Download Macrophages in Kidney Injury and Repair

Acta Nephrologica 26(2): 45-57, 2012
DOI: 10.6221/AN.2012009
45
Mini Review
Macrophages in Kidney Injury and Repair
Shuei-Liong Lin 1, 2 and Jeremy S. Duffield3
1
Renal Division, Department of Medicine, National Taiwan University Hospital
Graduate Institute of Physiology, College of Medicine, National Taiwan University
Taipei, Taiwan, Republic of China
3
Renal Division & Center for Lung Biology, Department of Medicine, & Institute of Stem Cell &
Regenerative Medicine, University of Washington, Seattle, WA, USA
2
Macrophages: Tissue Effector Cells of the
Monocyte Lineage
severities (28-32).
Activation of Macrophages
Macrophages (Mφs) are monocyte-derived tissue
effector cells from hematopoietic stem cells in bone
marrow via circulation. Their expansion in tissues
can either be due to local proliferation of resident
cells or infiltration from the circulation, depending
on the stimulus (1-3). Beyond their traditional role in
protecting the host from invading pathogens, Mφs
have been shown to play expansive roles as regulators
of development, tissue homeostasis, remodeling, and
repair (4). Although the key role of Mφs in kidney
repair after injury is gaining considerable appreciation,
along with neutrophils, Mφs have a reputation of
being leukocytes which drive tissue injury and fibrosis
in immunological and chronic inflammatory kidney
diseases (5-8). During disease, Mφs expand even in
the absence of pathogens, and disperse after repair or
recovery (8). However, a large population of Mφs
persists after expansion in chronic progressive kidney
disease (3, 9). In many human kidney biopsy studies,
Mφs have been found in large numbers in both acute
diseases such as post-streptococcal glomerulonephritis
(GN), anti-neutrophil cytoplasmic antibody (ANCA)
associated GN, or in chronic diseases such as IgA
nephropathy or lupus nephritis, and also in acute and
chronic kidney transplant disease (10-17). Many
studies have shown that the tissue of Mφs are in fact
not merely passive bystander cells, but are activated
and involved in the pathogenesis of kidney diseases.
These observations hold true not only in immunological kidney diseases such as GN but also in nonimmunological diseases, such as diabetic nephropathy,
ischemic/vascular kidney disease, and all forms of
chronic kidney disease (18-27). In all of these diseases,
the cell numbers and activation status of Mφs have
been reported to correlate positively with disease
Activation of Mφs is a key element of initiating
an efficient immune response or participating the
tissue repair and regeneration. A wide range of
stimuli such as conserved microbial structures, socalled pathogen-associated molecular patterns
(PAMPs), have been shown to activate Mφs through
a limited number of pattern recognition receptors
(PRRs) which include toll-like receptors (33-36).
These PAMPs include natural ligands on microbes,
such as lipopolysaccharide (LPS), peptidoglycans as
well as nonessential proteins such as flagellin. In
addition to the natural ligands, several PRRs interact
with endogenous, host-derived ligands including
modified protein, carbohydrate, lipid, nucleic acids,
nuclear proteins and mitochondrial proteins broadly
known as dangers associated molecular patterns
(DAMPs) (37-42). Immune complexes (ICs) (comprising immunoglobulins, antigens, complement components, pentraxins and other plasma proteins of the
innate immune system) frequently deposit in the
glomerulus and ligate activating immunoglobulin Fc
receptors (FcRs) to complement receptors (CRs); they
also have the capacity to activate Mφs with broadly
similar activation and pattern of cytokine release
(43). Release of DAMPs may be central to regulating
the responses of Mφs to tissue injury. By activating
intracellular signaling pathways that include nuclear
factor κB and mitogen-activated protein kinase, Mφs
spew out a broad range of pro-inflammatory cytokines
including tumor necrosis factor α (TNFα), interleukin
(IL)-1β, IL-6, IL-12, IL-18, IL-23 and pro-inflammatory chemokines including macrophage inflammatory protein (MIP), monocyte chemotactic protein
and chemokine (C-X-C motif) ligand 1 (CXCL1,
Corresponding author: Dr. Shuei-Liong Lin, Graduate Institute of Physiology, College of Medicine, National Taiwan University, No. 1,
Jen-Ai Rd., Sec. 1, Taipei 10051, Taiwan, R.O.C. Tel: +886-2-23123456 ext. 88235, Fax: +886-2-23964350, E-mail: [email protected]
Received: March 13, 2012; Revised: April 9, 2012; Accepted: May 9, 2012.
