Ketamine enhances autophagy and endoplasmic reticulum stress in rats and SV-HUC-1 cells via activating IRE1-TRAF2-ASK1-JNK pathway.

Background Ketamine-related cystitis (KC) has been researched in many clinical studies, but its exact mechanism is ambiguous and needs further research. Methods We established a KC rat model and analyzed physiological, biochemical, and urodynamic parameters of ketamine (KET)-related bladder injury. Bladder histologic feature, reactive oxygen species (ROS), autophagy-, apoptosis-, and endoplasmic reticulum stress (ERS)-related markers were examined by hematoxylin and eosin staining, Masson staining, ROS kit, quantitative real-time polymerase chain reaction, and western blot. In vitro, effects of 0.01, 0.1, and 1 mM KET on cell vitality, apoptosis, ROS level, autophagy-, apoptosis-, and ERS-related markers were examined again. Effects of KET-1 and salubrinal on complex formation, autophagy-, apoptosis-, and ERS-related markers were examined by Co-Immunoprecipitation and western blot. After transfection with shIRE1, complex formation, cell biological behaviors, ROS level, autophagy-, apoptosis-, and ERS-related markers were examined again. Results KET induced bladder hyperactivity and injury. KET facilitated urinary frequency, ROS production, and induced bladder histologic injury by activating autophagy-, apoptosis-, and ERS-related markers in rats. In vitro, KET (0.01, 0.1, and 1 mM) restrained cell vitality and elevated apoptosis and ROS level via activating autophagy-, apoptosis-, and ERS-related markers. Moreover, salubrinal reversed the promotion of KET-1 on complex formation, autophagy-, apoptosis-, and ERS-related marker expressions. After transfection with shIRE1, shIRE1 weakened complex formation induced by KET-1, and the effects of KET-1 on cells were offset by shIRE1. Conclusion KET enhanced autophagy and ERS in vivo and in vitro via restraining IRE1-TRAF2-ASK1-JNK pathway.

LINC01410 leads the migration, invasion and EMT of bladder cancer cells by modulating miR-4319 / Snail1.

BACKGROUND: Several previous studies have implied the significance of lncRNA1410 (LINC01410) in gastric cancer, rectal cancer, and cervical cancer. Nevertheless, the potential of LINC01410 in bladder cancer (BC) development has not been addressed. METHODS: The related mechanisms were explored by qRT-PCR analysis, CCK-8 assay, cell transfection assay, Transwell assay, Western Blot analysis, Luciferase reporter assay and RNA pull-down assay. RESULTS: In the following study, LINC01410, characterized as an oncogene, exhibited high levels of expression in BC tissues as compared to normal tissues and its expression leads to a reduced prognosis of BC. Functional characterization of LINC01410 showed that knocking down LINC01410 could markedly reduce the invasion and proliferation capacity of T24 and 5637 cells. Mechanistically, LINC01410 served as a sponge for miR-4319 and the findings were further attested through luciferase reporter assay. Analysis of miR-4319 demonstrated its low expression in BC tissues as compared to normal tissues and knocking down LINC01410 significantly increased miR-4319. Data obtained from rescue assay discovered that silencing of miR-4319 in T24 and 5637 cells restored the proliferation and invasion capacity of LINC01410. CONCLUSIONS: Taken together, this study is the first report on the oncogenic potential of LINC01410 in BC development by upregulating Snail1 protein and downregulating miR-4319. Trial registration Retrospectively registered.

CircTHBS1 targeting miR-211/CCND2 pathway to promote cell proliferation and migration potential in primary cystitis glandularis cells.

