Overactive 1 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 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 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) was observed. Focal tubular injury in association with dilated tubular lumens containing proteinaceous material was noted. Many tubular epithelial cells contained prominent resorption droplets. In rats receiving JD5037, podocyte and tubular resorption droplets tended to be smaller, with a lesser degree of coalescence and more limited tubular epithelial simplification (Fig. S2). These latter findings are consistent with the observed decrease in proteinuria with JD5037 treatment (Fig. 1A). The limited histopathologic findings are not surprising in light of the relatively early sampling point (19 wk of age) because the more prominent diabetic histomorphologic changes were observed in older cohorts of ZDF rats (18).

Peripheral CB1R Antagonism Normalizes Increased Xanthine Oxidase Activity and Uric Acid Levels in ZDF Rat Kidney. Xanthine oxidase (or xanthine oxidoreductase, XO/XOR) is a potential source of re- active oxygen species (ROS) in diabetes (19), and is also involved in the metabolism of uric acid, a product of excessive purine ca- tabolism that can lead to the development of gout or nephropa- thy. As shown in Fig. 3A, renal cortex XO activity and expression were markedly increased and accompanied by elevated serum and kidney uric acid levels in ZDF compared with controls rats, and all of these changes were prevented by JD5037 treatment (Fig. 3A). Fig. 1. Peripheral CB1R antagonism prevents the development of diabetes A potential off-target, direct effect of JD5037 on XO activity and the associated nephropathy in ZDF rats. Blood and urine parameters (A) could be excluded by the failure of 10–100 nM JD5037 to in- μ and CB1R protein (scale bar, 20 m) and mRNA expression in the renal cortex fluence the activity of recombinant XO, which was completely (B) were analyzed in lean controls (open columns, body weight: 356.4 ± 6.7 g), ZDF rats treated with vehicle (gray, BW: 370.3 ± 29.6 g) or 3 mg·kg·d JD5037 from age 6–18 wk (black, BW: 427.3 ± 24.2 g, P < 0.05 compared to both lean control and vehicle-treated ZDF groups). Columns and bars repre- sponding value in lean (L) (*P < 0.05, **P > 0.01, ***P < 0.001) or vehicle- sent mean ± SEM (n = 20 rats per group). Significant difference from corre- treated ZDF rats (V) (##P < 0.01, ###P < 0.001).

Jourdan et al. PNAS | Published online November 24, 2014 | E5421 Downloaded by guest on October 1, 2021 Adiponectin has anti-inflammatory effects and recent studies suggest an inverse correlation between adiponectinemia, uric acid levels, and renal function (20, 21). Accordingly, plasma adipo- nectin levels as well as the mRNA for adiponectin receptors 1 and 2 in the kidneys were reduced in ZDF rats and normalized by JD5037 treatment (Fig. 3C).

Proinflammatory Cytokine Profile in the Diabetic Kidney Is Modulated

by Peripheral CB1R Blockade. As a further indication of inflammation in the diabetic kidney, the renal cortical expression of the proin- flammatory cytokines Tnf, Il-18,andIl-6, as well as the activity of the apoptotic caspase 3/7 were increased, and JD5037 treatment attenuated all these changes (Fig. S3A). However, contrary to our earlier findings of robust proinflammatory

Fig. 2. Markers of podocyte health and injury in kidneys of lean and ZDF rats and in ZDF rats treated with JD5037. (A) Podocytes identified in glomeruli of lean and vehicle- or JD5037-treated ZDF rats by WT-1 (scale bars, 50 μm) and podocin immunohistochemistry and podocalyxin/WT-1 double immunohisto- chemistry. Analysis of podocin protein by Western blotting in renal cortices

