Overactive cannabinoid 1 receptor in podocytes drives type 2 diabetic nephropathy Tony Jourdana,1, Gergo} Szandaa, Avi Z. Rosenbergb,c, Joseph Tama, Brian James Earleya, Grzegorz Godlewskia, Resat Cinara, Ziyi Liua, Jie Liua, Cynthia Jud, Pál Pachere, and George Kunosa,1 aLaboratory of Physiologic Studies, Section on Neuroendocrinology, and eSection on Oxidative Stress and Tissue Injury, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, bKidney Diseases Section, National Institute on Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; cThe Johns Hopkins University School of Medicine, Baltimore, MD 21205; and dSkaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045 Edited by Lutz Birnbaumer, National Institute of Environmental Health Sciences, Research Triangle Park, NC, and approved October 30, 2014 (receivedfor review October 17, 2014) Diabetic nephropathy is a major cause of end-stage kidney disease, effective as globally acting ones in reversing obesity and its and overactivity of the endocannabinoid/cannabinoid 1 receptor metabolic sequelae (13, 14), and also delay the onset of overt (CB1R) system contributes to diabetes and its complications. Zucker T2DM (15) without inducing any behaviors that are predictive of diabetic fatty (ZDF) rats develop type 2 diabetic nephropathy with adverse neuropsychiatric effects in humans (13, 14). Conse- albuminuria, reduced glomerular filtration, activation of the renin- quently, there is considerable interest in translating these find- angiotensin system (RAS), oxidative/nitrative stress, podocyte loss, ings to clinical practice by developing peripherally restricted and increased CB1R expression in glomeruli. Peripheral CB1Rblock- CB1R antagonists/inverse agonists for clinical use. In this study ade initiated in the prediabetic stage prevented these changes or we used the Zucker diabetic fatty (ZDF) rat, a well-established reversed them when animals with fully developed diabetic ne- model of type 2 diabetic nephropathy (16, 17), to explore the phropathy were treated. Treatment of diabetic ZDF rats with los- role of peripheral CB1R in diabetic kidney disease, and the artan, an angiotensin II receptor-1 (Agtr1) antagonist, attenuated therapeutic potential of its inhibition by a nonbrain-penetrant the development of nephropathy and down-regulated renal corti- CB1R inverse agonist. We report that selective antagonism of cal CB1R expression, without affecting the marked hyperglycemia. peripheral CB1R can both prevent the deterioration of kidney In cultured human podocytes, CB1R and desmin gene expression function when treatment starts in the prediabetic stage and re- were increased and podocin and nephrin content were decreased verse it when treatment is started after the full development of ′ by either the CB1R agonist arachydonoyl-2 -chloroethylamide, an- diabetic nephropathy. The results also indicate that increased giotensin II, or high glucose, and the effects of all three were endocannabinoid/CB1R signaling in podocytes plays a key role in antagonized by CB1R blockade or siRNA-mediated knockdown of CNR1 the development of nephropathy, which can be dissociated from (the cannabinoid type 1 receptor gene). We conclude that in- the hyperglycemia and is likely induced via the RAS. creased CB1R signaling in podocytes contributes to the develop- ment of diabetic nephropathy and represents a common pathway Results through which both hyperglycemia and increased RAS activity ex- CB R Antagonism Prevents the Development of Nephropathy in ert their deleterious effects, highlighting the therapeutic potential 1 Prediabetic ZDF Rats. Six-week-old male, prediabetic ZDF rats of peripheral CB Rblockade. 1 were started on a daily oral dose (3 mg/kg) of the nonbrain penetrant CB R inverse agonist JD5037 or vehicle for 90 d. As endocannabinoid | podocyte | angiotensin II | nephropathy | 1 illustrated in Fig. 1A, vehicle-treated ZDF rats developed extreme hyperglycemia Significance iabetic nephropathy, a highly prevalent and serious com- Dplication of both type 1 and type 2 diabetes mellitus and a leading cause of renal failure, is characterized by albuminuria, Diabetic nephropathy is the leading cause of chronic kidney decreased glomerular filtration rate (GFR), mesangial expan- disease in the United States, and one of the most significant sion, thickening of the glomerular basement membrane, and long-term complications of both type 1 and type 2 diabetes, glomerular sclerosis (1). Multiple mechanisms have been impli- which currently lack fully effective therapy. Hyperglycemia and cated in the development of diabetic nephropathy, including activation of the renin-angiotensin system (RAS) are