The pathogenesis of cystitis glandular (CG) is unclear, but it is generally considered to be a neoplastic lesion of urothelial hyperplasia formed by long-term chronic stimulation. There is growing evidence that circRNAs play important roles in a variety of cellular processes. However, there are few reports on the role and molecular mechanism of circRNA in CG. In the present study, we first isolated primary cells from CG tissues and adjacent normal tissues. Further experiments showed that CircTHBS1 was up-regulated in primary CG cells (pCGs). The results of CCK-8 showed that the overexpression of CircTHBS1 promoted the viability of pCGs, while the deletion of CircTHBS1 reduced the cell viability. Knocking out CircTHBS1 also inhibited the migration of pCGs. In addition, we demonstrated that CircTHBS1 played a role in the adsorption of miR-211 by "sponge" in pCG. In turn, miR-211 can directly target CYCLIN D2 (CCND2) 3'UTR to perform its function. Finally, we confirmed the role and mechanism of CircTHBS1/miR-211/CCND2 regulation axis in pCGs. In summary, our study is the first to reveal the role and underlying mechanism of CircTHBS1 in CG, providing a potential biomarker and therapeutic target for human CG.

Phosphodiesterase-5 Inhibitors for Premature Ejaculation: Systematic Review and Meta-Analysis of Placebo-Controlled Trials.

The purpose of this analysis is to assess the efficacy and safety of phosphodiesterase-5 inhibitors (PDE5Is) for the treatment of premature ejaculation (PE). A comprehensive search was performed to ascertain from trials about PDE5Is for the treatment of PE and compare the results, including intravaginal ejaculatory latency time (IVELT), score of sexual satisfaction scale, and side effects, between the group treated with PDE5Is and that treated with placebo. Seven studies involving a total of 471 patients were included in this meta-analysis. This analysis showed that patients who were treated with PDE5Is had significantly increased IVELT (mean difference [MD] 2.60; 95% CI [1.85, 3.36]; p < .00001) and score of sexual satisfaction scale (MD 2.04; 95% CI [0.78, 3.30]; p = .002) compared with the group on placebo. More patients had side effects while taking PDE5Is, such as headache, dizziness, flushing, and nasal congestion. PDE5Is were significantly more effective than placebo in the treatment of PE. Side effects were more common among patients who were treated with PDE5Is.

Ketamine induces endoplasmic reticulum stress in rats and SV-HUC-1 human uroepithelial cells by activating NLRP3/TXNIP aix.

Many clinical studies have been conducted on ketamine-associated cystitis. However, the underlying mechanisms of ketamine-associated cystitis still remain unclear. Bladder tissues of rats were stained by Hematoxylin and Eosin (HE). The viability of human uroepithelial cells (SV-HUC-1 cells) was determined by cell counting kit-8 (CCK-8). Apoptosis and reactive oxygen species (ROS) were examined by flow cytometry. Additionally, the expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß and IL-18 were respectively determined by reverse transcription quantitative (RTq)-PCR and enzyme-linked immunosorbent assay (ELISA). The mRNA and protein levels of B-cell lymphoma/leukemia-2 (Bcl2), Bcl-2-associated X protein (Bax), cleaved caspase 3, glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), NOD-like receptor 3 (NLRP3), thioredoxin-interacting protein (TXNIP), Catalase and MnSOD were examined by RT-qPCR and Western blot. Small interfering RNA target TXNIP transfection was performed using Lipofectamine™ 2000. We found that ketamine effectively damaged bladder tissues of rats and promoted apoptosis through regulating the expression levels of GRP78, CHOP, Bcl-2, Bax and cleaved Caspase-3 proteins in vivo and in vitro. NLRP3 inflammatory body and TXNIP were activated by ketamine, which was supported by the changes in TNF-α, IL-6, IL-1 and IL-18 in vivo and in vitro. Furthermore, knocking down TXNIP reversed the effects of ketamine on apoptosis and NLRP3 inflammatory body in SV-HUC-1 cells. Meanwhile, the changes of Catalase and MnSOD showed that ROS was enhanced by ketamine, however, such an effect was ameliorated by down-regulation of TXNIP in SV-HUC-1 cells. Ketamine promoted cell apoptosis and induced inflammation in vivo and in vitro by regulating NLRP3/TXNIP aix.

miR-30c-5p Reduces Renal Ischemia-Reperfusion Involving Macrophage.

