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, 94 (4), 756-772

Targeted Inhibition of the Type 2 Cannabinoid Receptor Is a Novel Approach to Reduce Renal Fibrosis

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Targeted Inhibition of the Type 2 Cannabinoid Receptor Is a Novel Approach to Reduce Renal Fibrosis

Lili Zhou et al. Kidney Int.

Abstract

The cannabinoid receptor type 2 (CB2) is a G protein-coupled seven transmembrane receptor that transmits endogenous cannabinoid signaling. The role of CB2 in the pathogenesis of kidney injury and fibrosis remains poorly understood. Here we demonstrate that CB2 was induced, predominantly in kidney tubular epithelium, in various models of kidney disease induced by unilateral ureteral obstruction, adriamycin or ischemia/reperfusion injury. In vitro, forced expression of CB2 or treatment with a CB2 agonist was sufficient to trigger matrix gene expression, whereas knockdown of CB2 by siRNA abolished transforming growth factor-β1-induced signaling and fibrogenic responses in kidney tubular cells. CB2 also mediated fibroblasts and macrophage activation in vitro. Mice with genetic ablation of CB2 were protected against kidney injury after ureteral obstruction, validating a pathogenic role of CB2 in renal fibrosis in vivo. By using in silico screening and medicinal chemistry modifications, we discovered a novel compound, XL-001, that bound to CB2 with high affinity and selectivity and acted as an inverse agonist. Incubation with XL-001 inhibited in a dose-dependent fashion the fibrogenic response induced by CB2 overexpression, CB2 agonist or transforming growth factor-β1. In vivo, intraperitoneal injections of XL-001 ameliorated kidney injury, fibrosis and inflammation in both the obstruction and ischemia/reperfusion models. Delayed administration of XL-001 was also effective in ameliorating kidney fibrosis and inflammation. Thus, CB2 is a pathogenic mediator in kidney fibrosis and targeted inhibition with the novel inverse agonist XL-001 may provide a strategy in the fight against fibrotic kidney diseases.

Keywords: CB2; cannabinoid; chronic kidney disease; inverse agonist; renal fibrosis.

Conflict of interest statement

Conflict of Interest

L.Z and P.Y are co-inventors of a patent involving XL-001 treatment for CKD.

