Impact of Abdominal Fat Distribution on Mortality and Its Changes Over Time in Patients Undergoing Hemodialysis: A Prospective Cohort Study.

OBJECTIVE: Measures of fat distribution and visceral fat accumulation maintain a direct association with mortality in the general population. However, among patients undergoing hemodialysis (HD), there are few reports of this association. This study aimed to investigate the impact of computed tomography (CT)-measured abdominal fat levels, including the visceral fat area (VFA) and subcutaneous fat area (SFA), on all-cause mortality in patients undergoing HD and investigate whether there are sex-specific particularities regarding the associations between the abovementioned parameters. METHODS: A total of 258 participants were selected from the population of patients undergoing stable HD. The baseline characteristics were collected by records and interviews. The following variables were assessed at baseline and every year: body mass index, abdominal circumference, VFA, and SFA. Abdominal circumference and body fat distribution were assessed at the level of the umbilicus via CT. All CT scans were performed on a nondialysis day with the subject in a supine position. The primary end point was the 5-year all-cause mortality. RESULTS: This prospective cohort study revealed that age, cardiothoracic ratio, %VFA (VFA/[VFA + SFA]), and albumin were independent predictors of death via multivariable analyses. Regarding the %VFA, its area under the curve (0.599), which did not suffice to predict mortality, was higher than that of VFA, SFA, and body mass index. Also, the effect was recognized mainly in male patients. The %VFA of patients who survived for 60 months increased over time. CONCLUSION: These data suggest that patients (especially men) with a high VFA-to-abdominal fat ratio have a high risk of death. Thus, more attention should be paid to such patients.

KATNB1 is a master regulator of multiple katanin enzymes in male meiosis and haploid germ cell development.

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits.

Serum soluble (pro)renin receptor level as a prognostic factor in patients undergoing maintenance hemodialysis.

The (pro)renin receptor [(P)RR)] is a multifunctional protein that is cleaved to generate the soluble (P)RR [s(P)RR], reflecting the status of the tissue renin-angiotensin system and/or activity of the (P)RR. The serum s(P)RR level is associated with arteriosclerosis, independent of other risk factors, in patients undergoing hemodialysis (HD). This study was conducted to investigate whether the s(P)RR level was associated with new-onset cardiovascular events or malignant diseases and poor prognosis in patients undergoing HD. Overall, 258 patients [70 (61-76) years, 146 males] undergoing maintenance HD were prospectively followed up for 60 months. We investigated the relationships between s(P)RR levels and new-onset cardiovascular events/ malignant diseases and mortality during the follow-up period using Cox proportional hazard analyses. The cumulative incidence of new-onset cardiovascular events (P = 0.009) and deaths (P < 0.001), but not of malignant diseases, was significantly greater in patients with higher serum s(P)RR level (≥ 29.8 ng/ml) than in those with lower s(P)RR level (< 29.8 ng/ml). A high serum s(P)RR level was independently correlated with cardiovascular mortality (95% CI 1.001-1.083, P = 0.046). The serum s(P)RR level was associated with cardiovascular events and mortality, thus qualifying as a biomarker for identifying patients requiring intensive care.

Identification and characterization of the antigen recognized by the germ cell mAb TRA98 using a human comprehensive wet protein array.

TRA98 is a rat monoclonal antibody (mAb) which recognizes a specific antigen in the nuclei of germ cells. mAb TRA98 has been used to understand the mechanism of germ cell development and differentiation in many studies. In mice, the antigen recognized by mAb TRA98 or GCNA1 has been reported to be a GCNA gene product, but despite the demonstration of the immunoreactivity of this mAb in human testis and sperm in 1997, the antigen in humans remains unknown, as of date. To identify the human antigen recognized by mAb TRA98, a human comprehensive wet protein array was developed containing 19,446 proteins derived from human cDNAs. Using this array, it was found that the antigen of mAb TRA98 is not a GCNA gene product, but nuclear factor-κB activating protein (NKAP). In mice, mAb TRA98 recognized both the GCNA gene product and NKAP. Furthermore, conditional knockout of Nkap in mice revealed a phenotype of Sertoli cell-only syndrome. Although NKAP is a ubiquitously expressed protein, NKAP recognized by mAb TRA98 in mouse testis was SUMOylated. These results suggest that NKAP undergoes modifications, such as SUMOylation in the testis, and plays an important role in spermatogenesis.

