Ketamine-induced Bladder Fibrosis Involves Epithelial-to-Mesenchymal Transition Mediated By TGF-?1

BackgroundLong-term ketamine abuse wo?ld induce interstitial cystitis.Ketamine-induced cystitis(KC)is characterized by severe voiding frequency,urgency,pelvic pain,dysuria,and urge incontinence.Bladder wall fibrosis is a major complication of KC.Fibrosis is characterized by an increased accum?lation of extracell?lar matrix(ECM)due to collagen synthesis.As ECM accum?lates in bladder wall,bladder contracture occurs and bladder compliance decreases.The fibrosis wo ? Id eventually lead to bladder dysfunction and renal insufficiency,which can be life-threatening.However,the underlying pathogenesis bladder fibrosis in KC is poorly understood.Epithelial-to-mesenchymal transition(EMT)is a process by which differentiated epithelial cells lose their phenotypic features while,at the same time,acquiring mesenchymal characteristics.Mounting evidence indicates that EMT is an important pathway that contributes to the generation of activated fibroblasts in various organs,including the lung,liver,heart,and kidney.EMT has become an important target in the treatment of organ fibrosis,however,to our knowledge,the role of EMT in bladder fibrosis remains unknown.The purpose of this study is to determine whether ketamine activates TGF-?1 signaling,induces EMT,and subsequently contributes to bladder fibrosis in vivo and in vitro experiments.Methods1.Establishment of KC rat model and bladder fibrosis assessmentExperiments were performed on specific pathogen-free(SPF)female Sprague-Dawley rats weighing between 180 and 210 g.18 Rats were randomly distributed into three groups that received intraperitoneal injections of 0.9%saline(control group),low dose(30 mg/kg,LK group),or high dose(60 mg/kg,LK group)for a period of 16 weeks.Rats were housed in metabolic cages for 24 hours to record water intake and urine output.To assess the effect of ketamine on urinary frequency,spontaneous voiding spot assays were performed weekly.To confirm that ketamine induced bladder urinary dysfunction,we performed cystometry on anesthetized rats.The recorded cystometrography parameters included the frequency of voiding and non-voiding contractions,intravesical pressure(baseline,threshold,and peak micturition pressure),voided volume,bladder volume,and bladder compliance.All rats were sacrificed by spine dislocation after cystometry,and the bladders were removed.The bladder specimens were fixed overnight in 4%paraformaldehyde,embedded in paraffin blocks,and sectioned at 4 ?m.For histopathologic evaluation,sections were stained with hematoxylin and eosin(H&E)and masson trichrome stain(MTS).Immunohistochemistry was performed to analyze the expression of E-cadherinm,Collageon I,Vimentin,Fibronectin in bladder tissue.For identification of EMT in bladder epithelium,immunofluorescent double-labeling of E-cadherin-FSP1 was performed according to previously described methods.2.The effect of TGF-?1 blocker SB505124 on EMT and bladder fibrosis in KC rats.24 SD rats were randomly distributed into four groups that received intraperitoneal injections of 0.9%saline for 16 weeks(control group),ketamine(60 mg/kg daily)for a period of 16 weeks(KET group),ketamine(60 mg/kg daily)for a period of 16 weeks combined with daily administration of a TGF-P receptor 1 inhibitor,SB505124(10 mg/kg)(SB group),or ketamine(60 mg/kg daily)for a period of 12 weeks followed by 4 weeks of abstinence(abstinence group).spontaneous voiding spot assays,cystometry,and immunohistochemistry experiments were performed as part 1.Immunofluorescent double-labeling of E-cadherin-FSP1 and E-cadherin-a-SMA were performed to assess EMT in bladder epithelium.3.The effect of ketamine on TGF-?1 signaling pathway and EMT in SV-HUC-1 cellsThe SV-HUC-1 cell line was used for experiments.SV-HUC-1 cells were stim?lated with ketamine(5 to 125 ?g/mL)for 24 h and 48 h,and then CCK-8 assay was used to determine cell viability.SV-HUC-1 cells in c?lture were exposed either to control medium(DMEM/F12 containing 0.5%FBS)or to DMEM/F12 containing 0.5%FBS with ketamine(125?g/mL)for 72 hours.The effect of ketamine on the morphology of SV-HUC-1 cells was analyzed by phase contrast microscopy.Cells were stim?lated with ketamine(125?g/mL)for 24 hours.Real time quantitative PCR was performed to assess the mRNA levels of E-cadherin,FSP1,vimentin,fibronectin,and a-SMA.The cells were stim?lated with different concentrations of ketamine(5 to 125?g/mL)for 48 hours.The expression levels of E-cadherin,fibronectin,and a-SMA were determined by Western blot.Cells were stim?lated with ketamine(5 to 125 ?g/mL)for 36 hours for TGF-?1 production.Cell c?lture supernatants were then collected,and total TGF-?1 was measured by using quantitative sandwich ELISA kits(eBioscience,San Diego,CA,USA)according to the manufacturer's instructions.SV-HUC-1 cells were pre-treated with 2 ?M of the T?R inhibitor SB505124 for 1 h and then stim?lated or not stim?lated with ketamine(125 ?g/mL)for 48 hours.The expression levels of E-cadherin and?