BACKGROUND Ischemia-reperfusion (I/R) leads to kidney injury. Renal I/R frequently occurs in kidney transplantations and acute kidney injuries. Recent studies reported that miR-30 stimulated immune responses and reductions in renal I/R related to anti-inflammation. Our study investigated the effects of miR-30c-5p on renal I/R and the relationship among miR-30c-5p, renal I/R, and macrophages. MATERIAL AND METHODS Sprague Dawley rats received intravenous tail injections of miR-30c-5p agomir. Then a renal I/R model were established by removing the left kidney and clamping the right renal artery. Serum creatinine (Cr) was analyzed using a serum Cr assay kit, and serum neutrophil gelatinase associated lipocalin (NGAL) was measured using a NGAL ELISA (enzyme-linked immunosorbent assay) kit. Rat kidney tissues were analyzed using hematoxylin and eosin staining. THP-1 cells treated with miR-30c-5p agomir and miR-30c-5p antagomir were measured with quantitative reverse transcription-polymerase chain reaction. Protein levels were analyzed by western blot. RESULTS MiR-30c-5p agomir reduced serum Cr, serum NGAL, and renal I/R injury. MiR-30c-5p agomir inhibited the expression of CD86 (M1 macrophage marker), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-alpha (TNF-alpha) and promoted the expression of CD206 (M2 macrophage marker), interleukin (IL)-4, and IL-10 in rat kidneys. MiR-30c-5p agomir reduced the expression of CD86 and iNOS, and increased the expression of CD206 and IL-10 in THP-1 cells. CONCLUSIONS We preliminarily demonstrated that miR-30c-5p agomir might decrease renal I/R through transformation of M1 macrophages to M2 macrophages and resulted in changes in inflammatory cytokines.

CTCF regulates the FoxO signaling pathway to affect the progression of prostate cancer.

The present research focuses on the influence of CCCTC-binding factor (CTCF) on prostate cancer (PC) via the regulation of the FoxO signalling pathway. A bioinformatics analysis was conducted to screen out target genes for CTCF in LNCaP cells and to enrich the relevant pathways in LNCaP cells. It was found that the FoxO pathway was enriched according to the ChIP-seq results of CTCF. The expression of CTCF, pFoxO1a, FoxO1a, pFoxO3a and FoxO3a was tested by RT-qPCR and Western blot. Inhibition of CTCF could lead to the up-regulation of the FoxO signalling pathway. The rates of cell proliferation, cell invasion and apoptosis were examined by MTT assay, cell invasion assay and flow cytometry under different interference conditions. Down-regulation of CTCF could suppress cell proliferation, cell invasion and facilitate cell apoptosis. Lastly, the effect of CTCF on tumour growth was determined in nude mice. Inhibition of CTCF regulated the FoxO signalling pathway, which retarded tumour growth in vivo. In conclusion, CTCF regulates the FoxO signalling pathway to affect the progress of PC.

Ketamine induces reactive oxygen species and enhances autophagy in SV-HUC-1 human uroepithelial cells.

This study was aimed at exploring the underlying mechanisms of ketamine in the SV-40 immortalized human ureteral epithelial (SV-HUC-1) cells. The viability and apoptosis of SV-HUC-1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis-related proteins (B-cell lymphoma 2 [Bcl-2] and Bax) and autophagy-associated proteins (light chain 3-I [LC3-I] and LC3-II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3-methyladenine (3-MA) or N-acetyl-l-cysteine (NAC), the biological functions of SV-HUC-1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK-8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV-HUC-1 cells and accelerated apoptosis of SV-HUC-1 cells through regulating the expression level of IKBα (phospho), nuclear factor ÐºB (P65), Bcl-2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine-treated SV-HUC-1 cells treated with ketamine. Ketamine-induced autophagosomes in SV-HUC-1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV-HUC-1 cells in a dose-dependent and time-dependent manner. Additionally, cotreatment of NAC with 3-MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV-HUC-1 cell autophagy and impaired the cell viability of SV-HUC-1 cells by inducing ROS.

