Invited AJP Renal Review: F-00016-2008R1

MCP-1/CCL2: a new diagnostic marker and therapeutic target for

progressive renal injury in diabetic nephropathy.

Tesch GH1,2

Department of Nephrology1 and Monash University Department of Medicine2, Monash Medical

Centre, Clayton, Victoria, Australia.

Details for Correspondence:

Dr Greg Tesch.

Department of Nephrology,

Monash Medical Centre,

246 Clayton Road, Clayton

Victoria 3168, Australia.

Ph: (613) 95943527

Fax: (613) 95946530

Email: greg.tesch@med.monash.edu.au

Key Words: monocyte chemoattractant protein-1 (MCP-1), CC-chemokine ligand 2 (CCL2),

CC-chemokine receptor 2 (CCR2), diabetic nephropathy, macrophages, renal injury.

Running Title: MCP-1/CCL2 in diabetic nephropathy

Page 1 of 25Articles in PresS. Am J Physiol Renal Physiol (February 13, 2008). doi:10.1152/ajprenal.00016.2008

Copyright © 2008 by the American Physiological Society.

1

Abstract

Despite current therapies, many diabetic patients will suffer from declining renal function in

association with progressive kidney inflammation. Recently, animal model studies have

demonstrated that kidney macrophage accumulation is a critical factor in the development of

diabetic nephropathy. However, specific anti-inflammatory strategies are not yet being

considered for the treatment of patients with diabetic renal injury. This review highlights the

chemokine MCP-1/CCL2 as a major promoter of inflammation, renal injury and fibrosis in

diabetic nephropathy. Researchers have found that diabetes induces kidney MCP-1 production

and that urine MCP-1 levels can be used to assess renal inflammation in this disease. In addition,

genetic deletion and molecular blocking studies in rodents have identified MCP-1 as an

important therapeutic target for treating diabetic nephropathy. Evidence also suggests that a

polymorphism in the human MCP-1 gene is associated with progressive kidney failure in type 2

diabetes, which may identify patients at higher risk who need additional therapy. These findings

provide a strong rationale for developing specific therapies against MCP-1 and inflammation in

diabetic nephropathy.

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Introduction

The development of diabetes involves metabolic, endocrine and hemodynamic abnormalities

which can promote a state of chronic inflammation and vascular dysfunction in many tissues. In

the kidney, this can lead to the development of an innate immune response which is

predominantly characterized by the accumulation of kidney macrophages (8). Studies in human

and experimental diabetic nephropathy have shown that kidney macrophage accumulation is

associated with the progression of diabetes (hyperglycemia, HbA1c), the development of renal

injury (tissue damage, albuminuria) and kidney fibrosis (myofibroblast accrual, sclerosis), and

the decline in renal function, suggesting that it is an inflammatory-mediated disease (8, 9, 33).

This concept is supported by animal model studies which have demonstrated that genetic and

immunosuppressive strategies reducing kidney macrophage accumulation also suppress the

development of diabetic nephropathy (10, 11, 35). These findings have led researchers to

evaluate the mechanisms by which macrophages are recruited into diabetic kidneys in the hope

of finding new therapeutic targets. Recent evidence has highlighted the production of monocyte

chemoattractant protein-1 (MCP-1/CCL2) by diabetic kidneys as a major factor influencing

macrophage accumulation in this disease, which is the focus of this review.

Kidney cells produce MCP-1 in response to diabetes

MCP-1 is a secreted protein which specifically attracts blood monocytes and tissue macrophages

to its source, via interaction with its cell surface receptor, CCR2 (5). Kidney cells produce MCP-

1 in response a variety of proinflammatory stimuli (36), and predictably, its expression has been

identified in kidney diseases which involve significant inflammation (37), which include diabetic

nephropathy (1, 49). Elements of the diabetic milieu are known to induce MCP-1 mRNA

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synthesis and protein secretion by cultured renal parenchymal cells, suggesting that the onset of

diabetes can provoke renal macrophage recruitment (Fig 1).

