Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice
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- Chow, F.Y., Nikolic-Paterson, D.J., Ma, F.Y. et al. Diabetologia (2007) 50: 471. doi:10.1007/s00125-006-0497-8
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Tissue macrophage accumulation is thought to induce insulin resistance during obesity and stimulate the progression of diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) is a potent stimulator of macrophage recruitment. It is increased in adipose tissue during obesity and in diabetic kidneys, suggesting that inflammation of these tissues may be MCP-1-dependent. Based on these findings, the aim of this study was to examine whether a deficiency in MCP-1 would alter the development of type 2 diabetes and its renal complications.
Materials and methods
The role of MCP-1 in the progression of type 2 diabetes and its associated renal injury was assessed in obese db/db mice that were deficient in the gene encoding MCP-1 (Ccl2).
The incidence and development of type 2 diabetes were similar in Ccl2+/+ and Ccl2−/−db/db mice between 8 and 32 weeks of age. Body mass, hyperglycaemia, hyperinsulinaemia, glucose and insulin tolerance, plasma triacylglycerol and serum NEFA were not different between these strains. Pathological changes in epididymal adipose tissue, including increases in macrophage accumulation and Tnfa mRNA and reductions in Adipoq mRNA, were unaffected by the absence of MCP-1. In contrast, kidney macrophage accumulation and the progression of diabetic renal injury (albuminuria, histopathology, renal fibrosis) were substantially reduced in Ccl2−/− compared with Ccl2+/+db/db mice with equivalent diabetes.
Our study demonstrates that MCP-1 promotes type 2 diabetic renal injury but does not influence the development of obesity, insulin resistance or type 2 diabetes in db/db mice. MCP-1 plays a critical role in inflammation of the kidney, but not adipose tissue, during the progression of type 2 diabetes.
KeywordsAdipose Ccl2 db/db mice Diabetes Diabetic nephropathy Inflammation Insulin resistance Macrophage MCP-1
chemokine CC motif receptor 2
macrophage-colony stimulating factor
monocyte chemoattractant protein-1
monocyte chemoattractant protein-3
urine albumin excretion
Recent studies have implicated tissue macrophage accumulation in the development of obesity-related insulin resistance [1, 2] and type 2 diabetic nephropathy [3, 4]. Macrophage accrual in visceral adipose tissue is a feature of chronic obesity which is associated with the onset of insulin resistance [1, 2]. Indeed, animal studies have shown a correlation between macrophage numbers in epididymal fat and obesity, adipocyte size and reduced insulin sensitivity [1, 5]. Current perception is that macrophages may interact with adipocytes, resulting in the secretion of factors that act in a paracrine or endocrine manner to promote insulin resistance in adipose tissue, liver and muscle. During obesity, macrophages are the major source of TNF-α in adipose tissue, and TNF-α is known to reduce insulin signalling and insulin-responsive glucose transport in adipocytes [6, 7]. Macrophages in diabetic animals also produce nitric oxide , which can promote insulin resistance in muscle . Furthermore, mice lacking IκB kinase-β in myeloid cells are protected against systemic insulin resistance induced by obesity, suggesting that activation of nuclear factor-κβ in adipose macrophages may contribute to the induction of insulin resistance . In diabetic kidneys, the leucocytic infiltrate consists almost exclusively of macrophages and their accrual correlates with renal injury [4, 10]. In vitro studies have shown that elements of the diabetic milieu, such as hyperglycaemia and advanced glycation end-products, can stimulate macrophages to secrete substances which cause injury, cell proliferation or induce fibrotic responses in kidney cells [10, 11]. Furthermore, additional elements characteristic of type 2 diabetes, including hyperinsulinaemia, hyperlipidaemia and elevated levels of circulating adipokines, may also influence inflammatory responses in the kidney. Therefore, macrophages may be important promoters of both type 2 diabetes and its complications.
Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is known to affect the accumulation and function of macrophages [12, 13, 14]. MCP-1 levels are increased in plasma and adipose tissue in mouse models of obesity [15, 16], and correlate with the number of CD11b+ monocytes in these animals . Primary cultures of human preadipocytes constitutively produce MCP-1 but these levels are reduced upon maturation . Stimulation with TNF-α or insulin increases expression of the gene encoding MCP-1 (Ccl2) in adipocytes differentiated in vitro [15, 17]. Addition of MCP-1 to differentiated adipocytes in vitro decreases insulin-stimulated glucose uptake and the expression of several adipogenic genes, suggesting that MCP–1 may also affect adipocyte function . In diabetic kidneys, Ccl2 expression increases with macrophage accumulation and disease progression  and appears to be dependent on both the diabetic milieu  and activation of the renin–angiotensin system . In addition, urine MCP-1 levels correlate with albuminuria during human diabetic nephropathy [18, 20]. Therefore, MCP-1 may play a key role in the development of both type 2 diabetes and its associated nephropathy.
Our study examined whether MCP-1 plays a role in adipose and renal inflammation during the development of type 2 diabetes. Ccl2+/+ and Ccl2−/−db/db mice were used to determine the functional importance of MCP-1 in the development of obesity, inflammation, glucose and insulin tolerance and hyperglycaemia. A selected subgroup of these mice with equivalent diabetes in both strains was used to establish the role of MCP-1 in diabetic renal injury.
Materials and methods
Obese (db/db) and lean heterozygote control (db/+) mice were created from breeding pairs of C57BL/6 db/+ mice obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and were genotyped by PCR with restriction enzyme analysis for the mutated leptin receptor. Ccl2−/− mice, created by targeted gene disruption , were backcrossed eight times onto the C57BL/6J strain. These Ccl2−/− mice were then crossed with C57BL/6 db/+ mice to create db/+ Ccl2−/− mice, which were validated by PCR-based genotyping. The latter were then interbred to create homozygous db/dbCcl2−/− mice. Only male C57BL/6 db/db mice were used for experiments due to their higher incidence of diabetes . Experimental db/db mice were maintained on a normal diet and were examined from the initial appearance of hyperglycaemia (8 weeks of age) until the development of overt diabetes and renal injury was evident in half of all wild-type males (32 weeks of age). Although these animals develop more slowly progressing diabetes compared with the hybrid C57BL/KsJ db/db strain , their genetic purity is maintained after intercrossing with C57BL/6 knock-out strains, which makes them a more appropriate strain for identifying the effects of molecular deficiencies on a defined genetic background .
The development of obesity and hyperglycaemia was examined in groups of Ccl2+/+ and Ccl−/−db/db mice (n = 40–50) between 8 and 32 weeks of age. From each of these groups, 20 mice were randomly selected for assessment of glucose and insulin tolerance and fasting insulin levels at 8 and 32 weeks. Blood glucose levels were measured every 4 weeks by tail vein sampling using a Medisense glucometer (Abbott Laboratories, Bedford, MA, USA), and diabetes was defined as random morning blood glucose ≥16 mmol/l (300 mg/dl).
For assessment of adipose inflammation, groups of Ccl2+/+ and Ccl2−/−db/db mice (n = 10) were randomly selected and killed at 8 and 32 weeks to collect epididymal fat pads. For analysis of diabetic renal injury, subgroups of Ccl2+/+ and Ccl2−/−db/db mice (n = 10) with equivalent hyperglycaemia and obesity were selected and killed at 8 and 32 weeks. Groups of non-diabetic db/+ mice (n = 10) were killed at 8 and 32 weeks to obtain control tissue.
Approval for these animal studies was obtained from the Monash Medical Centre Animal Ethics Committee in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th edition (2004).
To genotype db/db mice, DNA was extracted from tails with proteinase K digestion . PCR analysis of genomic DNA was performed to identify the intact MCP-1 gene (Ccl2) or the neomycin resistance cassette (Neo) used in the disruption of Ccl2. The Ccl2 PCR involved thermal cycling conditions of 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 60 s and 72°C for 60 s. The Neo PCR used 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 120 s. The PCR primers used were: Ccl2 (5′ ACA GCT TCT TTG GGA CAC C 3′, 5′ GGA GCA TCC ACG TGT TGG C 3′); Neo (5′ATT GAA CAA GAT GGA TTG CAC 3′, 5′ CGT CCA GAT CAT CCT GAT C 3′). The resulting PCR products (Ccl2, 869 bp, Neo, 500 bp) were isolated by electrophoresis on a 2.2% agarose gel and detected with ethidium bromide (Sigma, St Louis, MO, USA).
