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In diabetic nephropathy agonism of CB 2 receptors reduces albuminuria and podocyte loss; however, the role of CB 2 receptors in obesity‐related nephropathy is unknown. The aim of this study was to determine the role of CB 2 receptors in a model of diet‐induced obesity (DIO) and characterize the hallmark signs of renal damage in response to agonism (AM1241) and antagonism (AM630) of CB 2 receptors.
Male Sprague Dawley rats were fed a high‐fat diet (HFD: 40% digestible energy from lipids) for 10 weeks. In another cohort, after 9 weeks on a HFD, rats were injected daily with either 3mg·kg−1 AM1241, 0.3mg·kg−1 AM630 or saline for 6 weeks.
Ten weeks on a HFD significantly reduced renal expression of CB 2 receptors and renal function. Treatment with AM1241 or AM630 did not reduce weight gain or food consumption in DIO. Despite this, AM1241 significantly reduced systolic BP, peri‐renal adipose accumulation, plasma leptin, urinary protein, urinary albumin, urinary sodium excretion and the fibrotic markers TGF‐β1, collagen IV and VEGF in kidney lysate. Treatment with AM630 of DIO rats significantly reduced creatinine clearance and increased glomerular area and kidney weight (gross and standardized for body weight). Diastolic BP, glucose tolerance, insulin sensitivity, plasma creatinine, plasma TGF‐β1 and kidney expression of fibronectin and α‐smooth muscle actin were not altered by either AM1241 or AM630 in DIO.
This study demonstrates that while agonism of CB 2 receptors with AM1241 treatment for 6 weeks does not reduce weight gain in obese rats, it leads to improvements in obesity‐related renal dysfunction.
This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc
Tables of Links
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson etal., 2014) and are permanently archived in the Concise Guideto PHARMACOLOGY 2013/14 (Alexander etal., 2013).
The prevalence of obesity has been steadily rising worldwide over the past few decades, with the incidence of obesity doubling since 1980 (World Health Organization, 2013), because of increased caloric intake and declining physical activity levels of individuals (Booth etal., 2005). Obesity poses a significant financial and social burden to societies worldwide, largely because of the deleterious effects of associated co‐morbidities (de Jong etal., 2002; Eknoyan, 2007), leading to targeted organ damage. Specifically for the kidneys, obesity is associated with hypertension, hyperlipidaemia, hyperleptinaemia, cardiovascular disease and insulin resistance, all of which may act to reduce renal function (Wolf etal., 2002; Decleves etal., 2011). However, even when confounding factors are taken into consideration, obesity remains an established and independent risk factor for the development and progression of chronic kidney disease (CKD) (Kramer etal., 2005; Mathew etal., 2011). Overweight individuals or individuals with obesity have an increased risk (40%) of kidney disease compared with individuals of normal weight (Wang etal., 2008). Furthermore, individuals with obesity have a reduced chance of successful kidney transplants and increased tendency of progression to end‐stage renal failure (Wang etal., 2008; Afkarian etal., 2013). Kidney damage caused by obesity typically leads to increased loss of protein (specifically albumin) in the urine, and a decrease in estimated glomerular filtration rate (eGFR), presumably via an increase in fibrotic damage driven by increased collagen deposition mediated by the cytokines TGF‐β1 and VEGF (Branton and Kopp, 1999; Qi etal., 2006; Ziyadeh, 2008).
Obesity leads to inflammation and activation of a number of key renal fibrotic cytokines and growth factors, including TGF‐β1 and VEGF (Ziyadeh, 2008). Increased production of TGF‐β1 mediates fibrosis, inducing the production of matrix and basement membrane proteins, such as collagen IV, fibronectin and α‐smooth muscle actin and interferes with albumin handling by the proximal tubules (Hryciw etal., 2004; Qi etal., 2006; 2008). VEGF acts to increase the permeability of endothelial cells, which increases glomerular filtration in the initial stages of nephropathy (Cha etal., 2000). Obesity has also been shown to increase circulating levels of the peptide hormone leptin, which is predominately produced by adipose tissue (Wolf etal., 2002). Leptin is cleared principally by the kidneys, and elevated leptin levels have been associated with the up‐regulation of collagen deposition and TGF‐β1 secretion by renal glomerular cells (Wolf etal., 1999; 2002; Briffa etal., 2013).