Figures

Figure 1
Figure 1
Renal expression of CB2 is induced in various models of CKD. (a, b) Quantitative real-time RT-PCR (qRT-PCR) showed the relative abundances of CB1 (a) and CB2 (b) mRNA in sham and UUO kidneys at 7 days after UUO. *P < 0.05 versus sham controls (n = 5 to 6). (c) Immunohistochemical staining demonstrated CB2 protein expression and localization in the obstructed kidneys at 7 days after UUO. Paraffin-embedded kidney sections were stained with CB2 antibody. Boxed area was enlarged. Arrow indicates positive tubular staining. Arrowhead denotes interstitial cells stained positively for CB2. Scale bar, 50 μm. (d, e) Western blotting analyses showed the renal expressions of CB2 protein in the obstructed kidneys at 7 days after UUO. Representative Western blot (d) and quantitative data (e) are presented. Numbers (1 ~ 4) indicate each individual animal in a given group. Relative CB2 levels (sham controls = 1.0) were presented after normalization with actin. *P < 0.05 versus sham controls (n = 5 to 6). (f, g) Renal CB2 expression is induced in adriamycin (ADR) nephropathy. CB2 expression was assessed in the kidneys at 3 weeks after ADR injection. Representative Western blot (f) and quantitative data (g) are presented. Numbers (1 ~ 4) indicate each individual animal in a given group. Relative CB2 levels (controls = 1.0) were presented after normalization with actin. *P < 0.05 versus controls (n = 5 to 6). (h, i) Western blotting analyses demonstrated renal CB2 expression at 11 days after ischemia/reperfusion injury (IRI). Representative Western blot (h) and quantitative data (i) are presented. Numbers (1 ~ 4) indicate each individual animal in a given group. Relative CB2 levels (sham controls = 1.0) were presented after normalization with actin. *P < 0.05 versus controls (n = 5).
Figure 2
Figure 2
CB2 mediates TGF-β1-induced fibrogenic responses in vitro. (a, b) Over-expression of CB2 induced fibronectin expression in vitro. HKC-8 cells were transfected with CB2 expression vector (pCMV-CB2) or empty vector (pcDNA3) as indicated. Numbers (1 to 3) represent triplicate wells in a given group. Representative Western blot (a) and quantitative data (b) are presented. *P < 0.05 versus pcDNA3 group. (c, d) Western blotting analyses show that TGF-β1 dose-dependently induced CB2 expressions in cultured tubular epithelial cells in vitro. Human kidney proximal tubular cells (HKC-8) were incubated with different concentrations of TGF-β1 as indicated. Representative Western blot (c) and quantitative data (d) are presented. *P < 0.05 versus controls. (e–g) Knockdown of CB2 inhibited TGF-β1-induced fibrogenic responses in vitro. HKC-8 cells were transfected with CB2-specific siRNA or control siRNA, followed by incubation with TGF-β1 (2 ng/ml). Cell lysates were immunoblotted with antibodies against CB2, fibronectin, α-SMA and GAPDH, respectively. Representative Western blot (e) and quantitative data on the relative abundance of fibronectin (f) and α-SMA (g) proteins in different groups are presented. *P < 0.05 versus controls. †P < 0.05 versus control siRNA in the presence of TGF-β1. (h) Representative images show immunofluorescence staining of fibronectin in different groups. Arrow indicates positive staining. Scale bar, 50 μm. (i) Western blotting analyses show that TGF-β1-induced signal transduction was significantly attenuated by knockdown of CB2. Human kidney proximal tubular cells (HKC-8) were transfected with CB2 siRNA, then incubated with TGF-β1. Quantitative data (j through m) are presented. *P < 0.05 versus controls. †P < 0.05 versus control siRNA in the presence of TGF-β1.
Figure 3
Figure 3
CB2 mediates the activations of fibroblasts and macrophages. (a, b) Western blotting analyses show that TGF-β1 induced CB2 expressions in fibroblasts in vitro. Rat normal kidney interstitial fibroblasts cells (NRK-49F) were incubated with TGF-β1 (2 ng/ml). Representative Western blot (a) and quantitative data (b) are presented. *P < 0.05 versus controls. (c–e) Knockdown of CB2 inhibited TGF-β1-induced fibrogenic responses in NRK-49F cells in vitro. NRK-49F cells were transfected with CB2-specific siRNA or control siRNA, followed by incubation with TGF-β1 (2 ng/ml). Cell lysates were immunoblotted with antibodies against fibronectin, collagen I and α-tubulin, respectively. Representative Western blot (c) and quantitative data on the relative abundance of fibronectin (d) and collagen I (e) proteins in different groups are presented. *P < 0.05 versus controls. †P < 0.05 versus control siRNA in the presence of TGF-β1. (f, g) Western blotting analyses show that lipopolysaccharide (LPS) induced CB2 expressions in macrophages in vitro. Mouse macrophage cell line (RAW264.7) was incubated with LPS (5 ng/ml). Representative Western blot (f) and quantitative data (g) are presented. *P < 0.05 versus controls. (h–j) Knockdown of CB2 inhibited LPS-induced M1-macrophage activation in vitro. RAW264.7 cells were transfected