Part 3:Cannabinoid Receptor Type 2 Promotes Kidney Fibrosis Through Orchestrating B-catenin Signaling
Mar 06, 2022
Contact: emily.li@wecistanche.com
However, it has rarely been reported that there is a relationship between CB2 and β-catenin.
CB2 is a member of the GPCR family. We previously found that CB2 is expressed in tubular cells, fibroblasts, and macrophages.' CB2 is also expressed in podocytes and mesangial cells.3The role of CB2 in CKD was contro-versial in the past.Despite the upregulation of CB2 mRNA and its main endogenous ligand 2-AG in CKD models, it was previously reported that a CB2 agonist could ameliorate renal fibrosis.'In contrast, our previous studies identified a deleterious role of CB2 in renal fibrosis through genetic knockout experiments and assays with the more selective CB2 inverse agonist XL-001, a novel CB2 inverse agonist that exhibits the strongest binding affinity to the CB2 receptor, with the best selectivity over the CB1 re-ceptor(2594-fold) and a Ki of 0.5 nM. Of note,β-catenin and its targets are suppressed by genetic ablation or pharmacologic inhibition of CB2.Given that CB2 is a member of the GPCR family, CB2 could modulate β-catenin. However, this hypothesis needs to be validated. In this study, we showed that CB2 mediates β-catenin activation through a GPCR signal wave.
Our study also confirms the emerging role of the endocannabinoid system in renal fibrosis. Of the 2 main endo-cannabinoids, anandamide (N-arachidonoylethanolamine)has a higher affinity for CBl, whereas 2-AG has a similar affinity for CB1 and CB2(its affinity for CB1 is slightly higher).Hence, 2-AG is the primary endocannabinoid agonist for the CB2 receptor. Notably, the kidney cortex exhibits similarly high basal levels of anandamide and2-AG; however, 2-AG levels are increased more in obstructive fibrosis of the kidney, accompanied by the decrease in anandamide levels, which results in the activation of both CB2 and CB1.'Of note, the detrimental role of CB2 in the pathogenesis of renal fibrosis was demonstrated by genetic approaches in our recent study,'' and is further confirmed in the present study.
Although a previous report showed that CB2 is expressed by both inflammatory cells and podocytes at a high basal level,8 CB2 and its main endogenous ligand 2-AG are actually upregulated after UUO and IRI.7Notably, in renal fibrosis of mice and a variety of human nephropathies, CB2 is predominantly expressed in renal tubular epithelium as well as in interstitial fibroblasts and leukocytes(Figure 2; Supplementary Figure S1), suggesting that the upregulation of CB2 is a common pathologic feature in the fibrotic kid-neys. In our animal studies, we adopted fluorescence in situ hybridization of CB2 to identify its expression pattern because of its low cost. The staining revealed that CB2 is mainly expressed in tubular epithelium. The reason behind this discrepancy may lie in the sensitivity of the fluorescence in situ hybridization technique in the detection of highly abundant mRNAs and the exposure time for imaging. We observed CB2 in not only the cell membrane, but also the tubular cytoplasmic area, suggesting receptor internalization into intracellular compartments, such as endosomes and lysosomes.6The specificity of the fluorescence in situ hybridization assay was confirmed with a primary CB2 antibody, and both were checked in CB2 knockout mice (Supplementary Figure S1). Interestingly, we observed heterodimer formation of CB2 with CB1 in UUO-affected kidneys and transforming growth factor-β-induced tubular cells (Supplementary Figure S4), suggesting that their stimulatory effects may be synergistic. However, CB1 expression was not affected by AM1241 or XL-001, which adds further complexity to the mechanisms. As previously mentioned regarding the mutually inhibitory effects of CB2 and CB1,'more studies should be performed to verify their interconnected effects, an investigation beyond the scope of the current study. Nevertheless, our previous and present findings consistently reveal that blockade of CB2 is a po-tential therapeutic intervention strategy to prevent renal fibrosis.Along with informing patients with CKD, our findings provide health warnings to the fast-growing group.
Figure 10| Cannabinoid receptor type 2(CB2) is a downstream target of β-catenin (β-cat).(a)Bioinformatics analysis revealed the presence of putative T-cell factor (TCF)binding sites (TBSs) in the promoter region of human and mouse CB2 genes. The sequences and positions of the putative TBSs in the CB2 genes are highlighted (red), whereas the TBS consensus sequence is given at the bottom of this panel. (b)Representative chromatin immunoprecipitation (ChlP) assay results showing the binding of TCF or lymphoid enhancer-binding factor(LEF)-1 to the CB2 gene promoter region. Human kidney proximal tubular (HKC)-8 cells were transfected with β-catenin expression plasmid (pFlag-β-cat)or pcDNA3 for 24 hours. Cell ysates were precipitated with an antibody against TCF, LEF-1, histone H3,or nonimmune lgG, and the chip assay was performed for CB2 gene promoters. The total diluted lysate was used as total genomic input DNA.(c) Graphic presentation of CB2 mRNA level. HKC-8 cells were transfected with pFlag-β-cat or pcDNA3 plasmid. Total RNA was analyzed by quantitative polymerase chain reaction.*P<0.05 versus the pcDNA3 group (n = 3). (d,e)Representative Western blot and quantitative data show overexpression of β-catenin-induced upregulation of CB2.*P<0.05 versus the pcDNA3 group (n =3). (f Representative graphs show β-catenin induced the formation of a CB2/β-arrestin 1(β-arr1)/ proto-oncogene Src complex. Cell lysates were immunoprecipitated (IP) with an antibody against CB2, followed by immunoblotting (B) with anti-β-arrestin 1(β-arr1), or anti-Src.(g-i)Representative Westem blot and quantitative data show the expression of CB2, β-arrestin 1(β-arr1), and Src. HKC-8 cells were transfected with pFlag-β-cat (or pcDNA3) plasmid in the presence or absence of CB2 small interfering RNA (siRNA).***P<0.001 versus the pcDNA3 group;ffP<0.001 versus the flag-β-catenin group alone (n=3),(Continued) of endocannabinoid consumers, for both recreational and medicinal purposes. Chronic heavy use could lead to the continuous activation of both CB1 and CB2 receptors, which may have negative effects on kidney health.
