SIGNIFICANCE

ABSTRACT

RESULTS

CB1R Antagonism Prevents the Development of Nephropathy in Prediabetic ZDF Rats.

Six-week-old male, prediabetic ZDF rats were started on a daily oral dose (3 mg/kg) of the nonbrain penetrant CB1R inverse agonist JD5037 or vehicle for 90 d. As illustrated in Fig. 1A, vehicle-treated ZDF rats developed extreme hyperglycemia by the end of the treatment period, accompanied by increased kidney weight, greatly elevated plasma creatinine and blood urea nitrogen levels, polydipsia, polyuria, a marked reduction 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). The same difference was evident for Cnr1 (the cannabinoid type 1 receptor gene) expression in the glomerulus, its diabetes-related increase being fully reversed 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 hyperglycemia, 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).

Fig. 1.

Peripheral CB1R antagonism prevents the development of diabetes and the associated nephropathy in ZDF rats. Blood and urine parameters (A) and CB1R protein (scale bar, 20 μm) and mRNA expression in the renal cortex (B) were analyzed in lean controls …

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).

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 …

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 protein resorption droplets (Fig. S2), although podocyte enlargement 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 reactive oxygen species (ROS) in diabetes (19), and is also involved in the metabolism of uric acid, a product of excessive purine catabolism that can lead to the development of gout or nephropathy. 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). A potential off-target, direct effect of JD5037 on XO activity could be excluded by the failure of 10–100 nM JD5037 to influence the activity of recombinant XO, which was completely blocked by the known XO inhibitor allopurinol (Fig. 3B). The concentrations of JD5037 used were relevant to the in vivo condition, as peak plasma concentration of JD5037 following a 3 mg/kg oral dose was 175 ± 40 nM (n = 3), of which >99% is protein bound.

Fig. 3.

Peripheral CB1R antagonism in ZDF rats prevents the increase in uric acid production and decrease in adiponectin and adiponectin receptor expression. (A) XOR activity in kidney tissue and plasma and kidney concentration of uric acid in lean control rats …

Adiponectin has anti-inflammatory effects and recent studies suggest an inverse correlation between adiponectinemia, uric acid levels, and renal function (20, 21). Accordingly, plasma adiponectin 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).

Peripheral CB1R Antagonism Prevents Activation of the Renin-Angiotensin System and Oxidative Stress in ZDF Rat Kidney.

Compared with lean rats, vehicle-treated ZDF rats had increased plasma aldosterone and angiotensin II levels, as well as increased phosphorylation of the p65 subunit of NF-κB and increased angiotensin II receptor-1 (Agtr1) mRNA in the renal cortex (Fig. 4A), suggesting up-regulation of the RAS, which is known to increase oxidative stress through activation of NADPH oxidases (22). Indeed, renal cortices from vehicle-treated ZDF rats displayed increased expression of ROS-generating NADPH oxidase isoforms, such as NADPH oxidase4 (Nox4), GP91Phox (Nox2), p47phox, and Nox1 (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).

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). …

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⋅d JD5037 for 4 wk to match the age of ZDF rats undergoing preventive treatment at the time of killing. The pronounced hyperglycemia of these animals, 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 increased 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 immunostaining (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.

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 markers of renal function were …

Fig. 6.

Effect of JD5037 treatment on relative kidney weight and the renal expression of genes implicated in podocyte loss (Cnr1, Agtr1, Nox4, Des) or survival (Nphs1, Nphs2) (A); on the presence WT-1+ podocytes in renal glomeruli (scale bars, 20 μm) …

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 receiving 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 decreased (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 vehicle for 28 d. Relative to lean controls, both vehicle-treated and losartan-treated ZDF rats developed extreme hyperglycemia by the end of the treatment period (Fig. 7, Top Left). However, losartan treatment improved renal function as reflected in a reduction in polyuria, albuminuria, blood urea nitrogen, plasma and urine creatinine and preservation of a higher GFR (Fig. 7). As expected, the elevated circulating levels of angiotensin II in ZDF rats were unaffected, whereas the elevated plasma aldosterone levels were normalized by losartan (Fig. 7, Bottom Left). Although losartan treatment did not reverse the increase in kidney weight, the increased expression of Agtr1 and Nox4, and the decreased expression of Nphs2 and Npsh1 in the renal cortex were normalized, and the elevated level of Cnr1 mRNA was reduced to levels below that in lean control rats (Fig. S6).

