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Ketamine is used clinically for anesthesia but is also abused as a recreational drug. Previously, it has been established that ketamine-induced bladder interstitial cystitis is a common syndrome in ketamine-abusing individuals. As the mechanisms underlying ketamine-induced cystitis have yet to be revealed, the present study investigated the effect of ketamine on human urothelial cell lines and utilized a ketamine-injected mouse model to identify ketamine-induced changes in gene expression in mice bladders. In the
Ketamine was first synthesized in 1962 (
To date, no specific treatment for patients with ketamine-induced cystitis has been established. In spite of the increase in the number of recreational users, investigating ketamine-induced cystitis in humans is not simple. As a result, a number of studies have used animal models for investigating the mechanisms of action and effects of ketamine. Previously, to the best of our knowledge, two mouse model (
Although the animal studies mentioned above provided notable insight into the mechanisms of ketamine-induced bladder damage, these effects remain to be fully elucidated. In the present study, three urothelial cell lines were used to study the cytotoxicity of ketamine and the barrier permeability affected by ketamine. In the
Three different urothelial cell lines, purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan) were used. The SV-HCU-1 cell line derived from normal human urothelial cells immortalized by the SV40 virus. The RT4 cell line is derived from a well-differentiated papillary tumor of the human bladder (
The cell number was determined by a colorimetric MTT assay. The cells were seeded in 96-well plates for 24 h, then were incubated with various concentrations of ketamine or normal saline for another 24–48 h. MTT was added into the medium for 2 h, then the medium was discarded and dimethyl sulfoxide was added to dissolve the formazan product. Each well was measured by light absorbance at 490 nm. The result was expressed as the percentage of the normal saline-treated control group.
The cells were seeded in 100-mm dishes. Following 24 h incubation, ketamine or normal saline was added. Following treatment for 24 and 48 h, the cells were trypsinized, centrifuged at 800 × g for 5 min and fixed with ice-cold 75% ethanol overnight at 4°C. Following removal of the ethanol, the cells were stained with a DNA staining solution [containing 1 mg/ml propidium iodide and 10 mg/ml RNase A dissolved in phosphate-buffered saline (PBS)] for 30 min at room temperature. The DNA content of the stained cells was measured using a FACScan flow cytometer. The cell doublets were removed by gating the left area of the FL2-W/FL2-A plot for analysis. The cell cycle data from flow cytometry were analyzed using ModFit LTTM software (Verity Inc. Sunnyvale, CA).
Approximately 4×104 SV-HUC-1 cells, 4×104 RT4 cells and 1×104 5637 cells were seeded on an Transwell insert with 0.4 μm pore size filter membrane (Millipore Corp. Billerica, MA, USA) and incubated for 24 h. Ketamine was added into the upper and lower chamber media at the same time. Following incubation for 19 or 43 h, green fluorescence-labeled antibodies (Alexa Fluor® 488 goat anti-mouse immunoglobulin G; Invitrogen Life Technologies) were added into upper chamber medium (9.6 μg/insert) and continued incubating for another 5 h. The total medium in the upper and lower chambers were collected for fluorescence analysis by a fluorescence microplate reader (excitation/emission: 488/519 nm).
Six-week-old male Balb/c mice were used in the present study and purchased from the National Laboratory Animal Center (Taipei, Taiwan). All of the animals were maintained at the qualified animal care facility of Biotechnology and Health Hall in National Chiayi University (Chiayi City, Taiwan, R.O.C) for one week prior to intraperitoneal (i.p.) injection. At seven weeks of age, the mice were divided into four groups (12 mice/group), including control-30 days (i.p. normal saline for 30 days), ketamine-30 days (i.p. 30 mg/kg/day ketamine for 30 days), control-60 days (i.p. normal saline for 60 days) and ketamine-60 days (i.p. 30 mg/kg day ketamine for 60 days). The mice were housed in polycarbonate cages, provided with food and water ad libitum and maintained on a 12 h light-dark cycle at 22±2°C. All of the experiments were approved by the Institutional Animal Care and Use Committee of National Chiayi University.
Following the 30- or 60-day treatment, the mice were euthanized and the bladder tissues were removed. A total of 20 bladders (five/group) were fixed in 10% neutral formalin for histological examination, three bladders/group were homogenized together and RNA was extracted, and the other bladders were stored under liquid nitrogen for future use. The bladder tissues in 10% neutral formalin were embedded in paraffin and then cut into 4-μm sections on glass slides. One slide from each mouse was stained with hematoxylin and eosin (H&E). Other slides were prepared for immunohistochemical analysis.