-SMA were determined by Western blot.Gene expression of TGF-?1 and EMT-activating transcription factors(Snail,Slug,and Twist)were assessed by qRT-PCR.Results1.After 16 weeks of ketamine administration,rats showed a significant increase in micturition frequency compared to controls.There were no significant differences in water intake and urine output between ketamine-treated rats and controls.The urodynamic examinations found that ketamine significantly increased the frequency of voiding contraction and non-voiding contraction and peak pressure,but decreased bladder volume and bladder compliance.The bladder dysfunction in HK group was worse.H&E and MTS staining showed that there were 3 to 5 layers of bladder epithelium and only sparse light blue stained collagen between smooth muscle bundles in the bladder tissue of the control group.In the LK and HK group,bladder tissue was associated with thinning bladder epithelium,denuded urothelial mucosa,and significantly increased collagen deposition in the muscle layers.Immunohistochemical staining revealed that the expression level of E-cadherin was significantly decreased,while the expression levels of vimentin and fibronectin in the LK and HK group were significantly increased,compared with the control.In immunofluorescent double-labeling experiments,bladder tissue from the rats treated with ketamine,but not those treated with physiological saline,was associated with the appearance of E-cadherin+ FSP1+ cells,indicating the existence of EMT in the bladder tissue of ketamine-treated rats.TGF-?1 expression was significantly up-reg?lated in the bladder tissue of ketamine-treated rats in both LK and HK groups2.There were no significant differences in water intake and urine output among the control group,ketamine group,SB group,and abstinence group.SB505124 treated rats showed a significant decrease in micturition frequency compared to ketamine treated rats.The urodynamic examinations found that SB505124 significantly decreased the frequency of voiding contraction and non-voiding contraction and peak pressure,and increased bladder volume and bladder compliance compared with ketamine group.H&E and MTS staining revealed that the SB group show decreased epithelial injury and decreased interstitial fibrosis,while abstinence group showed thinning urothelium,denuded urothelial mucosa,and sever interstitial fibrosis.Ketamine+SB505124 treatment resulted in a significant increase in E-cadherin expression and significant decline in collagen I,vimentin and fibronectin expression compared to rats treated with ketamine alone.In immunofluorescent double-labeling experiments,treatment with ketamine+SB505124 resulted in a statistically significant decrease in the total number of both E-cadherin+ FSP1+ cells and E-cadherin+?-SMA+ cells compared to rats treated with ketamine alone.Furthermore,4 weeks of abstinence does did reverse histological changes induced by ketamine.3.The results from CCK8 assay revealed that ketamine treatment does not affectSV-HUC-resultl cell viability,s?ggesting that there is no pronounced cytotoxicity in SV-HUC-1 cells.72 h after ketamine treatment,SV-HUC-1 cells underwent a morphological transition,with loose cell-cell contacts and an elongated fibroblast appearance.The mRNA expression of E-cadherin was markedly reduced upon ketamine treatment,whereas the expression levels of the mesenchymal markers FSP1,fibronectin,vimentin,and a-SMA were noticeably elevated in ketamine-treated SV-HUC-1 cells compared to controls.To confirm these findings,we performed Western blots to analyze the protein expression levels of E-cadherin,fibronectin,and a-SMA.Analogous to the real-time RT-PCR results,Western blot analysis showed that ketamine treatment drastically reduced the levels of E-cadherin and significantly increased both fibronectin and a-SMA in a concentration-dependent manner.Taken together,these results indicate that ketamine induces EMT in SV-HUC-1 cells.We further detected the expression levels of TGF-?1,TGF-?1 receptors(T?R-1 and T?R-2),and the transcription factors downstream of TGF-?1(Snail,Slug,and Twist)by real-time RT-PCR.Ketamine treatment significantly induced the secretion of TGF-?1 in SV-HUC-1 cells in a dose-and time-dependent manner.Consistent with the expression of TGF-?1,the expression of Snail,Slug,and Twist was also significantly up-reg?lated upon ketamine stim?lation.This result demonstratesthat ketamine probably induces EMT in SV-HUC-1 cells thro?gh the TGF-?1 signaling pathway.This possibility was confirmed by Western blot analysis showing that the TGF-?1 blocker SB505124 inhibits the ketamine-induced E-cadherin down-reg?lation and a-SMA up-reg?lation.Conclusion1.Long term ketamine induced bladder dysfunction as decreased bladder volume,non-voiding contraction,and decreased bladder compliance.The typical pathological bladder changes were epithelial injury and interstitial fibrosis.2.Our study provided the first evidence of EMT in the development of ketamine-induced bladder fibrosis.Treatment with TGF-? receptor inhibitor SB505124 in vivo prevents EMT associated with interstitial fibrosis.3.Ketamine treatment(5-125 ?g/ml)did not affect SV-HUC-1 cell viability.TGF-?1 signaling might have an important role in ketamine-induced EMT in SV-HUC-1 cells.4.Ketamine treatment up-reg?lated the expression of transcription factors downstream of TGF-?1(Snail,Slug,and Twist).5.T?R-1 may serve as a novel target to protect against bladder fibrosis in ketamine abusers.