High levels of glucose have been shown to stimulate MCP-1 production by human and

mouse mesangial cells, through a pathway which involves activation of protein kinase C (PKC),

increased levels of oxidative stress and the activation/nuclear translocation of the transcription

factor nuclear factor-kappa B (NF-kB) (20, 24, 55). This stimulatory effect of high glucose in

mesangial cells is further enhanced by the presence of advanced glycation end products (AGEs)

or mechanical stretch (2, 17). A variety of AGEs are also capable of stimulating MCP-1

production by mesangial cells, which involves interaction with the receptor for AGEs (RAGE)

with subsequent generation of oxidative stress via activation of peroxisome proliferator-activated

receptor-gamma (PPAR-g) (2, 29, 53).

Kidney epithelial cells, including glomerular podocytes and tubular cells, also make

MCP-1 in response to high glucose and AGEs. Exposure to high glucose rapidly induces MCP-1

mRNA and protein release by cultured mouse podocytes, which is inhibited by treatment with

all-trans retinoic acid (22). Glycated-BSA and carboxymethyllysine (CML)-BSA also stimulate

MCP-1 gene and protein expression in mouse podocytes, which occurs through a mechanism

involving RAGE, oxidative stress and activation of extracellular signal-regulated kinase (ERK)

and NF-kB, and is inhibited by pravastatin (18, 19). Similarly, cultured rat proximal tubular cells

secrete increased levels of MCP-1 in response to both high glucose and AGE-BSA (11).

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Urine MCP-1 levels correlate with kidney macrophage accumulation in diabetic

nephropathy

Analysis of patient biopsies and animal models by in situ hybridization or immunostaining has

identified expression of MCP-1 mRNA and protein in diabetic kidneys (glomerular podocytes,

mesangial cells and tubules), which correlates with the accumulation of CD68+ macrophages (8,

11, 49). Animal model experiments also show that kidney MCP-1 is increased early in disease,

being most abundant in tubules, and progresses with the development of diabetic nephropathy

(11). Serum levels of MCP-1 are sometimes elevated in diabetic patients; however, this is not

associated with the development of albuminuria, kidney macrophage accumulation or

nephropathy (2, 28, 49). In contrast, urine levels of MCP-1 closely reflect kidney MCP-1

production and correlate significantly with levels of albuminuria, serum glycated albumin, urine

N-acetylglucosaminidase (NAG), and kidney CD68+ macrophages in human and experimental

diabetic nephropathy (2, 11, 12, 32, 49). This has led to the suggestion that proteinuria during

diabetes may itself aggravate tubular injury and accelerate nephropathy by increasing tubular

MCP-1 production and the inflammatory response. This concept is supported by in vitro and in

vivo studies indicating that protein overload can induce tubular expression of MCP-1 (13, 44).

However, during diabetes, it appears more likely that tubular MCP-1 is initially induced by the

diabetic milieu, since increases in tubular MCP-1 and interstitial macrophages coincide with the

development of hyperglycemia and precede a rise in albuminuria in type 1 diabetic nephropathy

in mice (11). In comparison, a rat albumin overload model, which develops instant proteinuria,

takes two weeks to induce an increase in kidney MCP-1 mRNA levels (13), suggesting that

excreted forms of albumin cannot independently promote rapid tubular production of MCP-1 in

vivo.

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Urinary levels of MCP-1 can be used to assess the efficacy of treatments in reducing

diabetic renal inflammation. Treatment of patients with the angiotensin-converting enzyme

(ACE) inhibitor Lisinopril reduced urine MCP-1 in type 2 diabetic nephropathy and this

correlates with the decline in proteinuria (1). Similarly, type 2 diabetic patients with nephropathy

have elevated urine MCP-1 levels which correlate with urine 8-iso-prostoglandin F2alpha (a

marker of renal oxidative stress), and both of these indicators are decreased by aldosterone

blockade with spironolactone (45). In a rat model of type 1 diabetic nephropathy, treatment with

retinoic acid reduces urine MCP-1, which correlates with a decline in immunostaining for MCP-

1 and CD68+ macrophages in the kidney (22). These findings suggest that urine MCP-1 may

have significant diagnostic value in evaluating the renal inflammatory response in patients with

diabetic nephropathy. However, whether this will be better than albuminuria at predicting

diabetic renal inflammation is unknown. It is possible that urinary levels of MCP-1 may respond

more quickly to anti-inflammatory therapies and predict later changes in albuminuria, which

could be clinically useful.

