Peripheral Cannabinoid-1 Receptor Blockade Ameliorates Cystitis Severity

Background: The endocannabinoid system (ECS) plays a key physiological role in bladder function and it has been suggested as a potential target for relieving lower urinary tract symptoms (LUTSs). Whereas most studies indicate that activating the ECS has some beneficial effects on the bladder, some studies imply the opposite. In this study, we investigated the therapeutic potential of peripheral cannabinoid-1 receptor (CB1R) blockade in a mouse model for LUTSs. Materials and Methods: To this end, we used the cyclophosphamide (CYP; 300 mg/kg, intraperitoneal)-induced cystitis model of bladder dysfunction, in which 12-week-old, female C57BL/6 mice were treated with the peripherally restricted CB1R antagonist, JD5037 (3 mg/kg), or vehicle for three consecutive days. Bladder dysfunction was assessed using the noninvasive voiding spot assay (VSA) as well as the bladder-to-body weight (BW) ratio and gene and protein expression levels; ECS tone was assessed at the end of the study. Results: Peripheral CB1R blockade significantly ameliorated the severity of CYP-induced cystitis, manifested by reduced urination events measured in the VSA and an increased bladder-to-BW ratio. Moreover, JD5037 normalized CYP-mediated bladder ECS tone imbalance by affecting both the expression of CB1R and the endocannabinoid levels. These effects were associated with the ability of JD5037 to reduce CYP-induced inflammatory response, manifested by a reduction in levels of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα), in the bladder and serum. Conclusions: Collectively, our results highlight the therapeutic relevance of peripheral CB1R blockade in ameliorating CYP-induced cystitis; they may further support the preclinical development and clinical use of peripherally restricted CB1R antagonism for treatment of LUTSs.


Introduction
Lower urinary tract symptoms (LUTSs) include increased frequency, urgency, nocturia, incontinence, recurrent urinary tract infections, and even renal dysfunction. 1 LUTSs affect *16% of the population aged over 40 years and their prevalence increases with age. 2 LUTSs have a major impact on patients' quality of life due to the tremendous suffering and withdrawal of these individuals from social and professional activities.
The etiology of LUTSs is varied and includes neurogenic conditions (e.g., spinal cord lesions, multiple sclerosis, and diabetic neuropathy), and more commonly, patients present without any defined neurological disorder. 1 In addition, hemorrhagic cystitis, manifested by dysuria and hematuria as a complication of cyclophosphamide (CYP) therapy, is also largely involved in development of LUTSs in oncological patients. 3 CYP, a chemotherapeutic drug used to treat different types of cancers, is metabolized to acrolein by the liver and accumulates in the bladder, resulting in hemorrhagic cystitis and LUTSs, especially in immunocompromised patients. 4 Unfortunately, there is no proven treatment to manage this type of condition, which can severely degrade the patient's quality of life and may possibly lead to renal failure and even death.
The endocannabinoid system (ECS) consists of the two main (most studied) endocannabinoids (eCBs): N-arachidonoylethanolamine (anandamide [AEA]) and 2-arachidonoylglycerol (2-AG), and their synthesizing and degrading enzymes, as well as their G-proteincoupled cannabinoid receptors, CB 1 R and CB 2 R. Both cannabinoid receptors have been recently discovered in human and animal bladders, urothelial cells, detrusor muscle, and nerve fibers innervating the bladder. [5][6][7][8] Most studies show the beneficial effects of ECS activation in ameliorating LUTSs (reviewed in Ref. 9 ). Specifically, it was reported that intravenous (IV) injection of the AEA transport inhibitor, VDM-11, into anesthetized rats, which increases AEA concentrations, increased the micturition interval and threshold pressure. This effect was blocked by a CB 1 R antagonist (AM251), but not by a CB 2 R antagonist (AM630). 10 Others have shown that IV injection of a fatty acid amide hydrolase (FAAH) inhibitor into conscious rats also elevates AEA levels and enhances intercontraction intervals, micturition volume, bladder capacity, and threshold pressure. 11 On the other hand, elevated bladder AEA levels can also activate other off-target receptors such as the transient receptor potential cation channel subfamily V member 1 (TRPV1), which may evoke hyperreflexia and hyperalgesia and increase the micturition volume. 7,[12][13][14] In recent years, there has been a growing interest in blocking CB 1 R in peripheral organs for treatment of many pathologies mainly associated with metabolic syndrome (reviewed in Refs. 15,16 ). Moreover, CB 1 R antagonism was suggested as a potential therapeutic intervention for acute cisplatin-induced renal dysfunction, 17 a disease associated with enhanced inflammation, oxidative/nitrosative stress, and cell death.
In this study, we show (for the first time) that peripheral CB 1 R antagonism ameliorates CYP-induced cystitis by reducing micturition events, restoring the bladder-to-body weight (BW) ratio, normalizing bladder ECS tone, and reducing inflammation. These results indicate the therapeutic potential of peripherally restricted CB 1 R antagonism against LUTSs.
On the 4th day, mice were euthanized by cervical dislocation under anesthesia, the bladder was removed and weighed, and it was either snap-frozen or fixed in 4% buffered formalin. Trunk blood was collected, and serum was separated and stored at À80°C until processed for biochemical evaluation. The number of samples in each experiment was determined according to tissue availability, which was limited due to the small size of a mouse bladder.
Voiding spot assay: noninvasive assessment of bladder dysfunction On the 3rd day, bladder dysfunction was assessed using a noninvasive voiding spot assay (VSA); each mouse was placed in a single cage with an absorbent filter paper and was allowed to move freely for 4 h; during this time, the micturition events were captured and retained as void spots on the paper.
Normal mice tend to urinate only a few times in the cage corner, whereas CYP-treated mice urinate numerous times all over the filter paper. Urine spots were illuminated with ultraviolet (UV) light using the 2UV Transilluminator (UVP, USA), and the number and area of urine spots were analyzed using ImageJ software (NIH, Bethesda, MD).
Serum and bladder tumor necrosis factor alpha analyses Serum and bladder levels of tumor necrosis factor alpha (TNFa) were measured by an ELISA kit (MHSTA50; R&D Systems) according to the manufacturer's protocol.

