The Mechanism Of Ketamine And Its Analogue Methoxetamine Inducing Bladder Dysfunction

BackgroundAs a N-methyl-D-asparticacid receptor antagonist, Ketamine is active in the field of clinical anesthesia and pain management for half a century. However, due to the ability of causing strong euphoria, hallucination and feeling of impending death, it soon became a popular drug abuse for entertainment. Early studies of katemine abuse is mainly concentrated in the mental area and nervous system changes like other drugs. Since Shahani found 9 youths suffered from severe LUTS(Lower urinary tract symptoms) that caused by katemine abuse in 2007. Soon numbers of cases were reported in Europe and Asia, ketamine associated urinary dysfunction(KAUD) gradually attracted people’s attention.The patients complained about severe LUTS. The progression of KAUD could led to bladder contracture and upper urinary tract involvement, which affecting the quality of life seriously. But because of its clinical features and mechanism is not clear yet, there is no standardized treatment guidelines.The disease is widely found for 8 years, numbers of patients with severe symptoms gradually increased, conservative treatment for such kind of patients has not much effects. Clinical stagingcan determine the progression of the disease to some extent, also provides guidance to disease’s evaluation and selections of medical treatments, perfect for avoiding delays and excessive medical treatment. KAUD has its special complexity, in addition to the bladder change, upper urinary tract will be in varying degrees with the progression of disease. Existing reports different results from various treatments, progression of the disease is likely to be one factor, based on policies and experience to judge the timing of treatment is not absolutely reliable, about the timing of surgical intervention attracted experts attention. To our knowledge, there has not been research on the progression of the disease that try to stage and evaluate the ketamine-related urinary system damage, In second part of this study, we presented a scoring staging system of KAUD based on ketamine abusing history, laboratory test results, and imaging findings. It is the first clinical staging according to the progress of the disease, and provide the basis for a stepwise treatment strategy.Methoxetamine (MXE) is a structural analogue of ketamine,, also NDMA receptor antagonist. In addition, MXE can bind serotonin transporter (serotonin transporter, SERT), reducing reuptake of serotonin, causing serotonin levels in the brain increased, and confirmed that it can increase dopamine release and blocking reuptake of dopamine. As a new synthetic psychotropic drugs, MXE research history is not long. Its effect was first reported in 2010, similar to ketamine, including euphoria, calm, hallucinations, sensory strengthen, physical separation, near-death experience and so on. The potential harmful effects of MXE has not been widely recognized today, only nine countries banned the use of MXE, in addition to eight countries in European and American countries, in Asia only Japan issued a ban on the MXE. Therefore, the purchase of MXE in most countries does not require any authorization or credentials, because of the convenience of the Internet, MXE is even easier to get than KET. At first, MXE intended to prevent the urotoxicity associated with ketamine and to be tested as an antidepressant, however, some scholars used mice to exposed to MXE in 2004, and found that MXE has a certain toxicity to the bladder and kidneys. At this stage, studies about MXE chemical, pharmacology and toxicology is still inadequate, then is there a real damaging effect on urinary system caused by MXE, more data to further study is required. In the second part of this study, we were led by mature model of ketamine induced cystitis. SD rats were given introperitoneal injection of KET and MXE, to explore the urinary toxicity of MXE comparing with ketamine induced cystitis, for describing the similar mode of action on urinary system of MXE and KET. In the third part of the study, we used human urothelium to study the proinflammatory cytokine and chemokine secretion after MXE and KET administration.Methods1. The staging evaluation of ketamine associated urinary dysfunction based on a scoring systemWe examined the medical records of KAUD patients admitted to Nanfang Hospital from September 2009 to September 2014.All the cases