Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

Abstract Kidney is one of the most important organs in maintaining the normal life activities. With the high abundance of mitochondria, renal tubular cell plays the vital role in functioning in the reabsorption and secretion of kidney. Reports have shown that mitochondrial dysfunction is of great importance to renal tubular cell senescence and subsequent kidney ageing. However, the underlying mechanisms are not elucidated. Cannabinoid receptor 2 is one of the two receptors responsible for the activation of endocannabinoid system. CB2 is primarily upregulated in renal tubular cells in chronic kidney diseases and mediates fibrogenesis. However, the role of CB2 in tubular mitochondrial dysfunction and kidney ageing has not been clarified. In this study, we found that CB2 was upregulated in kidneys in 24‐month‐old mice and d‐galactose (d‐gal)‐induced accelerated ageing mice, accompanied by the decrease in mitochondrial mass. Furthermore, gene deletion of CB2 in d‐gal‐treated mice could greatly inhibit the activation of β‐catenin signalling and restore the mitochondrial integrity and Adenosine triphosphate (ATP) production. In CB2 knockout mice, renal tubular cell senescence and kidney fibrosis were also significantly inhibited. CB2 overexpression or activation by the agonist AM1241 could sufficiently induce the decrease in PGC‐1α and a variety of mitochondria‐related proteins and trigger cellular senescence in cultured human renal proximal tubular cells. CB2‐activated mitochondrial dysfunction and cellular senescence could be blocked by ICG‐001, a blocker for β‐catenin signalling. These results show CB2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing. The intrinsic mechanism may be related to its activation in β‐catenin signalling.


| INTRODUC TI ON
Kidney is responsible for the vital physiological activities such as excretion, filtration, reabsorption and secretion in body. 1 Renal tubular cell is fundamental to maintain the normal renal function. 2 To meet the extraordinary needs of energy, through its respiratory chain complexes, high abundance of mitochondria in renal tubular cells conduct oxidative phosphorylation to produce ATP. 3 Hence, the abnormal mitochondria in renal tubular cells would certainly lead to the damage of the metabolic state and also greatly affect the normal renal function. Large reports have shown that mitochondrial dysfunction is highly correlated with the decline of renal function. 4-In the multiple aetiologies, ageing is undoubtedly the very important mediator of mitochondrial dysfunction and the subsequent cell senescence in renal tubular cells 6 and decline of renal function. 7 In aged group, the excessive loss of mitochondrial mass 6,8 is accompanied by the high morbidity of kidney diseases. 9 To mitigate mitochondrial reactive oxygen species (ROS) production not only protects against mitochondrial dysfunction and tubular cell senescence, but also retards age-related renal fibrosis, 6 suggesting the central state of mitochondrial quality control in kidney ageing. However, the underlying mechanisms of mitochondrial dysfunction in renal tubular cells are not clearly determined.
Cannabinoid receptors belong to G-protein coupled seven-span transmembrane receptor (GPCR), including CB1 and CB2. 10 As the receptors in endocannabinoid system, they are highly related to immunity, development, memory, metabolism and other important physiological processes. [11][12][13] The most recent studies show that cannabinoid receptors also participate in the pathogenesis of many diseases such as cardiovascular diseases, neurodegenerative diseases and especially kidney diseases. 1,[14][15][16] Both CB1 and CB2 were found to be important mediators in the progression of renal fibrosis. 15,17 In our recent publication, CB2 was proved to induce the fibrotic change in renal tubular cells through β-catenin-activated pathway. 15 Notably, β-catenin activation is a very important mediating factor in renal tubular cell senescence and mitochondrial dysfunction, and age-related renal fibrosis. 6 However, the role of CB2 in the mitochondrial dysfunction of renal tubular cells has not been delineated.
Moreover, the relationship between CB2 and tubular cell senescence should also be clarified.
In this study, we examined the expression of CB2 in aged kidneys and identified the mediative role of CB2 in the accelerated ageing.
We also proved that CB2 is an important player in promoting mitochondrial dysfunction in tubular cells. Our study provides a new clue for the pathogenic role of endocannabinoid system in kidney health.