Investigation into potential therapeutic targets for obesity have identified that the endocannabinoid system may potentially play a role in a number of diseases including CKD (Janiak etal., 2007; Matias etal., 2008; Mukhopadhyay etal., 2010; Jenkin etal., 2012; Nam etal., 2012). The endocannabinoid system is an endogenous lipid‐signalling network comprised of cannabinoid receptors, primarily CB1 and CB2 receptors (Wang and Ueda, 2009; Muccioli, 2010). The endocannabinoid system modulates physiological functions, which can influence the progression of obesity by regulating energy balance, adipogenesis and glucose metabolism (Di Marzo, 2008). Clinical and experimental studies have demonstrated that antagonism of CB1 receptors can reduce body weight and improve glucose metabolism in overweight individuals and animal models of obesity (Van Gaal etal., 2005; Gary‐Bobo etal., 2006; Rosenstock etal., 2008). However, because of adverse neurological effects of whole‐body CB1 receptor antagonists (Van Gaal etal., 2005; Rosenstock etal., 2008), additional endocannabinoid targets are currently under investigation as anti‐obesity therapeutics.
Emerging research has shown that modulation of the CB2 receptor may be able to ameliorate renal damage associated with disease pathologies (Munoz‐Luque etal., 2008; Barutta etal., 2011). Specifically, agonism of CB2 receptors in a mouse model of Streptozotocin‐induced diabetes reduced albuminuria and glomerular podocyte loss (Barutta etal., 2011). Furthermore, activation of CB2 receptors attenuated inflammation by reducting cytokines, preventing of immune cell infiltration and attenuating fibrotic factors in diabetic nephropathy, specifically the C‐C motive receptor 2 (CCR2 receptor, also known as monocyte chemoattractant protein‐1 receptor) (Barutta etal., 2011). Thus, activation of CB2 receptors has been demonstrated in this model of diabetic nephropathy to reduce renal damage potentially via the modulation of fibrotic pathways, leading to reduced loss of urinary albumin. To add to this knowledge, we have previously demonstrated in proximal tubule cells in vitro, internalization of albumin significantly reduces the expression of CB2 receptors (Jenkin etal., 2013). Thus, altered renal expression of CB2 receptors may contribute to the increased production of collagen, and other fibrotic markers, potentially leading to the progression of fibrotic damage (Wohlfarth etal., 2003). Importantly, despite the close link between obesity and diabetes, it is yet to be elucidated if therapeutically targeting CB2 receptors may modulate renal fibrotic changes associated with diet‐induced obesity (DIO)‐linked CKD.
Therefore, the main aim of this study was to characterize the renal changes in response to modulation of CB2 receptors in rats following DIO. Specifically, we wanted to determine if the expression of CB2 receptors was altered in the obese rat, and to identify if the CB2 receptor was a therapeutic target for obesity‐linked renal disease. We characterized the morphology of the glomerulus and tubules, in addition to the level of fibrotic markers in obese rats treated with a CB2 receptor agonist and antagonist.
Animal experimental procedures were approved by Howard Florey Animal Ethics Committee (AEC 11–036). Seven‐week‐old male Sprague Dawley rats were individually housed in cages (dimensions width 27.5 × length 41 × height 25.5cm) in an environmentally controlled laboratory (ambient temperature 22–24°C and relative humidity 35–55%) with a 12h light/dark cycle (07:00–19:00). Following the conclusion of the DIO or CB2 agonist/antagonist treatment, rats were deeply anaesthetized with either vaporized 3% isoflurane (Abbott, Botany, NSW, Australia) or by an i.p. injection of 100mg·kg−1 sodium pentabarbitone (Virbac, Milperra, NSW, Australia), then killed via cardiac puncture. Kidneys and surrounding peri‐renal adipose tissue were then removed, weighed individually and stored at −80°C for further analysis. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny etal., 2010; McGrath etal., 2010).
Rats were randomly assigned to receive either a high‐fat diet (HFD; containing 40% digestible energy from lipids; sourced from Specialty Feeds, Glen Forrest, WA, Australia) or a lean control diet (standard rodent chow; containing 10% digestible energy from lipids; sourced from Barastoc, Ltd., Melbourne, Vic., Australia) (Cornall etal., 2011) for a period of 10 weeks. Animals allocated to the lean (n = 10) or HFD groups (n = 14) had measurements of body weight, BP, fat composition and urinary analysis at 9 weeks. A previous study indicated that after 9 weeks of consuming a HFD, a significant increase in proteinuria and decreased creatinine clearance is observed (Pinhal etal., 2013). Ad libitum access to food and water was maintained throughout the duration of the study.