that had a comprehensive profile and was fitted for inclusion and exclusion criteria, was include in the further analysis.The patients had been required to complete bladder diary, pelvic pain and urgency/frequency (PUF) questionnaire, interstitial cystitis problem index (ICPI) and interstitial cystitis symptom index (ICSI). Each patient’s records were reviewed for a complete medical history, entries in a 3-day bladder diary, symptom scores, laboratory tests for liver and renal function, ultrasonography findings, CT scan, urodynamics and radioisotope renography. There all total 135 KAUD cases included. The patients were categorized into three clinical stages based on bladder and upper urinary tract involvement and treated with a step-wise strategy. Stage Ⅰ patients were managed with a behavioral modification program that included the mandatory cessation of ketamine use, dietary restrictions, and bladder training. Stage I patients also received pain management instruction and pharmacotherapy with antibiotics, glucocorticoids, antihistamines, anticholinergics, and antioxidants. Stage II patients received additional hydrodistension by intravesical perfusion with heparin, lidocaine, and sodium bicarbonate. Stage III patients received augmentation cystoplasty or cystectomy with urinary diversion if pharmacotherapy and hydrodistension were ineffective after a three-month follow-up period. Voided volume, micturition interval, and nocturnal void frequency as recorded in their 3-day bladder diary, PUF, ICPI and ICSI score after treatment in one month, were compared with their baseline data prior to treatment. Ketamine abusing history, renal and liver function, bladder and upper urinary tract changes, urodynamics and radioisotope renography correlation with stages were analyzed. A scoring system based on ketamine abusing history, renal and liver function, bladder and upper urinary tract changes, urodynamics and radioisotope renography abnormality was established and the concordance between the clinical staging and model staging of each patient was compared. Receiver operating characteristic curve was employed to testify the sensitivity and specificity. Area under roc curve and 95% CI was calculated.2. Ketamine analogue methoxetamine induced bladder dysfunction and cystitis36 female Sprague-Dawley rats weighing 180-220g, were randomly divided into six groups as control 1 month, control 3 months, MXE 1 month, MXE 3 month, KET 1 month, KET 3 month. Each rat received intraperitoneal injection of 30mg/kg MXE or KET. All rats were weighed weekly to adjust the quantity of durg administration. The rats were housed in metabolic cages for 24 hours for recording water intake. Meanwhile, paper of urine staining was placed under metabolic cages to observe urinary frequency and pattern for two hours. The rats were anesthetized by intraperitoneal injection of urethane. The PE50 tube was placed and secured in the bladder via urethra. After bladder emptying with a syringe, the tube was connected with an infusion pump with an pressure transducer. The saline was instilled into bladder at 12ml/min. During the infusion, the bladder pressure change with or without voiding was observed. Each rat was recorded for at least five void cycle with MMS SOLAR system. After the last void, the residual urine volume was measured by a syringe. The parameters assessed were threshold pressure, peak pressure, capacity, residual urine volume, micturition efficiency, bladder compliance, and the frequency of non-voiding contractions. All experimental rats were sacrificed after cytometry and the bladder tissues were removed, frozen in liquid nitrogen and stored at-80℃. The collected samples were fixed with 4% paraformaldehyde, embedded in paraffin and then sectioned to 4 μm slides for histological analysis. Hematoxylin and eosin (HE), alcian blue, toluidine blue and masson’s trichrome staining were processed as standard methods. Western Blots was used to measure the expression of α-SMA, Collagen I and Fibronectin in bladder tissue. Real time quantitative PCR was performed to examine the mRNA expression of proinflammatory cytokines, chemokine and fibrotic related products (IL-1, IL-6, CCL-2, CXCL-1, CXCL-10, NGF, TGF-β,α-SMA, Collagen I, Collagen III and Fibronectin).3. The effect of ketamine analogue methoxetamine to human bladder urothelial cellsThe SV-HCU-1 was seeded in 96-well plates(5000 cells/well) for 24 hours, and stimulated with various concentration