MCP-1 promotes inflammation and progressive injury in diabetic kidneys

In overt diabetic nephropathy, the predominant source of MCP-1 is the cortical tubules (11),

which is similar to other inflammatory kidney diseases (46, 47). Macrophage accumulation

around these tubules is associated with tubular injury and myofibroblast accumulation, which

leads to declining renal function (8, 9) (Fig. 1).

The importance of MCP-1 in the early development of diabetic nephropathy has been

determined using animal models incorporating genetically deficient mice or therapeutic blockade

of the MCP-1 receptor (CCR2) (11, 12, 25). In a model of streptozotocin-induced type 1 diabetic

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nephropathy, mice genetically deficient in MCP-1 were found to have reduced renal injury

compared to wild type mice with equivalent hyperglycemia (11). In this study, the absence of

MCP-1 resulted in the prevention of albuminuria and elevated plasma creatinine at 18 weeks of

diabetes, which coincided with a marked reduction (≥50%) in glomerular and interstitial CD68+

macrophages, and histological damage (11). In addition, a smaller percentage of macrophages in

MCP-1 deficient diabetic kidneys had co-expression of CD169 and inducible nitric oxide

synthase (iNOS), suggesting that these macrophages were less activated (11). Therefore, this

study demonstrates that MCP-1 promotes macrophage recruitment and activation and the

development of renal injury in diabetic kidneys. Similar findings have also been obtained in the

db/db mouse model of type 2 diabetic nephropathy. MCP-1 deficient db/db mice are not

protected from the development of obesity, adipose inflammation or type 2 diabetes (12).

However, at 32 weeks of age, these db/db mice have striking reductions (≥50%) in albuminuria,

plasma creatinine, macrophage recruitment and activation, and histological injury compared to

their wild type diabetic controls (12). These findings obtained from diabetic nephropathy studies

in MCP-1 deficient mice have recently been supported by a study using both pharmacological

treatment and gene therapy to block MCP-1 function in mice which spontaneously develop type

1 diabetes (25). In this study (25), MCP-1 action was blocked in diabetic mice for 12 weeks by

either food supplementation with propagermanium (a CCR2 antagonist) or muscle transfection

with a plasmid containing the 7ND gene (a mutant of MCP-1). Both these strategies were found

to reduce glomerular macrophage infiltration and glomerulosclerosis during diabetes, however,

the effect on albuminuria was not reported.

In vitro evidence also indicates that MCP-1 may promote macrophage activation in

diabetic kidneys (Fig. 1). Stimulation with MCP-1 rapidly induces the activation of ERK and

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JNK signaling pathways in cultured macrophages, which are known promoters of macrophage

inflammatory response (42). Furthermore, blockade of ERK activity with PD98059 prevents

MCP-1 stimulated macrophage activity by inhibiting actin polymerization and TNF-α production

(42). In addition, MCP-1 induces iNOS mRNA and nitric oxide release by peritoneal

macrophages, which is dependent on signaling through phosphoinositol-3-kinase, PKC and ERK

(4). This data supports a direct role for MCP-1 in macrophage activation; however, it is unclear

whether this will play an important role in the diabetic kidney compared to the activation

induced by high glucose, AGEs, oxidative stress and proinflammatory cytokines.

MCP-1 stimulates renal fibrosis in diabetic kidneys

Evidence from animal models and in vitro experiments suggests that MCP-1 can directly and

indirectly promote renal fibrosis during diabetes (Fig. 1). The initial studies of MCP-1 deficient

diabetic kidneys showed that a lack of MCP-1 reduces the accumulation of interstitial

myofibroblasts and the deposition of glomerular and interstitial collagen type IV (11, 12).

Subsequent examination has shown that MCP-1 deficiency in type 1 diabetes also reduces

glomerular deposition of fibronectin as well as mRNA and protein expression of fibronectin and

transforming growth factor-β1 (TGF-β1) in the total kidney (16). In addition, therapeutic

blockade of MCP-1 in type 1 diabetic mice reduces glomerular deposition of TGF-β1 and

collagen IV and the mesangial matrix fraction (25). Notably, in each of these studies, a

deficiency or blockade of MCP-1 was associated with a marked decline in the number of kidney

macrophages, suggesting that MCP-1-mediated fibrosis may be due to the recruitment of

macrophages.

