The Journal of Urology
Volume 181, Issue 3 , Pages 940-944, March 2009

This Month in Investigative Urology

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Angiogenic Factors for Early Detection of Bladder Cancer 

Many angiogenic factors have been identified and shown to be produced by various cancer cells including bladder cancer. Among these factors are vascular endothelial growth factor, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and angiogenin. Eissa et al (page 1353) from Cairo, Egypt adapted and evaluated the measurement of these factors using enzyme-linked immunosorbent assay (ELISA), and compared the results with Western blot and voided urine cytology. The study included 240 patients diagnosed with bladder carcinoma, 108 with benign bladder lesions and 110 healthy individuals who served as the control group. All participants underwent serological schistosomiasis antibody assay in serum, urine cytology and estimation of angiogenic factors in voided urine. The intra-assay and interassay coefficients of variation of the investigated markers ranged from 10.3 to 12.3 and 10 to 13.7, respectively. The concordance rate with Western blot was 97.5%. The levels and positive rates of urinary angiogenic markers and urine cytology were significantly higher in the malignant group than in the benign and healthy groups. bFGF increased significantly in bladder squamous cell carcinoma cases. Moreover, bFGF and HGF were significantly correlated with tumor grade. The markers showed a significant association with clinical stage. The authors concluded that quantitative measurement of urinary angiogenic factors in voided urine samples by ELISA was reliable. The sensitivity of bFGF and HGF was the highest among the investigated markers and cytology in low grade and early stage cases, suggesting their convenience as sensitive, noninvasive diagnostic and screening tools for bladder cancer.

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Erythropoietin Attenuates Renal Injury 

In addition to its hematopoietic effects, erythropoietin (EPO) has multiple paracrine and autocrine functions that coordinate local responses to injury by attenuating primary (apoptosis) and secondary (inflammatory) causes of cell death. EPO was recently shown to exert important cytoprotective and anti-apoptotic effects in injury models of the brain, heart and kidney. Chang et al (page 1434) from Daejeon, Republic of Korea examined whether EPO also attenuates renal injury in a rat model of unilateral ureteral obstruction (UUO). Sprague-Dawley rats were divided into 4 groups of vehicle treated with sham operation, vehicle treated with UUO for 3 days, EPO treatment with sham operation and EPO treatment for UUO for 3 days. EPO treatment was given intraperitoneally and administered daily. Competitive reverse transcriptase-polymerase chain reaction, Western blot, hematoxylin and eosin staining and immunohistochemistry data were compared.

The authors found that transforming growth factor-β, tumor necrosis factor-α, monocyte chemoattractant protein-1, osteopontin and Fas mRNA levels in the EPO treated UUO group were significantly lower than in the UUO group. The Bcl-2 mRNA level in the EPO treated UUO group was significantly higher than in the UUO group. The activity of caspase-3 in the EPO-UUO group was significantly lower than in the UUO group. Interstitially infiltrated inflammatory cells were significantly decreased in the EPO treated UUO group vs the UUO group, and the EPO treated UUO group showed significantly fewer immunoreactions for transforming growth factor-β, endothelin-1 and caspase-3 compared to the UUO group. EPO treatment in rats with UUO significantly decreased the number of TUNEL positive cells. Chang et al concluded that EPO exerts renoprotective effects in an experimental UUO rat model via anti-apoptotic and anti-inflammatory actions.

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Receptor Isoforms That Mediate Estrogen and Progesterone Action 

Estrogen and progesterone action is mediated by high affinity intracellular receptors. The main estrogen receptor (ER) isoforms studied have been ERα and ERβ, and the main progesterone receptor (PR) isoforms studied have been PR-A and PR-B. Bladder symptoms can be ameliorated by sex steroids but the mechanism of action is unknown. Previous studies of steroid receptor expression in the bladder did not indicate receptor subtype expression. Tincello et al (page 1474) from Leicester, United Kingdom report on the distribution of ER and PR isoforms in the female lower urinary tract. Prospectively recruited women undergoing routine urogynecological or gynecological surgery provided cold cup biopsy samples from the bladder dome, trigone, and proximal and distal urethra. The samples were immediately frozen or fixed in formalin. Following RNA extraction transcripts for ERα, ERβ, PR-A and PR-B were noted on reverse transcriptase-polymerase chain reaction using isoform specific primers. The precise cellular localization of receptor proteins and their relative levels were assessed by immunochemistry in formalin fixed tissue sections with isoform specific antibodies. ERα, ERβ, PR-A and PR-B transcripts were detected in whole bladder extracts. Nuclear ERα immunoreactivity was present in squamous epithelium but absent from transitional epithelium. ERβ immunoreactivity was expressed in squamous and transitional cell epithelia. Nuclear PR expression was present in urethral squamous epithelium only. Thus, ERα and ERβ genes are transcribed in bladder tissue but only ERβ is translated into protein, suggesting that the urothelium responds to endogenous estrogen via ERβ. PR expression is confined to urethral squamous epithelium and the major isoform is PR-A.

