Part 1: Cannabinoid Receptor Type 2 Promotes Kidney Fibrosis Through Orchestrating β-catenin Signaling

Contact: emily.li@wecistanche.com

Shan Zhou, et al

State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, China;' Department of Nephrology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China;>Department of Nephrology, Huadu District People's Hospital, Southern Medical University, Guangzhou, China; Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; and Bioland Laboratory (Guangzhou Regenerative Medicine and Health, Guangdong Laboratory), Guangzhou. China

The endocannabinoid system has multiple effects. Through interacting with cannabinoid receptor type 1 and type 2, this system can greatly affect disease progression.

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Previously, we showed that activated cannabinoid receptor type 2(CB2)mediated kidney fibrosis. However, the underlying mechanisms remain underdetermined. Here, we report that CB2 was upregulated predominantly in kidney tubular epithelial cells in unilateral urinary obstruction and ischemia-reperfusion injury models in mice, and in patients with a variety of kidney diseases. CB2 expression was closely correlated with the progression of kidney fibrosis and accompanied by the activation of β-catenin.

Furthermore, CB2 induced the formation of a β-arrestin 1/Src/β-catenin complex, which further triggered the nuclear translocation of β-catenin and caused fibrotic injury. Incubation with XL-001, an inverse agonist to CB2, or knockdown of β-arrestin 1 inhibited CB2-triggered activation of β-catenin and fibrotic injury. Notably, CB2 potentiated Wnt1-induced β-arrestin 1/β-catenin activation and augmented the pathogenesis of kidney fibrosis in mice with unilateral ischemia-reperfusion injury or folic acid-induced nephropathy. Knockdown of β-arrestin 1 inhibited the CB2 agonist AM1241-induced β-catenin activation and kidney fibrosis. By promoter sequence analysis, putative transcription factor binding sites for T-cell factor/lymphoid enhancer factor were found in the promoter regions of the CB2 gene regardless of the species. Overexpression of β-catenin induced the binding of T-cell factor/lymphoid enhancer factor-1 to these sites, promoted the expression of CB2, β-arrestin 1, and the proto-oncogene Src, and triggered their accumulation. Thus, the CB2/β-catenin pathway appears to create a reciprocal activation feedback loop that plays a central role in the pathogenesis of kidney fibrosis.

Translational Statement

Cannabinoid receptors are involved in tissue fibrosis. Although cannabinoid receptor type 1 activation has been reported to mediate renal fibrosis, the role of cannabinoid receptor type 2(CB2) remains to be demonstrated in detail. This study provides evidence for the detrimental role of CB2 in the pathogenesis of renal fibrosis. The findings highlight a novel CB2/β-catenin pathway that promotes renal fibrosis. Blockade of CB2 is a potential therapeutic intervention to prevent renal fibrosis in a variety of nephropathies. The findings also provide health warnings to the fast-growing group of endocannabinoid consumers, for both recreational and medicinal purposes.

The endocannabinoid system plays a critical role in cardiovascular, neurodegenerative, and metabolic diseases.-Through interacting with cannabinoid receptors type 1(CB1)and type 2(CB2), the endocannabinoid system could also affect the prognosis of kidney diseases. CB1 plays a detrimental role in renal fibrosis.-However, the role of CB2 in renal fibrosis remains elusive.

CB2 was previously reported to be involved in the immune system."Recently, CB2 was found to be expressed in kid-neys.5,1-1 In early diabetic nephropathy and podocytes,CB2 plays a protective role in cellular integrity. However, in fibrotic injury, CB2 is upregulated in tubular cells and involved in fibrogenic responses, as reported in our previous work. CB2 is also expressed in interstitial fibroblasts and macrophages and mediates their activation. Of note, the levels of 2-arachidonoylglyceril (2-AG), the main endogenous ligand of CB2, are also increased in obstructive kidneys. Although a previous study pointed out that the activation

Figure 1| Cannabinoid receptor type 2 (CB2) is time-dependently induced in injured kidneys and associated with fibrogenesis. (a)Representative micrographs showing CB2 expression and collagen deposition in kidneys after ischemia-reperfusion injury(IRl). IRI surgery was performed in mice, and mice were sacrificed at the indicated time points. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2 (top), and parattin sections were subiected to Masson's trichrome staining for collagen deposition (bottom). Arrows indicate positive staining.For CB2 FISH staining, bar =75 um; for Masson's trichrome staining, bar = 50 um.(b,C) Linear reqression showing renal CB2 is positively correlated with the extent of fibrosis or neutrophil gelatinase-associated lipocalin (NGAL) expression in the IRl mouse model. Mice were sacrificed at 3 or 7 days after IRI. Renal mRNA levels of CB2 and NGAL were assessed by quantitative polymerase chain reaction.The degree of kidney fibrosis was analyzed by quantification of Masson's trichrome staining. The Spearman correlation coefficient (Rs)and P value are shown. (d-g) Representative Western blot and quantitative data showing renal expression of fibronectin, g-smooth muscle actin (SMA), and kidney injury molecule-1 (Kim-1) at indicated time points after IRl. Numbers (1-3) indicate each individual animal in a aiven group.**P<0.01 versus the sham control group; tP<0.01;t++P<0.001 versus the IRI 3d group (n = 5-6). (Continued)