BACKGROUND AND PURPOSEIn diabetic nephropathy agonism of CB2 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 CB2 receptors. EXPERIMENTAL APPROACHMale 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 3 mg·kg − 1 AM1241, 0.3 mg·kg −1 AM630 or saline for 6 weeks. KEY RESULTSTen weeks on a HFD significantly reduced renal expression of CB2 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. CONCLUSIONSThis study demonstrates that while agonism of CB2 receptors with AM1241 treatment for 6 weeks does not reduce weight gain in obese rats, it leads to improvements in obesity-related renal dysfunction. AbbreviationsCB1 receptor, cannabinoid receptor 1; CB2 receptor, cannabinoid receptor 2; CKD, chronic kidney disease; DIO, diet-induced obesity; eGFR, estimated glomerular filtration rate; HFD, high-fat diet; PAS, periodic acid-Schiff
Modulation of the endocannabinoid system as an anti-obesity therapeutic is well established; however, the direct effects of cannabinoid receptor 1 (CB 1 ) antagonism on renal function and structure in a model of diet-induced obesity (DIO) are unknown. The aim of this study was to characterise the renal effects of the CB 1 antagonist AM251 in a model of DIO. Male Sprague-Dawley rats were fed a low-or high-fat diet (HFD: 40% digestible energy from lipids) for 10 weeks to elicit DIO (nZ9). In a different cohort, rats were fed a HFD for 15 weeks. After 9 weeks consuming a HFD, rats were injected daily for 6 weeks with 3 mg/kg AM251 (nZ9) or saline via i.p. injection (nZ9). After 10 weeks consuming a HFD, CB 1 and megalin protein expression were significantly increased in the kidneys of obese rats. Antagonism of CB 1 with AM251 significantly reduced weight gain, systolic blood pressure, plasma leptin, and reduced albuminuria and plasma creatinine levels in obese rats. Importantly, there was a significant reduction in tubular cross-section diameter in the obese rats treated with AM251. An improvement in albuminuria was likely due to the reduction in tubular size, reduced leptinaemia and maintenance of megalin expression levels. In obese rats, AM251 did not alter diastolic blood pressure, sodium excretion, creatinine clearance or expression of the fibrotic proteins VEGFA, TGFB1 and collagen IV in the kidney. This study demonstrates that treatment with CB 1 antagonist AM251 improves renal outcomes in obese rats.
Endocannabinoids are products of dietary fatty acids that are modulated by an alteration in food intake levels. Overweight and obese individuals have substantially higher circulating levels of the arachidonic acid derived endocannabinoids, anandamide and 2-arachidonoyl glycerol, and show an altered pattern of cannabinoid receptor expression. These cannabinoid receptors are part of a large family of G protein coupled receptors (GPCRs). GPCRs are major therapeutic targets for various diseases within the cardiovascular, neurological, gastrointestinal, and endocrine systems, as well as metabolic disorders such as obesity and type 2 diabetes mellitus. Obesity is considered a state of chronic low-grade inflammation elicited by an immunological response. Interestingly, the newly deorphanized GPCR (GPR18), which is considered to be a putative cannabinoid receptor, is proposed to have an immunological function. In this review, the current scientific knowledge on GPR18 is explored including its localization, signaling pathways, and pharmacology. Importantly, the involvement of nutritional factors and potential dietary regulation of GPR18 and its (patho)physiological roles are described. Further research on this receptor and its regulation will enable a better understanding of the complex mechanisms of GPR18 and its potential as a novel therapeutic target for treating metabolic disorders.
The scavenger receptor megalin binds to albumin in the microvilli of the renal proximal tubule, and transports the ligand to the intravillar cleft for processing by endocytosis. Albumin endocytosis in the proximal tubule is regulated by protein complexes containing a number of transmembrane and accessory proteins including PDZ scaffolds such as NHERF1 and NHERF2. PDZ scaffold proteins bind to class I PDZ binding motifs (S/T-X-Φ) in the extreme C-terminus of targets. Megalin contains a functional PDZ binding motif (SDV) in its distal terminus, however a potential interaction with the NHERF proteins has not been investigated. As megalin associates with NHE3 in the microvilli and NHE3 is tethered to the intravillar cleft via its interaction with NHERF1, we investigated if there is a direct interaction between megalin and NHERF1 in renal proximal tubule cells. Using confocal microscopy we determined that megalin and NHERF1 co-localise in the apical region in proximal tubule cells. Immunoprecipitation experiments performed using rat kidney lysate indicated that megalin bound NHERF1 in vivo. Using fusion proteins and peptides, we determined that PDZ2 of NHERF1 bound to megalin and that this interaction was via the C-terminus of megalin directly and in the absence of any accessory protein. We next investigated which domain in megalin was regulating this interaction. Using GST fusion proteins we determined that the loss of the most distal C-terminus of megalin containing the PDZ binding motif (SDV) did not alter its ability to bind to NHERF1. Significantly, we then identified an internal NHERF binding domain in the C-terminus of megalin. Using peptide studies we were able to demonstrate that NHERF1 bound to an internal PDZ binding motif in megalin and that a loss of a single threonine residue abolished the interaction between megalin and NHERF1. Finally, in proximal tubule cells, silencing NHERF1 increased megalin expression. Therefore, we have identified a novel protein interaction in proximal tubule cells and specifically identified a new internal PDZ binding motif in the C-terminus of megalin.
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