Total RNA was isolated from three bladders in each group using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. The quality of RNA was examined using Agilent’s RNA LabChip kits on the 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The RNA samples from the four groups (control-30 days, ketamine-30 days, control-60 days and ketamine-60 days) were transferred to fluorescence-labeled antisense (a)RNA using OneArray Amino Allyl aRNA Amplification kit (Phalanx Biotech Group, Hsinchu, Taiwan) and Cy5 dye labeling (Amersham Pharmacia, Piscataway, NJ, USA). For global gene expression analysis, the fluorescent targets were hybridized to the Mouse Whole Genome OneArrayTM version MOA 2.0 (Phalanx Biotech Group), containing 27,295 mouse genome probes. One mixture sample was applied to two chips, and the normalized intensities were calculated from raw intensities by median scaling. Microarray image scanning and data analysis were achieved by Phalanx Biotech Group.
Reverse transcription was performed on 2 μg of total RNA by 5 μM random hexamer and RevertAidTM reverse transcriptase (Thermo Fisher Scientific, Fermentas, Pittsburgh, PA, USA), then 1/10 volume of reaction mixture was used for PCR with specific primers (keratin 6a forward 5′-TGCCAGGGGCAAGCTGGAAG-3′ and reverse 5′-ACGGGATTCTGCAGCCATGACA-3′; keratin 13 forward 5′-AGCTTGGAGGAGGCCGTAAT-3′ and reverse 5′-AAGCACTGTAGTCCCGCTCT-3′; keratin 14 forward 5′-TGGTGCAGAGCGGCAAGAGTG-3′ and reverse 5′-TGCGGATCTGGCGGTTGGTGG-3′) and β-actin forward 5′-CCTAAGGCCAACCGTGAAAAG-3′ and reverse 5′-TCTTCATGGTGCTAGGAGCCA-3′). The PCR products (keratin 6a, 486 bp; keratin 13, 375 bp; keratin 14, 399 bp; β-actin, 623 bp) were analyzed by 1% agarose gel.
After being washed in PBS, the slides were incubated in a blocking solution for 30 min and then with primary antibodies against keratin 14 (Genetex, Taipei, Taiwan) at a 1:100 dilution at 4°C overnight. The slides were then washed and incubated with secondary antibodies containing horseradish peroxidase at 25°C for 30 min. Following this treatment, the slides were washed with PBS and further incubated with 3,3′-diaminobenzidine for 5 min. Finally, the sections were rinsed in running water, treated with hematoxylin for ~10–15 sec and mounted for evaluation.
Numerical data (except gene expression microarray data) are expressed as the mean ± standard error. Statistical differences were analyzed by one-way analysis of variance analysis of variance followed by Tukey’s test. All statistics were calculated using SigmaState version 3.5 (Systat Software, San Jose, CA, USA)
Following ketamine treatment for 24 h, the IC50 value of ketamine was ~4, 2 and 3 mM in SV-HUC-1, RT4 and 5637 cells, respectively. At 48 h, the IC50 was ~3, 1.5 and 2 mM in the SV-HUC-1, RT4 and 5637 cells, respectively (
Due to the cytotoxicity of ketamine (
In addition to the
Gene expression microarray analysis of bladder tissue was applied to compare gene expression between the control and ketamine-treated animals. Upregulated genes with differential expression (fold change log 2 ≥ 1 and P<0.05) at 60 days and a statistical difference (only P<0.05) at 30 days were selected. Downregulated genes with differential expression (fold change log 2 ≤ −1 and P<0.05) at 60 days and statistical difference (only P<0.05) at 30 days were selected. Analysis revealed that 10 genes were upregulated (
Cytoskeletal keratins belong to intracellular intermediate filaments that connect to epithelial cell adhesion plaques in macula adherens and hemidesmosome sites. Numerous inherited skin-blistering diseases are caused by keratin gene mutations. There were 52 keratin family genes in the gene expression microarray chip. The majority of the keratins were downregulated by ketamine: 40% following 30 days and 52% following 60 days (
In the present
Ketamine demonstrated toxicity (
According to Yeung’s ICR mouse model (30 mg/kg/day for 1 and 3 months) (
Keratins are the major component of the fibrous intermediate filament in epithelial cells. Keratin 20 is a tumor marker of urothelial dysplasia (
In addition to decreases in the levels of various types of keratin, the cDNA array data indicated further mechanisms leading to the downregulation of urothelial barrier function. Firstly, the hemidesmosome, consisting of intracellular keratins, plectin plaque and adhesion molecules, such as the α6β4 integrin (
This study was supported by grants from the National Science Council of Taiwan (NSC101-2320-B-415-002-MY3) and from Chiayi Christian Hospital, Taiwan (R100-9).
fetal bovine serum
hematoxylin and eosin
propidium iodide
ribonuclease A
Cytotoxicity of ketamine on SV-HUC1, RT4 and 5637 cells. (A) Cytotoxicity of 0–8 mM ketamine. The cells were treated with 0–8 mM ketamine for 24 h (black bar) and 48 h (gray bar), and the cell viability was analyzed by an MTT assay. (B–D) Cell cycle distribution changed following incubation with 0–4 mM ketamine for 24 and 48 h. The cells were collected for cell cycle analysis following ketamine treatment in (B) SV-HUC1, (C) RT4 and (D) 5637 cells. Quantification performed from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, significant difference between the control and ketamine-treated cells.
