor, Scavenger receptor, CD163, Dectin-1

Th1 responses, tumor resistance Th2 responses, tissue repair

M2b

Immune complex ⫹ LPS

MHC-II, CD86

IL-1, IL-6, IL-12, TNF-␣, CCL2, CXCL9, CXCL10, CXCL11, iNOS CCL17, CCL18, Arginase-1, Ym1, FIZZ1, Stabilin 1, IGF1, Factor XIII-A IL-10, CCL1, SPHK1

M2c

IL-10, TGF-␤, Glucocorticoids, Apoptotic cells

Mannose receptor, B7-H4, SLAM (CD150)

IL-10, TGF-␤, CCL16, CCL18, Arginase-1

Immunoregulation, Th2 activation Immunoregulation, tissue remodeling

iNOS, inducible nitric oxide synthase; Ym1, a chitinase-like protein; FIZZ1, a resistin-like protein; IGF1, insulin-like growth factor 1; SPHK1, sphingosine kinase 1; SLAM, signaling lymphocytic activation molecule.

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surrounding microenvironments during injury, inflammation, fibrosis, and repair. Lee et al. found that kidney macrophages expressed pro-inflammatory markers during the initial phase of IRI, whereas macrophages displayed an alternatively activated phenotype during the repair phase (75). When M1 macrophages were adoptively transferred early after injury, they switched to an M2 phenotype within the kidney during the later recovery phase.

The Role of Macrophages in Kidney Injury and Inflammation Pro-Inflammatory Macrophages Contribute to Kidney Injury and Inflammation Acute kidney injury caused by ischemia reperfusion or cytotoxic drugs triggers a prominent infiltrate of neutrophils and natural killer cells within hours of tissue injury (72, 78, 93, 104). Monocytes infiltrate the injured kidney shortly after neutrophils, differentiate into macrophages, and contribute to early tubular injury (3). During this phase, the interstitial microenvironment in kidney tissue is dominated by pathogen-associated molecular patterns (PAMPs) derived from microorganisms as well as by damage-associated molecular patterns (DAMPs) released by necrotic cells (4, 107, 131). During infection, PAMPs activate resident macrophages as well as kidney parenchymal cells through innate pattern-recognition receptors, leading to the secretion of pro-inflammatory cytokines and chemokines that defend against pathogens and also cause nonspecific collateral tissue damage (6, 8). However, PAMPs are mostly absent in sterile kidney injury, and, in the sterile kidney inflammatory macrophage, infiltration is driven mostly by DAMPs (20, 69, 107). For example, biglycan, a small leucine-rich proteoglycan, which is released from kidney resident cells during early stages of IRI, directly activates macrophages through TLR4 and TLR2, which mediate rapid activation of NF-␬B and thereby stimulate the expression of inflammatory cytokines (110). Furthermore, IFN-␥, TNF-␣, and

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necessarily reflect their true phenotypes in vivo. It is generally believed that macrophages represent a spectrum of activated phenotypes rather than discrete stable subpopulations. For example, adipose tissue macrophages can convert themselves from a wound-healing phenotype to a classically activated phenotype during obesity-associated inflammation (84, 85). Macrophages are well recognized for their pathogenic role in kidney inflammation and fibrosis. In human studies, the degree of macrophage infiltration has been shown to correlate with the severity of kidney injury in patients with glomerulonephritis, suggesting their pathogenic role in kidney disease (27, 28, 50, 57, 135). A pathogenic role of macrophages has been demonstrated by depletion or repletion of resident kidney macrophages in different types of experimental kidney diseases (55, 62, 68, 75, 129). For example, depletion of kidney macrophages by liposomal clodronate (LC) significantly improves kidney injury and function in acute ischemia reperfusion injury (IRI) and unilateral ureteral obstruction (UUO) models (62, 68, 75). However, increasing evidence shows that macrophages also play a reparative role during the course of disease (12, 75, 80, 106, 116). Macrophages actively participate in clearance of apoptotic and necrotic cells to resolve injury and in remodeling of matrix to restore tissue in acute and chronic kidney disease (34, 53, 72, 112, 116). The diverse roles of macrophages, from inflammation and injury to tissue repair and remodeling, are not fully understood. Recently, Anders and Ryu proposed four types of in vivo macrophages, defined according to their predominant roles in various phases of kidney disease, namely pro-inflammatory, anti-inflammatory, profibrotic, and fibrolytic macrophages (6). Distinct subsets of macrophages can co-exist in kidney tissue, and particular subsets can dominate at different stages of disease, from the initiation of kidney injury to recovery (FIGURE 1). Phenotypic plasticity of macrophages allows their functional change in response to