from the same animals. (B) Colocalization of podocalyxin with CB1R, as ana- Fig. 3. Peripheral CB1R antagonism in ZDF rats prevents the increase in uric lyzed by double immunohistochemistry. Specificity of CB1Rlabelingisin- acid production and decrease in adiponectin and adiponectin receptor ex- −/− dicated by its absence in CB1R mice. (C) Nphs1, Tjp1,andDes mRNA levels in pression. (A) XOR activity in kidney tissue and plasma and kidney concen- renal cortices from the same three groups of rats. Symbols as described in the tration of uric acid in lean control rats (open columns) and ZDF rats treated legend of Fig. 1, n = 20 rats per group. Significance of differences indicated as with vehicle (gray columns) or JD5037 from age 6–19 (black columns). Im- # described for Fig. 1 ( P < 0.05). munoreactive XOR protein expression and renal Xdh mRNA levels are also shown. (Scale bars, 100 μm.) (B) JD5037 does not directly inhibit XO activity. The activity of recombinant XO was measured in vitro in the presence of blocked by the known XO inhibitor allopurinol (Fig. 3B). The vehicle, the XO inhibitor allopurinol (100 μM) or JD5037 (10 and 100 nM). (C) concentrations of JD5037 used were relevant to the in vivo Plasma adiponectin and renal levels of Adipor1 and Adipor2 mRNA in the same three groups of rats as in A. Columns represent means ± SEM from 20 condition, as peak plasma concentration of JD5037 following a animals per group; Significant difference from corresponding value in lean ± = > # 3 mg/kg oral dose was 175 40 nM (n 3), of which 99% is (*P < 0.05, **P < 0.01, ***P < 0.001), or vehicle-treated ZDF rats ( P < 0.05, protein bound. ##P < 0.01, ###P < 0.001).

E5422 | www.pnas.org/cgi/doi/10.1073/pnas.1419901111 Jourdan et al. Downloaded by guest on October 1, 2021 PNAS PLUS MEDICAL SCIENCES

Fig. 4. Chronic JD5037 treatment prevents the up-regulation of the RAS in ZDF rats (A) and the parallel increase in the renal expression of ROS-generating NADPH isoforms as well as 4-hydroxynonenal (4HNE) and 3-nitrotyrosine (3NT) levels in kidney tissue (B). RAS activity is reflected by plasma aldosterone and angiotensin II levels, Agtr1 mRNA, and phospho–NF-κB (Ser536) levels in kidney. Treatment groups, symbols, and statistical analyses as described in the legend of Fig. 1 (#P < 0.05). Columns and bars represent means ± SEM from 20 animals per group.

macrophage infiltration in pancreatic islets of ZDF rats (15), there was no evidence for macrophage infiltration or activation of the Nlrp3 inflammasome in the kidney, as reflected in unchanged expression of Cd68 and Nlrp3, unchanged caspase 1 activity, and IL-1b protein levels (Fig. S3B), and in the similar number of + CD68 macrophages in the three treatment groups (Fig. S3C). Fig. 5. Effects of JD5037 treatment on renal function and hyperglycemia in diabetic ZDF rats. ZDF rats were treated with daily oral doses of 3 mg/kg JD5037 or vehicle from age 15–19 wk. Blood glucose and plasma and urine Peripheral CB1R Antagonism Prevents Activation of the Renin- Angiotensin System and Oxidative Stress in ZDF Rat Kidney. Com- markers of renal function were measured at the beginning and end pared with lean rats, vehicle-treated ZDF rats had increased of treatment in lean (open columns), vehicle-treated ZDF (gray columns), and JD5037-treated ZDF rats (black columns). Significant differences plasma aldosterone and angiotensin II levels, as well as increased < κ from corresponding values in lean rats (***P 0.005), from correspond- phosphorylation of the p65 subunit of NF- B and increased an- ing values in vehicle-treated ZDF rats (#P < 0.05, ###P < 0.005), or giotensin II receptor-1 (Agtr1) mRNA in the renal cortex from corresponding pretreatment value is indicated as (&P < 0.05, &&P < (Fig. 4A), suggesting up-regulation of the RAS, which is known 0.01, &&&P < 0.005).