China) and bred in Experimental Animal Center of Southern Medical
University under pathogen-free conditions. Gene deletion of CB2 was confirmed by RT-PCR. For the accelerated ageing model, unilateral nephrectomy was carried out in age-matched CB2 +/+ and CB2 -/male mice on C57BL/6 background. All mice undergoing surgeries were treated with general anaesthesia. One week after surgery, the mice were administered subcutaneous injections of d-gal (G0750; Sigma-Aldrich) at 150mg/kg/day for 6 weeks. Kidney tissues were collected for various analyses. The detailed experimental designs were shown in Figure 3A. The animal experiments were approved by the Animal Ethic Committee at Southern Medical University.

| Transmission electron microscopy
Kidney cortex was collected and fixed in 1.25% glutaraldehyde/0.1 M phosphate buffer followed by resin embedding and ultrafine section making. Slides were subjected to assess kidney tubular mitochondrial ultrastructure under an electron microscope (JEOL JEM-1010).

| Quantifications of staining
Slides stained with Sirius red, immunohistochemical, immunofluorescence and SAβ-gal staining were observed at high-powered (x400, x1000) fields from randomly selected fields. Each section contained 10 fields. Quantification of fibrotic lesions or positive staining was assessed by a computer-aided technique (Image Pro Plus).

| Statistical analyses
All of the data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was carried out using SPSS 20.0 (SPSS Inc, Chicago, IL). Comparisons were made by the Student t test or t' test when comparing two groups or via one-way analysis of variance (one-way ANOVA) followed by the Least Significant Difference or Dunnett's T3 procedure for comparison of more than two groups.
A value of p < 0.05 was considered to be statistically significant.

| CB2 is upregulated in aged kidneys
To investigate the role of CB2 in ageing, we assessed the expression of CB2 in the kidneys from naturally aged mice. The expression of CB2 was first tested by fluorescence in situ hybridization (FISH). As shown in Figure 1A, CB2 was highly upregulated in renal tubules in 24-month-old mice. The similar result was also observed when CB2 was tested by Western blotting ( Figure 1B,C). We next assessed the expression of CB2 in d-gal-treated mice, an accelerated ageing model. 6,18 As shown in Figure 1D-F, CB2 expression was also greatly increased and primarily upregulated in renal tubules. To identify the location of CB2 in renal tubules, the co-staining for CB2 with different segment-specific tubular cell markers was performed in the kidneys in 24-month-old mice. As shown in Figure 1G, CB2 was co-localized with lotus tetragonolobus lectin (LTL), a marker of proximal tubules, and peanut agglutinin (PNA), a marker of distal tubules. While CB2 is barely co-stained with dolichos biflorus agglutinin (DBA), a marker of collecting ducts (data not shown). To assess the correlation between CB2 and mitochondrial function, the expression fashion of translocase of outer mitochondrial membrane 20 (TOMM20, a mitochondrial marker) and CB2 was assessed by immunofluorescence and FISH, respectively. As shown in Figure 1H, compared with the high expression of TOMM20 in the kidneys in 2-month-old mice, TOMM20 expression was dramatically decreased in the aged kidneys in 24-month-old mice. Interestingly, CB2 expression presented an opposite fashion ( Figure 1H), suggesting that CB2 plays a critical role in ageing and is related to mitochondrial dysfunction.

| CB2 gene ablation does not affect kidney ageing or mitochondrial function in young mice
The gene ablation efficiency of CB2 was first testified by RT-PCR and quantitative real-time PCR (Figure 2A,B). We next analysed the expression of the fibrosis-related genes fibronectin, α-smooth muscle actin (α-SMA), collagen type 1 alpha 1 (COL1a1) and collagen type 3 alpha 1 (COL3a1). As shown in Figure 2C-F, there is no difference of these genes in wild-type and CB2 knockout mice. Furthermore, the results of Periodic acid-Schiff (PAS) and Sirius red staining also showed that there was no obvious change in tubular damage and fibrotic injury after CB2 gene ablation ( Figure 2G). We next tested the senescence in two groups of mice. As shown in Figure 2G, L and M, the results of the senescence-associated β-galactosidase (SAβ-gal) staining, and analysis of mRNA expression of p16 INK4A and γH2AX, showed that CB2 gene ablation did not affect kidney ageing. We next assessed mitochondrial function. The protein expression of peroxisome proliferator-activated receptor-C coactivator-1α (PGC-1α), TOMM20 and mitochondrial transcription factor A (TFAM), the mitochondrial biogenesis-related transcription factors, was analysed by Western blotting. As shown in Figure 2H-K, they were also not changed by CB2 gene ablation. We also tested the expression of β-catenin and its signalling-related proteins, such as MMP7, Snail1 and AT1. There were no changes in these mice after CB2 gene deficiency ( Figure 2N-R).