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Urodynamic Measurements by Radiotelemetry in Freely Moving Dogs 

Preclinical cystometric measurements are usually performed in anesthetized or conscious animals implanted with a bladder catheter to fill the bladder while measuring intravesical pressure. It is well-known that anesthesia alters the urodynamics, and in conscious animals restraint is often necessary which evokes stress and limits the period of continuous urodynamic assessment. A more physiological and chronic method of evaluating pharmacological responses on urodynamics is described by McCafferty et al (page 1444) from King of Prussia, Pennsylvania. In adult female beagle dogs radiotelemetry transmitters enabling urodynamic/hemodynamic recordings were implanted into the bladder. Telemetered urodynamics were compared to those measured in anesthetized dogs receiving bladder infusion of saline. The responses to diuresis with furosemide and the M3 selective antimuscarinic darifenacin were evaluated.

The evaluations showed that saline infused, anesthetized dogs had a lower peak micturition pressure and higher threshold pressure than conscious, freely moving telemetered dogs. In telemetered dogs a single dose of furosemide increased the voiding frequency and average volume per void. Darifenacin decreased peak voiding pressure without affecting voiding frequency. The authors concluded that telemetry enables the continuous monitoring of urodynamics under more physiological conditions (conscious animals, normal filling rates, no restraint). This new model facilitates the evaluation of the chronic efficacy of new urological therapies and provides the potential to significantly decrease animal use.

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Mucosal Muscarinic Receptors Enhance Bladder Activity in Feline Interstitial Cystitis 

The origin and mechanisms involved in the pathophysiology of interstitial cystitis (IC) remain unclear. Research has been hampered by the limited number of animal models that mimic the idiopathic nature of the disease. However, domestic cats have been shown to have a syndrome called feline IC (FIC) which shares many characteristics seen in humans with IC. Ikeda et al (page 1415) from Columbus, Ohio examined Ca2+ transients in cross-sections of full-thickness bladder strips from normal cats and cats with FIC using optical mapping of Ca2+ transients and recording tension. They also compared responses of Ca2+ activity and detrusor contractions to pharmacological interventions, and the pharmacological responses were compared in mucosa denuded preparations. Optical mapping revealed that FIC bladders had significantly more spontaneous Ca2+ transients in the mucosal layer than control bladders. Optical mapping also demonstrated that FIC bladders were hypersensitive to muscarinic receptor stimulation when the mucosal layer was intact. The hypersensitivity was markedly decreased in mucosa denuded bladder strips. Thus, in FIC bladders there is increased Ca2+ activity and sensitivity of muscarinic receptors in the mucosal layer. Such changes may contribute to the symptoms of FIC.

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Spontaneous Contractions Evoke Afferent Nerve Firing in Bladders With Detrusor Overactivity 

Spontaneous contractions of detrusor smooth muscle may generate afferent nerve activity which can contribute to several lower urinary tract symptoms including urgency, frequency and incontinence. McCarthy et al (page 1459) from Pittsburgh, Pennsylvania investigated the correlation between afferent nerve firing and spontaneous bladder contractions in spinal cord transected (SCT) mice using optical mapping, single unit recording and tension measurements. Bladder nerve preparations (bladder sheets and the associated L6-S2 pelvic nerves) were dissected from normal and SCT mice exhibiting overactivity (determined by cystometry). Bladder sheets were mounted horizontally in a temperature regulated chamber to simultaneously record Ca2+ transients across the mucosal surface, single unit afferent nerve firing and whole bladder tension. Single unit afferent fibers showed a graded response to von Frey stimulation and a frequency of afferent firing that increased as a function of the degree of stretch. Optical maps of Ca2+ transients in control bladders exhibited multiple initiation sites that resulted in high frequency, low amplitude spontaneous contractions. In maps of the bladders of SCT mice Ca2+ transients arose from 1 or 2 focal sites, resulting in low frequency, high amplitude contractions and concomitant afferent firing. The authors concluded that large amplitude, spontaneous bladder contractions evoke afferent nerve activity, which may contribute to incontinence and other lower urinary tract symptoms.