The Sertoli cell expressed gene secernin-1 (Scrn1) is dispensable for male fertility in the mouse.

BACKGROUND: Male infertility is a prevalent clinical presentation for which there is likely a strong genetic component due to the thousands of genes required for spermatogenesis. Within this study we investigated the role of the gene Scrn1 in male fertility. Scrn1 is preferentially expressed in XY gonads during the period of sex determination and in adult Sertoli cells based on single cell RNA sequencing. We investigated the expression of Scrn1 in juvenile and adult tissues and generated a knockout mouse model to test its role in male fertility. RESULTS: Scrn1 was expressed at all ages examined in the post-natal testis; however, its expression peaked at postnatal days 7-14 and SCRN1 protein was clearly localized to Sertoli cells. Scrn1 deletion was achieved via removal of exon 3, and its loss had no effect on male fertility or sex determination. Knockout mice were capable of siring litters of equal size to wild type counterparts and generated equal numbers of sperm with comparable motility and morphology characteristics. CONCLUSIONS: Scrn1 was found to be dispensable for male fertility, but this study identifies SCRN1 as a novel marker of the Sertoli cell cytoplasm.

Association between serum soluble (pro)renin receptor level and worsening of cardiac function in hemodialysis patients: A prospective observational study.

The (pro)renin receptor ((P)RR) is cleaved to generate soluble (P)RR (s(P)RR), which reflects the status of the tissue renin-angiotensin system. Hemodialysis (HD) patients have a poor prognosis due to the increased prevalence of cardiovascular diseases. The present study aimed to investigate whether serum s(P)RR level is associated with the worsening of cardiac function in HD patients. A total of 258 maintenance HD patients were recruited and serum s(P)RR concentration was measured. Background factors in patients who survived (S group) and patients who died (D group) during the 12-month follow-up period and relationships between serum s(P)RR level and changes in cardiac function during the follow-up period in the S group were investigated. The median serum s(P)RR value at baseline was 29.8 ng/ml. Twenty-four patients died during the follow-up period. Cardiothoracic ratio, human atrial natriuretic peptide (hANP), brain natriuretic peptide (BNP), and E over e-prime were significantly higher in the D group. In the S group, changes in hANP or BNP were significantly greater in the higher serum s(P)RR group than in the lower serum s(P)RR group. High serum s(P)RR level was significantly correlated with changes in BNP, independent of other factors. High serum s(P)RR level was associated with increases in BNP, independent of other risk factors, suggesting that an increased expression of (P)RR may be associated with a progression of heart failure in HD patients and that serum s(P)RR concentration could be used as a biomarker for selecting patients requiring intensive care.

Haprin-deficient spermatozoa are incapable of in vitro fertilization.

Haprin (TRIM36) is a ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin-deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility-related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin-deficient mice having poorer sperm morphology and motility than wild-type mice. Interestingly, haprin-deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.

CRISP2 Is a Regulator of Multiple Aspects of Sperm Function and Male Fertility.