| β-catenin signalling is inhibited by CB2 gene ablation in D-gal-treated mice
To investigate the potential role of CB2 in kidney ageing and mitochondrial dysfunction, we adopted an accelerated ageing mouse model. 6,18 The wild-type and CB2 knockout mice were first performed unilateral nephrectomy and then injected with d-gal at 150mg/kg/day for 6 weeks ( Figure 3A). We first tested the expression of CB2 by FISH and qRT-PCR. As shown in Figure 3B,C, although administration of d-gal induced the upregulation of CB2 in tubules, CB2 expression was nearly completely inhibited in d-galtreated CB2 knockout (CB2 −/− ) mice. As β-catenin signalling is an important mediator of kidney ageing, 6 we next analysed the expression of β-catenin and its downstream targets MMP7 and AT1. 19,20 As shown in Figure 3D, E, H and I, d-gal treatment induced the upregulation of β-catenin and its activation, and however, gene ablation of CB2 largely blocked these changes. The similar results were observed when MMP7 and AT1 expression levels were assessed by qRT-PCR ( Figure 3F,G). These findings suggest that CB2 could regulate kidney ageing through activating β-catenin signalling.

| CB2 deficiency protects renal mitochondrial homeostasis in the accelerated ageing mice
To identify the role of CB2 in mitochondrial dysfunction, we first investigated the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), a key factor modulating mitochondrial biogenesis, and TOMM20 by immunostaining. As shown in Figure 4A-C, d-gal treatment triggered the downregulation of PGC-1α and TOMM20, while gene ablation of CB2 significantly restored their expression. Similar results were observed when PGC-1α and TOMM20 were assessed by Western blotting analyses ( Figure 4E-G). Furthermore, the expression of cytochrome b (Cytb), an mtDNAencoded OXPHOS complex III subunit, was also restored by CB2 gene deletion in d-gal-treated mice ( Figure 4E and H). To further assess the role of CB2 in mitochondrial dysfunction, we analysed the production of adenosine triphosphate (ATP). As shown in Figure 4D, administration of d-gal decreases the production of ATP, and however, the gene ablation of CB2 could significantly promote the increase in ATP levels.
The mitochondrial ultrastructure was also inspected by transmission electron microscopy (TEM). As shown in Figure 4I, compared with the normal rod-shape and well-arranged mitochondria in control mice, d-gal treatment induced the breakdown of mitochondrial ultrastructure, leading to the most mitochondria to be swelling, fragmented and disorganized, while CB2 gene knockout largely preserved the normal structure of mitochondria. These results show that CB2 plays a central role in renal tubular mitochondrial dysfunction.