REVIEWS granulocyte-macrophage colony-stimulating factor secreted by infiltrating Th1 T cells and natural killer cells promote full activation of the pro-inflammatory tissue macrophage, mirroring what has been referred to as “M1 macrophage activation” in in vitro stimulation with IFN-␥, TNF-␣, and lipopolysaccharide (6, 72). Inflammatory macrophages secrete TNF-␣, IL-1␤, IL-6, IL-23, reactive oxygen species (ROS), and other pro-inflammatory mediators and further amplify intrarenal inflammation and injury in a positive feedback loop

(FIGURE 2), as has been discussed in ischemiareperfusion injury (36, 61, 75), cisplatin nephrotoxicity (18, 103), anti-GBM glomerulonephritis (5, 55), lupus nephritis (7, 97, 98), renal allograft injury (63, 89), and adriamycin nephropathy (15, 129). Pro-inflammatory macrophages also produce metalloproteases (MMPs) to enable their migration through basement membranes and interstitial ECM networks (117). IRI. As mentioned above, macrophages are the predominant infiltrating cells that accumulate in

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FIGURE 1. Phase-dependent macrophage phenotypic change during the progress of kidney diseases The phenotypic switch of macrophages is determined by microenvironment during the course of acute and chronic kidney disease. A: acute kidney injury triggers recruitment of neutrophils and natural-killer (NK) cells within several hours of tissue injury. Inflammatory monocytes infiltrate to the site of tissue injury shortly after neutrophils, where they differentiate into macrophages and are polarized into proinflammatory macrophages (M1) by various inflammatory mediators, such as IFN-␥, that are released from neighboring inflammatory cells, including neutrophils, NK cells, and T effector cells (predominantly Th1/17). The activated M1 macrophages can further exacerbate tissue inflammation and cause substantial tissue damage. Subsequently, Th2 and Tregs are recruited into kidney and regulate immune responses, including macrophage switch to an anti-inflammatory (M2) phenotype following through uptake of apoptotic cells and stimulation by regulatory cytokines. M2 macrophages predominate at this stage and contribute to resolution of inflammation resolution and tissue repair. Fibrosis may occur, depending on the severity of injury and whether pathogenic factors continue to be expressed. B: in chronic kidney disease, M1 macrophages are increased in kidney tissue following neutrophil, NK cells, and Th1/17 infiltration in the early injury and inflammation. Due to progressive injury and persistent inflammation, M1 macrophages persistently surround sites of damaged tissue. Subsequently, small numbers of Th2 cells and Tregs are recruited into kidney to regulated local immune responses. It is likely that antiinflammatory (M2) macrophages coexist in small numbers or are absent due to a persistently inflammatory kidney microenvironment. Persistent inflammatory and fiborotic factors in chronic kidney disease promote renal fibrosis.

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FIGURE 2. Distinct macrophage subsets regulating the balance of renal injury, inflammation, repair, and fibrosis Triggers of kidney injury cause subsequent recruitment of monocytes that differentiate into different macrophage phenotypes in response to the local microenvironment. During the early phase of tissue damage, the kidney interstitial microenvironment is dominated by micro-organism-derived pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), as well as pro-inflammatory cytokines, which promote full activation of the pro-inflammatory M1 macrophage. Pro-inflammatory macrophages produce a large amount of TNF-␣, reactive oxygen species (ROS) and other proinflammatory mediators that amplify inflammation and promote additional injury in a positive feedback loop. Moreover, pro-inflammatory M1 macrophages also induce renal fibrosis by secretion of MMP-9. In contrast, the uptake of apoptotic cells and anti-inflammatory cytokines drive macrophage polarization toward an anti-inflammatory M2 phenotype, which in turn promotes renal repair through secretion of trophic factors such as Wnt7b, heme-oxygenase-1 (HO-1), and chitinase-like protein BRP-39. M1 macrophages may convert to M2 macrophages in response to tissue factors within the kidney during the recovery phase. Anti-inflammatory M2 macrophages also suppress kidney inflammation and injury via secretion of anti-inflammatory cytokines such as IL-10 and TGF-␤. In addition, Galectin-3 and TGF-␤ produced by M2 macrophages promote kidney fibrosis directly. The existence of fibrolytic macrophages has yet to be demonstrated unequivocally in kidney disease. 186