Histopathological analyses
Paraffin-embedded bladder sections (3 lm) from each group were stained with hematoxylin and eosin. Panoramic bladder images were captured with a Zeiss Axio-Cam ICc5 color camera mounted on a Zeiss Axio Scope A1 light microscope at · 5 and · 40 magnifications.

Immunohistochemistry
Bladder tissues from Veh-and CYP-injected mice (five animals per group) were fixed in 4% buffered formalin for 48 h and then embedded in paraffin. Sections were deparaffinized and hydrated. Heat-mediated antigen retrieval was performed with 10 mM citrate buffer, pH 6.0 (Thermo Scientific, IL, USA). Endogenous peroxide was inhibited by incubating with a freshly prepared 3% hydrogen peroxide (H 2 O 2 ) solution in methanol (MeOH).
Stained sections were photographed as described above. Positive areas were quantified using ImageJ software with a minimum of four random images of the detrusor muscle or urothelium per mouse.
Densitometry was quantified using Bio-Rad CFX Manager software. Quantification was normalized to the anti-b-actin antibody (#ab49900; Abcam).

Real-time PCR
Total bladder messenger RNA (mRNA) was extracted using Bio-Tri RNA lysis buffer (Bio-Lab, Israel), followed by DNase I treatment (Thermo Scientific), and reverse transcribed using the iScript cDNA kit (Bio-Rad). Real-time PCR was performed using the iTaq Universal SYBR Green Supermix (Bio-Rad) and the CFX connect system (Bio-Rad).
The primers used to detect mouse genes are listed in Table 1. Mouse genes were normalized to Ubc.
Sample preparation and endocannabinoid measurements by liquid chromatography with tandem mass spectrometry eCBs were extracted, purified, and quantified from bladder lysates. In brief, bladders were homogenized in ice-cold Tris buffer using the Bullet Blender and zirconium oxide beads (Next Advance, Inc.); protein concentration was determined by the bicinchoninic acid (BCA) assay. Samples were then supplemented with an ice-cold extraction buffer [1:1 methanol/Tris buffer + an internal standard (IS)] and chloroform/methanol (2:1), vortexed, and centrifuged.
The lower organic phase was transferred into borosilicate tubes; this step was repeated three times by adding ice-cold chloroform to the samples and transferring the lower organic phase into the same borosilicate tubes. The samples were dried and kept overnight at À80°C, then reconstituted with ice-cold chloroform and acetone, kept at À20°C for 30 min, and then centrifuged to precipitate proteins.
Next, the supernatant was dried and reconstituted in an ice-cold liquid chromatography with tandem mass spectrometry grade methanol and analyzed on an AB Sciex (Framingham, MA) QTRAP Ò 6500 + mass spectrometer coupled with a Shimadzu (Kyoto, Japan) ultra high-performance liquid chromatography (UHPLC) System. Liquid chromatographic separation was achieved using 5-lL injections of samples into a Kinetex 2.6-lm C18 (100 · 2.1 mm) column from Phenomenex (Torrance, CA). The autosampler was set at 4°C, and the column was maintained at 40°C during the entire analysis.
The gradient elution mobile phases consisted of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). eCBs were detected in a positive ion mode using electron spray ionization and the multiple reaction monitoring mode of acquisition, using d 4 -AEA as IS. The collision energy, declustering potential, and collision cell exit potential for the monitored transitions are presented in Table 2.
The levels of AEA and 2-AG in the samples were measured against standard curves and normalized to the bladder lysate protein concentration.