Fig. 7.

Chronic losartan treatment of ZDF rats improves renal function without affecting hyperglycemia. ZDF rats were treated from age 12–16 wk with daily oral doses of 20 mg/kg losartan (hatched columns) or vehicle (gray columns). Age-matched lean rats …

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 nephropathy (24). First, we analyzed the potential interaction between 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 anandamide and 2-arachidonoylglycerol, the two major endocannabinoids, 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 expression of both AGTR1 and CNR1, and both losartan and JD5037 prevented these changes. Furthermore, NPHS2 and NPHS1 expression were significantly decreased, whereas DESexpression 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).

Fig. 8.

The effects of losartan or JD5037 treatment on angiotensin II-induced changes in cellular endocannabinoid levels and (A) CNR1, AGTR1, DES, NPHS2, and NPHS1-expession in cultured human podocytes (B). Cells were incubated with vehicle, losartan (10 µM), …

We also examined the relationship between the effects of high glucose and CB1R activation in cultured human podocytes. Exposure 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 expression 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 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 attenuate 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 uricosuria and polyuria suggest additional effects of CB1R signaling on proximal and distal tubular functions, respectively, which is also compatible with the presence of CB1R on both the proximal and distal convoluted tubules (27). Nephropathy in ZDF rats was also associated with a marked up-regulation of CB1R in the diabetic kidney, which was similarly reversed by chronic treatment with the CB1R inverse agonist. This finding suggests that increased signaling by renal CB1R plays a key role in the development of diabetic nephropathy, and also indicates autoinduction of CB1R expression, as documented earlier in other tissues (28–30).

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 treatment 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 nephropathy, further highlighting the therapeutic potential of such compounds in diabetic nephropathy, which is notoriously difficult to treat (31).

Another important finding in the present study is the dissociation of nephropathy from the hyperglycemia, the former being reversed and the latter remaining unaffected by peripheral CB1R blockade (Fig. 6). The improvement of established glomerulopathy, 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 reabsorption. 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 CD68+, proinflammatory macrophages that express the Nlrp3 inflammasome and release active IL-1β and IL-18 (15), whereas there was no evidence for macrophage infiltration in the kidney of the same rats, which displayed a proinflammatory cytokine profile very different from that in β-cells (Fig. S3). Although others have documented macrophage infiltration of the kidneys of ZDF rats, this phenomenon and the associated nephrosclerosis were detected in much older (>36 wk) animals (18), whereas ZDF rats of similar age as used here were reported to be devoid of either renal macrophage infiltration or significant nephrosclerosis (33).

A second implication of the dissociation of hyperglycemia from diabetic nephropathy is that podocyte injury, which plays a key role in diabetic glomerulopathy (34), can be driven by multiple factors. Indeed, our in vitro findings in primary cultured human podocytes suggest that overactivation of CB1R may represent a common pathway mediating podocyte injury triggered by different stimuli. Activation of CB1R by ACEA increased the expression of genes linked to podocyte injury, such as DES and NOX4, and decreased NPHS2 and NPHS1 expression, which are required for normal podocyte function (Fig. S7A). These changes, as well as the increased expression of CNR1 (Fig. S7A), are similar to those we have observed in the diabetic compared with normal kidney. Exposure of podocytes to high glucose caused similar effects, which were similarly prevented either by CB1R blockade or by siRNA-mediated knockdown of CNR1 (Fig. S7B). Knockdown of podocyte CNR1 has been reported to prevent high glucose-induced Akt and NF-kB activation (25) and up-regulation of collagen and plasminogen activator inhibitor-1 (PAI1) (9). Together, these observations indicate the obligatory role of CB1R in mediating podocyte injury induced by high glucose. A role for CB1R in high glucose-induced apoptosis has also been documented in retinal pigment epithelial cells (35). Also, a link between CB1R and diabetic nephropathy is suggested by the reported association between a polymorphism in the CNR1 gene and nephropathy in patients with T2DM (36).