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using LC at the time of initial injury protected against kidney injury in IRI mice, suggesting that macrophages play a critical role in mediating tissue injury (21, 62). However, administration of diphtheria toxin abolished the protective effects of LCmediated macrophage ablation in IRI mice, suggesting that there may be a protective population of mononuclear phagocytes in the kidney in the aftermath of LC treatment (33). In addition, adoptive transfer of in vitro modulated M1 macrophages aggravated kidney injury, indicating their pathogenic role in IRI (75). Cisplatin nephrotoxicity. Cisplatin is a major antineoplastic drug and widely used to treat solid tumors, but it has dose-dependent kidney toxicity characterized by acute tubular necrosis and inflammation. A positive correlation between the number of interstitial macrophages and the degree of kidney injury was identified in cisplatin nephrotoxicity, suggesting that kidney macrophages play a pathogenic role in this situation (65, 76, 103). Cisplatin causes direct necrosis and apoptosis of proximal tubule cells but also induces a series of inflammatory changes that mediate kidney injury. In vitro cisplatin induced enhanced expression of TLRs and their associated signaling molecules in macrophages (121). Cisplatin-treated macrophages were more susceptible to different TLR ligands, such as PAMPs and DAMPs in injured kidney, produced large amounts of nitric oxide and pro-inflammatory cytokines, and demonstrated increased activation of NF-␬B and mitogen-activated protein kinases (MAPK) (18). Ramesh found that inhibition of p38 MAPK activation led to decreased production of TNF-␣ in macrophages and resulted in less kidney injury in cisplatin nephrotoxicity (103). Several other studies found that rosiglitazone (a PPAR␥ agonist) and alpha-lipoic acid (an anti-oxidant) decreased infiltration of interstitial macrophages and reduced kidney dysfunction and tubular injury in cisplatin nephrotoxicity (65, 76). All of these data indicate that macrophages display a pro-inflammatory phenotype and contribute to cisplatin-induced acute kidney injury. Anti-glomerular basement membrane glomerulonephritis. Glomerular macrophages display a pro-inflammatory phenotype and contribute to glomerular damage and inflammation in anti-glomerular basement membrane (GBM) glomerulonephritis. Early studies showed that macrophage accumulation in glomeruli is a direct response to the deposition of antibody in anti-GBM glomerulonephritis, and inhibition of macrophage accumulation by anti-macrophage serum significantly prevented progression of glomerulonephritis, thereby implicating macrophages as mediators of glomerular injury and inflammation (51, 52). Macrophage depletion by clodronate

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the outer medulla of the postischemic kidney. Macrophage accumulation in the injured kidney involves chemokine receptor expression on circulating monocytes (38, 39, 79). C-C motif chemokine receptor 2 (CCR2), the main chemokine receptor for monocyte chemoattractant protein-1 (MCP-1/ CCL2), is expressed on a subset of monocytes. CCL2/CCR2 signaling is critical for monocyte recruitment to the site of inflammation. Recruitment of circulating monocytes into the kidney was significantly reduced 24 h after IRI in CCR2 knockout mice, resulting in less functional tissue and tissue injury, indicating that macrophage infiltration is part of the innate immune response, which contributes to kidney IRI (39, 79). Pro-inflammatory macrophages contribute to the initiation of IRI by secretion of pro-inflammatory cytokines, recruitment of neutrophils, and induction of epithelial cell apoptosis. For example, TNF-␣ has a critical role in mediating the kidney injury. TNF-␣ has an autocrine effect on macrophage activation (134); it induces apoptosis, and it coordinates the activation of a network of cytokines and chemokines in the kidney (25, 108, 113). Therefore, depletion of macrophages before or during the early stages of IRI reduces kidney injury and improves tissue repair. For example, depletion of kidney macrophages

