Altered detrusor contractility and voiding patterns in mice lacking the mechanosensitive TREK-1 channel.

BACKGROUND: Previously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice. METHODS: Adult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals. RESULTS: TREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice. CONCLUSIONS: Our results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the "mixed" voiding phenotype in TREK-1 KO mice.

The Expression of Transcription Factors Mecp2 and CREB Is Modulated in Inflammatory Pelvic Pain.

Early activation of transcription factors is one of the epigenetic mechanisms contributing to the induction and maintenance of chronic pain states. Previous studies identified the changes in a number of nociception-related genes, such as calcitonin gene-related peptide (CGRP), substance P (SP), and brain-derived neurotropic factor (BDNF) in the pelvic organs after transient colonic inflammation. The gene and protein expression of these neuropeptides could be modulated by transcription factors Methyl-CpG-binding protein 2 (Mecp2) and cAMP response element-binding protein (CREB). In this study, we aimed to evaluate time-dependent changes in the expression levels of Mecp2 and CREB in the lumbosacral (LS) spinal cord and sensory ganglia after inflammation-induced pelvic pain in rat. Adult Sprague-Dawley rats were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce transient colonic inflammation. LS (L6-S2) spinal cord segments and respective dorsal root ganglias (DRGs) were isolated from control and experimental animals at 1, 2, 6, 24 h and 3 days post-TNBS treatment. Immunohistochemical (IHC) labeling and Western blotting experiments were performed to assess the expression of Mecp2, CREB and their phosphorylated forms. Total Mecp2 expression, but not phosphorylated p-Mecp2 (pS421Mecp2) expression was detected in the cells of the spinal dorsal horn under control conditions. Colonic inflammation triggered a significant decrease in the number of Mecp2-expressing neurons in parallel with elevated numbers of pS421Mecp2-expressing cells at 2 h and 6 h post-TNBS. The majority of Mecp2-positive cells (80 ± 6%) co-expressed CREB. TNBS treatment caused a transient up-regulation of CREB-expressing cells at 1 h post-TNBS only. The number of cells expressing phosphorylated CREB (pS133CREB) did not change at 1 h and 2 h post-TNBS, but was down-regulated by three folds at 6 h post-TNBS. Analysis of DRG sections revealed that the number of Mecp2-positive neurons was up-regulated by TNBS treatment, reaching three-fold increase at 2 h post-TNBS, and eight-fold increase at 6 h post-TNBS (p ≤ 0.05 to control). These data showed early changes in Mecp2 and CREB expression in the dorsal horn of the spinal cord and sensory ganglia after colonic inflammation, suggesting a possible contribution Mecp2 and CREB signaling in the development of visceral hyperalgesia and pelvic pain following peripheral inflammation.

Transurethral Instillation Procedure in Adult Male Mouse.

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Impact of Regular Cannabis Use on Biomarkers of Lower Urinary Tract Function.

OBJECTIVE: To evaluate the differences in the composition and quantities of urine peptides in regular cannabis users and nonusers by liquid chromatography tandem mass spectrometry analysis. MATERIALS AND METHODS: Urine specimens from healthy control subjects and cannabis users were utilized to identify the differences in the number and quantity of urine proteins by liquid chromatography tandem mass spectrometry analysis. Significantly altered proteins were determined by a permutation testing statistical method. Heat map, dendrogram, pathway, and network analyses were performed to assess the degree of expression and the potential relationships between proteins in both groups. RESULTS: A total of 1337 proteins were detected in both groups with 19 proteins being significantly altered in cannabis users. Innate immunity and carbohydrate metabolic pathways were highly linked with upregulated proteins in the cannabis group. Additionally, 91 proteins were present and 46 proteins were absent only in cannabis users in comparison with the control cohort. Our results suggest that regular use of cannabis is associated with significant alterations in a number of urinary peptides, with a large number of proteins present or absent only in cannabis users. Pathway analyses demonstrated an increased immune response in cannabis users compared with controls. CONCLUSION: Our observations potentially indicate activation (or inhibition) of specific signaling pathways in the lower urinary tract during chronic exposure to exogenous cannabinoids. Our study provides initial proteomic knowledge for future investigations on the potential role of exocannabinoids in the development of intravesical therapies to treat lower urinary tract disorders.

Altered expression and modulation of the two-pore-domain (K2P) mechanogated potassium channel TREK-1 in overactive human detrusor.

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.

Fertility Preservation Options for Men and Women With Cancer.

