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. 2008 Feb;172(2):288-98.
doi: 10.2353/ajpath.2008.070726. Epub 2008 Jan 17.

Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis

Neil C Henderson  1 , Alison C Mackinnon, Sarah L Farnworth, Tiina Kipari, Christopher Haslett, John P Iredale, Fu-Tong Liu, Jeremy Hughes, Tariq Sethi
Affiliations

Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis

Neil C Henderson et al. Am J Pathol. 2008 Feb.

Abstract

Macrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a beta-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-gamma/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-beta expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3(-/-) macrophages did, however, restore the fibrotic phenotype in galectin-3(-/-) mice. Cross-over experiments using wild-type and galectin-3(-/-) macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis.

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Figures

Figure 1
Figure 1
Galectin-3 expression is up-regulated in a mouse model of progressive renal fibrosis (UUO). Galectin-3 expression in tubular epithelium in sham-operated mouse kidney (a) and after 14 days of UUO (b). c: Real-time PCR quantification of galectin-3 expression in whole kidney homogenates from control sham-operated (day 0) and at 3, 7, and 14 days after UUO. ***P < 0.0001 compared with control. Scale bar = 100 μm.
Figure 2
Figure 2
Absence of galectin-3 protects against renal fibrosis. Mice underwent control (sham operation) or UUO and were sacrificed at 7 days (n = 6 mice in each group). a–c: Renal collagen deposition was examined by picrosirius red staining (a, b) and quantified using digital image analysis (c). c: Significantly reduced collagen deposition was observed in the galectin-3−/− mice compared with WT 7 days after ureteral ligation (*P < 0.05). d: Furthermore, transcripts for procollagen (I) were also reduced in the galectin-3−/− group compared with WT animals (*P < 0.05). e and f: Immunohistochemistry revealed markedly reduced α-SMA positivity (a marker of myofibroblast activation) in galectin-3−/− compared with WT mice 7 days after ureteral ligation. g: α-SMA was quantified using digital image analysis, and significantly less α-SMA staining occurred in the galectin-3−/− mice compared with WT (*P < 0.05). h: α-SMA mRNA transcripts, as assessed by real-time RT-PCR, were significantly decreased in the galectin-3−/− mice compared with WT animals (*P < 0.01). Scale bars = 100 μm.
Figure 3
Figure 3
Macrophage ablation by DT reduces renal fibrosis after UUO. Mice underwent UUO and received DT or control vehicle after ureteric ligation (n = 6 mice in each group) as described in Materials and Methods. Kidneys were harvested at day 7, and immunostaining was performed for the specific macrophage marker F4/80 (a, b), α-SMA (d, e), and collagen (g, h) in vehicle control-treated (left) and DT-treated (right) mice. c: Quantification of F4/80 staining (macrophage infiltration) by digital image analysis. Real-time RT-PCR quantitation of α-SMA (f) and procollagen (I) expression (i) in vehicle- and DT-treated mice. *P < 0.05 compared to vehicle treated mice. Scale bars = 100 μm.
Figure 4
Figure 4
Macrophage ablation by DT does not affect recruitment of CD34+/ColI+ and CD45+/ColI+ fibrocytes after UUO. a: CD34; b: ColI; c: merged. Arrows indicate CD34+/ColI+ fibrocytes. d: The number of infiltrating fibrocytes (CD34+/ColI+ and CD45+/ColI+) increased at day 7 after UUO, with no significant difference in infiltrating fibrocyte numbers between non-DT-treated (white bars) and DT-treated (black bars) mice. Day 7 UUO DT−CD34+/ColI+, 26.33 ± 2.8 per mm2; day 7 UUO DT+CD34+/ColI+, 29.83 ± 4.7 per mm2 (n = 6 mice in each group, P = NS). Day 7 UUO DT−CD45+/ColI+, 35.67 ± 3.2 per mm2 (n = 6), day 7 UUO DT+ CD45+/ColI+, 37.3 ± 4.1 per mm2 (n = 6 mice in each group, P = NS). Values are the mean ± SEM. Scale bars = 100 μm.
Figure 5
Figure 5
Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO or macrophage proinflammatory cytokine profiles in response to IFN-γ/LPS. a–d: H&E staining of kidneys from WT and galectin-3−/− mice 3 days after control sham operation or UUO. Renal macrophages were stained with F4/80 (e–h) and quantitated by digital image analysis (i). Macrophage recruitment was similar in WT and galectin-3−/− mice at all time points studied after UUO (days 0, 3, 7, and 14) (n = 6 mice in each group, P = NS). BMDMs and peritoneal macrophages were stimulated with IFN-γ (100 U/ml) for 48 hours, and LPS (100 ng/ml) was added for the last 24 hours. BMDM supernatants were assayed for IL-6 (j) and TNF-α (k) (galectin-3−/−, open bars; WT, filled bars). Peritoneal macrophage supernatants were assayed for IL-6 (l) and TNF-α (m) (galectin-3−/−, open bars; WT, filled bars). The results represent the mean ± SEM of three experiments (P = NS). Scale bars = 100 μm.
Figure 6
Figure 6
Disruption of the galectin-3 gene does not affect TGF-β expression or Smad 2/3 phosphorylation in obstructed kidneys. WT and galectin-3−/− mice underwent UUO, and kidneys were harvested at days 3, 7, and 14. a–c: RNA was extracted, and TGF-β gene expression was measured as described in Materials and Methods by real-time RT-PCR. TGF-β mRNA transcripts were markedly elevated compared to control after UUO at days 3, 7, and 14 (P < 0.01 compared to control kidney). However, there was no significant difference in renal TGF-β mRNA expression between WT and galectin-3−/− mice after UUO at any of the time points studied. P = NS. d: Western blot of pSMAD2 and pSMAD3 expression in lysates from control and UUO kidneys at day 7 (each lane represents a different mouse). e: Quantification by optical densitometry using Image J.
Figure 7
Figure 7
Macrophage-derived galectin-3 drives myofibroblast accumulation/activation in the kidney after UUO. a: Galectin-3 immunofluorescence staining of WT BMDMs. Nuclei are labeled with 4,6-diamidino-2-phenylindole. b: Representative Western blot of galectin-3 expression in WT BMDM lysate and supernatant. c–m: Galectin-3−/− mice underwent UUO, and mature WT or galectin-3−/− BMDMs (5 × 106) were adoptively transferred at days 1, 3, and 5 after ureteric ligation (n = 6 in each group) and kidneys were harvested at day 7 after UUO. c: Staining of WT macrophages with galectin-3 antibody at day 7 after UUO demonstrating recruitment and engraftment of adoptively transferred WT macrophages. d: Control staining of galectin-3−/− macrophages with galectin-3 antibody. e and f: Fluorescence microscopy demonstrating Cell Tracker Orange-labeled WT (e) and galectin-3−/− (f) macrophages recruited and engrafted at day 7 after UUO. g and h: α-SMA staining of obstructed kidney after adoptive transfer of WT (g) or galectin-3−/− (h) macrophages (arrows indicate α-SMA staining of blood vessels). i and j: Collagen staining (picrosirius red stain) of obstructed kidney after adoptive transfer of WT (i) or galectin-3−/− (j) macrophages. k: Quantitation of recruitment and engraftment of WT and galectin-3−/− macrophages by digital image analysis (P = NS). l: Quantitation of α-SMA staining using digital image analysis. m: Quantitation of collagen (picrosirius red) staining using digital image analysis. n: Quantitation of procollagen (I) gene expression by real-time RT-PCR. *P < 0.01 compared to WT adoptively transferred macrophages. Scale bars = 100 μm.
Figure 8
Figure 8
Galectin-3-positive macrophages promote renal fibroblast activation in vitro. Mature BMDMs (1 × 106 cells) were plated in six-well tissue culture dishes, incubated in serum-free media, and conditioned media (0.5 ml) was collected after 48 hours. Galectin-3−/− renal fibroblasts were isolated by trypsin digestion as described in Materials and Methods. rG3 refers to recombinant mouse galectin-3 (30 μg/ml). Conditioned media (CM) from BMDMs (0.5 ml) or control media were added to 0.5 ml of renal fibroblasts (4 × 104 cells). WT BMDM-conditioned media were also added in the presence of 5 μmol/L galectin-3 inhibitor (G-3I) [bis-(3-deoxy-3-{3-methoxybenzamido}-β-d-galactopyranosyl)-sulfane]. Cells were incubated for a further 48 hours (final fetal calf serum concentration, 7.5%) before lysis. α-SMA expression in galectin-3−/− renal fibroblast lysates was measured by Western analysis and quantified by optical densitometry using Image J. Results represent the mean ± SEM of three to four independent experiments.
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