REVIEWS macrophages of type II phenotype (M2b) that express anti-inflammatory molecules IL-10 and osteopontin were identified during disease remission of LN (123). Strikingly, depletion of kidney macrophages by LC suppressed intraglomerular proliferative lesions and abrogated crescent formation in NZB/NZW F1 mice, suggesting that M2b macrophages mediate a dysregulated “tissue repair” program in poly (I:C)-induced LN. Recent studies revealed that kidney F4/80hi macrophages exhibited a unique hybrid activation phenotype with expression of both pro-inflammatory and anti-inflammatory mediators in chronic LN, indicating that the standard M1/M2 paradigm for macrophages is insufficient to explain chronic inflammation in lupus nephritis (9, 109). Adriamycin nephropathy. Adriamycin nephropathy (AN) is a rodent model of chronic kidney disease that mirrors human primary focal segmental glomerulosclerosis. Macrophage infiltration correlated with kidney structural and functional injury in this model, suggesting that the macrophages may have been responsible for glomerular and interstitial injury (77). Reduction of interstitial inflammation and tissue injury in AN mice by blockade of CCR1, CCL2, or CCL5 was associated with a remarkable reduction of macrophage infiltration, suggesting macrophages play a critical role in development of AN (125, 132, 140). Direct evidence for a pathogenic role of macrophages was shown by our group by the protection of macrophages depletion in AN against kidney functional and structural injury (128). Moreover, adoptive transfer of ex vivo activated M1 macrophages or inflammatory macrophages separated from AN kidney exacerbated kidney injury in AN mice (15, 129). These studies also suggest that the novel therapeutic strategies to treat chronic kidney disease should target pro-inflammatory macrophages in vivo. Recently, Wyburn et al. found that targeting IL-18 derived from activated macrophages by a neutralizing binding protein protected against the development of AN, with less interstitial inflammation, tissue injury, and kidney dysfunction (133).

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microspheres resulted in greatly reduced TNF-␣ and IL-1␤ release in anti-GBM glomerulonephritis, suggesting that inflammatory macrophages mediated glomerular damage through release of pro-inflammatory cytokines (24). The role of proinflammatory macrophages was investigated further by using adoptive transfer in anti-GBM glomerulonephritis. Nikolic-Paterson and his colleagues found that adoptive transfer of IFN-␥stimulated pro-inflammatory macrophages directly mediated glomerular cell proliferation and proteinuria in acute anti-GBM nephritis (55). Adoptive transfer of macrophages that were exposed to a specific Jun amino terminal kinase (JNK) inhibitor significantly reduced proteinuria and glomerular cell proliferation in this model (56). Moreover, early or late treatment with the JNK inhibitor improved kidney function and attenuated glomerular and tubulointerstitial damage in the chronic anti-GBM model (35, 86). These studies suggest that the JNK pathway is essential for macrophage-mediated kidney injury in antiGBM glomerulonephritis. A recent study showed that inhibition of the receptor for macrophage colony-stimulating factor eliminated macrophage infiltration and expression of pro-inflammatory molecules in glomeruli, suggesting a pro-inflammatory phenotype of glomerular infiltrated macrophages (46). In addition, the immunomodulatory effects of statins in anti-GBM glomerulonephritis appear to be mediated through downregulation of M1 macrophage-associated cytokines as well as upregulation of the M2 macrophage-associated molecules in glomerular macrophages (37). These studies indicate that targeting glomerular macrophages could be a potential approach to treat glomerulonephritis. Lupus nephritis. Following the deposition of immune complex in the kidney, infiltrating inflammatory cells lead to tissue injury in lupus nephritis (LN). Macrophages that produce pro-inflammatory cytokines and interact with autoreactive T cells are important mediators in the progression of LN (71). Production of IFN-␥ by local macrophages controls macrophage migration to kidney and regulates the development of glomerulonephritis in LN (17). Kidney-infiltrating macrophages exhibit increased expression of OX40L, CD80, and CD86, which are markers of disease onset and remission in LN (111). The pathogenic role of pro-inflammatory macrophages in LN has been revealed by blockade of CCL2 or CCR2 and by depletion of colony stimulating factor-1 (CSF-1) in MRL/lpr mice (70, 91, 99, 122). Increased expression of CSF-1 in tubular epithelial cells has been noted in LN. Deletion of CSF-1 significantly reduced macrophage infiltration with a remarkable reduction of tissue injury in MRL/lpr mice (60, 91). However,