of methoxetamine and ketamine(0、.0.125mM、 0.25 mM、0.5 mM、1 mM、2 mM、4 mM and 8 mM) for 24 and 48 hours. After 24 and 48 hours cells were added CCK-8 for 2 hours incubation before measurement of absorbance in 450 nm. Cell viability was determined as the percentage of PBS-treated cells. Real time quantitative PCR was performed to examine the mRNA expression of proinflammatory cytokines, and chemokine after MXE and KET administration (IL-1, IL-6, CCL-2, CXCL-1, CXCL-10, NGF, and TGF-β).Results1. There were 39,80 and 16 patients included in in each stage respectively. After subsequent strategy of treatment, Voided volume, micturition interval, and nocturnal void frequency, PUF, ICPI and ICSI were improved significantly. Age (P=0.018), ketamine abusing history (P=0.006), eGFR (P=0.001), ALT (P=0.037), y-GT (P< 0.001), bladder (P=0.013) and ureteral changes (P<0.001), hydronephrosis (P< 0.001) and radioisotope renography abnormality (P=0.01) were correlated with clinical stages. After subsequent strategy of treatment, Voided volume, micturition interval, and nocturnal void frequency, PUF, ICPI and ICSI were improved significantly.In total,107 cases’ stages based on scoring were accordant with clinical stages (30,65 and 12 cases in each stage respectively). AUC of the scoring model ROC was 0.922 on cut point between I and II stages and 95% CI was 0.879～0.965. AUC of the scoring model ROC was 0.978 on cut point between II and III stages and 95% CI was 0.952～1.000.2. After MXE and KET administration for 1 month and 3 months, body weights of rats were decreased comparing with control group, and it was more serious in MXE group. There are on differences in water intake and urine volume for 24 hours. Urine staining showed frequency in MXE and KET group and KET 3 month group was worse. The urodynamics of rats confirmed that long term abuse of MXE and KET led to frequency, more non-voiding contraction, increased bladder pressure, while bladder compliance, void volume, and efficiency of urination decreased with time effect of drug administration. In HE stain of rat bladder tissue, thinning or even missing urothelial, inflammatory cell infiltration and vessel dilation in lamina propria and submucosa were observed in MXE and KET group. The alcian blue stain showed glycosaminoglycan was less in MXE and KET group. Mast cell infiltration was demonstrated in MXE and KET group by toluidine blue. Comparing with control group, masson trichrome stain found collagen expression was up-regulated in muscle layer. Western Blot revealed that α-SMA, Collagen I, Fibronectin was increased in MXE and KET group rats. The up-regulation of IL-1, IL-6, CCL-2, CXCL-1, CXCL-10, NGF, COX-2, TGF-β, α-SMA, Collagen Ⅰ, Collagen Ⅲ and Fibronectin was verified by real time quantitative PCR and the scale in 3 month group was higher.3. After stimulation of MXE and KET for 24 and 48 hours in SV-HUC-1 cells, cell viability was decreasing along with durg concentration increasing. The cell viability was 80% when MXE and KET was 0.25 and 0.5mM. So this concentration was referred as non-fatal to cell and applied for further experiment. The up-regulation of IL-1, IL-6, CCL-2, CXCL-1, CXCL-10, NGF, TGF-β and COX-2 was verified by real time quantitative PCR.Conclusion1. We presented a scoring staging system of KAUD based on ketamine abusing history, laboratory test results, and imaging findings. It might be helpful to evaluate the progression on bladder and upper urinary tract, and provide a reference for treatment. This staging system based on scoring still needs further validation and improvement.2. Ketamine analogue methoxetamine induced bladder dysfunction as frequency, decreased bladder capacity, decreased bladder compliance and non-voiding contraction.3. The histopathology of rat bladder showed methoxetamine could led to thinning or even missing urothelial, glycosaminoglycan decreased, inflammatory cell infiltration, vessel dilation in lamina propria and submucosa and collagen deposition. The increase of fibrotic products α-SMA, Collagen I, Fibronectin was verified by WB and RT-qPCR. The mode of action on urinary system of MXE was similar with KET.4. Long term abusing methoxetamine led to bladder inflammatory injury with multiply proinflammatory cytokines and chemokine (IL-1,IL-6,CCL-2,CXCL-1,CXCL-10,NGF,TGF-β) increased.5. Methoxetamine and ketamine could induce human urothelial cells to up-regulate proinflammatory cytokines and chemokine expression.