Approximately 0.2% of Americans aged 20 to 39 years are childhood cancer survivors. Advances in cancer detection and therapy have greatly improved survival rates for young cancer patients; however, treatment of childhood cancers can adversely impact reproductive function. Many cancer patients report a strong desire to be informed of existing options for fertility preservation and future reproduction prior to initiation of gonadotoxic cancer therapies, including surgery, chemotherapy, and radiotherapy. This article discusses, in detail, the effects of cancer treatment on fertility in men and women, and outlines both current and experimental methods of fertility preservation among cancer patients.

Clomiphene citrate in the management of male infertility.

Clomiphene citrate, a selective estrogen receptor modulator, has been used to treat infertility in women and men for 50 years. In men, clomiphene citrate has been employed in the management of unexplained infertility, oligo and asthenospermia, hypogonadism, and nonobstructive azoospermia. The available evidence reveals mixed results and suggests that clomiphene citrate may be appropriate for the management of male infertility in specific clinical scenarios. Further research is needed to clarify when clomiphene citrate is indicated in the treatment of male infertility.

Effects of testosterone and progressive resistance exercise in healthy, highly functioning older men with low-normal testosterone levels.

CONTEXT: Aging in men is associated with reduced testosterone (T) levels and physiological changes leading to frailty, but the benefits of T supplementation are inconclusive. OBJECTIVE: We studied the effects of T supplementation with and without progressive resistance training (PRT) on functional performance, strength, and body composition. DESIGN, SETTING, AND PARTICIPANTS: We recruited 167 generally healthy community-dwelling older men (66 ± 5 years) with low-normal baseline total T levels (200-350 ng/dL). INTERVENTION: Subjects were randomized to placebo or transdermal T gel [2 doses targeting either a lower (400-550 ng/dL) or higher (600-1000 ng/dL) T range] and to either PRT or no exercise for 12 months. MAIN OUTCOME MEASURE: The primary outcome was functional performance, whereas secondary outcomes were strength and body composition. RESULTS: A total of 143 men completed the study. At 12 months, total T was 528 ± 287 ng/dL in subjects receiving any T and 287 ± 65 ng/dL in the placebo group. In the PRT group, function and strength were not different between T- and placebo-treated subjects, despite greater improvements in fat mass (P = .04) and fat-free mass (P = .01) with T. In the non-PRT group, T did not improve function but improved fat mass (P = .005), fat-free mass (P = .03), and upper body strength (P = .03) compared with placebo. There were fewer cardiovascular events in the T-treated groups compared with placebo. CONCLUSIONS: T supplementation was well tolerated and improved body composition but had no effect on functional performance. T supplementation improved upper body strength only in nonexercisers compared with placebo.

The use of in vitro fertilization in the management of male infertility: what the urologist needs to know.

Infertility affects approximately 10% to 20% of reproductive-age couples, many of whom may present initially to a urologist. Some couples may be treated medically to increase spontaneous conception rates; however, many will require more aggressive management with in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). IVF involves ovarian stimulation, oocyte retrieval, and fertilization outside of the body; ICSI involves injecting one sperm into the oocyte to promote fertilization. Here we provide a brief overview of IVF and ICSI along with a discussion of the risks involved to facilitate the counseling and care of the infertile couple.

Genome wide analysis of differentially expressed genes in HK-2 cells, a line of human kidney epithelial cells in response to oxalate.

Nephrolithiasis is a multi-factorial disease which, in the majority of cases, involves the renal deposition of calcium oxalate. Oxalate is a metabolic end product excreted primarily by the kidney. Previous studies have shown that elevated levels of oxalate are detrimental to the renal epithelial cells; however, oxalate renal epithelial cell interactions are not completely understood. In this study, we utilized an unbiased approach of gene expression profiling using Affymetrix HG_U133_plus2 gene chips to understand the global gene expression changes in human renal epithelial cells [HK-2] after exposure to oxalate. We analyzed the expression of 47,000 transcripts and variants, including 38,500 well characterized human genes, in the HK2 cells after 4 hours and 24 hours of oxalate exposure. Gene expression was compared among replicates as per the Affymetrix statistical program. Gene expression among various groups was compared using various analytical tools, and differentially expressed genes were classified according to the Gene Ontology Functional Category. The results from this study show that oxalate exposure induces significant expression changes in many genes. We show for the first time that oxalate exposure induces as well as shuts off genes differentially. We found 750 up-regulated and 2276 down-regulated genes which have not been reported before. Our results also show that renal cells exposed to oxalate results in the regulation of genes that are associated with specific molecular function, biological processes, and other cellular components. In addition we have identified a set of 20 genes that is differentially regulated by oxalate irrespective of duration of exposure and may be useful in monitoring oxalate nephrotoxicity. Taken together our studies profile global gene expression changes and provide a unique insight into oxalate renal cell interactions and oxalate nephrotoxicity.