Anti-Inflammatory Macrophages Mediate Kidney Repair and Regeneration Pro-inflammatory macrophages contribute to the initiation and progression of kidney disease by secretion of pro-inflammatory mediators and interaction with kidney resident cells. In contrast, increasing evidence has shown that macrophages also play a reparative role during the recovery phase of disease (most clearly in the ischemia/ reperfusion injury model) (19, 53, 75). Depletion of macrophages during kidney repair is associated with sustained kidney inflammation and injury, and impaired tubular cell proliferation and tissue

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kidney milieu determine the macrophage phenotype within the injured kidney. CSF-1 produced by tubular epithelial cells in IRI mice has been shown to polarize resident macrophages toward an M2 phenotype, which partially contributed to kidney repair and regeneration after IRI (2, 90, 137). In addition, anti-inflammatory macrophages can be induced by apoptotic cell-derived factors. Sola et al. showed that apoptotic cell-derived sphingosine-1-phosphate (S1P) polarized kidney macrophages to a reparative phenotype in the kidney of IRI mice (116). S1P-dependent neutrophil gelatinase-associated lipocalin (NGAL/Lcn-2) produced by these macrophages was identified as a regenerative mediator enhancing tubular epithelial cell proliferation in the repair phase of IRI. The anti-inflammatory macrophage-derived reparative molecules in IRI mice are poorly known. Lin found that macrophage-derived Wnt7b also plays a critical role in promoting kidney regeneration via epithelial cell-cycle progression and basement membrane repair after IRI (80). Reparative macrophages also secrete chitinase-like protein BRP-39, which has been shown to promote regeneration in kidney by limiting tubular apoptosis via activation of phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling (112). Heme-oxygenase-1 (HO-1), a protective and anti-inflammatory enzyme, is upregulated in the kidney in response to IRI. The expression of HO-1 by kidney macrophages has been shown to improve the outcome in kidney IRI, whereas downregulation of HO-1 expression in kidney macrophages of aging mice resulted in an increased susceptibility to kidney IRI (31, 45). Taken together, these studies show that macrophages undergo a switch from a proinflammatory to a trophic phenotype that supports the transition from kidney injury to kidney repair during the course of acute kidney injury. The specific M2 macrophage-dependent pathways or derived molecules that promote kidney repair and regeneration need further investigation. Macrophages modulated ex vivo to display an anti-inflammatory or reparative phenotype have been successfully used as a cell-based therapy in IRI. When administered at the time of I/R injury, IL-10-transfected macrophages trafficked to the post-ischemic kidney and reduced tubular injury and pro-inflammatory cytokine production within the kidney (64). The renoprotection of these IL-10expressing macrophages was dependent on the production of lipocalin-2, which protects against tubular apoptosis and stimulates their proliferation in an iron-dependent pathway. Heme-oxygenase-1 (HO-1) is an anti-inflammatory enzyme that has been shown to be beneficial in various models of kidney injury (10, 66). HO-1-overexpressing macrophages displayed an anti-inflammatory