Jourdan et al. PNAS | Published online November 24, 2014 | E5423 Downloaded by guest on October 1, 2021 displayed increased expression of ROS-generating NADPH oxi- dase isoforms, such as NADPH oxidase4 (Nox4), GP91Phox (Nox2), p47phox,andNox1 (Fig. 4B). Moreover, ZDF kidneys contained elevated 4-hydroxynonenal and 3-nitrotyrosine levels compared with lean rats (Fig. 4B), suggesting increased oxidative and nitrative stress. The development of all these changes was markedly attenuated by peripheral CB1R blockade (Fig. 4).

Peripheral CB1R Antagonism Reverses Fully Developed Diabetic Nephropathy Without Affecting the Associated Hyperglycemia. To test whether peripheral CB1R blockade can reverse T2DM and the associated nephropathy once it has developed, 15-wk-old ZDF rats were treated with 3 mg·kg·dJD5037for4wktomatch the age of ZDF rats undergoing preventive treatment at the time of killing. The pronounced hyperglycemia of these ani- mals, which is a result of β-cell loss (15), remained unaffected by JD5037 treatment, whereas their polyuria, albuminuria, uricosuria, increased plasma and urinary creatinine, and in- creased plasma levels of angiotensin II and aldosterone were decreased and their reduced GFR was increased significantly beyond pretreatment levels (Fig. 5). Additionally, plasma levels of two hormones known to affect water reabsorption in the distal tubules were also altered: vasopressin levels were decreased and levels of atrial natriuretic peptide increased in ZDF vs. control rats and both changes were reversed by JD5037 treatment (Fig. S4). Similarly, increases in relative kidney weight and the renal expression of Cnr1, Agtr1, Nox4, and Des and decreases in the expression of Nphs1 and Nphs2 were attenuated by JD5037 treatment of these animals (Fig. 6A). Interestingly, the proximal tubular marker megalin was also decreased in ZDF rats in a CB1R-dependent manner (Fig. 6A), which was similar to earlier findings in mice with diet-induced obesity/insulin resistance (14), and suggested that CB1R-mediated changes in proximal tubular functions may contribute to the nephropathy of ZDF rats. Finally, podocyte loss was also reversed, as indicated by WT-1 immu- nostaining (Fig. 6B), a widely used marker of podocytes in rodents (23). Thus, 4-wk treatment of diabetic ZDF rats with a peripheral CB1R antagonist reversed the nephropathy, but not the β-cell loss. Modest histopathological changes in sections from five ZDF rats treated with vehicle from 15 to 19 wk of age (Fig. S5) were similar to those described above for the vehicle-treated controls in the prevention paradigm. In five age-matched ZDF rats re- ceiving JD5037 during the same period, there were very mild decreases in protein droplet size and tubular injury with luminal dilation, and intraluminal protein was similarly very mildly de- creased (Fig. S5). Of note, this treatment effect is not surprising because few chronic features (i.e., synechia, glomerulosclerosis, or significant interstitial fibrosis and tubular atrophy) have developed at the point at which the cohort is sampled for histopathological examination.