Phyto-mediated synthesized multifunctional Zn/CuO NPs hybrid nanoparticles for enhanced activity for kidney cancer therapy: A complete physical and biological analysis.

Cancer in human society is one of the most problematic health issue responsible for outnumbered deaths worldwide. The consumption of developed NPs in cancer diagnosis is a rapidly emerging field of bio-medical nanotechnology. Recent years, greener synthesized metal oxide hybrid nanoparticles have attracted great attention in cytotoxicity to different cancer therapy. Herein, we report that Duchesnea indica plant mediated green synthesis plant extract mediated Zn doped CuO (Zn/CuO) nanoparticles (NPs) prepared by hydrothermal method and these physico-chemical properties were characterized by XRD, UV-DRS, FTIR, and SEM with EDAX analytical techniques. The XRD pattern findings indicated that the crystal structure of the base CuO matrix are not distorted by the substitution of Cu2+ (0.73 Å) ions by Zn2+ (0.65 Å) ions. The average crystallite size of undoped and Zn/CuO NPs samples are found to be in between the range of 23 to 36 nm. And we can see that the Zn/CuO NPs are large aggregates, containing small particles with sizes of 100-300 nm with spherical shaped morphology by SEM and TEM microscopic images. The normal cell viability and cancer cell inhibition results on A-498 cancer cells and also normal human epithelial cells exhibited that no significant changes in the cell viability with normal kidney epithelial cells and doped NPs given excellent cell inhibition treated on A-498 kidney tumor cells.

LncRNA HULC promotes epithelial and smooth-muscle-like differentiation of adipose-derived stem cells by upregulation of BMP9.

AIMS: Adipose-derived stem cells (ADSCs), a source of mesenchymal stem cells, are able to differentiate into numerous cell lineages, including epithelial and smooth muscle cells. The use of ADSCs in tissue engineering technology has become the most promising therapeutic approach for urethral reconstruction. This study aimed to explore the effect of lncRNA highly upregulated in liver cancer (HULC) on the induction of ADSCs to differentiate into epithelial and smooth-muscle-like cells. METHODS: ADSCs were isolated from a male dog, and the expression of HULC in ADSCs was overexpressed by transfection with HULC expressing vector lentivirus. The transfected ADSCs were then incubated with 5 µM ATRA or 2.5 ng/ml TGF-ß1 and 5 ng/ml PDGF-BB for 21 days. The expression of epithelial differentiation and smooth-muscle-like differentiation markers were monitored. Besides, cross-regulation between HULC and BMP9 was detected in the differentiated epithelial cells and smooth-muscle-like cells. RESULTS: HULC increased cell viability of ADSCs, but has no impact on ADSCs apoptosis. HULC promotes ADSCs to differentiate into epithelial and smooth-muscle-like cells, as evidenced by the increases in the expression of Uroplakin-II, AE1/AE3, α-SMA, SM-MHC, Calponin, and SM-22α. In addition, HULC could positively regulate BMP9, and BMP9 silence abolished HULC-promoted ADSC's differentiation. Furthermore, HULC activated Wnt/ß-catenin pathway while deactivated Notch pathway. CONCLUSION: HULC was demonstrated to be a promoter during the epithelial and smooth-muscle-like differentiation of ADSCs via the BMP9/Wnt/ß-catenin/Notch network. This study provides the first in vitro evidence that HULC-based therapy could be a valuable approach to promote urethral reconstruction.

Cathelicidin LL37 Promotes Epithelial and Smooth-Muscle-Like Differentiation of Adipose-Derived Stem Cells through the Wnt/ß-Catenin and NF-κB Pathways.