Nine weeks of a HFD induced DIO, with rats exhibiting significant increases in body weight, body fat composition, hypertension and reduced renal function. Consequently, rats were fed a HFD for 9 weeks and then matched according to weight, body composition and BP, and were put into either obese control (n = 9), CB2 receptor agonist, AM1241 (n = 9) or CB2 receptor antagonist, AM630 (n = 10) treatment groups (AM1241 and AM630 compounds sourced from Cayman Chemicals, Ann Arbour, MI, USA). AM1241 is a highly selective CB2 receptor ligand with a high affinity for CB2 receptors (K i = 3.4 ± 0.5nm) and low affinity for CB1 receptors (K i = 280 ± 41nm) (Ibrahim etal., 2003). AM630 is a highly selective CB2 receptor antagonist/inverse agonist, with a K i for rat CB2 receptors of 2.3nm (Mukherjee etal., 2004) and human CB2 receptors of 31.2 ± 12.4nm (Ross etal., 1999), and a weak agonist of human CB1 receptors with a K i = 5152 ± 567nm (Ross etal., 1999). In this study, for 6 weeks, rats were maintained on the HFD and injected i.p. daily with either vehicle control 0.9% isotonic saline solution containing 0.75% Tween 80, or 3mg·kg−1 of AM1241 (Shoemaker etal., 2007; Curto‐Reyes etal., 2010; Barutta etal., 2011) or 0.3mg·kg−1 AM630 in saline with 0.75% Tween 80. The concentration of AM1241 is the same as that used in previously published studies conducted in mice, where the drug was administered via an i.p. injection for 14 weeks, 30 days or acutely (LaBuda etal., 2005; Shoemaker etal., 2007; Curto‐Reyes etal., 2010; Barutta etal., 2011). AM630 has also been administered i.p. in rats at dosages of 0.1 and 1mg·kg−1 for 21 days (Idris etal., 2008), while Ting etal. (2015) demonstrated that acute exposure to 0.3mg·kg−1 AM630 injected i.p. altered food intake in rats, which was the dosage chosen in the current study. Ad libitum access to food and water occurred throughout the duration of the study.
Pretreatment measurements were recorded at week 9, before the administration of saline or CB2 compounds, and post‐treatment measurements were recorded at week 15 in the final week of the pharmacological intervention. Rat weight and food consumption were recorded daily. Body composition of rats was analysed using an EchoMRI™ Whole Body Composition Analyzer (EchoMRI‐900, Houston, TX, USA). Body fat composition was calculated by determining total fat (g) divided by total body weight (g) and expressed as a percentage. Measurements for systolic and diastolic BPs were obtained from conscious rats using a non‐invasive tail‐cuff method with volume pressure recording software CODA 2 (Kent Scientific, Torrington, CT, USA). Glucose tolerance tests and insulin sensitivity tests were conducted as previously described (Jayasooriya etal., 2008; Xia etal., 2011) with minor modifications as glucose and insulin were administered via an i.p. injection following an overnight and 2h fast period respectively. Blood glucose in response to glucose (2g·kg−1) or insulin (0.75U·kg−1) load was analysed as AUC (Le Floch etal., 1990). Body composition and BP were analysed in the final week of the 10 week DIO treatment and at pre‐ and post‐treatment time points in the CB2 receptor agonist/antagonist‐treated DIO model. Glucose tolerance and insulin sensitivity were tested in week 10 only of the DIO treatment.
Urinary protein, albumin and sodium excretion were evaluated using 24h urine samples collected using metabolic cages in week 10 for the DIO rats or weeks 9 and 15 (pre‐ and post‐treatment periods). In addition creatinine clearance was performed on pre‐ and post‐treatment period samples. Changes in renal function, calculated by post‐treatment subtracted from pretreatment values were analysed and are represented as Δ. At time of death, plasma was collected via cardiac puncture. Renal functional measurements for urinary protein (Thermo Scientific, Rockford, IL, USA), albumin (ALPCO Diagnostics, Salem, NH, USA) and creatinine (Cayman Chemical Company, Ann Arbor, MI, USA) were determined using commercially available kits, according to the manufacturer's instructions. Urinary sodium was analysed using the COBAS Integra® 400plus system (Roche Diagnostics, Rotkreuz, Switzerland), urine samples were analysed for sodium content undiluted. Creatinine clearance (mL·min·kg−1) was calculated via the formula [urinary vol (mL·min−1) × urinary creatinine concentration (mg·L−1)]/[plasma creatinine (mg·L−1)] and standardized for body weight (Keenan etal., 2000).
