The Study Of The Pathogenesis Of Ketamine Cystitis

Background and ObjectionSince2007it has been known that long-term ketamine abuse can affect the urinary system, resulting in lower urinary tract syndrome, frequency, nocturia, urgency, dysuria, suprapubic discomfort and occasional hematuria. Cystoscopy findings and histological changes in bladder biopsies from ketamine abusers are similar to those patients with interstitial cystitis. Intravenous urography and urodynamic studies in a small group (n=6) of chronic recreational ketamine users identified various changes in bladder function and appearance. These subjects had narrowing of both ureters, contracted bladder syndrome with mild bilateral hydronephrosis, detrusor instability and urinary leakage. It remains unclear how ketamine abuse leads to these interstitial cystitis-like, functional and histological changes. It has been postulated that the accumulation of ketamine and/or its metabolites in the urine might cause direct toxic effects on the urinary tract. However, apart from the pathological changes caused by long-term ketamine abuse that are seen clinically and in animal models, no direct evidence has been found that gives a causal relationship between ketamine toxicity and the development of cystitis. In addition, there are currently no studies describing the effects of the urinary metabolites of ketamine on bladder function. The aim of this study was to fully explore the pathogenesis of ketamine cystitis in vivo, in vitro and in clinic.Methods and materialsSv-HUC-1cells was cultivated in vitro to the logarithmic growth phase. Then, Sv-HUC-1cells was exposed to100nM、1μM、10μM、100μM dose of ketamine respectively. MTT assay was used to measure the proliferative activity of the urothelial cells with the effect of ketamine. Amplex Red assay was used to measure the generation of free radicals produced by the urothelial cells when exposed to ketamine.Twenty-four2-month-old male Sprague-Dawley (SD) rats weighing180-200g had free access to standard laboratory rodent chow and water. Groups of six rats were randomly assigned to control, normal saline (NS), low-dose (5mg.kg-1) and high-dose (50mg.kg-1) ketamine groups. The two experimental groups received a single intraperitoneal (i.p.) injection of ketamine hydrochloride dissolved in500μL saline at09:00h each day. The NS group received an i.p. injection of vehicle the same time:rats in the control group were untreated. All rats were weighed weekly to adjust the quantity of ketamine administrated.Urinary frequency was determined by housing the rats individually in modified metabolic cages that had a fine stainless steel mesh, which retained feces but allowed urine to pass, through its base. Paper, impregnated with saturated copper sulfate solution (CuSO45H2O) and dehydrated at200℃for1h, was placed under the base of the cage. The urine falling onto this paper, rehydrated the anhydrous CuSO4, turning it blue. The number of urinations was determined by counting the number of blue spots observed. Baseline urinary frequency was measured3days before ketamine administration, and then at consecutive3day periods after4,8and16weeks of treatment. Urine samples were collected from the metabolic cage, but with the CuSO4paper removed. The short-term effect of ketamine was assessed by analyzing urine samples for APF and NO. A6h baseline urine sample was collected immediately before the first ketamine injection and subsequent urine samples were collected at6h intervals for30h. The longer term effects of ketamine were monitored using Gp-51and potassium measurements at baseline and after4,8,16weeks of treatment. Urine was collected for24h, immediately before daily ketamine administration and thereafter24-h urinary volume was recorded daily. Aliquots of urine were stored at-80℃and were thawed once before analysis. The levels of APF and Gp51in urine were measured using an ELISA assay. Levels of NO were estimated via the Griess reaction. Urinary potassium concentration was determined using an ion selective electrode technique in an automated chemistry analyzer.Rats were killed by spine dislocation, and the bladders were excised. Some of the bladder specimens were stained with hematoxylin and eosin (H&E), and examined under a light microscope. Immunohistochemical studies were performed using the DAKO ChemMateTM EnVisionTM Detection Kit. The following primary antibodies were used:anti-iNOS, anti-occludin antibody and anti-ZO-1. The integrated optical density of cytoplasm and cell membranes containing brown-yellow granules was detected by using Image-Pro Plus v6.0software. Western blot analysis was used to examinate the expression of iNOS, Occludin and ZO-1in the bladder. Protein signals were quantified by scanning densitometry using a FluorChem Q system. The results of western blotting were quantified by Quantity One4.4.0software.43ketamine-associated cystitis patients (male29cases, female14cases) were analyzed.32patients without indwelling urinary catheter were categorized as group A, while the other11patients with indwelling urinary catheter were in group B. The therapy regimes consisted of anti-inflammatory, antioxidant, relieving spasm and pain, improving the microcirculation and repairing the bladder epithelium barrier.30healthy adults were selected as the controls. Urinary potassium(K), sodium (Na) and creatinine (Cr) were determined in24h urine samples from all groups before and after treatments.24h urinary Cr was used as the internal standard.24h urinary K and Na in the individual patients were calculated as concentrations relative to the Cr concentrations. The pelvic pain and urgency/frequency patient symptom (PUF) were used for evaluation before and after the treatments. The differences of urinary K were compared with in each group and between groups before and after treatments. In addition, relationship of urinary K and PUF were assessed by statistics.In part two, statistical analysis was performed using SPSS13.0software. One-way ANOVA, with LSD test were used to test for differences between groups. In