Ureter reconstruction is a difficult procedure in urology. Adipose-derived stem cells (ADSCs), along with multipotency and self-renewal capacity, are a preferred choice for tissue engineering-based ureteral reconstruction. We explored the synergic role of cathelicidin LL37 (LL37) in epithelial and smooth-muscle-like differentiation. ADSCs were separated from adipose tissues of mouse and characterized by flow cytometry. The ADSCs were then stably transfected with pGC-FU-GFP (pGC) or pGC containing full-length LL37 (pGC-LL37), respectively. Cell viability and apoptosis were respectively estimated in the stably transfected cells and non-transfected cells. Then, qRT-PCR and Western blot analysis were used for determinations of epithelial marker expressions after induction by all-trans retinoic acid as well as smooth-muscle-like marker expressions after induction by transforming growth factor-ß1. Then, possibly involved signaling pathways and extracellular expression of LL37 were detected. Cell viability and apoptosis were not changed after LL37 overexpression. Expression levels of epithelial and smooth-muscle-like markers were significantly upregulated by LL37 overexpression. Moreover, expressions of key kinases involved in the Wnt/ß-catenin pathway as well as epithelial marker were upregulated by the LL37 overexpression, while it was reversed by Wnt/ß-catenin inhibitor. Likewise, expressions of key kinases involved in the nuclear factor κB (NF-κB) pathway as well as smooth-muscle-like markers were upregulated by LL37 overexpression, which was reversed by NF-κB inhibitor. LL37 was found in the culture medium. LL37, which could be released into the medium, had no impact on cell proliferation and apoptosis of ADSCs. However, LL37 promoted epithelial and smooth-muscle-like differentiation through activating the Wnt/ß-catenin and NF-κB pathways, respectively.

The role of CRP and ATG9B expression in clear cell renal cell carcinoma.

The purpose of the study is to investigate the correlation between the expression of C-reactive protein (CRP) and autophagy-related 9B (ATG9B) and pathological features of clear cell renal cell carcinoma (CCRCC) patients. We also intended to explore the effects of manipulated expression of CRP and ATG9B on the apoptosis and cell cycle progression of CCRCC cell line. ATG9B expression in CCRCC tissues and adjacent renal tissues was analyzed by immunohistochemistry (IHC). Gene expression was determined at transcription and translational levels using real-time quantitative PCR (RT-qPCR) and Western blot. The association between CRP/ATG9B expression and clinical-pathological parameters including age, gender, pathological grades, TNM stage and distant metastasis of the patients was assessed by correlation analysis. siRNA and overexpression plasmids construction were used to manipulate the expression of CRP in human CCRCC cell line 786-O. Cell apoptosis and cell cycle progression were determined using flow cytometry (FCM) and Hoechst 33258 staining. CRP expression correlates with ATG9B expression. The expression of CRP and ATG9B are significantly correlated with TNM staging, distant metastasis, and survival time of CCRCC patients. A high-level of CRP indicates a poor overall survival (OS). In addition, CRP expression influences cell cycle and apoptosis of CCRCC cells. The study reveals that CRP might be a CCRCC development promoter. In addition, there is a close relationship between CRP and ATG9B in CCRCC carcinogenesis.

miR-182-5p improves the viability, mitosis, migration, and invasion ability of human gastric cancer cells by down-regulating RAB27A.

We investigated the effect of miR-182-5p on the viability, proliferation, invasion, and migration ability of human gastric cells by regulating the expression of RAB27A. Real-time PCR assay was used to detect the expression of miR-182-5 and RAB27A in human gastric carcinoma tissues, para-carcinoma tissues, and different cell lines. Western blotting was also used to determine the RAB27A expression in both tissues and cell lines. We chose the HGC-27 cell line as experiment subject as it demonstrated the highest miR-182-5p level. HGC-27 cells were transfected with different vectors and the cell viability, mitosis, invasion, and migration ability were measured through MTT assay, flow cytometry (FCM) analysis, Transwell assay, and wound healing assay. In comparison with the normal tissues, miR-182-5p is expressed at a higher level in gastric cancer (GC) tissues, while RAB27A is expressed at a lower level in cancerous tissues. The down-regulation of miR-182-5p and up-regulation of RAB27A can significantly decrease the viability, migration, invasion, and mitosis of HGC-27 cells. The target relationship between miR-182-5p and RAb27A was confirmed through a dual-luciferase reporter gene assay and Western blot assay. miR-182-5p enhances the viability, mitosis, migration, and invasion of human GC cells by down-regulating RAB27A.