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S1P/S1P2 Signaling Induces Cyclooxygenase-2 Expression in Wilms Tumor 

Cyclooxygenase (COX), the key enzyme that converts arachidonic acid into prostaglandins, occurs in 2 isoforms. COX-1 is constitutively expressed and COX-2 is induced by various factors. Interestingly COX-2 has been found to be over expressed in various human cancers and may have a crucial role in carcinogenesis. COX-2 has been reported to be ubiquitously expressed in Wilms tumor, the most common malignant renal tumor in children. The regulation mechanism of COX-2 expression remains unexplored. However, S1P has been shown to induce its expression in some cell types. Li et al (page 1347) from Nashville, Tennessee performed quantitative real-time polymerase chain reaction and Western blot to detect COX-2 mRNA and protein expression in WiT49 cells on stimulation by S1P, as well as S1P2 and COX-2 mRNA expression in 10 freshly frozen Wilms tumor tissues and matched normal tissues. S1P induced COX-2 mRNA and protein expression in WiT49 cells in a concentration dependent manner. Over expression of S1P2 in WiT49 cells led to a significant increase in COX-2 mRNA and protein expression as well as subsequent prostaglandin E2 synthesis. In 10 Wilms tumor specimens S1P2 mRNA was greatly increased. The authors concluded that S1P induced COX-2 expression in Wilms tumor, and that this effect was mediated by S1P2. This finding extends the biological function of S1P2 and provides the biochemical basis for the development of inhibitors targeting the S1P/COX-2 signaling pathway.

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Protecting the Rectum During Prostate Cancer Irradiation 

Radiotherapy strategies for the curative management of prostate cancer (CaP) have become more common in the last decade. The primary complication of external beam radiotherapy is related to rectal damage. The risks are accentuated by the treatment methods of intensity modulation and hypofractionation, and there is a need for improved protection of the rectum. Ben-Yosef et al (page 1401) from Tel Aviv, Israel describe an implantable, biodegradable, inflatable preshaped triangular balloon made of commercially used poly(L-lactide-co-ε-caprolactone) co-polymer developed to provide separation between the prostate and the rectum. In several in vivo studies the authors evaluated the biocompatibility and degradability of the balloon implanted subcutaneously or perineally, and in the context of transperineal implantation and local irradiation.

The device was biocompatible in subcutaneously implanted rabbits (up to 42 days), in a transperineally implanted dog (up to 12 months) and in 8 transperineally implanted pigs (up to 6 months). Upon inflation in situ the balloon separated the tissues, remained inflated for several months and subsequently biodegraded. No systemic or local toxicity was noted on histopathological evaluation. Three-month followup in irradiated pigs (that received 15 Gy in 3 fractions 1 week apart) showed a stable balloon position with no local or systemic side effects. This novel device was safe and effective for its intended use of separating tissues for a desired duration. A clinical study will commence to evaluate the safety and efficacy of this device in the irradiation of prostate cancer.

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α2-Adrenoceptors and Glutamate Mechanisms in the External Urethral Sphincter Continence Reflex 

There are few drugs available to treat stress urinary incontinence, mainly due to insufficient knowledge about possible targets for pharmacological control of urethral closure mechanisms under stress conditions. Furuta et al (page 1467) from Tokyo, Japan investigated the role of α2-adrenoceptors (ARs) and glutamate mechanisms of the urethral continence reflex in response to abdominal pressure increases. In urethane anesthetized spinal cord transected (T8-9) female rats external urethral sphincter (EUS) electromyogram activity was evaluated during lower abdominal wall compression before and after intravenous application of test drugs. The effects of MK-801, an N-methyl-D-aspartate glutamate receptor antagonist, or medetomidine, an α2-AR agonist, on EUS activity were examined. In addition, idazoxan, an α2-AR antagonist, was administered before or after the application of MK-801. Idazoxan was also administered after the application of duloxetine, a serotonin/norepinephrine reuptake inhibitor. MK-801 and medetomidine dose dependently decreased EUS activity whereas idazoxan significantly increased EUS activity by 64%. The increase in EUS activity after idazoxan was abolished by MK-801 but idazoxan did not reverse the inhibitory effects of MK-801. In addition, idazoxan significantly potentiated the duloxetine effects on EUS activity by 120%. These findings indicate that glutamate is a major excitatory neurotransmitter in the urethral continence reflex response to abdominal pressure increases, and that α2-AR activation suppresses EUS activity, probably via presynaptic inhibition of glutamate release. Therefore, α2-AR antagonists could be beneficial for the treatment of stress urinary incontinence, alone or in combination with serotonin/norepinephrine reuptake inhibitors.