part three, statistical analysis was performed using Prism version5.0. Repeated measures ANOVA, with Dunnett’s correction, was used to test for differences from the control values. In part four, statistical analysis was performed using SPSS13.0software. The data were analyzed by using t test-way ANOVA and Pearson product-moment correlation analysis.ResultsSv-HUC-1cells was cultivated to the logarithmic growth phase. After treated with100mM、1μM、10μM、100μM of ketamine respectively for24h and48h, MTT assay was used to measure the proliferative activity of the Sv-HUC-1cells between ketamine treated groups and control group. Although the results had statistical difference, no obvious inhibitory effects of ketamine on the proliferation of cells were observed. It means that ketamine was not cytoxic to Sv-HUC-1cells. Amplex Red assay was used to measure the generation of free radicals produced by the urothelial cells when exposed to ketamine. The results showed that, in60mins duration, with cells and no cells plates had similar increased fluorescent rates with time. There were no significant difference between with cells and no cells groups, which indicated that ketamine had no effect on the generation of free radicals in Sv-HUC-1cells.Rats presented with cataleptic immobility within1min of administration of50mg.kg-1ketamine i.p. This was followed by ataxia (head and body swaying) after about10minutes, and falling over and staying still for approximately1h until recovery. Rats in the LK group became mildly excited and active within15min of ketamine injection. Three of six rats in the HK group developed hematuria after16weeks of ketamine treatment. At Week12, high dose ketamine significantly increased micturition frequency compared with that in the NS group (24-hour frequency20.4±0.9vs16.4±0.6, P<0.01), and both low and high-dose ketamine groups showed a significant increase in frequency at Week16(24-hour frequency21.0±0.7vs16.4±0.8, P<0.05;28.6±1.4vs16.4±0.8, P<0.001; n=6). Weight gain at Week16was significantly slower in the HK group than in the NS group (15.6±4.4g vs53.1±4.0g,P<0.05;n=6).Urinary nitric oxide levels were measured in triplicate samples immediately before and6h after ketamine administration. The fractional change in NO in relative to un-injected controls was1.19±0.05in the NS group, LK1.73±0.09in the LK group and2.77±0.06in the HK group. There was no significant difference in NO levels between control and NS groups. Rat urinary APF was measured at6h intervals for42h. The mean APF concentration at the initial collection point before injection was22.3±0.13pg.mL-1, with no differences being seen between groups. Neither the control nor the NS injected animals deviated from this over the42h collection period, whereas, both low and high dose ketamine increased APF concentrations, with the high dose producing a more rapid response. After12h a similar magnitude of response was seen with both doses of ketamine and the response lasted until the end of the collection period.Urinary potassium:creatinine ratios significantly decreased with time in both the HK and LK groups (HK:at0weeks46±0.8vs.8weeks36±0.6and16weeks31.5±1.4P<0.05, P<0.001; NK:at0weeks:44.7±0.7vs16weeks36.8±0.8, P<0.05,n=6). There were no changes in the ratios in either the control or NS groups. Urinary GP-51levels were significantly reduced from baseline levels after8and16weeks of treatment with low and high dose ketamine.(HK:initial38.98±0.35pg/ml vs.8weeks32.83±0.33pg/ml and16weeks15.74±1.22pg/ml P<0.05, P<0.001; LK:initial37.35±0.49pg/ml vs.8weeks33.68±0.32pg/ml and16weeks34.40±0.30pg/ml P<0.01, P<0.05, n=6). There were no significant changes over time in GP-51levels in the control or NS groups.Rats receiving high dose ketamine had a thicker and more compact bladder epithelial layer compared to control rats. Inflammatory cell infiltration and collagen deposition (fibrosis) was present in the bladder submucosa in the HK group. The normal loose connective tissue within the submucosal layer was diminished in the HK group. In both the LK and HK groups there was increased staining for iNOS in the bladder epithelial layer as shown by quantified immunohistochemistry and Western blot densitometry. NS rats also presented increased iNOS expression compared with control rats. The tight junction protein ZO-1was chiefly confined to the bladder epithelial layer, but the staining density and the relative protein expression of ZO-1was decreased in the LK and HK groups compared to the NS group. Expression of occludin mainly occurred in the endothelial cells of the bladder submucosa. The staining density and the relative protein expression of occludin were significantly increased in both LK and HK rats compared to NS rats.Conclusion Ketamine, in the dose between100nM to100μM, had not cytoxic effect on Sv-HUC-1cells. Moreover, it can not induce oxidative stress reaction on the cells. Therefore, we thought it was its metabolites rather than ketamine itself affect the urinary system.Due the results of the rat model of ketamine cystitis, we hypothesized that metabolites of ketamine in urine had a direct toxic effect on bladder epithelial cells, generating nitric oxide and APF. The cumulative toxicity and sustained release of APF impaired the integrity of the bladder epithelial barrier resulting in decreased expression of glycoprotein GP-51and ZO-1within the bladder epithelial layer. The resulting increase in permeability enabled urine constituents, such as urea, and potassium to penetrate the bladder interstitium and muscle layers, resulting in mononuclear cell infiltration, fibrosis, angiogenesis and hypervascularity. Potassium diffusion depolarized nerves and muscles causing symptoms, such as frequency, hematuria and pain. With further development our rat model of ketamine induced cystitic symptoms may help to uncover the etiology and evaluate new methods for treating IC.In clinic, urinary potassium measurement had a role in evaluating the disease status and efficacy of treatments of patients who suffered from ketamine-associated cystitis.