Following kidney removal, a portion of kidney tissue was freshly frozen in Optimal Cutting Temperature Compound (OCT; Tissue‐Tek, Torrance, CA, USA), 5μm thick sections were cut using an HM 550 Cryostat (Thermo Fisher, Soresby, Vic., Australia) and sections were stained using haematoxylin and eosin and periodic acid–Schiff (PAS) staining. Sections were imaged at 200× magnification (Carl Zeiss microscope, Jena, Germany) and at least 50 random cortical glomeruli and renal tubule sections were analysed. In order to analyse glomerular area, the outer edges of all glomerular tufts were traced on a captured image, and the encircled area was determined using image analysis software (Axiovision 4.8, Zeiss, Jena, Germany). Tubular diameter was analysed at the widest point for cross‐sectional diameter on captured images using Axiovision 4.8 image analysis software.
Following cardiac puncture, blood was transferred into 10mL EDTA BD Vacutainer® tubes (McFarlene Medical, Surrey Hills, Australia) and kept on ice until samples were centrifuged at 4000× g for 10min at 4°C. The plasma layer was aspirated and stored at −80°C for further analysis. Plasma levels of TGF‐β1 (Promega, Madison, WI, USA) and leptin (Quantikine, R&D Systems, Minneapolis, MN, USA) were analysed according to manufacturer's instructions.
Protein was isolated from rat kidney as described previously (Jenkin etal., 2010). Aliquots (25–100μg) of protein lysate were separated on a 7.5–20.0% SDS‐PAGE gel and transferred to a nitrocellulose membrane. CB2 receptors (Cayman Chemicals, Ann Arbour, MI, USA), TGF‐β1, collagen IV, VEGF (Abcam, Cambridge, UK), α‐smooth muscle actin (Santa Cruz Biotechnology, Dallas, TX, USA) and fibronectin (BD Pharmigen, Oxford, UK) were detected using Western blot analysis from kidney lysate using specific antibodies with β‐actin (Sigma Aldrich, St Louis, MO, USA) as a loading control. Secondary anti‐mouse and anti‐rabbit antibodies were purchased from Sigma Aldrich. Band densitometry was analysed using Image Lab software (Bio‐Rad Laboratories, Hercules, CA, USA).
A power calculation was performed to determine the minimum number of animals per group based on the parameters of plasma leptin and proteinuria, using the G*power software.
The Statistical Package for the Social Sciences (SPSS) statistical package software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analysis. Data were analysed for normal distribution. All data are presented as mean ± SEM. Differences among treatment groups were individually analysed compared with obese rats using independent samples t‐test for two group direct analyses or two‐factor repeated measures anova (treatment was the between subjects factor, and time point was the within subjects factor) for analysis of pre‐ and post‐treatment measurements between treatment and obese controls. Tukey's post hoc test was used to identify statistical differences in data sets between groups. Data sets which were not normally distributed were analysed using Mann–Whitney U‐test or Kruskal–Wallis test. Significance was set at P < 0.05.
In rats fed HFD for 10 weeks, there was a significant increase in body weight and body fat composition compared with rats fed a control chow diet (P < 0.05 Table1, n = 10–21). Further, at 10 weeks there were significant increases in diastolic (P < 0.05) and systolic BP (P < 0.05) (P < 0.05, n = 10–21). No significant differences were observed between groups for glucose tolerance or insulin sensitivity (Table1, n = 6–13). DIO rats exhibited significantly worse renal function than lean rats as measured by significantly elevated urinary protein (P < 0.05, n = 8–10), and urinary albumin (P < 0.05, n = 8–10), and significantly reduced urinary sodium (P < 0.05, n = 8–10) compared with lean chow‐fed rats (Table1).
In whole‐kidney lysate, CB2 receptor protein was significantly reduced in rats fed a HFD at 10 weeks relative to standard chow‐fed rats (P < 0.05, Figure1, n = 7–8). No significant differences between lean and HFD groups were observed in protein expression of collagen, fibronectin, TGFβ1 and α‐smooth muscle actin in whole‐kidney lysate (Figure1, n = 7–8). However, VEGF protein expression was significantly lower in rats fed a HFD compared with lean standard chow‐fed animals (P < 0.05, Figure1, n = 7).