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Lin and Duffield
neutrophil chemoattractant). Mφs can also generate
reactive nitrogen and oxygen species, including nitric
oxide. This pattern of activation is broadly known as
classical or M1 activation and can be detected by cell
surface upregulation of TREM-1, Ly6C, FcγRI or
expression of inducible nitric oxide synthase (iNOS)
or IL-1β proteins (44, 45). By contrast, certain pathogens, such as amoebae and schistosomes, activate
Mφs in a different manner. In response to these
pathogens, Mφs generate high levels of transforming
growth factor β (TGFβ), IL-10, IL-13 and chemokines
including chemokine (C-C motif) ligand 17 (CCL17)
and CCL22. This alternative pattern of activation
is known as M2 activation and has been associated
in vivo with wound healing. In the presence of a cell
surface ED3 antigen (CD163) in rats or the cell surface markers galectin 3 (Mac2), scavenger receptors
including the mannose receptor, MARCO, and the
transferrin receptor in mice have been implicated as
markers of M2 type (wound healing) Mφs in tissues.
Certain DAMPs, such as adenosine, have now been
reported to drive Mφ activation towards an M2
phenotype and such polarized Mφs promote woundhealing functions. However, most DAMPs broadly
identified activate Mφs in a M1-type activation and it
is likely that additional signals at the Mφ cell surface
are important in determining cell phonetype.
Importantly, it is worth stating that these imposed
classifications of Mφ activation states are an oversimplification and may not hold up across different
organs and disease settings. In any event, the capacity
of Mφs to engage in and perform injurious or woundhealing functions probably depends on specific and
efficient activation.
In addition to the production of cytokines and
chemokines, a major role for Mφs is their capacity for
phagocytosis (46). In addition to invading microbes,
Mφs scavenge aged erythrocytes, dying leukocytes,
cellular debris, pathological matrix, and ICs (47). In
all these circumstances, phagocytic clearance can
occur without cellular activation and in the context of
kidney disease would be beneficial to tissue remodeling and regeneration following injury (48-51). In
fact, when healthy, most monocytes do not become
significantly activated but rather clear things away
(erythrocytes, neutrophils, ICs), thus, it is likely that
the major normal function of monocytes/Mφs is a
wound-healing non-inflammatory role, and it is only
under overwhelming circumstances that cellular
activation occurs and pro-injurious factors are
liberated. In this context it is very striking that in
models of single kidney injury and repair such as the
ischemia reperfusion model (a model of human acute
tubular necrosis (ATN)), inflammatory Mφs are
recruited during the repair phase and these Mφs
correlate numerically with repair (7, 8). One collec-
tive interpretation of these observations is that the
physiological role of Mφs is one of an attempt to
initially sterilize and debride a tissue, followed by
repair of the tissue (52, 53). However, in repetitive
or chronic injury states, the Mφs are driven to become excessively or aberrantly activated with deleterious effects.
How Macrophages Activated
in Injured Kidney?
Since Mφs are present in diverse kidney diseases,
there has been debate about how Mφs are activated
(Fig. 1) (3, 54-57). Several studies in mouse models
of GN (nephrotoxic nephritis (NTN) and the antiglomerular basement membrane (anti-GBM) disease)
have indicated that Mφs are secondary effectors
regulated by CD4 + T lymphocytes reactive against
foreign antibodies planted in the glomerulus (54). It
is likely that this type of T lymphocyte-directed
activation of Mφs takes place through paracrine
signaling by interferon γ (IFNγ), IL-12, IL-17, and
other Th1- or Th17-skewed lymphokines. While this
mechanism of Mφ activation is likely important in
certain contexts in human glomerular diseases, it is
unlikely that this is a major mechanism of Mφ activation in the kidney because there is relatively little
evidence of cell-mediated autoimmunity against the
kidney itself in diseases such as ANCA associated
GN, and lupus nephritis. The exception to this is antiGBM and Goodpasture’s syndrome (58-60). Many
‘immunological diseases’ of the kidney feature
deposition of ICs in the glomerulus. In these diseases,
the glomerulus in large part is a bystander, rather than
a target, of autoimmunity. ICs become trapped in
the glomerulus by virtue of its highly specialized
vasculature and sieving function. It is likely that the
major physiological function of neutrophils and
monocytes/Mφs in the glomerulus is safe, ‘nonphlogistic’, phagocytic clearance of formed ICs from
the glomerulus (61, 62). This innate immune function
is complex in that it involves many innate immune
proteins, receptors and regulated cell signaling
pathways, with many systems in place to prevent
myeloid leukocyte activation triggered by this
cleaning-up process, including complementing
proteins and pentraxins (47, 63). However this ‘nonphlogistic’ clearance of ICs is readily overwhelmed
with resultant consumption of plasma complement,
inappropriate myeloid leukocyte activation, and
liberation of local cytotoxic products and proinflammatory chemokines which contribute to local
tissue injury, activation of the coagulation cascade,
and recruitment of further leukocytes with consequent
loss of glomerular function (9, 54, 64-66). The consequence of this is that the net effect of the monocyte/
Macrophages in Kidney Injury and Repair
Mφ function is deleterious in several models of GN
(9, 66, 67). The activating Fcγ receptors (and FcαR)
and late complement components (including C5a)
have been strongly implicated in this activation process
(61, 68-72). In addition to IC GN, increasing understanding of the mechanisms of ANCA associated GN
presenting as rapidly progressive GN also suggest
that local activation of neutrophils and monocytes/
Mφs in glomerular capillaries by ANCA ICs on the
endothelial surface is an important part of the pathogenesis of glomerular injury in these diseases (73).