Angiotensin II Receptor Blockade Improves the Nephropathy of ZDF Rats. Twelve-week-old male ZDF rats were started on daily oral treatment (20 mg/kg) with the Agtr1 antagonist losartan or ve- hicle for 28 d. Relative to lean controls, both vehicle-treated and losartan-treated ZDF rats developed extreme hyperglycemia by Fig. 6. Effect of JD5037 treatment on relative kidney weight and the renal the end of the treatment period (Fig. 7, Top Left). However, expression of genes implicated in podocyte loss (Cnr1, Agtr1, Nox4, Des)or losartan treatment improved renal function as reflected in a re- + survival (Nphs1, Nphs2)(A); on the presence WT-1 podocytes in renal glomeruli duction in polyuria, albuminuria, blood urea nitrogen, plasma (scale bars, 20 μm) (B); on caspase 3/7 activity in kidney tissue (C); or in cultured and urine creatinine and preservation of a higher GFR (Fig. 7). human podocytes incubated with 30 mM glucose (D). Treatment groups and As expected, the elevated circulating levels of angiotensin II in statistical significance are indicated as described in the legend of Fig. 1 (#P < 0.05), BW: 297 ± 6.7 g (lean); 401.9 ± 15.2 g (ZDF + V); 413.2 ± 15.0 g (ZDF + JD). ZDF rats were unaffected, whereas the elevated plasma aldo- sterone levels were normalized by losartan (Fig. 7, Bottom Left). Although losartan treatment did not reverse the increase in to increase oxidative stress through activation of NADPH oxi- kidney weight, the increased expression of Agtr1 and Nox4, and dases (22). Indeed, renal cortices from vehicle-treated ZDF rats the decreased expression of Nphs2 and Npsh1 in the renal cortex

E5424 | www.pnas.org/cgi/doi/10.1073/pnas.1419901111 Jourdan et al. Downloaded by guest on October 1, 2021 were normalized, and the elevated level of Cnr1 mRNA was PNAS PLUS reduced to levels below that in lean control rats (Fig. S6).

Effects of CB1R Activation in Cultured Human Podocytes. Podocytes play a central role in maintaining normal glomerular function and podocyte damage contributes to diabetic nephropathy (24). CB1R are present in podocytes (Fig. 2B), where their activation has been shown to induce endoplasmic reticulum stress and apoptosis (25). Hyperglycemia and increased RAS activity are thought to be the two main pathogenic factors in diabetic ne- phropathy (24). First, we analyzed the potential interaction be- tween angiotensin II and the endocannabinoid/CB1R system in human podocytes. Exposure of the cells to 100 nM angiotensin II resulted in a significant increase in the cellular content of both and 2-arachidonoylglycerol, the two major endo- , and these effects were abrogated either by losartan (10 μM) or by JD5037 (100 nM) (Fig. 8A). Similar angiotensin II treatment of podocytes resulted in up-regulation of the expres- sion of both AGTR1 and CNR1, and both losartan and JD5037 prevented these changes. Furthermore, NPHS2 and NPHS1 ex- pression were significantly decreased, whereas DES expression was increased by angiotensin II, and these effects were also blocked by either losartan or JD5037, except for NPHS1, where JD5037 was ineffective (Fig. 8B). We also examined the relationship between the effects of high glucose and CB1R activation in cultured human podocytes. Ex- posure of the cells to either 30 mM glucose or 5 μM of the CB1R agonist arachidonyl-2’-chloroethylamide (ACEA) increased the expression of CNR1. Both stimuli decreased the expression of NPHS2 and NPHS1 and increased the expression of DES and NOX4, and similar changes were observed at the protein ex- pression level for podocin and nephrin. Interestingly, all of these effects induced either by glucose or ACEA were completely prevented by CB1R blockade by 10 nM JD5037 (Fig. S7A). The role of CB1R in these changes was further indicated by the ability of siRNA-mediated knockdown of podocyte CNR1 to attenuate MEDICAL SCIENCES or abolish the effects of both glucose and ACEA on the above parameters (Fig. S7B). ACEA activated the CB1R/Gi/o/cAMP pathway as indicated by its ability to inhibit forskolin-stimulated cAMP accumulation in a CB1R-dependent manner (Fig. S8A), and this signaling pathway is involved in the cellular effects of both ACEA and high glucose, as indicated by the inhibition of both by pertussis toxin (Fig. S8B). Discussion The development of nephropathy is a common and ominous complication of diabetes that reduces life expectancy and currently lacks fully effective therapy. Hyperglycemia is thought to be one of the major drivers of the glomerulopathy, and treatments aimed to achieve tight glycemic control can slow the progression and at- tenuate renal impairment (24). However, the presence of end- stage kidney disease in diabetic patients with good glycemic control suggests the role of additional pathogenic factors (26). An important conclusion of the present study is that renal CB1R play a critical role in diabetic nephropathy, as indicated by both in vivo and in vitro observations. Chronic in vivo treatment of ZDF rats with a novel, nonbrain-penetrant CB1R inverse agonist for 3 mo completely prevented pathologies related to glomerular dysfunction, including albuminuria, reduced GFR, and elevated plasma creatinine. The parallel prevention of uri- cosuria and polyuria suggest additional effects of CB1R signaling on proximal and distal tubular functions, respectively, which is also Fig. 7. Chronic losartan treatment of ZDF rats improves renal function compatible with the presence of CB R on both the proximal and without affecting hyperglycemia. ZDF rats were treated from age 12–16 wk 1 with daily oral doses of 20 mg/kg losartan (hatched columns) or vehicle (gray distal convoluted tubules (27). Nephropathy in ZDF rats was also columns). Age-matched lean rats were used as controls (open columns). Note associated with a marked up-regulation of CB1R in the diabetic that losartan treatment did not affect blood glucose or plasma angiotensin II kidney, which was similarly reversed by chronic treatment with the levels, but normalized the other parameters which are markers of ne- CB1R inverse agonist. This finding suggests that increased sig- # phropathy. Statistical significance as indicated for Fig. 1 ( P < 0.05). naling by renal CB1R plays a key role in the development of

