Blockade of CCR2 ameliorates progressive fibrosis in kidney

Abstract

Fibrosis is a hallmark of progressive organ diseases. Monocyte chemoattractant protein (MCP)-1, also termed as macrophage chemotactic and activating factor (MCAF/CCL2) and its receptor, CCR2 are presumed to contribute to progressive fibrosis. However, the therapeutic efficacy of MCP-1/CCR2 blockade in progressive fibrosis remains to be investigated. We hypothesized that blockade of CCR2 may lead to the improvement of fibrosis. To achieve this goal, we investigated renal interstitial fibrosis induced by a unilateral ureteral obstruction in CCR2 gene-targeted mice and mice treated with propagermanium or RS-504393, CCR2 inhibitors. Cell infiltrations, most of which were F4/80-positive, were reduced in CCR2 knockout mice. In addition, dual staining revealed that CCR2-positive cells were mainly F4/80-positive macrophages. Importantly, CCR2 blockade reduced renal interstitial fibrosis relative to wild-type mice. Concomitantly, renal transcripts and protein of MCP-1, transforming growth factor-beta, and type I collagen were decreased in CCR2-null mice. Further, this CCR2-dependent loop for renal fibrosis was confirmed by treatment with CCR2 antagonists in a unilateral ureteral obstruction model. These findings suggest that the therapeutic strategy of blocking CCR2 may prove beneficial for progressive fibrosis via the decrease in infiltration and activation of macrophages in the diseased kidneys.

Figures

Figure 1

Inhibition of CCR2 reduced renal pathology. a: Severe cell infiltration, tubular atrophy, and interstitial fibrosis were observed 14 days after ureteral ligation in the outer medulla of wild-type mice. In contrast, such lesions in the outer medulla were significantly reduced in CCR2 gene-targeted mice (b) and propagermanium-treated mice at the dose of 3 mg/kg (c). d: A sham-operated mouse kidney.

Figure 2

CCR2 deletion reduced interstitial fibrosis. a and d: Progressive interstitial lesions exhibited interstitial fibrosis in wild-type mice on day 14. In contrast, the mean interstitial fibrosis, expressed as percentage involvement of the field, was reduced in CCR2 gene-targeted mice (b, d) and mice treated with propagermanium (c, d) or RS-504393 (d) as compared with wild-type mice. Values are the mean ± SEM. PG(3) mice treated with propagermanium at the dose of 3 mg/kg; PG(8) mice treated with propagermanium (8 mg/kg); PG(8L), mice treated with propagermanium (8 mg/kg) from 4 days after ureter ligation; RS, RS-504393. *, P < 0.05 as compared with wild-type mice. Original magnifications, ×200.

Figure 3

CCR2 deletion reduced type I collagen expression. a: Type I collagen was detected by immunohistochemical analysis. b: Type I collagen expression was reduced in the interstitium in CCR2 gene-targeted mice and propagermanium-treated mice compared with that of wild-type mice. c: The up-regulated mRNA expression of type I collagen in diseased kidneys was reduced by CCR2 blockade, whereas mRNA expression of type I collagen was faintly detected in normal kidneys. Values are the mean ± SEM. PG(3) mice treated with propagermanium at the dose of 3 mg/kg. *, P < 0.05 as compared with wild-type mice. Original magnification, ×400.

Figure 4

Reduced expression of renal MCP-1 by CCR2 deletion. a: MCP-1 protein was detected mainly in tubular epithelial cells and infiltrates at day 14. b: MCP-1 protein expression was reduced in the interstitium in CCR2 gene-targeted mice and propagermanium-treated mice (3 mg/kg) compared with that of wild-type mice. Values are the mean ± SEM. *, P < 0.05 as compared with wild-type mice. c: Transcripts of MCP-1 were faintly detected from normal kidneys by real-time RT-PCR. In contrast, transcripts of MCP-1 in diseased kidneys were up-regulated in wild-type mice, which were reduced by CCR2 blockade. Original magnification, ×400.

Figure 5

Renal TGF-β expression was reduced by the deletion of CCR2. a: Up-regulation of TGF-β1 protein was detected mainly in tubular epithelial cells in diseased kidneys in wild-type mice at day 14. In contrast, TGF-β1 protein was reduced of interstitium in CCR2 gene-targeted mice (b) and propagermanium-treated mice (3 mg/kg) (c). TGF-β1 immunoreactivity was not detected in sections incubated with the blocking peptide (d). e: The percentage of TGF-β1-positive area in one field is shown. f: Transcripts of TGF-β were faintly detected in normal kidneys by real-time RT-PCR. In contrast, transcripts of TGF-β in diseased kidneys were up-regulated in wild-type mice. The levels of these transcripts were reduced by CCR2 blockade. In wild-type mice, CCR2-positive cells were visualized with fluorescein isothiocyanate (g) and TGF-β1-positive cells with Cy3 (i). h: Some of the infiltrating CCR2-positive cells were also positive for TGF-β1 in injured kidneys at day 14. Values are the mean ± SEM. *, P < 0.05 as compared with wild-type mice. Original magnifications, ×400.

Figure 6

Interstitial F4/80-positive macrophages, most of which were positive for CCR2, were reduced. a: The number of F4/80-positive cells was reduced in CCR2 gene-targeted mice and mice treated with propagermanium or RS-504393 7 and 14 days after ureteral ligation compared with that of wild-type mice. F4/80-positive cells were visualized with Cy3 and CCR2-positive cells with fluorescein isothiocyanate. In wild-type mice, CCR2 (b)- and F4/80 (d)-positive cells were detected in the outer medulla 14 days after ureteral ligation. c: Most infiltrated F4/80-positive cells were also positive for CCR2 in injured kidneys. e: The number of CCR2-positive cells was similarly reduced in mice treated with propagermanium or RS-504393 7 and 14 days after ureteral ligation compared with that of wild-type mice. Values are the mean ± SEM. PG(3), mice treated with propagermanium at the dose of 3 mg/kg; PG(8), mice treated with propagermanium (8 mg/kg); PG(8L), mice treated with propagermanium (8 mg/kg) 4 days after ureter ligation; RS, RS-504393. *, P < 0.05 as compared with wild-type mice. Original magnifications, ×400.