Therefore similar arguments for the factors and
mechanisms involved in activation of leukocytes in
IC diseases such as lupus nephritis, probably hold
true for ANCA associated GN too (9, 61, 67).
In addition to the immunological kidney diseases
many other kidney diseases feature the involvement
of glomerular and interstitial Mφs. Diabetic nephropathy, chronic kidney disease of any initial etiology,
acute interstitial nephritis (AIN), and ATN all feature
marked recruitment of interstitial Mφs (47). While it
has been easier to understand how glomerular Mφs
clearing ICs or those adjacent to activated T lymphocytes might become activated, it is less clear how
interstitial or glomerular Mφs in non-immunological
kidney disease become activated. One possibility is
that natural killer (NK) cells, recruited to sites of
injury also liberate IFNγ (Fig. 1). Another possibility
is that chemokines such as CXCL1 released from
injured parenchymal cells not only recruit Mφs but
also activate them (74). However, in many diseases
of the kidney there are very few NK cells and data
showing that chemokine ligation of monocyte
chemokine receptors triggers activation has not been
forthcoming (54). It is more likely that factors released
from injured parenchymal cells or extracellular factors
oxidized or modified during injury function act as
ligands for activating receptors on Mφs (Fig. 1) (75).
These DAMPs have been increasingly described as
binding to receptors of the innate immune response
known as PRRs triggering leukocyte activation, which
has a similar pattern of activation as PAMPs and ICs.
This group of danger molecules includes advanced
glycation endproducts, high mobility group box 1,
S100A8, S100A9, adenosine, Histone 4, mitochrondrial DNA containing CpG sequences, ribnucleoproteins SAP130 and others (37-42).
Heterogeneity of Macrophages
As an increasing number of cell-surface Mφ
markers have been identified using antibodies; labeling
studies have shown different populations of Mφs in
the kidney and elsewhere (3, 5, 8, 9, 29, 47, 76-89).
These Mφ subpopulations have been found to produce
different cytokines and behave differently. In vitro
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studies indicate that Mφs can be polarized to phenotype M1 or M2 after activation by different cytokines.
However in vitro studies are highly artificial and may
not reflect the scenario in vivo. Not only are the
cytokines IFNγ, LPS, IL-4, or IL-13 which generate
different subpopulations of Mφs less abundant in an
injured kidney, but the correlation between in vitro
markers and in vivo function is also poor (3, 47).
Hence the classification of Mφ subpopulations has
been modified to reflect the controversy (Fig. 1). The
M1 or classical activated population of Mφs can
generate a broad range of pro-inflammatory cytokines,
chemokines, and reactive nitrogen and oxygen species,
including nitric oxide. This pattern of activation is
broadly known as classical or M1 activation. The M2
population of Mφs may be better described as woundhealing since depending on the injury and organ
context the M2 Mφs may promote wound healing,
angiogenesis, or fibrosis. In addition, exposure to
apoptotic cells, the anti-inflammatory cytokine IL-10
or the ICs can generate Mφs that produce high levels
of IL-10 themselves and are actively involved in the
suppression of immune responses. This Mφ subpopulation might be better identified as a regulatory Mφ
(Mreg) (47, 90, 91). This Mreg macrophage is defined
by high levels of IL10 production. IL10 may be an
effector of function in certain settings but in many
settings IL10 is merely a marker of regulatory function.
Other genes including CXCL9, -10, and -11 may play
roles in the Mreg phenotype.
Three hypotheses explaining heterogeneity of
monocytes/Mφs have been proposed: (1) monocytes
differentiate into infinite subpopulations depending
on the environment (Hume Hypothesis) (92); (2) preexisting subpopulations of monocytes are functionally
prescribed (Geissmann/Randolph Hypotheses) (9395); (3) discrete functional subpopulations can switch
from one to the other (52). Evidence supports all
three of these hypotheses. In vitro cultured Mφs acquire different phenotypes transcriptionally depending
on the cytokine mixture applied, suggesting infinite
possibilities. Furthermore, in vivo studies demonstrate
that monocytes not only sense danger or injury but
also respond to the tissue specific environment and
acquire multiple phenotypes (3, 96-98). In contrast,
studies from the 1990’s revealed two or more discrete
subpopulations of human monocytes and studies by
Geissmann and Randolph et al. provided evidence of
clear functional differences between subpopulations
of circulating monocytes in mice, in keeping with the
second hypothesis (93, 99-101). Our own studies
using the marker Ly6C to define Mφ subpopulations
confirm the second hypothesis but show that the third
hypothesis holds true in vivo. That is, a single monocyte subset differentiates sequentially into functionally
discrete populations rather than infinite phenotypes
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Lin and Duffield
TH1 cell
NK cell
IFNγ
APC
IFNγ
DAMPs
TNFα
Classically activated (M1)
macrophage
Granulocyte
IL-4/IL-13
IL-10
Pentraxin-2
Prostaglandin
GPCR ligands
Regulatory T cell
IL-10
IL-4/IL-13
TH2 cell
Wound-healing (M2)
macrophage
Regulatory macrophage
Fig. 1. Subpopulations of macrophages in vivo. Schematic showing three different types of macrophages, and factors that regulate their
activation/differentiation in sterile inflammation in vivo. Although local factors can regulate recruited monocytes to differentiate
into different macrophage subpopulations, regulatory macrophages also differentiate from classically activated (M1) and
wound-healing (M2) macrophages, triggered by mechanisms that are poorly understood (3, 47).