The cysteine-rich secretory proteins (CRISPs) are a group of proteins that show a pronounced expression biased to the male reproductive tract. Although sperm encounter CRISPs at virtually all phases of sperm development and maturation, CRISP2 is the sole CRISP produced during spermatogenesis, wherein it is incorporated into the developing sperm head and tail. In this study we tested the necessity for CRISP2 in male fertility using Crisp2 loss-of-function mouse models. In doing so, we revealed a role for CRISP2 in establishing the ability of sperm to undergo the acrosome reaction and in establishing a normal flagellum waveform. Crisp2-deficient sperm possess a stiff midpiece and are thus unable to manifest the rapid form of progressive motility seen in wild type sperm. As a consequence, Crisp2-deficient males are subfertile. Furthermore, a yeast two-hybrid screen and immunoprecipitation studies reveal that CRISP2 can bind to the CATSPER1 subunit of the Catsper ion channel, which is necessary for normal sperm motility. Collectively, these data define CRISP2 as a determinant of male fertility and explain previous clinical associations between human CRISP2 expression and fertility.

Katanin-like 2 (KATNAL2) functions in multiple aspects of haploid male germ cell development in the mouse.

The katanin microtubule-severing proteins are essential regulators of microtubule dynamics in a diverse range of species. Here we have defined critical roles for the poorly characterised katanin protein KATNAL2 in multiple aspects of spermatogenesis: the initiation of sperm tail growth from the basal body, sperm head shaping via the manchette, acrosome attachment, and ultimately sperm release. We present data suggesting that depending on context, KATNAL2 can partner with the regulatory protein KATNB1 or act autonomously. Moreover, our data indicate KATNAL2 may regulate δ- and ε-tubulin rather than classical α-ß-tubulin microtubule polymers, suggesting the katanin family has a greater diversity of function than previously realised. Together with our previous research, showing the essential requirement of katanin proteins KATNAL1 and KATNB1 during spermatogenesis, our data supports the concept that in higher order species the presence of multiple katanins has allowed for subspecialisation of function within complex cellular settings such as the seminiferous epithelium.

LRGUK1 is part of a multiprotein complex required for manchette function and male fertility.

Infertility occurs in 1 in 20 young men and is idiopathic in origin in most. We have reported that the leucine-rich repeat (LRR) and guanylate kinase-like domain containing, isoform (LRGUK)-1 is essential for sperm head shaping, via the manchette, and the initiation of sperm tail growth from the centriole/basal body, and thus, male fertility. Within this study we have used a yeast 2-hybrid screen of an adult testis library to identify LRGUK1-binding partners, which were then validated with a range of techniques. The data indicate that LRGUK1 likely achieves its function in partnership with members of the HOOK family of proteins (HOOK-1-3), Rab3-interacting molecule binding protein (RIMBP)-3 and kinesin light chain (KLC)-3, all of which are associated with intracellular protein transport as cargo adaptor proteins and are localized to the manchette. LRGUK1 consists of 3 domains; an LRR, a guanylate kinase (GUK)-like and an unnamed domain. In the present study, we showed that the GUK-like domain is essential for binding to HOOK2 and RIMBP3, and the LRR domain is essential for binding to KLC3. These findings establish LRGUK1 as a key component of a multiprotein complex with an essential role in microtubule dynamics within haploid male germ cells.-Okuda, H., DeBoer, K., O'Connor, A. E., Merriner, D. J., Jamsai, D., O'Bryan, M. K. LRGUK1 is part of a multiprotein complex required for manchette function and male fertility.

A novel transcriptional factor Nkapl is a germ cell-specific suppressor of Notch signaling and is indispensable for spermatogenesis.

Spermatogenesis is an elaborately regulated system dedicated to the continuous production of spermatozoa via the genesis of spermatogonia. In this process, a variety of genes are expressed that are relevant to the differentiation of germ cells at each stage. Although Notch signaling plays a critical role in germ cell development in Drosophila and Caenorhabditis elegans, its function and importance for spermatogenesis in mammals is controversial. We report that Nkapl is a novel germ cell-specific transcriptional suppressor in Notch signaling. It is also associated with several molecules of the Notch corepressor complex such as CIR, HDAC3, and CSL. It was expressed robustly in spermatogonia and early spermatocytes after the age of 3 weeks. Nkapl-deleted mice showed complete arrest at the level of pachytene spermatocytes. In addition, apoptosis was observed in this cell type. Overexpression of NKAPL in germline stem cells demonstrated that Nkapl induced changes in spermatogonial stem cell (SSC) markers and the reduction of differentiation factors through the Notch signaling pathway, whereas testes with Nkapl deleted showed inverse changes in those markers and factors. Therefore, Nkapl is indispensable because aberrantly elevated Notch signaling has negative effects on spermatogenesis, affecting SSC maintenance and differentiation factors. Notch signaling should be properly regulated through the transcriptional factor Nkapl.