Urine was collected from mice housed in metabolic cages for 24 h at 8, 16, 24 and 32 weeks of age. At the end of experimentation, heparinised whole blood and serum were collected from anaesthetised mice after fasting (6 h) by cardiac puncture. HbA1c levels and plasma levels of creatinine (creatininase method), triacylglycerol and cholesterol were analysed by the Biochemistry Department at the Monash Medical Centre. ELISA kits were used to measure plasma insulin (Linco Research, St Charles, MO, USA), urine albumin (Bethyl Laboratories, Montgomery, TX, USA) and urine MCP-1 (R&D Systems, Minneapolis, MN, USA). Serum NEFA were assessed with a colorimetric assay kit (Wako Pure Chemical Industries, Osaka, Japan).
Glucose and insulin tolerance tests
To determine glucose tolerance, mice were injected intraperitoneally with d-glucose (1 g/kg) after a 12 h fast. For examining insulin tolerance, mice were injected intraperitoneally with human insulin (2 U/kg; Actrapid, Novo Nordisk, Bagsvaerd, Denmark) after a 6 h fast. For both tests, blood samples were collected at 0, 30, 60, 90 and 120 min and blood glucose was measured using the glucose oxidase method.
Real-time RT-PCR analysis
RNA was extracted from snap-frozen epididymal fat and whole kidney using a RiboPure RNA isolation kit (Ambion, Austin, TX, USA). cDNA was prepared from total RNA by reverse transcription using random hexamer primers and Superscript II (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time RT-PCR was performed using primers and 6-carboxyfluorescein (FAM)-labelled minor groove binder (MGB) probes for mouse Ccl2, Tnfa and Adipoq. In each sample, ribosomal 18S was amplified using a commercial kit containing a VIC-labelled probe plus primers (Applied Biosystems, Foster City, CA, USA). PCR was performed over 50 cycles on a Rotor-Gene 3000 system (Corbett Research, Sydney, NSW, Australia) using a RealMastermix Probe kit (Eppendorf, Hamburg, Germany), UDG (New England Biolabs, Beverly, MA, USA) and VIC plus FAM-labelled primer/probe combinations. Each PCR involved thermal cycling conditions of 37°C for 10 min, 95°C for 5 min, followed by 50 cycles of 95°C for 15 s, 60°C for 20 s and 68°C for 20 s. The primer pairs and probes used were: Ccl2 (forward, 5′ GAC CCG TAA ATC TGA AGC TAA 3′; reverse, 5′ CAC ACT GGT CAC TCC TAC AGA A 3′; probe, 5′ ACA ACC ACC TCA AGC AC 3′); Tnfa (forward, 5′ GGC TGC CCC GAC TAC GT 3′; reverse, 5′ TTT CTC CTG GTA TGA GAT AGC AAA TC 3′; probe, 5′ TCA CCC ACA CCG TCA G 3′); and Adipoq (forward, 5′ CAT TCC GGG ACT CTA CTA CT 3′; reverse, 5′CTG ATA CTG GTC GTA GGT GAA G 3′; probe, 5′ GAA AGA TGT GAA GGT GA 3′). The relative abundance of each mRNA was calculated using the comparative Ct (ΔCt) method and normalised against 18S.
To verify deficiency of Ccl2 mRNA in Ccl2−/−db/db mice, qualitative RT-PCR analysis was performed on cDNA samples prepared from the epididymal fat mRNA of Ccl2+/+ and Ccl2−/−db/db mice. Each PCR was run for 35 cycles using the same Ccl2 and 18S primers and thermocycling conditions as in real-time RT-PCR. The resulting PCR products (Ccl2, 79 bp; 18S, 100 bp) were isolated by electrophoresis on a 2.2% agarose gel and detected with ethidium bromide (Sigma).