Bone marrow
Capillary
Monocytes
Kidney
Ly6Chigh Mφ
Injury
Ly6Clow Mφ
MIP1α, MIP2, IL-1β, FcγRI
Ly6Cint Mφ
Ly6Clow Mφ
Ly6Clow Mφ
PDGF, IGF-1, CCL17,
CCL22, Galectin 3, Ang2
Fig. 2. Model showing Ly6Chigh monocytes are preferentially recruited to the injured kidney compared with Ly6Clow monocytes from
capillary, which differentiate into three populations of kidney macrophages, Ly6Chigh, Ly6Cint and Ly6Clow. A pool of mature
Ly6Chigh monocytes is released from bone marrow in response to kidney injury. These kidney macrophage subpopulations
generate discrete M1-biased (Ly6Chigh) and M2-biased (Ly6Clow) cytokines in vivo. The Ly6Cint subpopulation comprises
both macrophages derived from activation of resident macrophages and also macrophages in transition with Ly6Chigh and
Ly6Clow subpopulations (3).
Macrophages in Kidney Injury and Repair
(Fig. 2). In order for this to occur, either cellular activation must engage a transcriptional program which
regulates a phenotypic switch, or environmental
triggers within the injured tissue activate a phenotypic
switch. Several studies support the idea that a phenotypic switch is triggered by environmental factors.
Local release of adenosine in injured tissues binds
adenosine receptors on Mφs and can trigger polarization of Mφs (3, 74). Further studies to define the
role of locally released adenosine, DAMPs, and their
cognate receptors in this switch are required. It
would also be interesting to study the role of the
cross talk between the influxed monocytes and local
cells in determining the phenotypic switch (102-104).
Lessons from Genetic Models in Rodents
Until recently, the function of Mφs in tissue
injury has largely been inferred by their presence in
injured tissues and the cytokines that they can generate
in vitro when activated. A limited number of studies
have use polyclonal anti-Mφ sera suggested deleterious
functions for Mφs in glomerular diseases, but these
have to be interpreted with caution due to lack of
specificity of such preparations (66, 85). Liposomal
encapsulated clodronate has been developed recently
to ablate Mφs in vivo. This strategy relies on the
selective uptake of liposomes by monocytes and Mφs,
delivering toxic levels of the bisphosphonate clodronate. However, liposomes are endocytosed by many
cells including neutrophils and endothelial cells, and
clodronate has anti-inflammatory effects.
Genetic Models for Macrophage Ablation in Mice
We developed a genetic approach to ablate Mφs
in vivo, relying on the selective susceptibility of
human cells but not mouse cells to the toxic effects of
diphtheria toxin (DT) (9, 105). Humans are more than
1000 fold more susceptible to DT than rodents due to
the cell surface expression of the human heparin
binding epithelial growth factor receptor which is a
receptor for DT (DTR) and transports DT to the
cytosol where it is extremely lethal. We generated a
mouse model CD11b-DTR where the DTR was
expressed under a Mφ-specific promoter for the
integrin CD11b. Although CD11b is expressed by
other cells including neutrophils, only monocytes,
Mφs, dendritic cells, and NK cells are susceptible to
DT (6). Using this model we have been able to target
monocytes and Mφs specifically in models of kidney
disease, at different time-points. In a model of crescentic GN induced by nephrotoxic serum, Mφ ablation
prevents disease progression including interstitial
fibrosis and tubular atrophy. In a second model of
cyroglobulinemia-associated membranoproliferative
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GN, the overall effect of Mφ ablation was amelioration
of disease (62). Hence, Mφs are deleterious in these
models of glomerular disease.
In order to explore the role of Mφs in fibrosis
progression further in ‘non-immunological’ disease
of the kidney, we used a simple model of mechanical
injury caused by obstruction of the ureter of the
kidney that results in inflammation and fibrosis (3,
47). We discovered that Mφs also promote fibrosis in
response to mechanical injury, indicating the
generalized role for Mφs in fibrosis progression but
also indicating that much of the interstitial disease
seen in immunological kidney disease may be in
response to secondary cellular injury rather than
glomerular ICs.