Although a previous report showed that β-arrestin 1 plays a role in renal fibrosis and activation of β-catenin,"we further clarify the functional mechanisms of the β-arrestin 1-mediated CB2/β-catenin pathway in detail. We found β-arrestin 1 is more abundant than β-arrestin 2 in IRI mice (data not shown). It is well known that β-arrestin 1 is responsible for GPCR activation and downstream signal transduction via Src, AKT, extracellular signal-regulated protein kinase (ERK1/2), and other kinases.2 that activation of Src could mediate several pro-fibrosis pathways, such as epidermal growth factor receptor and signal transducer and activator of transcription (STAT)3," we could not exclude the possibility that these pathways are also the downstream pathways of CB2. Nevertheless, our observations suggest that β-catenin signaling is mediated by the CB2-induced β-arrestin 1/Src complex. In addition, β-arrestin 1 is a master checkpoint regulator, and it serves as a scaffold protein to initiate the activation of β-catenin by translocating it to the nucleus. Another novel finding is that CB2 is a target of β-catenin. Several targets of β-catenin have been previously CB2 was identified in this study. Hence, it is notable that the CB2/β-catenin pathway appears to create a reciprocal activation loop that cooperatively pro-motes the pathogenesis of renal fibrosis.
In summary, our results suggest that aberrantly expressed CB2 is critically involved in renal fibrosis through β-arrestin 1-induced β-catenin activation. β-catenin triggers the expression of CB2, which facilitates receptor activation and transmission of the signal wave. These results(i) illustrate a novel CB2/β-catenin pathway that promotes renal fibrosis and (ii) implicate that targeted inhibition of CB2 or β-arrestin 1 may protect against fibrosis through inhibition of the reciprocal activation loop.
CONCISE METHODS
For detailed methods, see the Supplementary Methods.
Animal models
Male C57BL/6 mice were purchased from the Experimental Animal Center of Southern Medical University(Guangzhou, China). For the IRI model, bilateral pedicles were clipped by microaneurysm clamps for 32 minutes at 37.5°C during the ischemia and recovery period. The UUO model was established by triple-ligating the left ureter with 4-0 silk after an abdominal midline incision. For the UIRI model, the left pedicles were clipped for 35 minutes using micro-aneurysm clamps, and the contralateral kidney was removed 1 day before sacrifice. For the folic acid-induced nephropathy model, male C57BL/6 mice were treated with folic acid via a single intraperitoneal injection of 25 mg/kg body weight. In vivo expression of Wnt1 or CB2 or knockdown of β-arrestin 1 in mice was carried out by a hydrodynamic-based gene delivery approach.",5 The detailed experimental designs are shown in Figures 5a, 7a, and 9a. The animal experiments were approved by the Animal Ethics Committee at Southern Medical University, Guangzhou, China. Breeding pairs of the CB2 null mice(CB2/)aged 6-8 weeks, in C57BL/6N background, were purchased from Cyagen Biosciences (stock no. KOCMP-21582-Cnr2; Cyagen Biosciences, Guangzhou, China). CB2 receptor deficiency was confirmed by reverse transcription-PCR.
Human kidney biopsy samples and cell culture
Human kidney sections（3 μm）were obtained from renal biopsies performed at the Nanfang Hospital and the Huadu District People's Hospital, Southern Medical University, Guangzhou, China. Normal control biopsies were derived from nontumor kidney tissues of patients who had renal carcinoma. The studies involving human kidney sections were performed with informed patient consent and were approved by the Institutional Ethics Committee at Nanfang Hospital and the Huadu District People's Hospital. Human proximal tubular epithelial cells(HKC-8) were provided by Dr. Lorraine C. Racusen (Johns Hopkins University, Baltimore, MD). Nuclear and cytoplasmic fractions were separated with a commercial kit (BestBio, Shanghai, China).
Determination of serum creatinine and blood urea nitrogen levels
Serum creatinine and blood urea nitrogen levels were determined by an automatic chemistry analyzer (AU480 Chemistry Analyzer, Beckman Coulter, Atlanta, GA).
In situ hybridization
CB2 probes were purchased from Boster Technology, Wuhan, China. Kidney sections（8 μm）were fixed with 4% formaldehyde and assessed by fluorescence staining of CB2 using a commercial kit (MK2530, Boster Biological Technology, Wuhan, China).
Histology and immunohistochemical and immunofluorescence staining
Paraffin-embedded kidney sections were stained using periodic acid-Schiff and Masson's trichrome staining to identify injured tubules and determine collagen deposition. The lesions were quantified using a computer-aided technique. Immunohistochemical and immunofluorescence staining was performed in paraffin and frozen sections, respectively. Antibodies are described in the Supplementary Methods.