Formalin-fixed kidney sections (2 μm) were stained with periodic acid–Schiff reagent to identify structure and haematoxylin to distinguish cell nuclei. Digital images of the tissue sections were obtained from microscopy (magnification ×400). Glomerular cellularity, glomerular volume and tubular atrophy were determined by image analysis as previously described . All scoring was performed on blinded slides.
Antibodies used in this study were: rat anti-mouse CD68 (FA-11; Serotec, Oxford, UK); rat anti-mouse F4/80 (Serotec); rat anti-mouse CD169 (a macrophage activation marker ; Serotec); goat anti-mouse collagen IV (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and fluorescein-conjugated anti-α-smooth muscle actin (1A4, Sigma). Normal rabbit and goat serum and isotype-matched irrelevant rat IgGs were used as negative controls.
Immunoperoxidase staining was used to detect expression of CD68, CD169 and collagen IV in 2% paraformaldehyde–lysine–periodate-fixed kidney cryostat sections and F4/80 and α-smooth muscle actin in formalin-fixed sections . Activated macrophages were detected by two-colour immunostaining for CD68 and CD169 .
Quantitation of immunohistochemistry
Adipose macrophage accumulation, expressed as cells per mm2, was assessed by counting the number of F4/80+ macrophages in 20 fields (magnification ×250) in both epididymal fat pads of each mouse. Kidney macrophage accrual was assessed by immunostaining with CD68, because most glomerular macrophages are not detected with the F4/80 antibody . Glomerular macrophage accumulation was determined by counting the number of immunostained CD68+ cells in 20 hilar glomerular tuft cross-sections per animal (magnification ×400). Immunostained interstitial macrophages, expressed as CD68+ cells per mm2, were counted in 25 consecutive interstitial fields (magnification ×250), representing 30–40% of kidney cortex in the cross-section. Expression of collagen IV and α-smooth muscle actin was assessed as the percentage area stained within the glomerular tuft or the cortical interstitium, using image analysis software (Image Pro Plus; Media Cybernetics, Silver Spring, MD, USA). All scoring was performed on blinded slides.
Statistical differences between two groups were analysed by the unpaired Student’s t test and differences between multiple groups were assessed by one-way ANOVA using Tukey’s multiple comparison test. Correlation analyses were performed using Pearson’s coefficient. Data were recorded as the means±SEM and values of p < 0.05 were considered significant. All analyses were performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA).
MCP-1/Ccl2 expression is increased in obese db/db mice
Characteristics of db/+ and db/db strains
Epididymal fat (g)
0.62 ± 0.03
1.69 ± 0.12a
1.53 ± 0.11a
1.36 ± 0.07
1.40 ± 0.22
1.53 ± 0.12
Blood glucose (mmol/l)
8.2 ± 0.4
14.1 ± 0.8a
14.7 ± 0.9a
8.5 ± 0.2
21.4 ± 2.0a
20.7 ± 2.0a
3.3 ± 0.2
4.9 ± 0.3a
4.6 ± 0.2a
3.9 ± 0.1
6.3 ± 0.3a
6.0 ± 0.4a
Plasma insulin (pmol/l)
37 ± 2
327 ± 30a
325 ± 49a
47 ± 10
405 ± 122a
515 ± 113a
Plasma cholesterol (mmol/l)
2.1 ± 0.2
2.3 ± 0.2
2.3 ± 0.1
2.0 ± 0.2
3.0 ± 0.2a
2.9 ± 0.2a
Plasma triacylglycerol (mmol/l)
0.9 ± 0.1
1.0 ± 0.2
0.9 ± 0.1
0.7 ± 0.1
1.7 ± 0.3b
1.5 ± 0.4c
Serum NEFA (mmol/l)
0.68 ± 0.04
0.69 ± 0.07
0.64 ± 0.05
0.51 ± 0.06
0.78 ± 0.03a
0.78 ± 0.05a
MCP-1 deficiency does not affect adipose inflammation in diabetic db/db mice
MCP-1 deficiency reduces renal inflammation in diabetic db/db mice
Equivalent diabetes at 32 weeks of age in db/db strains undergoing assessment for renal injury
Ccl2+/+db/db (n = 10)
Ccl2−/−db/db (n = 10)
Body weight (g)
44.0 ± 3.7
48.4 ± 3.6
Blood glucose (mmol/l)
36.7 ± 4.4
37.2 ± 2.9
8.9 ± 0.7