| CB2 gene ablation ameliorates kidney ageing
We further assessed the role of CB2 in kidney ageing. As shown in Figure 5A,B d-gal treatment strongly induced the upregulation of γH2AX, a marker for DNA double-strand breaks and an important senescence marker, while it was nearly completely inhibited in CB2 knockout mice. We next assessed the cellular senescence by SAβ-gal staining. The senescent tubules, which appeared the bright-blue granular staining, were greatly induced by d-gal administration, but evidently inhibited by gene ablation of CB2, as shown in Figure 5A and C. Similar results were observed when γH2AX, p16 INK4A and p19 ARF , the important factors triggering senescent pathway, were assessed by Western blotting (Figure 5D-G). Klotho is an anti-ageing protein, 21 which is a protector for mitochondrial function in renal tubular cells. 5 We next assessed the expression of Klotho. As shown in Figure 5H and I, CB2 gene ablation could also largely preserve the expression of Klotho. 3.6 | CB2 deficiency retards age-related kidney fibrosis Furthermore, we tested serum creatinine (Scr) and blood urea nitrogen (BUN) levels in mice injected with d-gal, but there were no significant changes among three groups ( Figure 6A,B). We then tested renal fibrosis. As shown in Figure 6C-E d-gal treatment induced the upregulation of fibronectin and α-SMA, the two fibrosis markers, while they were largely blocked in CB2 knockout mice. The similar result was observed when fibronectin was assessed by immunostaining ( Figure 6F,G). Furthermore, the fibrotic lesions were also analysed by Sirius red staining. As shown in Figure 6F and H, the quantification of fibrotic lesions further confirmed the protective role of CB2 gene deficiency in age-related kidney fibrosis.

| CB2 induces mitochondrial dysfunction and cellular senescence in vitro
We then assessed the role of CB2 in tubular cell senescence in vitro. The human proximal tubular epithelial cell line (HKC-8) was cultured and transfected with CB2 expression plasmid. The expression of CB2 was first testified by Western blotting (Figure 7A,B). We next assessed the expression of mitochondria-related proteins. The key transcription factor of mitochondria biogenesis, PGC-1α, was greatly decreased by CB2 overexpression (Figure 7A

| β-catenin plays a mediative role in CB2-induced mitochondrial dysfunction and cellular senescence
We further assessed the role of CB2 in kidney ageing using XL-001, a novel inverse agonist of CB2 invented by our group. 14 As shown in Figure 8A-H, pretreatment with XL-001 could largely inhibited AM1241-induced mitochondrial dysfunction and cellular senescence.
In our recent study, we found β-catenin plays a vital role in kidney ageing and renal tubular cell senescence 6,22 and mediates CB2-induced GPCR signal wave, 15 and we then tested its role in CB2-induced kidney ageing. HKC-8 cells were pretreated with ICG-001, a small molecule inhibiting β-catenin-mediated gene transcription, and then transfected with CB2 expression plasmid. As shown in Figure 8I-L, ICG-001 pretreatment largely inhibited CB2induced decrease in PGC-1α, Cytb and TOMM20. In addition, the expression of p16 INK4A was also tested. As shown in Figure 8M,N, CB2-induced increase in p16 INK4A was greatly blocked by ICG-001 pretreatment.

| CB2 plays a central role in the accelerated ageing in renal tubular cells
HKC-8 cells were pretreated with XL-001 and then treated with d-gal. As shown in Figure 9A  and cotreated with ICG-001. As shown in Figure 9K-M, AM1241 further decreased d-gal-downregulated PGC-1α and TOMM20 expression, but this was significantly blocked by co-treatment with ICG-001. Furthermore, d-gal-induced increase in p14 ARF was further elevated by AM1241, and however, co-treatment with ICG-001 could greatly inhibit this effect ( Figure 9K and N). We observed the similar results in cultured cells. SAβ-gal activity staining showed that D-gal and AM1241 aggravated tubular cell senescence in dgal-treated cells, but treatment with ICG-001 largely inhibited it ( Figure 9O,P). These results further clarify that CB2 plays a central role in kidney ageing and is related with activation of β-catenin.