Jourdan et al. PNAS | Published online November 24, 2014 | E5425 Downloaded by guest on October 1, 2021 Earlier studies have documented the nephroprotective effect of globally acting CB1R inverse agonists (8–11), but therapeutic development of this class of compounds has been halted because of neuropsychiatric side effects (12). Peripherally restricted CB1R antagonists, which in rodents are devoid of behavioral effects secondary to blockade of CB1R in the CNS (13, 14), are unlikely to produce such side effects. Furthermore, JD5037 treat- ment started after the full development of diabetic nephropathy and continued for 4 wk improved the indicators of renal function beyond their level at the start of treatment. By reversing the hyperglycemia-induced increase in apoptotic mechanisms such as caspase 3/7 activity, CB1R blockade could preserve the function and prevent further loss of podocytes. Thus, peripheral CB1R blockade can not only prevent but also reverse established ne- phropathy, further highlighting the therapeutic potential of such compounds in diabetic nephropathy, which is notoriously diffi- cult to treat (31). Another important finding in the present study is the disso- ciation of nephropathy from the hyperglycemia, the former being reversed and the latter remaining unaffected by peripheral CB1R blockade (Fig. 6). The improvement of established glomerulop- athy, including albuminuria, reduced GFR, and elevated plasma creatinine may be due to the observed reversal of podocyte loss by chronic CB1R blockade (Fig. 7B), whereas the reduction in polyuria in the face of unchanged hyperglycemia is likely related to reversal of some distal tubular dysfunction affecting water reab- sorption. Indeed, both increased plasma atrial natriuretic peptide, which is similar to that found in another rat model of T2DM (32), and reduced vasopressin can contribute to water loss, and both effects were reversed by JD5037 treatment (Fig. S4). The resistance of the hyperglycemia to CB1R blockade in the same animals has interesting implications. First, it suggests that different pathologies drive these two manifestations of T2DM. Indeed, pancreatic islets of ZDF rats are heavily infiltrated by

Fig. 8. The effects of losartan or JD5037 treatment on angiotensin II- induced changes in cellular endocannabinoid levels and (A) CNR1, AGTR1, DES, NPHS2,andNPHS1-expession in cultured human podocytes (B). Cells were incubated with vehicle, losartan (10 μM), or JD5037 (100 nM) for 1 h and then exposed to 100 nM angiotensin II for 24 h. Columns represent means ± SEM from 12 independent replicates. Significant difference from corresponding value in vehicle (*P < 0.05, **P < 0.01, ***P < 0.001), or angiotensin II-treated cells (#P < 0.05, ##P < 0.01, ###P < 0.001).