Statistics
Values are expressed as the meanstandard error of mean (SEM). Unpaired two-tailed Student's t-test was used to determine differences between the two groups. Results of multiple groups were compared by one-way analysis of variance, followed by one-sided Tukey's test, using GraphPad Prism, v6 for Windows (San Diego, CA). Significance was set at p < 0.05.

CYP-induced cystitis exacerbates micturition events and bladder inflammation
To evaluate the severity of CYP-induced cystitis in our mouse model (Fig. 1A), noninvasive VSA was conducted. Significant increases in urine spots (Fig. 1B, C) and the bladder-to-BW ratio (Fig. 1D) were found in the CYP-injected mice. The cystitis was associated with enhanced bladder mRNA expression levels of the inflammatory cytokines, Cxcl10, Ccl2, Il18, Tgfb, and Tnfa (Fig. 1E-I).
In contrast, expression levels of only the fibrotic marker Col3 (Fig. 1J), and not Col1 and Fn1 (Fig. 1K, L), were enhanced following CYP injection, as expected from an acute model. Nevertheless, histological examination of bladders revealed a typical appearance of cystitis with excessive submucosal edema and hemorrhage (Fig. 1M).
CYP-induced cystitis upregulates CB 1 R and TRPV1 expression CYP-induced cystitis did not significantly change the mRNA expression levels of Cnr1 or Cnr2 (Fig. 2A,  B); however, the protein expression levels of CB 1 R were markedly elevated (Fig. 2C, D), indicating an elevated ECS tone. CB 2 R protein levels were not examined due to the lack of a reliable antibody for this receptor. Since the ECS can also activate other off-target receptors, such as the TRPV1, we examined its expression     and detected significant upregulation in its protein expression levels (Fig. 2E, F) in the CYP-treated mice.
Elevated CB 1 R protein expression levels were further validated using immunohistochemistry, revealing significant elevated expression levels in urothelial cells, particularly in the umbrella cells, and the lamina propria, as well as in detrusor muscle cells (Fig. 2G-I).
Peripherally restricted CB 1 R antagonism attenuates CYP-induced cystitis and inflammation Treatment with the peripherally restricted CB 1 R antagonist, JD5037, a day before, in combination with, and a day after CYP injection (Fig. 3A) significantly reduced the elevated urine spots (Fig. 3B, C) and the bladder-to-BW ratio (Fig. 3D) in CYP-injected mice. Histologically, JD5037 reduced the area affected by edema and hemorrhage (Fig. 3E).
Moreover, CB 1 R antagonism resulted in normalization of elevated mRNA (Fig. 3F) and protein (Fig. 3G) levels of the inflammatory cytokine, TNFa, as well as its circulating levels in the serum (Fig. 3H). Taken together, these results indicate that peripheral CB 1 R antagonism attenuates cystitis severity.
Peripherally restricted CB 1 R antagonism normalizes bladder ECS tone To assess the involvement of the ECS in CYP-induced cystitis and its reversal by JD5037, we assessed changes in expression of the receptors and ligands in this system in our model. Interestingly, the improvements in bladder function by JD5037 were accompanied by downregulation of CB 1 R (Fig. 4A, B), but not the TRPV1 expression levels (Fig. 4A, C).
We next determined the mRNA levels of degrading and synthesizing enzymes as well as the amount of AEA and 2-AG in the bladder. Whereas CYP-injected mice displayed no significant changes in the expression levels of AEA's synthesizing enzyme, N-acyl phospha-tidylethanolamine phospholipase D (Fig. 4D), a significant reduction in the expression of its degrading enzyme, FAAH, (Fig. 4E) was measured.
These changes were found to be in accordance with the actual amount of AEA found in the bladder (Fig. 4F). Interestingly, JD5037 treatment normalized AEA levels in the treated mice (Fig. 4F), although it did not affect the expression of its related enzymes. In contrast to AEA, decreased synthesis and enhanced catabolism of 2-AG, resulting in reduced bladder levels, were found in mice injected with CYP, as manifested by changes in expression levels of 2-AG's synthesizing enzyme, diacylglycerol lipase beta, and its degrading enzyme, monoacylglycerol lipase (Fig. 4G, H), respectively.
Yet, similar to the effect of JD5037 on AEA levels, the treatment also normalized the amount of 2-AG in the bladder (Fig. 4I) without affecting the expression of its related enzymes. Both AEA and 2-AG levels were normalized to the protein levels in each bladder (Fig. 4J), which were not affected by CYP or JD5037 treatment. These findings indicate that peripheral CB 1 R antagonism can restore bladder ECS tone.