| DISCUSS ION
The population ageing is becoming a big problem to the economic and health care society in the whole world. 23 In the future 30 years, the aged group of population would occupy up to one-fifth of the population. 24 Ageing is one of the strongest inducer in the decline of organ function. 25 of metabolites such as acetone into tricarboxylic acid cycle. 28 Furthermore, the oxidative respiratory chain in the inner layer of mitochondrion carries out a chain reaction to produce energy through the electron transportation. 29 As an organ with vigorous metabolism, kidney parenchymal cells, especially renal tubular cells, possess the high abundance of mitochondria to produce ATP for executing the reabsorption and excretion function. 30 Renal tubular cell mitochondrial dysfunction is highly related to the damaged cell function. 31 Our previous reports show that to protect against mitochondrial dysfunction could inhibit cellular senescence and age-related fibrosis in kidneys. 6 In aged organs, the damaged mitochondria are characterized with loss of mitochondrial mass, ROS production, fragmentation 6 and abnormal calcium influx, 32 etc., which further lead to the reduced production of ATP to form a vicious cycle.
Therefore, mitochondrial quality control has been thought to stand at the centre of anti-ageing strategies, while the underlying mechanisms of mitochondrial dysfunction in kidney ageing have not been elucidated.
Wnt/β-catenin signalling is a conserved development pathway, 33 and however, it is reactive in multiple kinds of kidney diseases. 34 Interestingly, we have found that it is also overactivated in aged kidneys. 6 The activation of β-catenin is accompanied by the loss of mitochondria mass and overproduced ROS, suggesting the highly intimate correlation of β-catenin with mitochondrial dysfunction. β-catenin could be activated by Wnt-triggered inhibition on its degradation 35 and also could be stimulated by GPCR-induced signal wave. 15,36,37 Interestingly, we have found several GPCRs such CXCR4 and CB2 induce the kidney injury depending on β-catenin activation. 15,36 CB2 was recently found to be primarily upregulated in the proximal and distal tubules in renal fibrosis model and promoted kidney fibrosis by orchestrating β-catenin signalling, as reported by our recent work. 14,15 Notably, our recent findings show the accelerated cellular senescence in tubular cells plays the vital role in the progression of renal fibrosis. 22 This suggests there would be a high correlation between CB2 and tubular cell ageing. However, this has not been experimentally proved. In this study, we have verified the internal correlation between CB2 and kidney ageing. We first assessed the expression of CB2 in the kidneys in naturally ageing mice and d-gal-accelerated ageing mice. We found that CB2 is highly increased in aged kidneys and correlated with the decrease in mitochondrial mass ( Figure 1).
We next performed the study in CB2 knockout mice ( Figure 2).
The results show that gene ablation of CB2 could inhibit β-catenin signalling (Figure 3), preserve mitochondrial function (Figure 4), retard cellular senescence ( Figure 5) and greatly ameliorate agerelated kidney fibrosis ( Figure 6). The induction of CB2 on tubular cell ageing and mitochondrial dysfunction was also proved in cultured proximal tubular cell line (Figure 7). Notably, ICG-001, a small molecular compound which inhibits β-catenin activation, could greatly block CB2-induced cell ageing and mitochondrial damage ( Figure 8). Furthermore, the high correlation between tubular cell ageing and mitochondrial dysfunction with CB2/β-catenin pathway was also testified in d-gal-treated tubular cell culture ( Figure 9). All of these results reveal the central role of CB2 in renal tubular mitochondrial dysfunction and kidney ageing. CB2 belongs to GPCR and was previously thought to be responsible for immunity modulation. 38 Its role in kidney diseases was recently proved by our group. Through the gene deletion of CB2, we found that CB2 plays a detrimental role in the progression of renal fibrosis 14 and also clarified the internal interlaced relationship between CB2 and β-catenin pathway. 15 As β-catenin pathway plays a vital role in kidney ageing and the promotion of mitochondrial dysfunction, 5,6 we hypothesize that CB2 could also act as an important mediator in kidney ageing. Through our findings, we conclude that the activation of endocannabinoid system, especially CB2, would undoubtedly affect the kidney function through an ageing-promoted pathway. Considering the increasing use of exocannabinoid products for medial and other purposes, 39 our studies provide an important health alert that the long-term stimulation of this system would impose a heavy burden to the lifespan of organs.

| CON CLUS ION
We have uncovered CB2 is the important mediator in kidney ageing. The study, for the first time, identified CB2 plays a central role in kidney ageing and clarified CB2 could promote mitochondrial dysfunction in renal tubular cells and be related to β-catenin activation. Given that the increased use of exocannabinoid products for medical and recreational purposes, our studies provide a warning that needs high attention that the long and heavy activation of CB2 receptor would accelerate kidney ageing and lead to the damage on the mitochondria, one of most important organelle in the body.