In DIO control rats compared with treatment groups, weight gain was found to have a significant main effect (P < 0.05, n = 9–10), indicating that body weight significantly increased over the treatment period; however, no significant interactions between time and group was observed (Obese Control vs. AM1241; P = 1.00, Obese Control vs. AM630 P = 0.302, n = 9–10), indicating that treatment with either compound does not significantly affect weight gain (Figure2). Similarly, no significant interactions were observed in food consumption (Obese Control vs. AM1241; P = 0.412, Obese Control vs. AM630 P = 0.943, n = 9–10), glucose tolerance (Obese Control vs. AM1241; P = 0.165, Obese Control vs. AM630 P = 0.912, n = 9–10), or insulin sensitivity (Obese Control vs. AM1241; P = 0.912, Obese Control vs. AM630 P = 0.365, Figure2, n = 9–10). Plasma TGF‐β1 concentrations compared with obese controls were not altered by either treatment (Table2). No significant interactions between diastolic BP and treatment groups compared with obese control were observed (Obese Control vs. AM1241; P = 0.067, Obese Control vs. AM630 P = 0.423, Table2, n = 9–10). However, systolic BP in obese rats treated with AM1241 was significantly reduced across the treatment period compared with obese control rats (main effect P = 0.158, interaction P < 0.05, Table3, n = 9–10). No significant differences in systolic BP for obese rats treated with AM630 were observed compared with obese control (main effect P = 0.973, interaction P = 0.257, Table2, n = 9–10). In obese rats treated with AM1241, plasma leptin was significantly lower compared with obese controls (P < 0.05, n = 9–10); however, AM630 treatment did not alter leptin in obese rats compared with obese control animals (Table2, n = 9–10).
Histological analysis showed that in DIO rats (Figure3, n = 9–10), treatment with the CB2 receptor agonist, AM1241, did not affect glomerular area compared with obese control rats. Treatment of DIO rats with AM1241 did, however, significantly reduce renal tubular diameter (P < 0.05, Table3 and Figure3, n = 9–10) and peri‐renal adipose tissue standardized for body weight (P < 0.05, Table3 and Figure3, n = 9–10) compared with obese controls. While in AM630‐treated rats, renal tubular diameter and peri‐renal adiposity were not altered compared with obese control rats (Table3, n = 9–10); however, glomerular area was significantly increased in DIO rats treated with AM630 compared to obese rats treated with AM1241 (P < 0.05, Table3, n = 9–10). No glomerular lesions were apparent in DIO rats treated with either compound or in the control group (Figure3, n = 9–10). Furthermore, DIO rats treated with AM630 also exhibited significantly higher kidney weight (gross weight and standardized for body weight) compared with obese control rats (P < 0.05, Table3, n = 9–10), while this was not affected in DIO rats treated with AM1241.
Functional renal measurements analysed as the Δ change between pre‐ and post‐treatment measures found that AM1241 treatment in DIO rats significantly reduced urinary excretion of protein, albumin and sodium compared with the obese control rats (P < 0.05, Figure4, n = 9–10). However, the pretreatment level of total urinary protein was significantly higher in the DIO‐treated AM1241 group (23.11 ± 2.44μg·mL−1) compared with both the obese control rats (15.34 ± 1.15μg·mL−1) and AM630‐treated obese rats (14.10 ± 1.46μg·mL−1, P < 0.05, n = 9–10). In DIO rats, treatment with AM630 did not significantly alter urinary protein, albumin or sodium excretion (Figure4, n = 9–10). Plasma creatinine was not altered in DIO rats by either treatment (Figure3, n = 9–10). eGFR, as estimated by creatinine clearance standardized to body weight was significantly reduced by AM630 treatment (P < 0.05, n = 9–10), but not by AM1241 treatment in obese rats compared with obese saline controls (Figure4, n = 9–10).
In obese rats, plasma TGF‐β1 was not altered by treatment with AM1241 or AM630 compared with obese control (Table2, n = 9–10). Circulating levels of leptin were significantly reduced in obese rats treated with AM1241 (P < 0.05, n = 9–10) compared with obese controls, but were not altered by AM630 treatment in obese rats (Table2, n = 9–10).