Discussion
The present study reveals, for the first time, the relevance of CB 1 R antagonism in ameliorating hemorrhagic cystitis. We found that peripheral CB 1 R antagonism reduces the elevated micturition events, bladder edema, and inflammation as well as restores the bladder ECS tone. Our findings are unique since most publications in this field indicate that activation of CB 1 R or CB 2 R is an effective strategy to decrease LUTSs in animal models (reviewed in Ref. 9 ).
These studies mostly refer to the modulatory action of CB 1 R on the sensory neurons innervating the bladder as well as to the anti-inflammatory effect of CB 2 R agonism. In contrast, our findings suggest an alternative pathway by which bladder dysfunction can be ‰ FIG. 3. Peripherally restricted CB 1 R antagonism attenuates CYP-induced cystitis severity and inflammation. A CYPinduced cystitis model with CB 1 R antagonism-experimental design (see the Materials and Methods section; A). Treatment with the peripherally restricted CB 1 R antagonist, JD5037 (3 mg/kg, IP), significantly reduced micturition events (B, C), the bladder-to-BW ratio (D), and submucosal edema and hemorrhage, as shown in the representative panoramic H&E staining of bladders from each group, (original magnification · 5, scale bar-500 lm; and · 40 scale bar-50 lm; E), as well as normalized the elevated bladder mRNA (F) and protein (G) levels of TNFa and its circulating levels (H). Data represent the mean -SEM. For (C), n = 16 mice per group; for (D), n = 27 mice per group; for (F), n = 7 mice per group; for (G), n = 7 mice for the Veh group and 17 mice for the CYP-treated groups; and for (H), n = 18 mice for the Veh group and 22 mice for the CYP-treated groups. **p < 0.01, ***p < 0.001, and ****p < 0.0001. CYP, cyclophosphamide; IP, intraperitoneal; JD, JD5037; TNFa, tumor necrosis factor alpha; Veh, vehicle.