A major function for Mφs is phagocytosis of
dying cells and debris and this may be considered a
component of the repair process. Furthermore, Mφs
may produce cytokines including IL-10, hepatocyte
growth factor, vascular endothelial cell growth factor,
and WNT ligands, which may drive both repair and
regeneration of parenchymal cells, and also limit
activation of leukocytes. In a model of kidney injury
followed by repair, the ischemia reperfusion injury
model (IRI), Mφ recruitment coincides with repair
not injury (7, 8). In the CD11b-DTR mouse model,
ablation of Mφs during the repair phase after IRI does
indeed prevent normal repair (8), and these findings
have been confirmed by abalation sutides using
liposomal clodronate. Studies are currently underway
to dissect the mechanisms by which Mφs promote
repair in single injury followed by repair and regeneration whereas they promote cell loss and fibrosis in
repetitive injury or chronic injury.
Rodent Congenics
Strains of inbred mice and rats have widely differing susceptibility to kidney diseases. These differences have been exploited to identify the genes
that determine disease susceptibility (106, 107). The
Wistar Kyoto rat strain is extremely susceptible to the
model of IC GN induced by nephrotoxic serum (NTN).
This type of rat was crossed with Lewis rats which
are resistant to developing disease. By back-crossing
and testing for disease susceptibility, two novel disease
susceptibility genes have been identified and they
are both monocyte/Mφ genes. One Fcγ receptor 3
(FcγRIII) is present in an alternate form in susceptible
rats. This renders its Mφs unable to efficiently
phagocytose ICs, and renders them more active by
pathways other than FcγRIII (61). The other disease
susceptibility gene is JunD, a transcription factor in
the activator protein-1 family that regulates activation
of Mφs (67). These studies serve to both highlight the
central role of Mφs in nonphlogistic clearance of ICs
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Lin and Duffield
and intracellular regulation of Mφ activation as key
facets that regulate disease progression. Furthermore,
rodent FcγRIII is analogous to human FcγRIIA, a
major activating FcγR. This receptor has many polymorphisms that determine susceptibility to the development of systemic lupus erythematosis (SLE)
and lupus nephritis (108, 109). In congenic studies in
mice that develop spontaneous SLE, several disease
susceptibility chromosomal loci have been identified.
The mouse Sle1 locus is syntenic with the human SLE
susceptibility loci containing genes involved in the
complement cascade and FcγRs, which are present on
monocytes and Mφs. It also contains genes for B-cell
survival signals, regulatory T cells and complement
receptor 2, and regulates the development of autoantibody and nephritis (106, 110-112). These studies
therefore also place monocytes and Mφs at the center
of the immune response in these models of lupus
nephritis. Collectively, these powerful genetic studies
place the regulation of Mφ activation and signaling
through Mφ FcγRs at the center of the immune response
in the glomerulus.
Mechanisms of Macrophage-Mediated
Fibrosis
In recent studies, we have identified a primary
role of Mφs in kidney fibrosis. Together with reports
of similar roles in other organs, this pro-fibrotic role
of Mφs is suggested to be a stereotyped response
to chronic injury or repetitive injury (3, 9, 105, 113120). Multiple mechanisms by which Mφs cause
fibrosis have been proposed in different disease contexts (98, 121, 122). Arginase generated by Mφs may
directly promote fibrosis by hydrolyzing arginine to
ornithine which can be used to generate polyamines
glutamate and proline which are necessary for collagen
synthesis (98, 123). IL-13 generated by Mφs directly
drives myofibroblasts to generate a collagenous
matrix. In lung diseases, production of IL-13 and
YM1 by Mφs has been strongly implicated in directly
driving myofibroblast activation (96, 124). However,
in contrast to the lung and skin, Mφs do not generate
IL-13, Fizz1 or YM1 in the injured kidney (3). The
restricted nature of these Mφs cytokines and factors
in the lung (and skin) reveals another facet of Mφs
biology: although Mφs have stereotyped responses to
injury, these Mφs respond to the local environment
and have an organ biased transcriptional profile. The
cytokine TGFβ is implicated in Mφ-driven fibrosis in
many organs (125, 126). In the kidney however,
epithelial cells, rather than Mφs, may play a major
role in generating the active form of TGFβ by α vβ 6
integrin mediated cleavage of the inactive molecule
(127-129). Although TGFβ can activate pericytemyofibroblast transition, it also induces cell cycle
arrest and cell death in epithelial cells, which is also
deleterious to the kidney (129). Therefore, although
Mφ-TGFβ may be playing a role in fibrogenesis, it is
not likely to be the major Mφ effector cytokine.
Fibrocytes, it has been suggested that myeloid
cells, which express CD34 and high levels of class II
MHC and also generate collagenous matrix directly,
play a role in fibrosis of many organs (130-133).
Despite reports providing evidence for the presence
of fibrocytes in models of kidney disease, our recent
exhaustive studies of these cells indicates that in
mice, at least, collagen producing myeloid leukocytes
are rare in kidney disease and do not contribute
directly to fibrogenesis (134). It is more likely that
fibrocytes serve as antigen presenting cells and recruit
more monocytes/Mφs which directly or indirectly
signal myofibroblasts and their precursor’s pericytes
via cellular (paracrine) cross talk (134).