In whole‐kidney tissue of DIO rats, treatment with AM1241 significantly reduced collagen IV, unprocessed TGF‐β1, mature TGF‐β1 and VEGF protein expression compared with obese controls (P < 0.05, Figure5, n = 9–10). In DIO rats, AM630 treatment did not significant alter renal expression of collagen IV or VEGF. However, treatment with AM630 did significantly reduce TGF‐β1 expression in obese rats compared with obese controls (P < 0.05, Figure5, n = 9–10). No differences between obese controls and the treated groups were observed for whole‐kidney protein expression of fibronectin and α‐smooth actin (Figure5, n = 9–10).
Our model of DIO in male Sprague Dawley rats has demonstrated that 10 weeks of consuming a HFD is sufficient to increase overall body fat and BP, typical characteristics of the obese phenotype (Keenan etal., 2000). This occurred in addition to impaired renal function, a co‐morbidity strongly associated with obesity. Furthermore, CB2 receptor expression was significantly down‐regulated in the kidneys of obese animals. Significantly, our novel data have clearly demonstrated that in rats with DIO, activation of CB2 receptors with AM1241 can ameliorate the progression of obesity‐related CKD as measured by urinary protein, urinary albumin and renal sodium excretion rates, conversely, antagonism of obese rats with the CB2 receptor antagonist AM630 reduced creatinine clearance, indicating accelerated decline in renal function.
In our model of DIO, 10 weeks on a HFD led to reduced renal function, which is reflected by altered handling of protein, albumin and sodium. Mechanistically, reduced urinary sodium excretion is associated with hypertension (Wright and Cavanaugh, 2010). Proteinuria and albuminuria observed in obesity‐related renal damage is hypothesized to be due to excess metabolic excretory load and structural renal remodelling including increased fibrosis (Cignarelli and Lamacchia, 2007).
Treatment with the CB2 receptor agonist and antagonist did not significantly alter weight gain, food consumption, glucose tolerance or insulin sensitivity in our model. This suggests that any alterations in renal function in response to modulation of CB2 receptors occurred as a result of the treatment rather than a positive side effect associated with weight loss. In mice, central overexpression of CB2 receptors in the hypothalamus has been shown to lead to a lean phenotype via reduction in food consumption, body weight and the orexogenic factors proopiomelanocortin (ACTH) and galanin (Romero‐Zerbo etal., 2012). Conversely, peripheral modulation of the CB2 receptor with a range of CB2 receptor agonists and antagonists, changes food consumption only under particular experimental conditions. Specifically, in response to CB2 receptor‐acting compounds, alteration in food intake is dependent upon animal strain and food availability (Onaivi etal., 2008). Here we have shown that in Sprague Dawley rats, the CB2 receptor compounds AM1241 and AM630 do not alter food consumption in obese animals with access to food ad libitum. As such, the consequences of pharmacologically modulating CB2 receptors in obesity to regulate food intake and body weight are still not clearly understood. However, activation of CB2 receptors has been shown to effectively ameliorate a number of pathophysiological processes associated with obesity including inflammation, fibrosis and cardiovascular dysfunction, which are important steps in the progression of organ damage associated with obesity (Pacher etal., 2005; Mukhopadhyay etal., 2010; Servettaz etal., 2010; Pacher and Mechoulam, 2011).
Additionally, we have shown that treatment with the CB2 receptor agonist AM1241 significantly reduces systolic BP in rats with DIO in the absence of any weight change. Hypertension leads to changes in renal haemodynamics, which advances the damage to the kidneys in obesity (Kramer etal., 2005). Systolic BP specifically has been demonstrated to be a stronger predictor of renal disease than diastolic BP in male adults (He and Whelton, 1999). The CB2 receptor is thought to play a limited role in regulating the cardiovascular system in normal conditions, but may have a protective role in a pathophysiological setting (Pacher and Mechoulam, 2011). We have shown that treatment with AM1241 significantly reduced BP in rats with DIO, while treatment with AM630 had no effect, congruent with previously reported data (Hanus etal., 1999).