The antioxidant, MnTE-2-PyP, prevents side-effects incurred by prostate cancer irradiation.

Prostate cancer is the most commonly diagnosed cancer, with an estimated 240,000 new cases reported annually in the United States. Due to early detection and advances in therapies, more than 90% of patients will survive 10 years post diagnosis and treatment. Radiation is a treatment option often used to treat localized disease; however, while radiation is very effective at killing tumor cells, normal tissues are damaged as well. Potential side-effects due to prostate cancer-related radiation therapy include bowel inflammation, erectile dysfunction, urethral stricture, rectal bleeding and incontinence. Currently, radiation therapy for prostate cancer does not include the administration of therapeutic agents to reduce these side effects and protect normal tissues from radiation-induced damage. In the current study, we show that the small molecular weight antioxidant, MnTE-2-PyP, protects normal tissues from radiation-induced damage in the lower abdomen in rats. Specifically, MnTE-2-PyP protected skin, prostate, and testes from radiation-induced damage. MnTE-2-PyP also protected from erectile dysfunction, a persistent problem regardless of the type of radiation techniques used because the penile neurovascular bundles lay in the peripheral zones of the prostate, where most prostate cancers reside. Based on previous studies showing that MnTE-2-PyP, in combination with radiation, further reduces subcutaneous tumor growth, we believe that MnTE-2-PyP represents an excellent radioprotectant in combination radiotherapy for cancer in general and specifically for prostate cancer.

Kidney injury molecule-1 is up-regulated in renal epithelial cells in response to oxalate in vitro and in renal tissues in response to hyperoxaluria in vivo.

Oxalate is a metabolic end product excreted by the kidney. Mild increases in urinary oxalate are most commonly associated with Nephrolithiasis. Chronically high levels of urinary oxalate, as seen in patients with primary hyperoxaluria, are driving factor for recurrent renal stones, and ultimately lead to renal failure, calcification of soft tissue and premature death. In previous studies others and we have demonstrated that high levels of oxalate promote injury of renal epithelial cells. However, methods to monitor oxalate induced renal injury are limited. In the present study we evaluated changes in expression of Kidney Injury Molecule-1 (KIM-1) in response to oxalate in human renal cells (HK2 cells) in culture and in renal tissue and urine samples in hyperoxaluric animals which mimic in vitro and in vivo models of hyper-oxaluria. Results presented, herein demonstrate that oxalate exposure resulted in increased expression of KIM-1 m RNA as well as protein in HK2 cells. These effects were rapid and concentration dependent. Using in vivo models of hyperoxaluria we observed elevated expression of KIM-1 in renal tissues of hyperoxaluric rats as compared to normal controls. The increase in KIM-1 was both at protein and mRNA level, suggesting transcriptional activation of KIM-1 in response to oxalate exposure. Interestingly, in addition to increased KIM-1 expression, we observed increased levels of the ectodomain of KIM-1 in urine collected from hyperoxaluric rats. To the best of our knowledge our studies are the first direct demonstration of regulation of KIM-1 in response to oxalate exposure in renal epithelial cells in vitro and in vivo. Our results suggest that detection of KIM-1 over-expression and measurement of the ectodomain of KIM-1 in urine may hold promise as a marker to monitor oxalate nephrotoxicity in hyperoxaluria.

The transcription factor SPDEF suppresses prostate tumor metastasis.

Emerging evidence suggests that the SAM pointed domain containing ETS transcription factor (SPDEF) plays a significant role in tumorigenesis in prostate, breast, colon, and ovarian cancer. However, there are no in vivo studies with respect to the role of SPDEF in tumor metastasis. The present study examined the effects of SPDEF on tumor cell metastasis using prostate tumor cells as a model. Utilizing two experimental metastasis models, we demonstrate that SPDEF inhibits cell migration and invasion in vitro and acts a tumor metastasis suppressor in vivo. Using stable expression of SPDEF in PC3-Luc cells and shRNA-mediated knockdown of SPDEF in LNCaP-Luc cells, we demonstrate for the first time that SPDEF diminished the ability of disseminated tumors cells to survive at secondary sites and establish micrometastases. These effects on tumor metastasis were not a result of the effect of SPDEF on cell growth as SPDEF expression had no effect on cell growth in vitro or subcutaneous tumor xenograft-growth in vivo. Transcriptional analysis of several genes associated with tumor metastasis, invasion, and the epithelial-mesenchymal transition demonstrated that SPDEF expression selectively down-regulated MMP9 and MMP13 in prostate cancer cells. Further analysis indicated that forced MMP9 or MMP13 expression rescued the invasive phenotype in SPDEF expressing PC3 cells in vitro, suggesting that the effects of SPDEF on tumor invasion are mediated, in part, through the suppression of MMP9 and MMP13 expression. These results demonstrate for the first time, in any system, that SPDEF functions as a tumor metastasis suppressor in vivo.