To understand the mechanisms of Mφ-mediated
fibrogenesis further, we have recently defined subpopulations of Mφs in the kidney by the cell surface
marker Ly6C (Fig. 2) (3, 47). In chronic kidney
disease models including the ureteral obstruction
model, three populations of kidney Mφs can be
identified (Ly6C high , Ly6C int , Ly6C low ). All three
may derive from a single population of circulating
inflammatory Ly6C high monocytes. Ly6Chigh Mφs in
the kidney are activated, producing pro-inflammatory
cytokines including IL-1β and chemokines including
MIP1α, MIP2. These Mφs are similar to M1 activated
Mφs, but occur in sterile injuries without ICs, implicating the role of DAMPs in the process of Mφ
activation. In stark contrast, although Ly6C low Mφs
derive from Ly6Chigh Mφs, they generate low levels of
IL-1β and MIP2, but instead produce M2 type cytokines CCL17, CCL22, platelet-derived growth factor
(PDGF), insulin-like growth factor 1 (IGF-1), angiopoietin 2 (Ang2), TGFβ and IGFBP5, all of which
have been associated with fibrogenesis. Myofibroblasts have receptors for PDGF, IGF-1 and type 2
chemokines including CCR1 and CCR5. While it is
possible that one Mφ-derived factor is responsible for
the pro-fibrotic biology of Mφs, far more likely is that
many cytokines converge on myofibroblasts or pericytes to drive fibrosis. Nevertheless, the Ly6C low
Mφs, by virtue of their M2-skewed transcriptional
profile, fulfill several of the criteria for paracrine
signaling, and are therefore a target for therapy.
Several other mechanisms by which Mφs cause fibrosis
potentially exist. Firstly, Ly6C high Mφs produce M1
type cytokines including TNFα that may directly
signal to myofibroblasts; secondly, if Ly6C high Mφs
were prevented from becoming activated then it is
likely that Ly6Clow Mφs would not be activated since
the latter derive from the former. Therefore, targeting
activation of Ly6Chigh Mφs may also have therapeutic
Macrophages in Kidney Injury and Repair
gains. Thirdly, we have recently discovered that
myofibroblasts derive from pericytes, a newly described cell type in the kidney (134, 135). Pericytes
are perivascular cells of capillaries, derived from
metanephric mesenchyme during development, and
are necessary for angiogenesis and vascular stability
through two-way signaling between pericytes and
endothelial cells (136). It is therefore possible that
Mφ-signaling to endothelial cells, directs pericyte
migration, and differentiation into myofibroblasts.
In that context, Ly6Chigh Mφs generate high levels of
Ang2 which may signal deleteriously to endothelial
cells with the consequence of triggering fibrosis
through signaling from the endothelium.
Mechanisms of Macrophage-Mediated
Cellular Loss
Our studies and those of others have additionally
shown that in chronic or repetitive kidney diseases,
Mφs not only promote fibrosis but also promote loss of
epithelial cells and microvasculature (9, 129, 137,
138). The loss of epithelial cells has been detected by
the presence of increased cellular apoptosis, but this
cell death occurs at the same time as the proportion of
epithelial cells engaged in the cell cycle increases.
Thus, in these diseases, Mφs drive both the cell cycle
and apoptotic cell death (9). The mechanisms by which
this occurs have not been completely elucidated.
Possible roles for iNOS and TNFα have been explored,
and M1-skewed Mφs are implicated in this process, but
no consistent cytokine signals have been clearly defined
(139). Regardless of the specific cellular cross talk,
two important facets of Mφ biology are highlighted.
Firstly, Mφ activation is required for epithelial cell
death and secondly, a common theme emerges by
which Mφs provoke cells into the cell cycle and target
their untimely death at DNA cell cycle checkpoints.
It is possible that Mφs function in this context to test
cell health by triggering epithelial cells into the cell
cycle. Cells will pause at a DNA checkpoint due to
stress, inadequate energy or resources, or excessive
damage to DNA, and are more susceptible to apoptotic
cell death. Therefore, Mφs function as policemen
checking for health and driving rapid death and clearance if the stress testing does not go well (4). Although
this kind of function might appear desirable, excessive
cell loss can ensue leading to tubule atrophy and
peritubular capillary rarefaction in chronic inflammation with persistent activation of Mφs. New studies
are required to understand the molecular mechanisms
underlying these observations.