CB 1 R BLOCKADE AND CYSTITIS 629
mitigated through reducing the enhanced activity of the CB 1 R present on urothelial cells 5,8 and the detrusor muscle. 8 The ECS, ubiquitously present in humans and animals, acts both centrally and peripherally to maintain cellular and organ homeostasis. Thus, changes in ECS tone, evidenced by modulation in expression of the cannabinoid receptors, their functional activity (upregulated or downregulated), and the relative number of eCBs, may render the subject susceptible to different diseases. 19 The analgesic effects of CB 1 R agonists are well established in many chronic disorders. 20 Upregulation of CB 1 R during cystitis revealed here could be one of the compensatory mechanisms to ameliorate pain and inflammation in acute hemorrhagic cystitis in mice. Indeed, bladder cystitis is characterized by an imbalanced ECS tone, as manifested here by enhanced bladder expression of CB 1 R and altered AEA and 2-AG levels, as well as changes in their corresponding synthesizing and degrading enzymes. , as shown in the representative immunoblots. CYP did not induce any significant changes in the mRNA expression levels of Nape-pld (D); however, it did induce a significant reduction in Faah (E). Accordingly, the actual amount of AEA increased in the CYP-injected mice and was normalized by JD5037 (F). CYP induced a significant reduction in the bladder mRNA levels of Daglb (G) and significant upregulation of Magl (H). Accordingly, the actual amount of 2-AG was significantly reduced in the CYPinjected mice and normalized by JD5037 (I). Bladder protein concentrations ( J). Data represent the mean -SEM. For (B-E and G, H), n = 7 mice per group, and for (F, I, J), n = 9 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. 2-AG, 2-arachidonoylglycerol; AEA, anandamide; Daglb, diacylglycerol lipase b; ECS, endocannabinoid system; Faah, fatty acid amide hydrolase; Magl, monoacylglycerol lipase; Nape-pld, N-acyl phosphatidylethanolamine phospholipase D.
These findings are in accordance with those reporting elevated AEA levels and bladder inflammation in rats, but not in mouse models for cystitis. 14,21,22 However, in contrast to others, they showed no changes in 2-AG levels in a CYP-induced mouse model for cystitis. 22 These discrepancies may be related to differences in the model established, the number of mice used in each experiment, the eCB extraction method, and the normalization method used for calculating eCB levels.
Regarding eCB levels, we used normalization to the bladder's protein content rather than the bladder weight since its increased weight could arise from edema and water absorption, resulting in higher chances for errors in calculating the eCB levels. Nevertheless and surprisingly, we found that peripheral CB 1 R blockade results in a modulatory effect on the ECS, and normalizes its action under these conditions, both regarding the receptor expression levels and the amount of ligands produced in the bladder.
Although CB 1 R blockade normalized AEA and 2-AG levels, the mRNA expression levels of their synthesizing and degrading enzymes were not significantly changed; these contradicting results may be explained by changes in their protein expression levels or their activities. Future experiments would need to assess these assumptions experimentally.
In attempting to determine the specific effects of AEA and 2-AG on bladder function, we suggest that the two ligands may play opposing roles. Whereas some studies imply that AEA increases the micturition interval and threshold pressure 10,11 and may decrease pain behavior and bladder hypersensitivity, 21,22 others have shown that elevated bladder AEA levels may activate pronociceptive TRPV1 channels 7,14,23,24 and induce pain.
Our results support the latter observations since we found that CYP elevates AEA levels and TRPV1 expression. In fact, normalization of AEA levels by JD5037 therefore has great importance in ameliorating pain sensation and further suggests that peripheral CB 1 R blockade may have an indirect analgesic effect in this model; however, this hypothesis should be further tested experimentally with behavioral and pain tests.
Regarding the role of 2-AG in the bladder and its opposite pattern to AEA, reduced inflammation in CB 1 R antagonist-treated mice may be due to the recovery of 2-AG (preferential a CB 2 R ligand) levels, and it may support the anti-inflammatory role of CB 2 R that was well established in cystitis. 25,26 Since we have not measured the protein expression of CB 2 R (due to lack of a valid antibody), we cannot comment further on its role in our settings.
Peripheral CB 1 R overactivation is strongly related to metabolic inflammation 27 and its peripheral antagonism has great therapeutic potential in reducing it. Specifically, CB 1 R activation has been associated with increased TNFa secretion in human bladder carcinoma cells, 28 whereas CB 1 R antagonism was reported to inhibit TNFa production in the inflamed small intestine in rats. 29 Moreover, CB 1 R genetic deletion reduces TNFa secretion by Kupffer cells, 30 and pharmacological CB 1 R antagonism or its genetic deletion inhibits TNFa expression in diabetic cardiomyopathy. 31 Moreover, we have previously shown that peripheral CB 1 R antagonism or its genetic deletion from renal proximal tubule cells normalizes the elevated TNFa levels in both type 1 diabetes and diet-induced obesity mouse models. 32,33 In agreement with all of these studies, we show here that peripheral CB 1 R antagonism also normalizes bladder TNFa levels as well as systemic inflammation.
Although CYP-induced cystitis is not defined as a chronic metabolic disease, there is ample evidence that acute pathologies are also accompanied by an imbalanced ECS tone and enhanced CB 1 R activity. 17,34 In fact, this upregulation may be manifested as a compensatory effect by the damaged organ to restore its function. However, this imbalance may promote unwanted effects such as a destructive inflammatory response.
In conclusion, hemorrhagic cystitis is characterized by bladder dysfunction, inflammation, and altered ECS tone. Peripheral CB 1 R blockade has the potential to ameliorate these characteristics and to restore normal bladder function. These findings support the rationale for clinical testing of peripheral CB 1 R blockers that are currently being developed in preclinical settings for the treatment of CYP-induced cystitis in oncologic patients as well as other systemic conditions associated with LUTSs.