Our model of DIO showed that obese rats exhibited increased circulating leptin and TGF‐β1 levels compared with lean animals. While plasma concentrations of TGF‐β1 were not affected by modulation of CB2 receptors, we have shown for the first time that activation of CB2 receptors with AM1241 in obese rats significantly ameliorated the progression of hyperleptinaemia compared with obese controls. The adipokine leptin is removed from the circulation by the kidneys and is associated with the promotion of renal fibrosis via the up‐regulation of TGF‐β1 secretion by renal tissue (Wolf etal., 1999). Hyperleptinaemia and increased adiposity has been observed previously in CB2 receptor knockout (CB2−/−) mice compared with age‐matched wild‐type mice (Agudo etal., 2010). However, in mice fed a HFD for 15 weeks, CB2 receptor knockout significantly reduced plasma leptin concentrations compared with HFD controls, which is likely to be the result of reduced adiposity (Deveaux etal., 2009), indicating that the role and mechanism of CB2 receptors regulating weight has yet to be elucidated. In our model of DIO there is not a reduction in adiposity in response to CB2 receptor agonism, yet there is a significant reduction in plasma leptin concentrations. Thus, there is likely to be some cross‐talk between leptin and the CB2 receptor, which may be important for a number of co‐morbidities associated with obesity (Deveaux etal., 2009; Agudo etal., 2010).
Hypertension and hyperleptinaemia associated with obesity can lead to a decline in renal function typically featuring increased urinary protein, albumin and sodium excretion via alterations to renal haemodynamics, modifications to water and sodium handling by the kidneys and activation of fibrotic and oxidative stress pathways (Wolf etal., 1999; Dobrian etal., 2000; Kramer etal., 2005; Amazonas and Lopes de Faria, 2006; Abassi etal., 2009; Briffa etal., 2013). In obese rats treated with AM630, there were no significant changes to BP or leptin; however, CB2 receptor antagonism with AM630 in DIO rats led to elevated urinary albumin excretion, indicating accelerated decline in renal function compared with obese controls. Furthermore, we have shown that AM630‐treated obese rats had a significantly higher glomerular cross‐sectional area, similar to our previous in vitro analysis where cells exposed to AM630 demonstrated an increased renal proximal tubular hypertrophy (Jenkin etal., 2010). These data taken together indicate that antagonism of CB2 receptors may exacerbate hypertrophic changes occurring in the kidney of obese rats and ultimately advance the progression of obesity‐related CKD.
The CB2 receptor has been identified in kidney tissue previously (Deutsch etal., 1997), with renal expression reduced in the glomeruli of patients with advanced diabetic nephropathy (Barutta etal., 2011). Also, treatment of STZ diabetic rats with AM1241 has been shown to ameliorate the progression of albuminuria, via the reduction of podocyte loss (Barutta etal., 2011). However, prior to this study, renal expression of CB2 receptors in a model of obesity had not been examined. Our data clearly demonstrate that after 10 weeks on a HFD, CB2 receptor protein expression in kidney tissue is significantly reduced. It is also likely that organ‐specific expression of CB2 receptors in DIO is likely to occur, with a previous study demonstrating CB2 receptor expression was enhanced in adipocytes in response to obesity (Deveaux etal., 2009).
Previous studies have investigated the role of CB2 receptors and specifically CB2 receptor agonism as a therapeutic to treat models of nephropathy (Mukhopadhyay etal., 2010; Barutta etal., 2011). However, these studies focused on the role of the glomerulus, with activation of CB2 receptors reducing glomerular podocyte loss and cytokine‐mediated cellular damage (Mukhopadhyay etal., 2010; Barutta etal., 2011; Pacher and Mechoulam, 2011). Our novel data here indicate that the CB2 receptor agonist AM1241 reduces tubular hypertrophy and accumulation of peri‐renal adipose tissue, which may lead to the reduction in renal damage observed. Furthermore, we have shown that in renal tissue of rats with DIO, markers of fibrosis including TGF‐β1, collagen IV and VEGF were significantly reduced in AM1241‐treated obese rats compared with obese controls. The findings of VEGF in this study are of particular interest. Specifically, VEGF has been shown to up‐regulate proliferative factors, and act as an anti‐apoptotic protein (Kanellis etal., 2002). Thus, in the AM1241‐treated obese rats, down‐regulation of VEGF can account for the reduction in tubular diameter observed. However the reduction in VEGF following 10 weeks of DIO, suggests that the increase in glomerular and tubular diameter observed is not mediated by VEGF.
The main aim of this study was to examine the effect of modulation of CB2 receptors in DIO. Once we had established that there was renal dysfunction in our model of DIO; as determined by albuminuria, proteinuria and altered sodium excretion, we then investigated if modulation of CB2 receptors may alter markers that are associated with renal fibrosis in this model. Renal fibrosis is typically characterized by epithelial cell dysfunction including dilation, leukocyte migration, increased extracellular matrix deposition, myofibroblast proliferation and activation (Liu, 2011). In addition, multiple signalling pathways including TGF‐β1 are implicated in the progression of epithelial cell dysfunction and fibroblast activation (Liu, 2011). Thus, future investigations should more comprehensively establish if prolonged exposure to an HFD up‐regulates fibrotic markers indicative of renal damage.