LRGUK-1 is required for basal body and manchette function during spermatogenesis and male fertility.

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

Sexual life of Japanese patients with erectile dysfunction taking phosphodiesterase type 5 inhibitors: an Internet survey using the Psychological and Interpersonal Relationship Scales-Short Form questionnaire.

OBJECTIVES: To investigate details of sexual function of erectile dysfunction in Japanese patients taking phosphodiesterase type 5 inhibitors. METHODS: A Japanese version of the Psychological and Interpersonal Relationship Scales-Short Form was used to carry out a nationwide survey using the Internet. A total of 556 erectile dysfunction patients (age 30-70 years) who had been prescribed a phosphodiesterase type 5 inhibitor and had attempted sexual intercourse within the past 6 months were included in this survey. Scores were compared in relation to the phosphodiesterase type 5 inhibitors most frequently taken within the past 6 months. RESULTS: In the subdomains of self-confidence and spontaneity of the Psychological and Interpersonal Relationship Scales-Short Form, scores for vardenafil and tadalafil were significantly higher than those for sildenafil. In the subdomain of time concern of the Psychological and Interpersonal Relationship Scales-Short Form, the score for tadalafil was significantly lower than that for others. CONCLUSIONS: Our findings support the hypothesis that Japanese patients with erectile dysfunction have high sexual self-confidence, spontaneity and low time concerns when taking tadalafil. These characteristics of tadalafil could be associated with high patient satisfaction and high preference.

Finger taps and constipation are closely related to symptoms of overactive bladder in male patients with Parkinson's disease.

OBJECTIVES: To assess which motor and non-motor symptoms are closely related to overactive bladder severity in male patients with Parkinson's disease. METHODS: A total of 160 male patients (mean age 71.4 ± 8.2 years) diagnosed with Parkinson's disease were included in the present study at Osaka University and affiliated hospitals. The severity of Parkinson's disease was classified as stage 3, 4 or 5 based on the Hoehn and Yahr staging system. Disease duration was 8.9 ± 5.1 years. Age, seven items from the Unified Parkinson's Disease Rating Scale motor section part III and three non-motor symptoms were assessed by multivariate analysis for their impact on the overactive bladder symptom score, a specific questionnaire for overactive bladder. RESULTS: Overactive bladder symptom score was significantly higher in the group with severe motor symptoms related to finger taps and gait than in the group with mild motor symptoms related to these two factors. Furthermore, overactive bladder symptom score of patients with erectile dysfunction and constipation was significantly higher than that in patients without these symptoms. Multivariate analysis identified only finger taps and constipation as factors independently associated with overactive bladder symptom score. CONCLUSIONS: Although a study on a larger scale is required to further assess the association of Parkinson's disease symptoms with overactive bladder symptom score, information on finger taps and severity of constipation should be obtained when assessing urological patients with Parkinson's disease.

Is low testosterone concentration a risk factor for metabolic syndrome in healthy middle-aged men?