thought to activation of the renin-angiotensin system (RAS) (2), increase in be the two main drivers of this pathology. We have recently oxidative (3) and nitrosative/nitrative stress (4), as well as an shown that selective blockade of peripheral cannabinoid increase in local inflammation (5). receptor-1 (CB1R) delayed and attenuated the development of The endocannabinoid system plays a well-documented role in type 2 diabetes in a rat model. Here we show that the ne- obesity and its metabolic complications, including insulin re- phropathy-inducing effects of both hyperglycemia and activa- sistance and type 2 diabetes (T2DM). Globally acting cannabi- tion of the RAS involve CB1Ractivationinglomerularpodocytes, and that antagonism of peripheral CB1R could represent a novel, noid 1 receptor (CB1R) antagonists/inverse agonists improve obesity-related insulin resistance, dyslipidemia, fatty liver, and effective, and rational approach to both prevent and reverse β-cell loss, and attenuate obesity-related inflammatory changes diabetic nephropathy. both in preclinical models of diet-induced or genetic obesity and Author contributions: T.J., P.P., and G.K. designed research; T.J., G.S., J.T., B.J.E., G.G., R.C., in clinical trials in overweight subjects with metabolic syndrome Z.L., and J.L. performed research; C.J. contributed new reagents/analytic tools; T.J., A.Z.R., (reviewed in refs. 6 and 7). Global CB1R blockade also has and P.P. analyzed data; and T.J. and G.K. wrote the paper. beneficial effects in mouse models of type 1 and type 2 diabetic The authors declare no conflict of interest. – nephropathy (8 11). However, the therapeutic development of This article is a PNAS Direct Submission. this class of compounds has been halted because of adverse 1To whom correspondence may be addressed. Email: [email protected] or george. neuropsychiatric side effects in a small proportion of treated [email protected]. subjects (12). Recent studies in rodent models have demon- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. strated that peripherally restricted CB1R antagonists are as 1073/pnas.1419901111/-/DCSupplemental. E5420–E5428 | PNAS | Published online November 24, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1419901111 Downloaded by guest on October 1, 2021 hyperglycemia by the end of the treatment period, accompanied by PNAS PLUS increased kidney weight, greatly elevated plasma creatinine and blood urea nitrogen levels, polydipsia, polyuria, a marked re- duction in GFR, as well as increased excretion of albumin, glucose, and uric acid, compared with lean controls. Chronic treatment of ZDF rats with JD5037 completely or nearly completely normalized all these parameters (Fig. 1A).Thesamedifferencewasevident for Cnr1 (the cannabinoid type 1 receptor gene) expression in the glomerulus, its diabetes-related increase being fully re- versed by treatment with JD5037 (Fig. 1B). Body weight gain was similar in vehicle-treated and JD5037-treated ZDF rats during the first 4 wk of treatment, as reported earlier (15), but as the vehicle-treated rats got sicker with extreme hyperglyce- mia, their body weight plateaued, whereas JD5037-treated rats continued to grow, resulting in significantly greater weight in the latter by the end of the treatment period (Fig. 1). CB1R immunoreactivity was also detectable in proximal tubular cells (Fig. S1), in agreement with earlier findings in the mouse (14), and the specificity of immunostaining was evident in its absence −/− in preparations from CB1R mice (Fig. S1). Vehicle-treated ZDF rats also displayed a marked reduction in the number of podocytes compared with control lean rats based on podocin and Wilms’ tumor (WT-1) immunostaining as well as podocalyxin/WT-1 double immunostaining, which was largely prevented by JD5037 treatment (Fig. 2A). CB1R protein was tightly colocalized with podocalyxin in the perisomal region of podocytes, indicating its expression primarily in podocytes (Fig. 2B). Consistent with podocyte loss, renal cortices of ZDF rats had much lower podocin mRNA (Nphs2) and protein (Fig. 2A) and lower nephrin (Nphs1) and zonula occludens-1 (Tjp1) mRNA, but higher desmin (Des) mRNA than cortices of control rats, and these changes were prevented by peripheral CB1R antagonism (Fig. 2C). Histopathological findings in glomeruli and tubulointerstitium of vehicle-treated ZDF rats were subtle and no vascular changes were noted. Occasional glomeruli contained podocytes with prominent MEDICAL SCIENCES protein resorption droplets (Fig. S2), although podocyte enlarge- ment was more commonly observed. No evidence of chronicity (glomerulosclerosis, interstitial fibrosis or tubular atrophy)