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Macrophages recruited into a diabetic environment can promote renal fibrosis. Culturing

macrophages in the presence of diabetic serum or AGEs causes them to release interleukin-1 and

platelet derived growth factor which induces the proliferation of renal interstitial fibroblasts (9).

In addition, MCP-1 can directly stimulate macrophages to secrete increased levels of active and

total TGF-β1, which can subsequently promote production of extracellular matrix (50).

Therefore, MCP-1 has the ability to influence the macrophage contribution to fibrosis through

both direct and indirect mechanisms.

MCP-1 is also capable of inducing a fibrotic response in glomerular mesangial cells.

MCP-1 signaling through CCR2 on human mesangial cells has been shown to induce fibronectin

mRNA and protein synthesis by a mechanism involving TGF-β1 production and activation of

NF-kB in these cells (16). This may have importance in diabetic glomerulosclerosis, since MCP-

1 can promote glomerular TGF-β1 production without affecting glomerular macrophage

accumulation (39). Furthermore, TGF-β1 can itself stimulate mesangial expression of MCP-1,

which suggests the possibility of a self amplifying cytokine-loop mechanism for promoting

mesangial matrix accumulation (7).

MCP-1/CCR2 gene polymorphisms and diabetic nephropathy

Studies examining the influence of MCP-1 gene polymorphisms on the progression of kidney

disease have focused on the -2518 A/G single nucleotide polymorphism (SNP) in the distal

regulatory region of MCP-1 which is believed to regulate gene expression. This SNP was

originally identified as potentially important when it was shown that presence of a specific allele

was associated with greater production of MCP-1 by interleukin-1β stimulated peripheral blood

mononuclear cells (38). Subsequent clinical studies have since shown that the G allele, which is

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more highly expressed in Asian and Mexicans compared to Caucasians (38), is associated with a

reduced susceptibility to renal injury in some studies of lupus nephritis and IgA nephropathy (26,

31). However, the association of these particular kidney diseases with MCP-1 polymorphisms

remains controversial.

The presence of the MCP-1 A(-2518) allele has also been associated with the

development of diabetes and diabetic nephropathy. Frequencies of the A allele and A/A genotype

are reported to be significantly higher in Caucasian patients with type 1 diabetes compared to

normal controls (54). Similarly, in a large cohort of Caucasians, the G allele correlates with a

lower incidence of type 2 diabetes and is associated with decreased levels of plasma MCP-1 (41).

In contrast, a study performed in Korean patients showed no significant difference in the overall

distribution of -2518 A/G in the MCP-1 gene in patients with type 2 diabetes compared to

healthy controls (30). However, this study demonstrated a significant association of the A allele

with diabetic kidney failure, suggesting that in some populations, the A allele may be more

related to the development of diabetic nephropathy than the occurrence of diabetes itself.

Current evidence suggests that there are no associations of SNPs in CCR2 with the

development of diabetic nephropathy or other kidney diseases. One study in children has

identified an increased frequency of G to A substitution in the CCR2 gene at position 190

(CCR2-64I) which is associated with the development of type 1 diabetes (43), but subsequent

studies have been unable to confirm this finding in adults or show any associations with diabetic

complications (54).

Therapeutic strategies targeting MCP-1 in diabetic nephropathy

Despite current treatments including glycemic control, ACE inhibition, angiotensin receptor

blockers (ARBs) and statins, diabetic nephropathy continues to progress in many patients in

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association with kidney macrophage accumulation, suggesting the need for additional

immunotherapy (33). In support of this concept, a recent study in rats has shown that

immunosuppression with mycophenolate mofetil (MMF) provides added benefit when combined

with ACE inhibition in treating diabetic nephropathy (52). In this study, combined treatment

produced superior suppression of kidney macrophage recruitment in association with greater

reductions in renal MCP-1 and TGF-β1. This suggests that specific targeting of MCP-1 would

be beneficial as an adjunct therapy to patients with diabetic nephropathy.