Ketamine increases urothelium barrier permeability in SV-HUC1, RT4 and 5637 cells. The cells were treated with 0–4 mM ketamine for 24 or 48 h, then the upper and bottom chamber media were incubated with Alexa Fluor® 488 goat anti-mouse immunoglobulin G and analyzed by a fluorescence microplate reader. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, significant difference between the control and ketamine-treated cells.
Comparison of keratin family gene expression in the control and ketamine-treated mouse bladders. (A) The pie chart of keratin family genes reveals the percentage of downregulated, upregulated and ND keratin genes. (B) Polymerase chain reaction analysis of three keratin gene expression. The RNA was extracted from mouse bladders following normal saline (control) or 30 mg/kg/day ketamine (ketamine) i.p. injection for 30 and 60 days. The product sizes of keratin 6a, 13, 14 and β-actin are 486, 375, 399 and 623 bp, respectively. (C) Heat map of keratin gene expression. The map was created using the Cluster 3.0. The genes are arrayed from the most downregulated (left) to the most upregulated (right).
Keratin 14 protein expression in mouse bladder tissues by immunohistochemical analysis. The slides of bladder tissue were hybridized with anti-keratin 14 antibodies and then photographed under ×400 microscopy. Upper and lower images, representative images from two different mouse bladders.
Upregulated genes with differential expression (fold-change log 2 ≥ 1 and P<0.05) at 60-day ketamine treatment and statistical difference (P<0.05) at 30-day ketamine treatment in mouse bladders.
Normalized intensity | Ratio of change (%) | ||||||
---|---|---|---|---|---|---|---|
| |||||||
30-day | 60-day | (K-C)/C × 100% | |||||
| |||||||
Gene name | Accession number | C | K | C | K | 30-day | 60-day |
Hedgehog-interacting protein | NM_020259.4 | 296.8 | 602.8 | 183.8 | 847.5 | 103.1 | 361.1 |
Fucosyl-transferase 9 | NM_010243.3 | 165.3 | 226.6 | 78.2 | 330.7 | 37.1 | 322.9 |
Leucine rich repeat containing G protein coupled receptor 5 | NM_010195.2 | 97.3 | 143.4 | 149.1 | 435.0 | 47.4 | 191.8 |
Titin-cap | NM_011540.2 | 264.0 | 501.2 | 282.9 | 741.5 | 89.8 | 162 |
Family with sequence similarity 55, member C | NM_001134494.1 | 438.0 | 705.3 | 170.4 | 401.8 | 61.0 | 135.8 |
Toll-like receptor 12 | NM_205823.2 | 127.2 | 263.7 | 131.7 | 306.6 | 107.3 | 132.8 |
Transthyretin | NM_013697.5 | 8198.6 | 12323.3 | 5331.2 | 11324.8 | 50.3 | 112.4 |
Ras-related associated with diabetes | NM_019662.2 | 699.4 | 1682.9 | 427.9 | 836.2 | 140.6 | 95.4 |
Transformation related protein 53 inducible nuclear protein 1 | NM_021897.3 |
917.6 | 1624.4 | 528.7 | 1016.2 | 77.0 | 92.2 |
Claudin 23 | NM_027998.4 | 2564.6 | 3367.4 | 2346.5 | 4466.1 | 31.3 | 90.3 |
C, control mouse bladders; K, ketamine-injected mouse bladders.
Top ten downregulated genes with differential expression (fold change log 2 ≤ −1 and P<0.05) at 60-day ketamine treatment and statistical difference (P<0.05) at 30-day ketamine treatment in mouse bladders.