9.3 ± 0.6
MCP-1 deficiency decreases kidney damage in diabetic db/db mice
Histological injury in experimental mice at 32 weeks of age
Volume (μm3 × 104)
2.94 ± 0.14
8.46 ± 0.71a
5.04 ± 0.38c,d
34.4 ± 0.8
56.8 ± 2.6a
40.1 ± 1.9d
Collagen IV (% area)
16.4 ± 0.5
25.8 ± 1.3a
18.6 ± 1.2d
Atrophic tubules (%)
0.03 ± 0.01
16.4 ± 1.9a
7.6 ± 1.2b,d
α-SMA (% area)
1.1 ± 0.1
4.8 ± 0.9a
1.8 ± 0.2e
Collagen IV (% area)
17.2 ± 0.4
25.5 ± 1.4a
19.4 ± 0.6d
Using a gene knock-out approach, our study demonstrates that the development of type 2 diabetes in db/db mice is independent of MCP-1. Deficiency of MCP-1 had no effect on the incidence of diabetes or the levels of obesity, hyperglycaemia, hyperinsulinaemia and hyperlipidaemia. In addition, the impairment of glucose and insulin tolerance observed during the early and advanced stages of disease were unaffected by the absence of MCP-1, suggesting that MCP-1 does not contribute to peripheral insulin resistance. In contrast, MCP-1 deficiency provided marked protection against the development of renal injury in diabetic db/db mice.
This study indicates that chronic inflammation in white adipose tissue is independent of MCP-1 in db/db mice. Levels of Ccl2 mRNA were increased five- to sixfold in the epididymal fat of wild-type db/db mice at 8 and 32 weeks compared with lean db/+ mice, and correlated with body weight and epididymal fat mass. This suggests that obesity induces MCP-1 production in visceral adipose tissue. However, at 8 and 32 weeks of age, epididymal fat from Ccl2+/+ and Ccl2−/−db/db mice had a similar increase in macrophage numbers and Tnfa mRNA and a similar decrease in Adipoq mRNA, suggesting equivalent inflammation. These findings are also consistent with the development of glucose and insulin tolerance being similar in the two strains. In comparison, a human study recently showed that pioglitazone treatment improved insulin sensitivity in subjects with impaired glucose tolerance in association with reductions in MCP-1 and macrophages in subcutaneous adipose tissue, suggesting that these events were related . However, on the basis of our present findings, it now appears likely that pioglitazone may have affected insulin sensitivity, MCP-1 levels and macrophage accrual through different mechanisms.
Our data in db/db mice demonstrate that MCP-1 is redundant as a macrophage chemokine during adipose inflammation and that any direct functional effects of MCP-1 on adipocytes or adipose macrophage activation do not contribute to the development of type 2 diabetes. In comparison, two recent studies in a high-fat diet model of obesity have suggested that transgenic overexpression of Ccl2 in adipose tissue promotes adipose macrophage accumulation and insulin resistance [27, 28]. However, the aP2 promoter used in these experiments to drive transgenic Ccl2 expression is not specific for adipocytes and is also active in macrophages . Since MCP-1 is known to activate macrophages [13, 14], it is entirely feasible that the increased recruitment of macrophages into adipose tissue in these studies is facilitated by secretion of MCP-1 and other chemokines/cytokines by constitutively activated macrophages. One of these studies also found that a single intramuscular treatment with a plasmid encoding a human mutant form of Ccl2 (7ND), which dimerises with wild-type MCP-1 and inhibits its activity, was sufficient to reduce insulin resistance in obese db/db mice . Surprisingly, the effect of 7ND treatment on blood glucose levels and macrophage accumulation in adipose tissue was not examined in these mice. In addition, the selectivity of 7ND for the inhibition of MCP-1 compared with other related chemokines has yet to be established.