Fig. 9. Schematic representation of the proposed CB1R-mediated podocyte diabetic nephropathy, and also indicates autoinduction of CB1R loss in diabetic nephropathy. This figure was prepared using a template from expression, as documented earlier in other tissues (28–30). the Servier Medical Art website.

E5426 | www.pnas.org/cgi/doi/10.1073/pnas.1419901111 Jourdan et al. Downloaded by guest on October 1, 2021 + PNAS PLUS CD68 , proinflammatory macrophages that express the Nlrp3 was drastically reduced by losartan, supporting a direct link inflammasome and release active IL-1β and IL-18 (15), whereas between Agtr1 activation and Cnr1 expression. Our findings in there was no evidence for macrophage infiltration in the kidney of human podocytes provide additional evidence for this: angio- the same rats, which displayed a proinflammatory cytokine profile tensin II treatment increased endocannabinoid levels and very different from that in β-cells (Fig. S3). Although others have CNR1 expression in podocytes, and also promoted a “nephro- documented macrophage infiltration of the kidneys of ZDF rats, pathic” phenotype by decreasing NPHS2 and NPHS1 and in- this phenomenon and the associated nephrosclerosis were detected creasing DES expression. More importantly, the obligatory role in much older (>36 wk) animals (18), whereas ZDF rats of similar of CB1R signaling in the effects of angiotensin II is indicated by age as used here were reported to be devoid of either renal their absence in cells preincubated with JD5037 (Fig. 8). Similar macrophage infiltration or significant nephrosclerosis (33). to the present findings, activation of Agtr1 induced CB1R- A second implication of the dissociation of hyperglycemia mediated effects in other tissues via increasing the synthesis of from diabetic nephropathy is that podocyte injury, which plays the endocannabinoid 2-arachidonoyl glycerol (49, 50), whereas a key role in diabetic glomerulopathy (34), can be driven by CB1R blockade was found to down-regulate AGT1R (51). There multiple factors. Indeed, our in vitro findings in primary cultured is also evidence that CB1R involvement in angiotensin II- human podocytes suggest that overactivation of CB1R may induced pathologies may reflect Agtr1/CB1R heteromeriza- represent a common pathway mediating podocyte injury trig- tion, a process which amplifies Agtr1 signaling (52). gered by different stimuli. Activation of CB1R by ACEA in- In summary, the data presented indicate that enhanced CB1R creased the expression of genes linked to podocyte injury, such signaling in podocytes plays a key role in the pathogenesis of as DES and NOX4, and decreased NPHS2 and NPHS1 expression, diabetic nephropathy, which likely represents a final common which are required for normal podocyte function (Fig. S7A). pathway through which both hyperglycemia and increased RAS These changes, as well as the increased expression of CNR1 (Fig. activity promote the multiple pathological changes, including S7A), are similar to those we have observed in the diabetic increased oxidative/nitrosative stress, that culminate in the compared with normal kidney. Exposure of podocytes to high development of glomerulopathy, as schematically illustrated in glucose caused similar effects, which were similarly prevented Fig. 9. Peripherally restricted CB1R antagonists could represent either by CB1R blockade or by siRNA-mediated knockdown of a novel and rational therapeutic approach to treat this life- CNR1 (Fig. S7B). Knockdown of podocyte CNR1 has been threatening complication of diabetes. reported to prevent high glucose-induced Akt and NF-kB acti- vation (25) and up-regulation of collagen and plasminogen Materials and Methods activator inhibitor-1 (PAI1) (9). Together, these observations For a detailed description of materials and methods used, see SI Materials and indicate the obligatory role of CB1R in mediating podocyte in- Methods.SeeTable S1 for a list of primers. jury induced by high glucose. A role for CB1R in high glucose- induced apoptosis has also been documented in retinal pigment