Macrophages in Repair and Regeneration
A common theme throughout this article is that
51
the natural state of Mφs is non-phlogistic clearance of
the unwanted from the body, and liberation of safe
helpful cytokines that promote well-being. Chronic,
repetitive or severe injury overcomes the inbuilt mechanisms that prevent activation and then Mφs become
chronically activated leading to deleterious consequences. With this in mind, one might expect that
Mφs play an important role in tissue repair and
recovery. Indeed it is clear that Mφs are important in
non-inflammatory regeneration, angiogenesis and also
repair (7, 8, 140, 141). We recently found that after
single injury to the kidney followed by repair there is
intense recruitment of Mφs during the repair phase
(8). Ablation of Mφs in this repair phase hinders
tubule regeneration. At the current time, the factors
that dictate how Mφs become predominantly reparative
versus deleterious remain obscure. Our own studies
have identified the Wnt signaling pathway as an
important target of Mφs in driving regeneration (8),
but doubtless multiple mechanisms have been
uncovered. Consistent with previous findings, a recent
study also shows that iNOS+ pro-injurious (M1) Mφs
are recruited into the kidney in the first 48 hours after
IRI, whereas arginase- and mannose receptor-positive,
wound-healing (M2) Mφs predominate at later time
points (7). In vitro studies show that IFNγ-stimulated,
pro-injurious Mφs begin to express markers of M2
Mφs when cocultured with renal tubular epithelial
cells. Moreover, IL-4-stimulated, M2 skewed Mφs,
but not IFNγ-stimulated M1-skewed Mφs, promote
proliferation of renal tubular epithelial cells in vitro
and in vivo. Thus, during the normal repair of kidney
injured by ischemia, Mφs acquire regenerative functions to stimulate successful epithelial proliferation.
Monocytes and Macrophages as Targets for
Kidney Disease Therapy
Targeting Macrophage Activation
Inflammatory cytokines such as TNFα or IL-1β
alone, or pro-inflammatory chemokines do not activate
Mφs, although the lymphokine IFNγ has recognized
capacity to weakly activate Mφs. Increasing evidence
suggests that soluble factors and debris released from
injured tissues, known as DAMPs, bind to PRRs of
Mφs including toll-like receptors and other receptors
such as receptors for advanced glycation endproduct
(37, 41). Selective blocking myeloid cell activation
triggered by DAMPs while permitting activation
triggered by foreign pathogens is a highly attractive
approach to the treatment of chronic inflammation
since it does not pose the risk of increased infection
susceptibility.
We have recently discovered that a circulating
pentameric protein of the innate immune system,
52
Lin and Duffield
serum amyloid P (SAP), which is also known as
pentraxin-2 (PTX2), with structural similarities to Creactive protein is strongly anti-fibrotic in kidney
disease (47). PTX-2 deposits in injured tissues and
opsonizes dead cells and debris. Once it has opsonized
targets, it undergoes a conformational change converting to a high affinity ligand for the activating
FcρRs, hFcρRIIA and hFcγRIII (mFcγRIII and
mFcρRIV). Unlike cross-linking of FcγRs by immunoglobulin, cross-linking of FcγRs by PTX-2 does
not activate Mφs. Conversely it inhibits activation
mediated by other stimuli. Part of the mechanism by
which PTX-2 inhibits Mφ activation is through local
release of IL-10. IL-10 is an anti-inflammatory cytokine that is well recognized for inhibiting inflammation
and has direct anti-fibrotic effects on myofibroblasts.
By endocytosing and phagocytosing SAP-opsonized
debris, Mφs release IL-10 locally in the injured kidney
resulting in less activated Mφs (both M1 and M2
subtypes) that are unable to drive fibrosis. Importantly,
SAP does not inhibit activation triggered by the
bacterial cell wall lipoprotein LPS, implicating SAP
as a novel and safe endogenous inhibitor of sterile
inflammation and fibrosis. Recombinant PTX-2 is
currently in Phase 2 trials as an anti-fibrotic therapy
in lung fibrosis, and it clearly may have broad therapeutic indications (91). Although still in their infancy,
new PRRs and DAMPs that activate the innate immune
system are being identified and may become new
targets for therapy (38).
Targeting Monocytes
Rodent studies using liposomal clodronate or
the CD11b-DTR ablation system indicate that
monocyte/Mφ ablation is effective in limiting tissue
injury and fibrosis. Selective cellular ablation is
widely accepted in humans by monoclonal or polyclonal antibodies, such as anti-thymocyte globulin
(ATG), anti-CD3 antibodies (OKT3), anti-CD20
antibodies (rituxumab), or anti-CD52 (Campath)
antibodies, which target T cells or B cells. All of
these therapies are highly efficacious but come with
considerable risk of infection (except anti-CD20
antibodies). ATG likely also ablates monocytes in
addition to T cells. In addition, a wide range of other
therapies, including mycophenolic acid, cyclosporine
A, and cyclophosphamide, function either to ablate or
profoundly inhibit proliferation of lymphocytes. It is
quite likely therefore that monocyte specific ablative
therapies would be successful in humans, but there
may be unacceptable side effects, particularly if they
are considered in the treatment of chronic diseases.
Nevertheless, these therapies could find a role in the
management of acute inflammatory diseases of the
kidney including AIN and rapidly progressive GN.
The monocyte and Mφ receptor for colony
stimulating factor-1 (CSF1) or the monocyte colony
stimulating factor (M-CSF) is the CSF1 receptor.