Several novel therapies have recently been shown to indirectly reduce kidney expression

of MCP-1 and diabetic renal inflammation in animals models by targeting renal oxidative stress

or the downstream intracellular signaling pathways, which are induced by hyperglycemia. These

include the use of an analog of 1,25-Dihydroxyvitamin D3 (55), a PPAR-g agonist (3), a PKC-β

inhibitor (51), eicosapetaenoic acid (EPA) (21), and a flavonoid (34). However, the suppression

of the inflammatory response was only partially affected by these treatments, indicating the need

for more specific blockade of MCP-1.

The essentially normal phenotype of MCP-1 deficient mice suggests that the use of anti-

MCP-1 therapies in chronic diseases will not be harmful (11). There are a number of strategies

that selectively target MCP-1 which have been proven effective in rodent models of

inflammatory disease. Small molecular antagonists of CCR2 (INCB3344, propagermanium, RS-

504393) have been shown to suppress inflammation in mouse models of multiple sclerosis, renal

ischemia reperfusion injury, ureter obstruction and diabetic nephropathy and in a rat model of

arthritis (6, 15, 25, 27). Engineered biological antagonists of CCR2 have also proven effective

(14, 23, 40). Subcutaneous infusion of cells transfected with a vector expressing a truncated

inactive form of MCP-1 has been found to suppress the development of renal inflammation in a

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mouse model of lupus nephritis (23). Similarly, muscle transfection with 7ND (a mutant of

MCP-1) reduces renal inflammation in mouse models of renal ischemia reperfusion injury, lupus

nephritis and diabetic nephropathy (14, 40, 25). These studies provide the foundation for the

development of specific anti-MCP-1 therapies to treat human diseases, including diabetic

nephropathy.

Conclusions

Evidence from human studies and animal models demonstrates that kidney MCP-1 production

plays a critical role in the development of diabetic renal inflammation which leads to progression

of diabetic nephropathy. Current therapies can only partially reduce the impact of MCP-1 on this

disease, suggesting the need for additional treatment. Urinary levels of MCP-1 can be monitored

as a marker of diabetic renal inflammation, and this may prove to have significant diagnostic

value in assessing the effectiveness of novel or combined therapies. Selective targeting of MCP-

1 has been proven to be an effective treatment in suppressing animal models of kidney disease

which include diabetic nephropathy, however, such therapies have not yet been validated in

human diabetic nephropathy. Given that macrophages also play a significant role in the

development of non-renal diabetic complications (48), it is feasible that any new therapies

targeting MCP-1 may have broader clinical importance than treating diabetic nephropathy alone.

Acknowledgments

Dr G.H. Tesch is supported by a Career Development Award from the National Health and

Medical Research Council of Australia, Kidney Health Australia and the Australian and New

Zealand Society of Nephrology.

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Figure Legend

Figure 1: Postulated role for MCP-1 in diabetic nephropathy. Elements of the diabetic milieu

induce renal parenchymal cells to secrete MCP-1 which attracts monocytes into the kidney and

stimulates myofibroblast-like properties in mesangial cells. Further exposure of kidney

macrophages to MCP-1 and the diabetic milieu promotes macrophage activation resulting in the

release of reactive oxygen species (ROS), proinflammatory cytokines (eg. IL-1, TNF-α, MCP-1)

and profibrotic growth factors (eg. PDGF, TGF-β). The self-amplifying inflammatory response

causes injury and death to parenchymal cells and the fibrotic response induces myofibroblast

proliferation and enhanced production of extracellular matrix by fibroblasts and mesangial cells.

Together these responses promote the progression of diabetic nephropathy resulting in the

development of renal failure.

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MCP-1

Injury Apoptosis, Necrosis Inflammation

Progression of Diabetic Nephropathy

Activated Macrophages

ROS, proinflammatory

cytokines

Fibrosis Fibroblast Proliferation Matrix Deposition

profibrotic growth factors Myofibroblasts

Parenchymal Cells

Monocytes Diabetic Milieu

High Glucose AGEs

BloodMCP-1

Kidney

Figure 1

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