OBJECTIVE: To clarify the probability of low serum testosterone level as a risk factor for metabolic syndrome (MS) in middle-aged men, we measured serum total testosterone (TT) and assessed several metabolic factors because the direct risk for MS has not been investigated fully in men. METHODS: This study comprised 1150 men aged ≥30 years. Physical and laboratory variables were assessed. Analyses were conducted to determine the association between serum TT level and incidence risk of MS and MS factors by a separate logistic regression model. RESULTS: Mean (± standard deviation [SD]) serum TT level was 5.4 ± 1.7 ng/mL in the 1150 men, and only 92 men (8.0%) were classified as having MS by the Japanese criteria. In age-adjusted analyses, higher levels of serum TT were independently associated with a lower risk of MS (odds ratio, per SD decrement of TT, 2.3; 95% confidence interval [CI], 1.7-2.9). MS risk increased by lower quintile of TT: ORs were 15.1 (95% CI, 4.6-50.0) for first quintile, 8.8 (95% CI, 2.6-29.9) for second quintile, 5.8 (95% CI, 1.7-20.5) for third quintile, and 5.0 (95% CI, 1.4-17.9) for fourth quintile compared with highest quintile of TT. Age-adjusted ORs for the incidence of dichotomous components of MS per SD decrement of TT were 1.8 (95% CI, 1.5-2.3) for waist circumference, 1.6 (95% CI, 1.1-2.2) for dyslipidemia, and 1.5 (95% CI, 1.2-1.8) for hypertension. CONCLUSION: We found that higher probability of MS was associated with lower levels of serum TT level in relatively healthy middle-aged Japanese men.

Standardized diagram improves diagnostic accuracy and variability in the identification of testicular cells prepared for testicular sperm extraction and intracytoplasmic sperm injection.

OBJECTIVE: To examine the accuracy and variability of identification of human germ cells and validate the previously reported diagram referable to identifying human testicular cells, which was made to improve the identification. METHODS: Eighty-seven testicular cells obtained from azoospermic patients were stained with MitoTracker, and observed under phase contrast, fluorescent, and differential interference microscopy. The recorded image and movie data of phase contrast microscopy were assessed by 10 reviewers comprising embryologists and reproductive physicians 2 times, once without the diagram and 1 year later with use of it. True cell type identifications were determined as referenced by morphologic characteristics and MitoTracker staining. Variability between reviewers was assessed using multirater κ statistics, and changes of the concordance rates to the reference were examined. RESULTS: Multirater κ coefficients changed from 0.14 to 0.49 overall, from 0.10 to 0.34 for sperm-like cells, and from 0.044 to 0.46 in round-shaped cells before and after using the diagram, which represents a change from fair to substantial agreement overall for round-shaped cells and to moderate agreement for sperm-like cells. The concordance rates to the reference before and after the use of the diagram also significantly improved from 28.4% to 59.1% overall, from 38.9% to 54.6% for sperm-like cells, and from 19.4% to 59.1% for round-shaped cells, respectively. CONCLUSION: Identification of human germ cells by embryologists and reproductive physicians was not uniform or satisfactory. However, the diagram significantly improved identification such that it may be useful as an efficient checklist for the identification of germ cells.

Correlation between overactive bladder symptoms and quality of life in Japanese male patients: focus on nocturia.

OBJECTIVE: To elucidate the effects of nocturia, one of the most bothersome of symptoms, on health-related quality of life (QOL), we examined the correlation between nocturia-specific QOL and other lower urinary tract symptoms (LUTS). METHODS: Patients who visited our hospital complaining of LUTS were assessed retrospectively. A total of 259 men with LUTS answered the following questionnaires: International Prostate Symptom Score (IPSS), Overactive Bladder Symptom Score (OABSS), Nocturia QOL questionnaire (NQOL), and the Benign Prostatic Hyperplasia Impact Index (BII). The Spearman rank correlation coefficient was used to examine the correlation between NQOL total score and NQOL subdomain scores of sleep/energy and bother/concern and scores of other questionnaires. We then compared NQOL score in patients with or without OAB symptoms. RESULTS: The NQOL total score correlated significantly not only with IPSS total, IPSS storage symptoms, IPSS voiding symptoms, and QOL index but also with the OABSS and BII scores. The NQOL total score was significantly higher in the non-OAB vs OAB patients, indicating that OAB may deteriorate QOL as it relates to nocturia. In nocturia subgroups 0 to 2 (mild nocturia), NQOL score was significantly higher in non-OAB than in OAB patients, whereas in the nocturia subgroups 3 to 5 (severe nocturia), NQOL score was not significantly different between non-OAB and OAB patients. CONCLUSION: The NQOL total score correlated significantly with IPSS, OABSS, and BII scores. Symptoms of OAB and bother due to benign prostatic hyperplasia might affect QOL in patients with nocturia.