with CB2-specific siRNA or control siRNA, followed by incubation with LPS (5 ng/ml). Cell lysates were immunoblotted with antibodies against TNF-α, iNOS and GAPDH, respectively. Representative Western blot (h) and quantitative data on the relative abundance of TNF-α (i) and iNOS (j) proteins in different groups are presented. *P < 0.05 versus controls. †P < 0.05 versus control siRNA in the presence of LPS. (k–l) Knockdown of CB2 inhibited IL-4-induced M2-macrophage activation in vitro. RAW264.7 cells were transfected with CB2-specific siRNA or control siRNA, followed by incubation with IL-4 (20 ng/ml). Cell lysates were immunoblotted with antibodies against Mannose R (MR) and actin, respectively. Representative Western blot (k) and quantitative data on the relative abundance of MR (l) proteins in different groups are presented. *P < 0.05 versus controls. †P < 0.05 versus control siRNA in the presence of IL-4.
Figure 4
Figure 4
Mice with CB2 deficiency are protected against renal fibrosis after obstructive injury. (a) Representative micrographs show Masson’s trichrome staining (MTS) of the obstructed kidneys in CB2 null mice (CB2−/−) and wild-type controls (CB2+/+) at 7 days after UUO. Blue staining (arrow) indicates fibrotic collagen deposition. Scale bar, 50 μm. (b) Quantitative determination of the fibrotic area in CB2−/−null mice and CB2+/+ wild-type controls at 7 days after UUO. *P < 0.05 versus CB2+/+ group (n = 5 to 6). (c) Western blot analyses show that ablation of CB2 reduced renal expression of fibronectin and α-SMA after UUO. Numbers (1 to 4) represent different individual animals in a given group. (d, e) Graphic presentations of relative renal fibronectin (d) and α-SMA (e) protein levels in two groups as indicated. *P < 0.05 versus CB2+/+ group. (f) Immunostaining for renal fibronectin and α-SMA proteins in CB2+/+ and CB2+/+ mice as indicated. Frozen sections and paraffin sections were stained with different antibodies against fibronectin and α-SMA, respectively. Arrow indicates positive staining. Scale bar, 50 μm. (g) Western blot analyses show renal expression of β-catenin and its downstream MMP-7 after UUO. Numbers (1 to 4) represent different individual animals in a given group. (h, i) Graphic presentations of renal β-catenin (h) and MMP-7 (i) expressions in two groups as indicated. *P < 0.05 versus CB2+/+ mice. (j) Immunostaining for renal β-catenin proteins in CB2+/+ and CB2+/+ mice as indicated. Arrow indicates positive staining. Scale bar, 50 μm.
Figure 5
Figure 5
Discovery and characterization of compound XL-001, a novel inverse agonist of CB2. (a) Screening strategy for identification of XL-001. (b) The chemical structure of XL-001. (c) Binding affinity of XL-001 on the CB2 receptor (CB2 Ki = 0.5 nM). (d) Binding pose of XL-001 with the CB2 receptor. (e, f) The detailed 2D (e) and 3D (f) interactions between XL-001 and CB2. Hydrogen bonding interactions of XL-001 with Asn20 (2.20 Å) and Ser285 (3.67 Å) of CB2. The p-diehtylaminobenzene group formed strong hydrophobic interactions with Thr114 (3.74 Å), Trp194 (3.29 Å), and Trp258 (3.52 Å). Moreover, the p-methoxylbenzene group interacted strongly with Phe87 (3.46 Å) and Val113 (3.62 Å), while the p-methylbenzene group had a strong hydrophobic interaction with Phe183 (3.77 Å).
Figure 6
Figure 6
XL-001 inhibits CB2-mediated fibrogenic responses in vitro. (a) Western blot analyses show that XL-001 inhibited CB2 agonist-induced fibronectin and α-SMA protein expression. HKC-8 cells were incubated with specific CB2 agonist (AM1241) in the absence or presence of XL-001 for 24 hours. (b, c) Graphic presentations of the relative abundances of fibronectin (b) and α-SMA (c) proteins in different groups as indicated. *P < 0.05 versus controls; †P < 0.05 versus AM1241 alone. (d–e) XL-001 acted as a CB2 inverse agonist. HKC-8 cells were transiently transfected with CB2 expression vector (pCMV-CB2), and then incubated with AM1241 or XL-001 as indicated. Representative Western blotting (d) and quantitative data on fibronectin (e) and α-SMA (f) are presented. *P < 0.05 versus controls; †P < 0.05 versus pCMV-CB2 alone. (g) XL-001 also dose-dependently inhibited TGF-β1-induced fibronectin and α-SMA protein expression. HKC-8 cells were incubated with TGF-β1 (2 ng/ml) in the absence or presence of XL-001 for 24 hours. (h, i) Graphic presentations of the relative abundances of fibronectin (h) and α-SMA (i) proteins in different groups as indicated. *P < 0.05 versus controls; †P < 0.05 versus TGF-β1 alone. (j) Representative micrographs show the immunofluorescence staining of fibronectin expressions in different groups as indicated. Scale bar, 50 μm.
Figure 7
Figure 7