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Seminal Vesicle Remnant After Proximal Transection or Ligation 

Seminal vesiculectomy has traditionally been part of the radical prostatectomy procedure. However, dissection of the seminal vesicles (SVs) during radical prostatectomy has the potential to damage the pelvic plexus, thus compromising trigonal, bladder neck and cavernous innervation, and contributing to delayed gain of continence and erectile function. The rate of prostate cancer invasion into the SVs in currently operated patients is low and in most can be predicted preoperatively. This situation calls for SV sparing radical prostatectomy in select patients, leaving a distal remnant of the SVs in place. Rosenberg et al (page 1483) from Chicago, Illinois investigated the fate of the SV remnant after proximal transection or ligation in a rat model. The right SV was divided by suture ligation or by transection, and the left SV served as a control. The morphology of the SVs was evaluated macroscopically and microscopically 1, 2 and 4 weeks after division. Transected SVs were similar in weight and morphology to control contralateral glands. One week after SV ligation the remnants became significantly heavier and showed balloon dilatation of the hollow spaces, while the lining epithelium became significantly flattened. Two and 4 weeks after ligation half of the animals showed gland shrinkage and half demonstrated persistent dilatation. Thus, transection of the SV preserves the gland remnant in a relatively normal morphology while ligation leads to severe and inconsistent morphological changes. The authors recommend that when considering SV sparing radical prostatectomy, SV transection may be preferred to ligation.

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Urokinase-Type Plasminogen Activator Receptor and Epidermal Growth Factor Receptor in Patients With Prostate Cancer 

The prognosis of prostate cancer, the most frequently diagnosed malignant tumor in males in the Western population, is closely related to the local and distant spread of the cancer at the moment of diagnosis. Prostate cancer cell motility and invasion have been linked to up-regulated signaling of epidermal growth factor receptor (EGFR) and urokinase-type plasminogen activator receptor (UPAR). Milanese et al (page 1393) from Milan, Italy analyzed the expression of UPAR and EGFR in the serum of patients with clinical suspicion of CaP to evaluate their possible role as CaP markers. Serum from 79 consecutive patients referred for transrectal ultrasound guided prostate biopsy was collected and blood samples were obtained before prostate biopsy. Total UPAR and EGFR antigen in serum were measured by specific ELISAs. Patients with prostate adenocarcinoma underwent radical retropubic prostatectomy. The patients with CaP (30) had significantly higher levels of serum UPAR and EGFR than those without prostate cancer, but not higher levels of prostate specific antigen. UPAR and EGFR levels were closely correlated in the serum of patients with CaP. In a multivariate model high serum EGFR increased the probability of positive biopsy by 1.9 times. ROC analysis revealed that serum EGFR had a sensitivity of 93.3% and a specificity of 98% in the detection of CaP (cutoff 67.9 ng/ml). Thus, the measurement of UPAR and EGFR in the serum of patients with clinical suspicion of CaP might provide clinically relevant information on the state of the prostate gland. Measuring serum EGFR could help predict which patients have CaP while serum UPAR over expression seems to be related to tumor extraprostatic spread.

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Effect of 1,25-Dihydroxyvitamin D on Testicular Morphology and Gene Expression in Cryptorchidism 

1,25-dihydroxyvitamin D is a physiologically active metabolite of vitamin D, and there is accumulating evidence that vitamin D and its receptor, VDR, are important in reproductive tissues. Hirai et al (page 1487) from Osaka, Japan evaluated the morphological effect and alterations in gene expression caused by 1,25-dihydroxyvitamin D treatment in the mouse testis undergoing experimental cryptorchidism and subsequent orchiopexy. Mean modified Johnsen score and testicular weight were estimated after 4 weeks of treatment with a 1,25-dihydroxyvitamin D prodrug. Sites of vitamin D receptor and mRNA expression, and 1,25 dihydroxyvitamin D analogue accumulation were examined. Alterations in gene expression in cryptorchid mouse testis with or without 1,25-dihydroxyvitamin D administration were compared by testis specific cDNA microarray. Johnsen score and testicular weight increased nonsignificantly with 1,25-dihydroxyvitamin D treatment. VDR and its mRNA were positive in Sertoli's cells. The 1,25-dihydroxyvitamin D analogue accumulated mainly in Sertoli's cells. Of 2,483 testis specific genes 19 showed up-regulation by 1,25-dihydroxyvitamin D treatment. Of these genes adenosine triphosphate-binding cassette transporter 1 (Abca1), a regulator of cellular cholesterol homeostasis, was expressed mainly in Sertoli's cells. Synthesis of Abca1 protein increased with 1,25-dihydroxyvitamin D treatment in primary cultures of Sertoli's cells but not with follicle-stimulating hormone or testosterone treatment. Thus, 1,25-dihydroxyvitamin D contributes to spermatogenesis by up-regulating certain specific genes in Sertoli's cells. Testis specific cDNA microarray analysis and vitamin D supplementation may have implications in the management of male infertility.

PII: S0022-5347(08)03345-4

doi:10.1016/j.juro.2008.12.023

The Journal of Urology
Volume 181, Issue 3 , Pages 940-944, March 2009

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