Because podocyte injury also develops under normoglycemic conditions, factors other than glucose may also activate podocyte CB1R. A likely candidate is the RAS, the other major driver of diabetic nephropathy (37, 38), activation of which in ZDF rats is indicated by the elevated plasma angiotensin II and aldosterone levels and the increased renal expression of Agtr1 (Fig. 4A). The important role of the RAS in diabetic nephropathy is further supported by the existence of a positive feedback loop, whereby podocyte injury promotes the filtration of liver-derived angiotensinogen and the generation of angiotensin II in the kidney (39). Regardless of its renal or extrarenal origin, angiotensin II acting via Agtr1 has been implicated in pronephrotic changes similar to those detected in the kidney of diabetic ZDF rats. Thus, in addition to the well-established link between angiotensin II and the NF-kB–dependent NOX activation and ROS production (40, 41), Agtr1 activation has been shown to reduce plasma adiponectin (42), down-regulate adipoR1 (43), increase the plasma uric acid (44) and IL-18 (45), increase the gene expression of Il-6 (46) and TNF-α (47), and increase caspase 3/7 activity in kidney tissue (48).

The finding that all of the similar changes observed in ZDF rats were prevented by peripheral CB1R blockade suggests that angiotensin II acting via AGTR1 may be the glucose-independent signal that induces the increased CB1R signaling in podocytes, as a cause of podocyte injury and diabetic glomerulopathy. Indeed, losartan treatment of ZDF rats resulted in much improved renal functions without any improvement in hyperglycemia. Remarkably, Cnr1 expression in the renal cortex of ZDF rats was drastically reduced by losartan, supporting a direct link between Agtr1 activation and Cnr1 expression. Our findings in human podocytes provide additional evidence for this: angiotensin II treatment increased endocannabinoid levels and CNR1 expression in podocytes, and also promoted a “nephropathic” phenotype by decreasing NPHS2 and NPHS1 and increasing DES expression. More importantly, the obligatory role of CB1R signaling in the effects of angiotensin II is indicated by their absence in cells preincubated with JD5037 (Fig. 8). Similar to the present findings, activation of Agtr1 induced CB1R-mediated effects in other tissues via increasing the synthesis of the endocannabinoid 2-arachidonoyl glycerol (49, 50), whereas CB1R blockade was found to down-regulate AGT1R (51). There is also evidence that CB1R involvement in angiotensin II-induced pathologies may reflect Agtr1/CB1R heteromerization, a process which amplifies Agtr1 signaling (52).

In summary, the data presented indicate that enhanced CB1R signaling in podocytes plays a key role in the pathogenesis of diabetic nephropathy, which likely represents a final common pathway through which both hyperglycemia and increased RAS activity promote the multiple pathological changes, including increased oxidative/nitrosative stress, that culminate in the development of glomerulopathy, as schematically illustrated in Fig. 9. Peripherally restricted CB1R antagonists could represent a novel and rational therapeutic approach to treat this life-threatening complication of diabetes.

Fig. 9.

Schematic representation of the proposed CB1R-mediated podocyte loss in diabetic nephropathy. This figure was prepared using a template from the Servier Medical Art website.

MATERIALS AND METHODS

For a detailed description of materials and methods used, see SI Materials and Methods. See Table S1 for a list of primers.

SUPPLEMENTARY MATERIAL

ACKNOWLEDGMENTS

FOOTNOTES

REFERENCES