This tyrosine kinase dependent receptor drives
monocyte proliferation in tissues and may be an
alternative target for therapy. Several tyrosine kinase
inhibitors that are selective for the CSF1 receptor
have been developed and are in early trial phases
(142).
Targeting Macrophage Recruitment
Chemokines and their receptors are important
in monocyte recruitment. One problem encountered
with targeting monocyte recruitment is the redundancy
of chemokines and their receptors (143). Moreover,
chemokine receptors such as CCR2 have broader
roles in the release of monocytes from bone marrow
thus their blockades may pose additive risks of
infection. In addition, the finding that two or more
subpopulations of monocytes exist in the circulation,
one with a high CCR2 receptor, another with a high
CX3CR1 receptor, renders targeting strategies more
complicated since there may be redundancy of the
functions of these populations (144). However,
CCR2 + Ly - 6C high monocytes unexpectedly required
CX3CR1 in addition to CCR2 and CCR5 to accumulate
within atherosclerotic plaques. In many other inflammatory settings, these monocytes utilize CCR2, but
not CX3CR1, for trafficking. Thus, antagonizing
CX3CR1 may be effective therapeutically in ameliorating CCR2+ monocyte recruitment to some specific
inflammatory settings without impairing their CCR2dependent responses to inflammation overall (101).
New small molecules that provide broader blockades
of chemokine receptors, including compounds such
as BMS-813160 (Bristol-Myers Squibb, New York,
NY) which block both CCR2 and CCR5 in humans
and rodents, or combination inhibitors that block
individual receptors including CCR1, CCR2 and
CCR5, hold promise as new therapies in fibrosing
inflammatory diseases, not only in the kidney but also
in other organs (145-149). Since the innate cellular
immune response to pathogens is important in health,
and since almost all rodent studies are performed
nowadays in sterile facilities, safety and efficacy
studies will need to be completed before these compounds can be used in human diseases. In this context, the CCR1 antagonist CP-481715 (Pfizer, New
York, NY) is currently in phase I trials for rheumatoid
arthritis (150, 151).
Targeting Macrophage Differentiation
Several studies including our own work support
the model in which Mφs differentiate into a wound-
Macrophages in Kidney Injury and Repair
healing or pro-fibrotic Mφs (3, 37, 52, 90, 152). The
mechanisms by which this differentiation occurs
remain incompletely understood. Several candidate
factors driving such differentiation have been
described. One of which is local release of adenosine,
binding to adenosine receptors. Adenosine has been
ascribed as ‘anti-inflammatory’ since it can result in
wound healing Mφs and prevent pro-inflammatory
cytokine production. However, the role of adenosine
in generating pro-fibrotic Mφs has yet to be explored.
Mechanisms to selectively target adenosine receptors
or target the extracellular enzymes such as CD73
which generate extracellular adenosine should be
tested in models of sterile inflammation with fibrosis
to determine efficacy (153).
Other Potential Targets
The list of Mφ paracrine effector molecules is
extensive, but the precise roles these factors play
individually or in concert have been inadequately
tested. In Mφ cytokines PDGF, IGF-1, and Ang2,
prostaglandin E2 are all cytokines liberated by M2
Mφs in vivo that may be playing deleterious roles.
Our study has shown that blocking PDGF receptor
signaling using monoclonal antibody or imatinib
may attenuate kidney fibrosis through inhibiting
pericyte-myofibroblast transition in mice with progressive kidney fibrosis (154). However, promising
studies that target the PDGF receptor β, using tyrosine kinase inhibitors are currently underway in
humans to determine whether selective blockades of
this paracrine pathway will impact human fibrosis
progression.
Therapeutic Options in the Treatment of
Chronic Kidney Disease and Immune-mediated
Kidney Diseases
In summary, Mφs are widespread in kidney
diseases. Increasing evidence has shown that targeting
Mφs and their functions will improve outcome in
many kidney diseases. The final common pathway of
chronic kidney disease that leads to organ failure
increasingly appears to be driven, at least in part, by
chronically activated Mφs. New therapeutics targeting
Mφs are emerging and are undergoing investigation
in human trials as potential new therapies in a range
of kidney diseases.
Acknowledgments
The laboratory is funded by grants from the
National Science Council (99-2628-B-002-013,
S.L.L.), Ta-Tung Kidney Foundation, Mrs. Hsiu-Chin
Lee Kidney Research Foundation, and National
53
Institutes of Health (DK84077, and DK87389, J.S.
D.) and a Research in Progress from Genzyme and the
Nephcure Foundation (J.S.D.).
The authors thank Mark Lupher Jr (Promedior,
Malvern, PA), Richard A. Lang (Cincinnati Children’s
Hospital, Cincinnati, OH), Jeremy Hughes (University
of Edinburgh, Lothian, UK), Charles Alpers (University of Washington, Seattle, WA), Yung-Ming
Chen (National Taiwan University Hospital, Taiwan)
for valuable discussions and collaboration.
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