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repair (67, 75). The mechanisms underlying kidney macrophage phenotypic switch from pro-inflammatory to anti-inflammatory are not well understood. The reduction of DAMPs and PAMPs as well as the increase of apoptotic cells represent a change in the tissue environment that would promote phenotype change of tissue macrophages (40). Apoptotic cells, anti-inflammatory cytokines, as well as growth factors are likely to predominate in the induction of anti-inflammatory macrophages (FIGURE 2). For example, macrophage uptake of apoptotic cells increased production of anti-inflammatory cytokines such as transforming growth factor (TGF)-␤ and IL-10 (6, 26). In addition, regulatory T cells further promote the antiinflammatory macrophage phenotype via release of IL-10 and TGF-␤ and by suppressing effector T cells (81). Steroid-based treatments appear to reduce kidney inflammation and injury by promoting anti-inflammatory macrophages in vivo (58). Together, a microenvironment dominated by cell apoptosis and anti-inflammatory mediators can deactivate pro-inflammatory macrophages and/or directly promote polarization toward reparative and anti-inflammatory macrophages, which in turn contribute to tissue repair and regeneration (FIGURE 2). In contrast to the protective effect of macrophage depletion during early phase of kidney I/R, macrophage depletion during the later recovery phase impedes tissue repair and regeneration. For example, one study revealed that suppression of macrophage recruitment in osteopontin-knockout mice reduces tubulointerstitial fibrosis during the recovery process of IRI (100). Several other studies confirmed that macrophages are involved in the kidney repair after IRI in that macrophage ablation by either LC or diphtheria toxin 48 –72 h after IRI resulted in persistent kidney injury (67, 83, 127). Lee found that depletion of macrophages at the time of IRI (when M1 macrophages are predominant) attenuated kidney injury, whereas depletion during the repair phase (when M2 macrophages are predominant) delayed kidney repair (75). Interestingly, IFN␥-stimulated M1 macrophages injected during the repair phase switched toward an anti-inflammatory M2 phenotype within the kidney. In vitro coculture studies indicate that macrophage phenotypic change was induced by tubular cell-derived factors. These data suggest that kidney macrophages change their phenotype in response to dynamic signals from the local kidney environment. The mechanisms by which macrophages are polarized to an anti-inflammatory phenotype in the post-ischemic kidney are not well understood. It is likely that anti-inflammatory effect of phagocytosis of apoptotic cells and multiple signals in the local

REVIEWS phenotype, with increased phagocytosis of apoptotic cells and increased IL-10 production (32). Adoptive transfer of these genetically modified macrophages preserved kidney function and reduced microvascular platelet deposition in mice with IRI (32). Ranganathan et al. found that netrin-1 induced anti-inflammatory M2 macrophage polarization in vitro through activating peroxisome proliferator-activated receptor gamma (PPAR␥)dependent pathways (104). Adoptive transfer of netrin-1-treated macrophages suppressed inflammation and protected against kidney injury in IRI mice (105). Investigating transcriptional and chromatin-mediated control of macrophage polarization should identify novel targets and lead to the development of future macrophage-directed therapies.

The Role of Macrophages in Kidney Fibrosis

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Kidney fibrosis is a second-line healing program that only occurs if kidney repair is insufficient or consistently suppressed by ongoing tissue injury and inflammation. Traditionally, macrophages have been recognized as key players that contribute to kidney fibrosis. However, increased evidence has suggested an anti-fibrotic role of infiltrating macrophages in obstructive nephropathy. Triggers of kidney cell damage recruit circulating monocytes into interstitial compartments where they differentiate into M1 or M2 macrophages, depending on the local tissue milieu. Pro-inflammatory M1 macrophages release inflammatory mediators, including TNF-␣ and ROS, which cause tissue inflammation and subsequent kidney fibrosis. In contrast, anti-inflammatory M2 macrophages release anti-inflammatory mediators, including IL-10 and TGF-␤; the latter suppresses kidney inflammation yet promotes kidney fibrosis. Unilateral ureteral obstruction (UUO) is a well characterized model for investigating the factors that contribute to kidney fibrosis (23). Systemic macrophage depletion using LC 1 day before UUO decreased tubular cell apoptosis and kidney fibrosis, suggesting that the initial phase of macrophage infiltration may promote subsequent kidney fibrosis (118). Similarly, administration of LC selectively depleted both F4/80⫹ macrophages and F4/80⫹ dendritic cells, but not F4/80⫺ dendritic cells, in mice with UUO, resulting in attenuated tubular apoptosis and kidney fibrosis and decreased levels of the pro-fibrotic cytokine TGF-␤ (68). In addition, kidney fibrosis in murine obstructive nephropathy is attenuated by depletion of monocyte lineage in CD11b-DTR mice, but not dendritic cells in CD11c-DTR mice (87, 115). These findings suggest that F4/80⫹ macrophages/dendritic cells, but not