Low serum free testosterone level is associated with carotid intima-media thickness in middle-aged Japanese men.

In the present study, we measured carotid artery intima-media thickness (CIMT) and assessed several metabolic factors in middle-aged healthy Japanese men to clarify the relation between testosterone and atherosclerosis. The study comprised 176 male employees aged ≥40 years who visited Osaka University Healthcare Center for their annual health examinations. Serum total testosterone (TT) concentration was measured using radioimmunoassay (RIA) and serum free testosterone concentration was measured using analog ligand RIA (aFT). A multivariate model adjusted for age, body mass index, mean arterial pressure and treatment for hypertension demonstrated a significant association between aFT and CIMT. Even after adjustment for other clinically relevant factors, the significant association between aFT and CIMT was not attenuated. After adjustment for all other clinically relevant factors, both univariate and multivariate models ascertained the stepwise association that a level of aFT of ≤10.0 pg/mL was significantly associated with CIMT. However, the association between TT and CIMT was not significant in either univariate or multivariate models. We conclude that our finding showing that low serum aFT level is an influencing and independent risk factor for CIMT is of value in the clinical setting because no other studies, to our knowledge, have conducted multivariate analyses using the various metabolic factors included in the present analyses.

A single nucleotide polymorphism within the novel sex-linked testis-specific retrotransposed PGAM4 gene influences human male fertility.

BACKGROUND: The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes. METHODS AND FINDING: We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo. CONCLUSION: These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility.

Water avoidance stress induces frequency through cyclooxygenase-2 expression: a bladder rat model.

OBJECTIVES: Water avoidance stress is a potent psychological stressor and it is associated with visceral hyperalgesia, which shows degeneration of the urothelial layer mimicking interstitial cystitis. Cyclooxygenase-2 inhibitors have been recognized to ameliorate frequency both in clinical and experimental settings. We investigated the voiding pattern and cyclooxygenase-2 expression in a rat bladder model of water avoidance stress. METHODS: After being subjected to water avoidance stress or a sham procedure, rats underwent metabolic cage analysis and cystometrography. Real time reverse transcription polymerase chain reaction was carried out to examine cyclooxygenase-2 messenger ribonucleic acid in bladders of rats. Protein expression of cyclooxygenase-2 was analyzed with immunohistochemistry and western blotting. Furthermore, the effects of the cyclooxygenase-2 inhibitor, etodolac, were investigated by carrying out cystometrography, immunohistochemistry and western blotting. RESULTS: Metabolic cage analysis and cystometrography showed significantly shorter intervals and less volume of voiding in water avoidance stress rats. Significantly higher expression of cyclooxygenase-2 messenger ribonucleic acid was verified by reverse transcription polymerase chain reaction. Immunohistochemistry and western blotting showed significantly higher cyclooxygenase-2 protein levels in water avoidance stress bladders. Furthermore, immunohistochemistry showed high cyclooxygenase-2 expression exclusively in smooth muscle cells. All water avoidance stress-induced changes were reduced by cyclooxygenase-2 inhibitor pretreatment. CONCLUSIONS: Chronic stress might cause frequency through cyclooxygenase-2 gene upregulation in bladder smooth muscle cells. Further study of cyclooxygenase-2 in the water avoidance stress bladder might provide novel therapeutic modalities for interstitial cystitis.