Normalized intensity | Ratio of change (%) | ||||||
---|---|---|---|---|---|---|---|
| |||||||
30-day | 60-day | (K-C)/C × 100% | |||||
| |||||||
Gene name | Accession number | C | K | C | K | 30-day | 60-day |
WAP four-disulfide core domain 3 | NM_027961.1 | 102.6 | 46.6 | 528.3 | 59.7 | −54.6 | −88.7 |
Metallothionein 2 | NM_008630.2 | 1230.5 | 782.7 | 5276.6 | 751.2 | −36.4 | −85.8 |
Tissue inhibitor of metallo-proteinase 1 | NM_001044384.1 |
1445.2 | 551.3 | 3648.6 | 845.7 | −61.9 | −76.8 |
Solute carrier family 7, member 11 | NM_011990.2 | 624.4 | 403.7 | 2992.5 | 708.8 | −35.3 | −76.3 |
Keratin 14 | NM_016958.1 | 2972.3 | 1652.2 | 6385.7 | 1621.5 | −44.4 | −74.6 |
Glutamine fructose-6-phosphate transaminase 2 | NM_013529.3 | 329.1 | 224.3 | 806.2 | 211.3 | −31.8 | −73.8 |
Macrophage scavenger receptor 1 | NM_031195.2 | 457.3 | 283.3 | 891.6 | 281.0 | −38.0 | −68.5 |
Interleukin 33 | NM_001164724.1 |
1403.7 | 705.9 | 3731.8 | 1207.9 | −49.7 | −67.6 |
C-type lectin domain family 4, member d | NM_00116316.1 |
151.6 | 73.6 | 201.6 | 66.5 | −51.5 | −67.0 |
Neuregulin 1 | NM_178591.2 | 86.5 | 49.5 | 199.8 | 68.0 | −42.8 | −66.0 |
Keratin 78 | NM_212487.4 | 241.5 | 166.6 | 857.4 | 365.9 | −31.0 | −57.3 |
C, control mouse bladders; K, ketamine-injected mouse bladders.
Top ten downregulated keratin genes following ketamine treatment for 60 days.
Normalized intensity | Ratio of change (%) | ||||||
---|---|---|---|---|---|---|---|
| |||||||
30-day | 60-day | (K-C)/C×100% | |||||
| |||||||
Gene name | Accession number | C | K | C | K | 30-day | 60-day |
Keratin 6a | NM_008476.3 | 42.6 | 24.3 | 985.4 | 46.0 | −43.0 | −95.3 |
Keratin 13 | NM_010662.1 | 130.9 | 170.0 | 1165.9 | 181.6 | 29.9 | −84.4 |
Keratin 14 | NM_016958.1 | 2972.3 | 1652.2 | 6385.7 | 1621.5 | −44.4 |
−74.6 |
Keratin 23 | NM_033373.1 | 580.3 | 676.9 | 1926.2 | 722.6 | 16.6 | −62.5 |
Keratin 78 | NM_212487.4 | 241.5 | 166.6 | 394.9 | 185.3 | −31 |
−57.3 |
Keratin 5 | NM_027011.2 | 6344.0 | 6327.0 | 9821.2 | 5129.2 | −0.3 | −47.8 |
Keratin 6b | NM_010669.2 | 13.9 | 9.5 | 35.5 | 19.9 | (NA) | −44 |
Keratin 8 | NM_031170.2 | 13729.3 | 15504.3 | 20223.6 | 13109.0 | 12.9 | −35.2 |
Keratin 19 | NM_008471.2 | 9891.2 | 7572.7 | 15246.7 | 10048.5 | −23.4 | −34.1 |
Keratin 79 | NM_146063.1 | 104.4 | 100.4 | 163.7 | 108.2 | −3.9 | −33.9 |
P<0.05, indicates a significant difference between the control and ketamine-injected groups. Rosetta Resolver® was applied to detect signal noise for reducing the false positive, the signal was adjusted to NA if this sample was not qualified.
C, control mouse bladders; K, ketamine-injected mouse bladders.
Four gene expression change data in normal saline and ketamine treatment for 30 and 60 days.
Normalized intensity | Ratio of change (%) | ||||||
---|---|---|---|---|---|---|---|
| |||||||
30-day | 60-day | (K-C)/C×100% | |||||
| |||||||
Gene name | Accession number | C | K | C | K | 30-day | 60-day |
Integrin α6 | NM_008397.3 | 6661.3 | 9441.9 | 9028.3 | 7893.3 | 41.7 |
−12.6 |
Integrin β4 | NM_133663.2 |
802.7 | 739.2 | 1082.0 | 394.7 | −7.9 | −63.5 |
Claudin-1 | NM_016674.4 | 1146.1 | 1002.7 | 3597.1 | 1209.6 | −12.5 | −66.4 |
Keratin 20 | NM_023256.1 | 473.0 | 568.4 | 1954.1 | 708.4 | 20.2 | −63.7 |
P<0.05 between the control and ketamine-injected groups.
C, control mouse bladders; K, ketamine-injected mouse bladders.