CD11c⫹ DCs play a pivotal role in the development of kidney fibrosis following ureteral obstruction in mice. The mechanism underlying the pro-fibrotic role of macrophages in UUO has not been fully elucidated. Braga and colleagues found that M2 macrophages contributed to kidney fibrosis of UUO in a MyD88-dependent manner (11). Mediators released by injured tissue can activate infiltrating macrophages through toll-like receptors (TLRs) and MyD88 signaling pathways, which promote kidney fibrosis. These results suggest that targeting innate immune response signaling pathways of macrophages could be a possible therapeutic strategy against kidney fibrosis. Galectin-3 produced by kidney resident macrophages drives myofibroblast accumulation/activation and promotes kidney fibrosis in UUO (49). Our group found that MMP-9 were involved in epithelial mesenchymal transition (EMT) and thereby contributed to kidney fibrosis (120, 138). LPS/IFN-␥-activated M1 macrophages produced a large amount of MMP-9, which increased tubular cell EMT via the ␤-catenin pathway. Tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage of UUO, whereas TECs, macrophages, and myofibroblasts produced MMP-9 during late-stage UUO. Blockade of MMP-2/MMP-9 or MMP-9 alone significantly reduced tubular cell EMT and kidney fibrosis in UUO (119). It is noted that these studies on EMT were performed in vitro, whereas the occurrence of EMT in in vivo renal fibrosis remains subject to argument (54, 59, 74). In contrast, an inverse correlation between the number of interstitial macrophages and the degree of fibrosis has been shown recently in UUO, thereby suggesting there is a subpopulation of infiltrating macrophages with an anti-fibrotic role in the recovery phase of obstructive nephropathy. Nishida and colleagues demonstrated that interstitial macrophages display an anti-fibrotic role at day 14, but not at day 5, after UUO (95). They found that the angiotensin II type 1 receptor (Agtr1) on macrophage functions to attenuate kidney fibrosis in vivo. Their data suggest that angiotensin II affects the quantity and phagocytic activity of macrophages through Agtr1. The antifibrotic role of interstitial macrophages at a later stage (day 14) of UUO was confirmed by using cyclophosphamide-mediated macrophage depletion (96). The mechanisms underlying the antifibrotic role of interstitial macrophages in UUO have been studied recently. López-Guisa et al. demonstrated that mannose receptor 2 (Mrc2)expressing macrophages displayed a fibrosis-attenuating role through activating a lysosomal collagen turnover pathway in UUO (82). They found that Mrc2, a cell surface receptor that binds to and internalizes collagen, was upregulated on 189

REVIEWS macrophages and myofibroblasts in UUO, and that reduced Mrc2 expression significantly worsened kidney fibrosis in Mrc2-deficient mice with UUO. Zhang et al. showed that absence of scavenging receptors on uPAR⫺/⫺ macrophages led to delayed clearance of pro-fibrotic molecules, resulting in kidney fibrosis in late-stage UUO (136). Taken together, current data suggest a phase-dependent balance of profibrotic and antifibrotic effects of macrophages in UUO. Kidney fibrosis could be a consequence of kidney injury and inflammation, which involves macrophage infiltration. Pro-inflammatory (M1) and anti-inflammatory (M2) macrophages will accelerate or reduce kidney injury and inflammation respectively, to impact indirectly or directly on the degree of kidney fibrosis (FIGURE 2). In contrast, macrophages at the later stage of repair may become profibrotic or fibrolytic to respectively induce or resolve kidney fibrosis directly. However, existence of profibrotic and fibrolytic macrophages has yet to be demonstrated unequivocally in vivo CKD.