XL-001 reduces renal fibrosis in obstructive nephropathy. (a) Experimental design. (b) Representative micrographs show that XL-001 reduced renal interstitial collagen deposition and fibrosis. Kidney sections were subjected to Masson's trichrome staining (MTS). Arrow indicates positive staining. Scale bar, 50 μm. XL (10), XL-001 (10 mg/kg body weight); XL (20), XL-001 (20 mg/kg body weight). (c) Graphic presentation of kidney fibrotic lesions in different groups after quantitative determination. *P < 0.05 versus sham controls; †P < 0.05 versus UUO alone. (d) Representative macrographs show collagen I and α-SMA protein expression in different groups as indicated. Arrows indicate positive staining. Scale bar, 50 μm. (e) Western blotting analyses show renal protein levels of fibronectin, α-SMA, β-catenin, PAI-1 and Snail1 in different groups. Numbers (1 to 3) represent different individual animals in a given group. (f–i) Graphic presentations of renal fibronectin (f), α-SMA (g), β-catenin (h), PAI-1 and Snail1 (i) proteins in different groups as indicated. †P < 0.05 versus UUO alone.
Figure 8
Figure 8
XL-001 inhibits renal expression of pro-inflammatory cytokines and reduces renal inflammation after UUO. (a–d) qRT-PCR shows the relative abundances of renal TNF-α (a), MCP-1 (b), IL-6 (c), and RANTES (d) mRNA in different groups as indicated. *P < 0.05 versus sham controls. †P < 0.05 versus UUO alone (n = 5 to 6). The doses of XL-001 (0, 10, 20 mg/kg body weight) were indicated. (e) Representative micrographs show renal infiltration of CD3+ T cells and MR+ macrophages in different groups as indicated. Paraffin-embedded kidney sections were stained with antibodies against RANTES. Arrow indicates positive staining. Scale bar, 50 μm. (f) Western blot analyses show renal levels of the phosphorylated-p65 (p-p65) and total p65 in different groups as indicated. Numbers (1 to 3) represent different individual animals in a given group. (g, h) Graphic presentations show the relative protein levels of renal p-p65 (g) and total p65 (h) in different groups. †P < 0.05 versus UUO alone.
Figure 9
Figure 9
XL-001 reduces renal fibrosis after ischemia-reperfusion injury. (a) Diagram shows the experimental design. Red and black arrows indicate the starting point of IRI and unilateral nephrectomy (UNx). Green bar and arrowheads indicate XL-001 treatment (20 mg/kg body weight). (b) Serum creatinine levels were measured at 11 days after IRI. *P < 0.05 versus sham controls. †P < 0.05 versus IRI alone (n = 5 to 6). (c) Urinary albumin levels in different groups at 11 days after IRI. Urinary albumin was expressed as milligrams per milligram creatinine. *P < 0.05 versus sham controls. †P < 0.05 versus IRI alone. (d) Representative micrographs show collagen deposition in different groups as indicated. Paraffin sections were subjected to Masson trichrome staining (MTS). Arrow indicates positive staining. Scale bar, 50 μm. (e) Representative micrographs show fibronectin and α-SMA expression in different groups as indicated. Kidney sections were stained with different antibodies against fibronectin and α-SMA, respectively. (f) Western blotting show that XL-001 inhibited fibronectin and α-SMA expression after IRI. Numbers (1 to 3) represent different individual animals in a given group. (g, h) Graphic presentations show the relative abundance of fibronectin (g) and α-SMA (g) protein expression in different groups as indicated. *P < 0.05 versus sham controls. †P < 0.05 versus IRI alone (n = 5 to 6). (i, j) Western blotting show that XL-001 inhibited renal β-catenin expression. Numbers (1 to 3) represent different individual animals in a given group. Representative Western blot (i) and quantitative data are presented. *P < 0.05 versus sham controls. †P < 0.05 versus IRI alone (n = 5 to 6). (k) Representative micrographs show renal β-catenin expression in different groups as indicated. Arrow indicates positive staining. Scale bar, 50 μm.
Figure 10
Figure 10
XL-001 is effective in protecting against renal fibrosis and inflammation in an established obstructive nephropathy. (a) Experimental design. Red arrows indicate the time of UUO surgery. Green bars indicate XL-001 treatment. (b) Representative micrographs show kidney fibrotic lesions in different groups. Paraffin sections were used for Masson’s trichrome staining. Scale bar, 50 μm. (c) Western blot analyses show renal expression of fibronectin, α-SMA, β-catenin, PAI-1 and MMP-7 in different groups as indicated. Numbers (1 to 3) indicate individual animals in a given group. (d–h) Graphic presentations show the relative protein levels of fibronectin (d), α-SMA (e), β-catenin (f), PAI-1 (g) and MMP-7 (h) in different groups. *P < 0.05 versus controls; †P < 0.05 versus UUO alone (n = 5 to 6). (i) Western blotting analyses show p-p65 in different groups as indicated. Quantitative data (j) is presented. *P < 0.05 versus controls; †P < 0.05 versus UUO alone. (k, l) Graphic presentations show the relative levels of TNF-α (k) and RANTES (l) mRNA in different groups. (m) Representative micrographs show the immunofluorescence staining of mannose R expressions in the UUO kidneys after injections with vehicle or XL-001. Scale bar, 50 μm.

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