PUMA decreases the growth of prostate cancer PC-3 cells independent of p53.

PUMA (p53 upregulated modulator of apoptosis), a member of the B-cell lymphoma 2 (Bcl-2) protein family, is a pro-apoptotic protein. PUMA expression is modulated by the tumor suppressor p53. PUMA has a role in rapid cell death via p53-dependent and -independent mechanisms. To evaluate whether p53 is required for PUMA-mediated apoptosis in prostate cancer cells, p53 protein was silenced in human prostate cancer PC-3 cells by using p53 small interfering RNA (siRNA). The interference efficiency of p53 on RNA and protein levels was detected by reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation and p21 expression were subsequently examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and western blot analysis, respectively. p53-silenced or control PC-3 cells were transfected with pCEP4-(hemagglutinin)-PUMA plasmid, or non-carrier plasmid. Enzyme-linked immunosorbent assay was used to determine cell apoptosis by measuring histone release and caspase-3 activation, and MTT assay was used to measure cell viability. In addition, the expression of pro-apoptosis protein Bax and anti-apoptosis protein Bcl-2 were evaluated. The results of the present study revealed that p53 siRNA significantly suppressed p53 RNA and protein expression in PC-3 cells. Deficiency of p53 increased the cell growth rate and decreased p21 expression. However, PUMA overexpression remained able to induce apoptosis in p53-silenced and control cells by increasing Bax expression and decreasing Bcl-2 expression, leading to the activation of caspase-3. These results suggest that PUMA may mediate apoptosis of prostate cancer PC-3 cells, potentially independently of p53. Furthermore, PUMA gene treatment to induce cancer cell apoptosis may be more efficient compared with p53-dependent apoptosis, where loss of p53 expression or function may lead to limited efficacy of PUMA expression. Therefore, the present study proposes the significant hypothesis that increasing PUMA expression may be an effective approach for the treatment of prostate cancer, regardless of p53 status.

MicroRNA-294 Promotes Cellular Proliferation and Motility through the PI3K/AKT and JAK/STAT Pathways by Upregulation of NRAS in Bladder Cancer.

In our study we examined the role of microRNA-294 (miR-294) in bladder cancer and related mechanisms. Real-time polymerase chain reaction (RT-PCR) was performed to determine the expression level of miR-294. Western blot was used to determine the expression of NRAS, mainly factors in the PI3K/AKT and JAK/STAT pathways. Cell counting kit-8 assay, clonogenic assay, wound-healing assay, transwell and flow cytometry were used to explore, respectively, cell proliferation, survival, migration, invasion, and apoptosis of bladder cancer cell line T24. The expressions of miR-294 in bladder cancer cells including J82, HT1376, T24, and SW780 were significantly increased compared to those in human bladder epithelium cells (both HCV29 and SV-HUC-1). The proliferation rate, surviving fraction, migration, and invasion of T24 cells in miR-294 mimetic transfected group were significantly increased, while they were significantly decreased by miR-294 inhibitor transfection. Moreover, miR-294 suppression could increase the apoptotic rate of T24 cells. In addition, drug resistance of T24 cells to cisplatin was increased in miR-294 mimetic-treated group, while it was decreased by miR-294 inhibitor compared to empty control. Overexpression of miR-294 could upregulate NRAS expression in T24 cells and activate PI3K/AKT and JAK/STAT pathways. We found that miR-294 expression was positively related with proliferation and motility of T24 cells. Moreover, miR-294 suppression could promote the sensitivity of T24 cells to cisplatin. We also found miR-294 could upregulate NRAS and activate the PI3K/AKT and JAK/STAT pathways in T24 cells.