1. Diversity of Macrophages in Kidney Diseases Kidney macrophages display heterogeneity, which has been defined by different surface markers. We have identified two major subsets of macrophages, which are F4/80⫹CD11c⫺ and F4/80⫹CD11c⫹ cells in murine kidney (15). Even though CD11c has traditionally been considered to be a dendritic cell marker, both subsets showed major characteristics and functions of macrophages. However, they exhibited different distributions within kidney: F4/80⫹CD11c⫺ macrophages were scattered throughout whole kidney, whereas F4/80⫹CD11c⫹ macrophages were only distributed in the cortex but not in the medulla. Moreover, F4/80⫹CD11c⫹ macrophages had stronger phagocytic ability and produced more nitric oxide and IL-10 than did F4/80⫹CD11c⫺ macrophages (15). These results suggest that these macrophages may have sitespecific functions or differing potency for protective or destructive function in kidney diseases. The possible existence and importance of site-specific macrophages is not clear. For example, the functions of glomerular macrophages may differ from those of interstitial macrophages. Recent studies have emphasized the importance of tissue-resident macrophages and demonstrated that they selfmaintain locally without need for input from circulating monocytes (1, 47). It is important to define the role of tissue-resident macrophages in kidney, and differences between tissue-resident and monocyte-derived macrophages in kidney immunity, homeostasis, and repair. 190

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2. Transcriptional Control of Macrophages in Kidney Disease Phenotypic changes and functions of macrophages are dependent on microenvironments in disease conditions and are regulated by the signaling pathways of various genes. Transcription factors including JNK, MAPK, and NF-␬B have been demonstrated to be involved in defining the M1 phenotype. For the M2 phenotype, others including C/EBP␤, PPAR␥, IRF, and the STAT family have been reported to be important (73). However, the core genes that regulate macrophage phenotype and function are still unclear. Furthermore, specific genes important in regulating possible fibrotic and fibrolytic macrophages have not been defined. The methods to target specific genes of macrophages have been made possible by LysMCre mice; for example, deletion of IL-4Ra by LysMCre reveals distinct subsets of M2 macrophages controlling inflammation and fibrosis in chronic schistosomiasis (22, 124). The same strategy can be used to define transcriptional control elements in kidney macrophages.

3. Macrophages in Kidney Repair and Regeneration Macrophages have been shown to be important in would-healing processes, especially tissue repair and regeneration. Monocytes/macrophages are able to fuse with other cells for tissue regeneration and also to transform into other cell types, including neuronal, endothelial, and muscle cells (48, 126, 139). Furthermore, macrophages are able to secrete exosomes to aid recovery of injured cells (30). Macrophages are involved in the formation of the niches of stem cells and progenitor cells; without macrophage help, stem cells or progenitor cells are not able to proliferate and differentiate in

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Challenges And Future Directions

Kidney macrophages display phenotypic heterogeneity in kidney disease. M1 macrophages have been demonstrated in all types of kidney disease (6). However, M2 macrophages have been shown to exist in acute kidney injury such as ischemic kidney but not in most chronic kidney diseases (14). Also M1/M2 phenotypes do not fully mirror macrophage phenotypes in vivo. A newly suggested classification may more closely reflect phenotypes of in vivo macrophages; in this classification, macrophages have been defined as proinflammatory, anti-inflammatory, fibrotic, and fibrolytic (6). However, these proposed phenotypes need to be defined in various kidney disease models. More importantly, the applicability of these classification systems, namely the M1/M2 paradigm or the newer system four macrophage phenotypes, needs to be examined in human kidney disease.

REVIEWS injured tissues (29). However, it is unknown whether macrophages mainly promote kidney cell regeneration directly through cell fusion, through transformation, or via exosomes, or indirectly by helping stem cells and progenitor proliferation and differentiation. These questions need further investigation.

4. Therapeutic Potential of Macrophages

No conflicts of interest, financial or otherwise, are declared by the author(s). Author contributions: Q.C. and Y.W. conception and design of research; Q.C. performed experiments; Q.C. prepared figures; Q.C. drafted manuscript; D.C.H. and Y.W. edited and revised manuscript; D.C.H. and Y.W. approved final version of manuscript.

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