Studies of high-fat diet-induced obesity in mice deficient in chemokine CC motif receptor 2 (CCR2) are somewhat controversial, one study showing that these mice are unprotected from adipose inflammation and insulin resistance and another indicating protection [5, 30]. It is indeed possible that CCR2 deficiency may not duplicate the results of MCP-1 deficiency, because CCR2 is not an exclusive receptor for MCP-1, and other ligands capable of binding CCR2, such as MCP-3, are also increased in adipose tissue during obesity . In addition, adipose levels of MCP-1 and macrophage expression of CCR2 may not be equivalent in different mouse models of obesity, which may lead to variations in the severity and mechanisms of inflammation in adipose tissue.
The finding that adipose inflammation in obese db/db mice is independent of MCP-1 suggests that other mechanisms are responsible for adipose macrophage accumulation. Additional macrophage chemokines, including macrophage inhibitory protein-1α, macrophage migration inhibitory factor and macrophage-colony stimulating factor (M-CSF), are produced by adipocytes and may be important for adipose macrophage recruitment and activation [17, 31, 32]. In addition, M-CSF is known to promote tissue macrophage accrual by inducing local proliferation , and this may be a contributing mechanism for macrophage accumulation in adipose tissue.
Examination of Ccl2+/+ and Ccl2−/−db/db mice with equivalent diabetes showed that kidney macrophage accumulation (CD68+ cells) and activation (CD68+CD169+ cells) were MCP-1-dependent. In addition, kidney and urine MCP-1 correlated with kidney macrophage accrual in the Ccl2+/+db/db mice, suggesting that inflammation in diabetic kidneys can be monitored clinically through urine MCP-1 measurement. These findings are supported by studies showing that hyperglycaemia and advanced glycation end-products stimulate MCP-1 production by kidney cells and that MCP-1 promotes renal inflammation during streptozotocin-induced type 1 diabetes .
Although both epididymal fat and the kidney produce elevated levels of Ccl2 in db/db mice, Ccl2 was only associated with inflammation in the kidney. Ccl2 was increased early in the epididymal fat of db/db mice, coinciding with the onset of obesity, but preceding the development of diabetes. In the epididymal fat of db/db mice, Ccl2 levels did not correlate with macrophage accumulation, suggesting that MCP-1 is not an important contributor to this event. In contrast, kidney Ccl2 levels in db/db mice did correlate with kidney macrophage accumulation and the development of diabetes and were found to be functionally important for the renal inflammation. These findings, along with other in vitro studies , suggest that MCP-1 production is induced by different mechanisms in epididymal fat (obesity) and the diabetic kidney (hyperglycaemia, advanced glycation end-products), and these different environments may influence whether or not MCP-1 promotes inflammation.
This study supports a role for MCP-1-dependent macrophage accumulation in promoting type 2 diabetic renal injury. A reduction in kidney macrophage accumulation and activation in diabetic Ccl2−/−db/db mice was associated with a similar decline in glomerular pathology (hypertrophy, hypercellularity, and fibrosis) and interstitial pathology (tubular atrophy, myofibroblast accumulation and fibrosis). The significance of these effects was also detected clinically in terms of reductions in urine albumin excretion (62%) and plasma creatinine (65%) in diabetic Ccl2−/−db/db mice. However, it should be noted that creatinine measurements in these obese mice are not a particularly reliable measure of renal function, since significant muscle wastage occurs during disease progression .
In summary, this study has demonstrated that MCP-1 promotes macrophage accumulation in kidneys but not in epididymal fat during the development of type 2 diabetes in db/db mice. Although MCP-1 had no impact on the physiological characteristics of type 2 diabetes, it played a significant role in the progression of diabetic renal injury. Therefore, neutralising MCP-1 activity should be viewed as an important therapeutic strategy in the treatment of type 2 diabetic nephropathy.
This work was funded by project grants from the Diabetes Australia Research Trust and the National Health and Medical Research Council of Australia and fellowship support by Kidney Health Australia and the Australian and New Zealand Society of Nephrology.
Duality of interest
None of the authors has any duality of interest in connection with this study.