Involvement of mitochondrial pathway of apoptosis in urothelium in ketamine-associated urinary dysfunction.

BACKGROUND: Long-term use of ketamine results in ketamine-associated urinary dysfunction (KAUD), which is characterized by frequent micturition, urgent urination, urine pain, hematuria, dysuria and urge incontinence. This study aims to examine the effect of ketamine on the urothelium and investigate the underlying mechanisms responsible for KAUD. METHODS: A rat model of KAUD by ketamine injection was developed. Histological changes in bladder tissues were measured by hematoxylin and eosin staining, and apoptosis by the TUNEL assay. Primary bladder epithelial cells were treated with ketamine, and cell proliferation measured by a CellTiter 96 AQ cell proliferation assay, apoptosis assessed by TUNEL assay and levels of apoptosis-related proteins determined by Western blotting. RESULTS: Animals injected with ketamine displayed a decrease in body weight, behavioral signs of addiction, urinary dysfunction and damage to the epithelial layer of the bladder, particularly in the group receiving high-dose ketamine for 3 months. Ketamine increased apoptosis both in bladder tissues and bladder epithelial cells. In addition, ketamine induced the expression of Bax, cytochrome c and capase-3, but inhibited the expression of NF-κB and bcl-2. CONCLUSIONS: These results suggest that the mitochondrial pathway of apoptosis in bladder epithelium may contribute to KAUD.

Long-term ketamine abuse induces cystitis in rats by impairing the bladder epithelial barrier.

Long-term ketamine abuse is known to affect the lower urinary tract and produce symptoms of cystitis. However, the pathophysiology and causative mechanism of the changes in bladder function remain unclear. The present study aimed to investigate the existence of ketamine-induced cystitis in a rat model and characterize the underlining mechanisms. Rats were assigned to blank control, normal saline (NS), low-dose ketamine (LK, 5 mg/kg), and high-dose ketamine (HK, 50 mg/kg) groups. The two experimental groups received ketamine hydrochloride daily for 16 weeks. All rats were housed individually for assessment of urinary frequency and urine volume. Urinary biomarkers were measured at different time points. Rat bladders were excised for histopathology, immunohistochemistry, and western blot analysis. Ketamine-treated rats had increased urinary frequency compared to NS-treated rats at Week 16. Urinary nitric oxide and antiproliferative factor levels were increased in ketamine-treated rats within the first 30 h after administration. After long-term ketamine administration, urinary glycoprotein GP51 and potassium levels were decreased in the HK and LK groups compared to the NS group. Ketamine-treated rats showed thickened bladder epithelial layer, increased expression of inducible nitric oxide synthase and occludin, and decreased expression of zonula occludens-1 in the bladder wall. Ketamine, or its urinary metabolites, disrupted the proliferation of bladder epithelial cells, resulting in defected bladder epithelial barrier. Subsequent leakage of urinary potassium causes a stress response in the bladder and provokes cystitis.

Effects of long-term ketamine administration on rat bladder protein levels: a proteomic investigation using two-dimensional difference gel electrophoresis system.