The degree of tubular interstitial fibrosis is closely associated with decline of renal function (Qi etal., 2006), and experimental evidence suggests that proteinuria and albuminuria can lead to increased release of pro‐inflammatory and fibrotic cytokines from renal cells (Wohlfarth etal., 2003; Qi etal., 2006). In this study, AM1241 treatment in obese rats lead to both a significant reduction of fibrotic markers in renal tissue and improved proteinuria and albuminuria. In other fibrotic disease models, CB2 receptor agonism has been shown to attenuate the damage associated with the development or progression of fibrosis in animals (Munoz‐Luque etal., 2008; Servettaz etal., 2010). Interestingly, although AM630‐treated obese rats showed no significant changes in either VEGF or collagen IV protein expression, TGF‐β1 was found to be significantly reduced in renal tissue compared with obese controls. The precise mechanism for this effect is unclear; however, as a complex role between CB2 receptors and TGF‐β1 regulation has been highlighted in lymphocytes in vivo, where TGF‐β1 secretion and CB2 receptor expression displayed reciprocal modulation (Gardner etal., 2002). The CB2 receptor mediates a diverse range of immunological functions, given its high expression in immune cells (Klein etal., 2003; Cabral and Griffin‐Thomas, 2009), and agonism of CB2 receptors has been shown in models of nephropathy to limit inflammation through oxidative stress pathways (Mukhopadhyay etal., 2010; Barutta etal., 2011; Pacher and Mechoulam, 2011). Also, in line with a previous study that demonstrated the role of leptin in modulating TGF‐β1 expression (Wolf etal., 1999), our novel data suggest that the reversal of hyperleptinaemia may reduce renal TGF‐β1 levels in obese rats treated with AM1241. The effects of AM1241 in altering renal function of obese rats in this study may also be mediated through inhibition of inflammatory processes, with future studies investigating this avenue now warranted.
One limitation of this study is that before treatment with the specific CB2 receptor drugs, rats allocated to the AM1241 treatment group had a significantly higher urinary protein excretion compared with both obese control and AM630‐treated obese rats. Obese rats in this study were matched for weight, body composition and BP at 9 weeks of feeding on a HFD, before being placed into treatment groups. However, Sprague Dawley rats have a range of physiological responses to HFD, with some becoming obese while others exhibit a less severe phenotype (Levin etal., 1997). The rats in the AM1241 treatment group still showed significant reductions in proteinuria, despite being higher at the pretreatment time point. Further, significant reductions in albuminuria, a measurement closely tied with proteinuria were demonstrated by AM1241 administration in obese rats. This study has significant potential clinical implications for the treatment of obesity‐related renal damage. Importantly, we have shown that activation of CB2 receptors directly targets the kidneys to ameliorate the progression of CKD in a model of DIO, in addition to significantly reducing systolic BP, plasma leptin and fibrotic markers in renal tissue of obese rats in the absence of changes to weight, food consumption or glucose and insulin sensitivity. It is also noteworthy that antagonism of CB2 receptors may exacerbate the progression of obesity‐related renal damage. Thus, these data taken together suggest that CB2 receptor agonism in obesity‐related nephropathy is a potential therapeutic, adding to the previous research that CB2 receptor agonism may have a protective role in a number of models of nephropathy (Mukhopadhyay etal., 2010; Barutta etal., 2011; Pacher and Mechoulam, 2011; Jenkin etal., 2012).
This work was supported by the Allen Foundation (D. H. H., A. J. M.), and through the Australian Government's Collaborative Research Networks (C. R. N.) programme (A. J. M.). Scholarship funding by Australian Postgraduate Award (K. A. J., L. O.) and Australian Rotary Health (A. C. S.).
Jenkin K. A., O'Keefe L., Simcocks A. C., Briffa J. F., Mathai M. L., McAinch A. J., and Hryciw D. H. (2016) Renal effects of chronic pharmacological manipulation of CB 2 receptors in rats with diet‐induced obesity. Br J Pharmacol, 173: 1128–1142. doi: 10.1111/bph.13056.