Animal Treatment Protocols. Animal protocols were approved by the In- stitutional Animal Care and Use Committee of the National Institute on epithelial cells (35). Also, a link between CB1R and diabetic nephropathy is suggested by the reported association between Alcohol Abuse and Alcoholism, NIH. Male ZDF rats and their lean controls were obtained from Charles River Laboratories. Rats were individually housed a polymorphism in the CNR1 gene and nephropathy in patients and maintained under a 12-h light/dark cycle and fed ad libitum a standard with T2DM (36). laboratory diet (STD, NIH-31 rodent diet). MEDICAL SCIENCES Because podocyte injury also develops under normoglycemic As a preventive treatment, 6-wk-old prediabetic ZDF rats were placed on conditions, factors other than glucose may also activate podocyte a regimen of JD5037 (3 mg·kg·d) or vehicle [4% (vol/vol) DMSO + 1% Tween CB1R. A likely candidate is the RAS, the other major driver of 80 in saline] by oral gavage for 90 d (20 rats per group). To test whether diabetic nephropathy (37, 38), activation of which in ZDF rats is JD5037 treatment can reverse the pathology, 15-wk-old diabetic ZDF rats indicated by the elevated plasma angiotensin II and aldosterone were treated daily for 4 wk with the same dose of JD5037 or vehicle (10 rats levels and the increased renal expression of Agtr1 (Fig. 4A). The per group). Finally, 12-wk-old diabetic ZDF were treated orally with losartan important role of the RAS in diabetic nephropathy is further (TCI America, L0232) for 28 d (20 mg·kg·d). supported by the existence of a positive feedback loop, whereby podocyte injury promotes the filtration of liver-derived angio- Peripheral CB1R Antagonist. JD5037 was synthesized and its pharmacological properties analyzed as described previously (53). tensinogen and the generation of angiotensin II in the kidney (39). Regardless of its renal or extrarenal origin, angiotensin II acting Endocannabinoid Content. The endocannabinoid content of podocytes was via Agtr1 has been implicated in pronephrotic changes similar to quantified by liquid-chromatography/tandem mass spectrometry, as described those detected in the kidney of diabetic ZDF rats. Thus, in ad- previously (54). dition to the well-established link between angiotensin II and the NF-kB–dependent NOX activation and ROS production (40, 41), Statistics. Values are expressed as means ± SEM. Data were analyzed by Agtr1 activation has been shown to reduce plasma adiponectin ANOVA followed by the Tukey–Kramer post hoc test. Time-dependent varia- (42), down-regulate adipoR1 (43), increase the plasma uric acid bles were analyzed and results in multiple groups were compared by ANOVA (44) and IL-18 (45), increase the gene expression of Il-6 (46) and followed by Bonferroni test. (GraphPad Prism v6 for Windows). Significance TNF-α (47), and increase caspase 3/7 activity in kidney tissue (48). was set at P < 0.05. The finding that all of the similar changes observed in ZDF ACKNOWLEDGMENTS. We thank Drs. J. F. McElroy and R. J. Chorvat (Jenrin rats were prevented by peripheral CB1R blockade suggests that Discovery) for providing the cannabinoid 1 JD5037, angiotensin II acting via AGTR1 may be the glucose-independent Dr. J. Kopp (National Institute on Diabetes, Digestive, and Kidney Diseases, signal that induces the increased CB1R signaling in podocytes, as National Institutes of Health) for providing the human podocyte cell line a cause of podocyte injury and diabetic glomerulopathy. Indeed, and for helpful comments on the manuscript, Ms. J. Harvey-White for technical assistance, Dr. R. Kechrid for assistance with the animal studies, and losartan treatment of ZDF rats resulted in much improved renal Mr. Kris Ylaya for his help on histological preparation. This study was functions without any improvement in hyperglycemia. Re- supported by intramural funds from the National Institute on Alcohol Abuse markably, Cnr1 expression in the renal cortex of ZDF rats and Alcoholism, National Institutes of Health.

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