OBJECTIVES: Long-term ketamine abuse can affect the urinary system, resulting in interstitial cystitis-like syndrome. However, its pathogenesis remains unclear. In the present study, a proteomic approach of two-dimensional difference gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-light mass spectrometry was carried out to investigate the potential disease-associated proteins in a rat model of ketamine-associated cystitis. METHODS: Rats were randomly assigned to control, normal saline, low dose of ketamine (10 mg/kg) and high-dose of ketamine (50 mg/kg) groups with six rats in each group. The two experimental groups were given ketamine hydrochloride i.p. daily, whereas the normal saline group rats were treated with saline. After 16 weeks of treatment, all bladders were excised, and samples from normal saline and high dose of ketamine groups were resolved in two-dimensional difference gel electrophoresis. Differentially expressed spots were excised and identified by matrix-assisted laser desorption/ionization time-of-light mass spectrometry. Phosphoprotein and non-phosphoprotein purification, histopathology, immunohistochemistry, and western blot were carried out in all groups. RESULTS: Histological study showed hyperplastic epithelium and inflammatory cells infiltration in the high dose of ketamine-treated rat bladders. Two-dimensional difference gel electrophoresis revealed 30 altered expressions between the normal saline and high dose of ketamine-treated group. Among these proteins, two upregulated and two downregulated protein spots were all identified as smooth muscle protein-22/transgelin. Immunohistochemical staining and western blot analysis showed that the expression of total transgelin had no significant difference between groups. However, the expression of phosphorylated transgelin in the low-dose and high dose of ketamine groups was increased, whereas the non-phosphorylated transgelin was decreased when compared with the normal saline group. CONCLUSIONS: Long-term ketamine abuse induces phosphorylation of transgelin in the bladder wall, and this might play an important role in the pathogenesis of ketamine-associated cystitis.

Evaluation of upper urinary tract tumors by FISH in Chinese patients.

Upper urinary tract tumor (UUTT) usually presents a high grade and stage, and recurs frequently. The aim of this study was to evaluate the utility of a fluorescence in situ hybridization (FISH) assay on chromosomes 3, 7, 9, and 17 as a reliable and noninvasive method for the diagnosis of Chinese patients with UUTT. Urine specimens from 50 patients with UUTT and 25 donors without evidence of urothelial tumors were analyzed by cytology and FISH. Voided urine samples from 20 normal individuals were used to establish the cut-off values for FISH assay. The McNemar test was applied for sensitivity and specificity. The overall sensitivity of FISH was statistically significantly greater than that of cytology (84.0 vs. 40.0%, P = 0.000). The overall specificities of FISH and urine cytology were all 96.0% (P = 1.000). Polysomy in chromosomes 3, 7, and 17 were 38, 42, and 30%, respectively. Heterozygous and homozygous loss of the p16 locus was found in 36 and 32%, respectively. FISH analysis performed on cells collected from voided urine is feasible, and FISH could prove to be a reliable and less invasive ancillary test and improve the sensitivity of urine cytology in the diagnosis of UUTT.

[Synchronous squamous cell carcinoma of the renal pelvis and squamous cell carcinoma of the ureter: report of two cases and review of literature].

OBJECTIVE: To study the clinicopathological characteristics of synchronous squamous cell carcinoma (SCC) of the renal pelvis and SCC of the ureter. METHODS: The clinical data of two cases of synchronous SCC of the renal pelvis and SCC of the ureter were retrospectively reviewed and analyzed. In case 1, a 68-year-old man with hematuria for a month, imaging modalities revealed a right renal pelvis tumor and a right distal ureter tumor. The patient underwent nephroureterectomy and excision of the bladder cuff. Case 2, a 60-year-old man with the complaint of lower abdominal pain and left flank pain for a month, was diagnosed as left distal ureteral stone in another hospital. Ureterolithotomy was performed and a ureteral tumor was found at the lower site of the stone intraoperatively. The pathological report demonstrated SCC, and the patient was transferred to our hospital for further treatment. We found a left renal mass invading the left hemicolon during surgery, and nephroureterectomy was performed with a bladder cuff excision, left hemicolon resection, and also complete lymph node dissection. Neither of patients received adjuvant radiotherapy/chemotherapy. RESULTS: Moderately differentiated SCC was reported in both of renal pelvis and ureter in case 1 and the tumor invaded the subepithelial connective tissue in the renal pelvis and superficial muscle in the ureter. In case 2, moderately differentiated SCC of the left renal pelvis with colon metastasis and poorly differentiated SCC of the ureter was reported with two retroperitoneal lymph node metastases. The two patients died from tumor recurrence and metastasis 5 and 6 months after the surgery, respectively. CONCLUSION: Synchronous SCC of the renal pelvis and SCC of the ureter are rare and has high likeliness of early recurrence and metastasis, often with poor prognosis.