The computerized rounding report: implementation of a model system to support transitions of care.

OBJECTIVES: In response to ACGME work-hour restrictions, residency programs that require continuous inpatient clinical care for educational objectives will be forced to increase the proportion of junior resident experience involved in shift work. Maintaining the balance of education over service at these levels will be a challenge, where a considerable amount of time must be spent gathering data for morning rounds and signing out patients at shift change. Patient safety is an issue with this new paradigm. We hypothesized that computerized sign-out would improve resident efficiency. MATERIALS AND METHODS: A multidisciplinary clinical team collaborated to design a computerized rounding and sign-out (CSO) program to automate collection of clinical information in addition to a brief narrative describing ongoing care issues. Residents returned a self-administered questionnaire before (n = 168) and after implementation (n = 83) examining: pre-rounding time, missed patients, handoff quality, and duty hours. RESULTS: Residents reported spending 11 fewer min/d pre-rounding (P = 0.006). After implementation, residents missed fewer patients on rounds (P = 0.01). A majority (70%) of responders stated that the new program helped them with duty hours. CONCLUSION: The current study demonstrates the reproducibility of the University of Washington model system for rounding and sign-out at an independent site, using basic infrastructure and leadership common to all residency programs. Developing a CSO was associated with a modest reduction in pre-rounding time and fewer patients missed on rounds. Although automating resident tasks may improve workflow in an increasingly complex hospital environment, structured handoff education and other institutional changes are necessary.

Molecular aspects of renal cell carcinoma: a review.

Renal cell carcinoma (RCC) is a disease in which cancer cells form in the tubules of the kidney. RCC, the incidence of which is increasing annually, represents five percent of adult epithelial cancers. Clear cell carcinoma represents the most frequent histological subtype. RCC is characterized by a lack of early warning signs, diverse clinical manifestations. Incidentally detected tumors in asymptomatic individuals have been steadily increasing owing to the increased usage of various imaging technologies. Currently there are no recommendations for screening to detect and make an early diagnosis of renal cancer. But in recent years, the discovery of new molecular and cytogenetic markers has led to the recognition and classification of several novel subtypes of RCC, and the introduction of molecular-targeted therapy for advanced-stage RCC. We performed a literature review using PubMed and discuss current knowledge of epidemiology, pathophysiology, evaluation, treatment, and future research directions of RCC.

Potentiation of mitomycin C tumoricidal activity for transitional cell carcinoma by histone deacetylase inhibitors in vitro.

PURPOSE: Histone deacetylase inhibitors represent promising cancer treatments since they offer improved access to target DNA/protein complexes by cytotoxic agents. We hypothesized that histone deacetylase inhibitors would be most effective when combined with DNA damaging agents such as mitomycin C. Valproic acid is a safe, affordable histone deacetylase inhibitor. We examined the effect of the combination of valproic acid and mitomycin C on human bladder cancer cells in vitro and compared this to the effect of valproic acid or mitomycin C alone on the cells. MATERIALS AND METHODS: We used HTB5 and HTB9 cells derived from low and high grade bladder tumors, respectively. HTB5 and HTB9 cells were grown in modified Eagle's and RPMI medium, respectively. Cell growth and proliferation were measured by standard methods. Apoptosis was evaluated microscopically after dual staining of cells with annexin V-fluorescein isothiocyanate/propidium iodide. The change in protein expression was analyzed by Western blot. RESULTS: Treatment of HTB5 and HTB9 bladder cancer cells for 24 to 72 hours with valproic acid and mitomycin C resulted in concentration and time dependent decreases in viability and proliferation. HTB9 cells showed marked sensitivity to mitomycin C with a 48-hour 50% median inhibitory concentration of 1 µg. Cells were less sensitive to valproic acid alone with a 48-hour 50% median inhibitory concentration of 2.5 mM. The chromatin structure relaxation induced by valproic acid pretreatment sensitized the bladder cancer cell lines, augmenting the cytotoxic action of mitomycin C. Valproic acid potentiated the induction of cell death by mitomycin C in each cell line in synergistic fashion. The effect of combining the 2 drugs was greater than the sum effect of each drug alone. CONCLUSIONS: Results indicate that the valproic acid and mitomycin C combination is effective, likely due to synergistic mechanisms. Animal model validation is needed but early results suggest promising intravesical treatments for superficial bladder cancer.