- Email: [email protected]
This Month in Investigative Urology
This Month in Investigative Urology Identification of Tumor Modulators and Invasion Suppressor Genes in Bladder Cancer Epigenetic regulation of genes in carcinogenesis is well established and has been demonstrated to control the expression of oncogenic and tumor suppressor elements involved in different cancers. Epigenetic induced changes may involve the silencing of gene expression by methylation and acetylation/ deacetylation events, prompting efforts to restore normal gene expression profiles in cancer cells using demethylation agents and/or histone deacetylase inhibitors (HDACi), respectively. Gould et al (page 2395) from Massachusetts assessed the ability of different classes of histone deacetylase inhibitors to target tumor and invasive suppressor genes in a panel of bladder carcinoma cell lines using reverse phase protein arrays. They used 3 poorly, moderately and highly invasive cell lines which were exposed to HDACis, trichostatin A, valproic acid, apicidin and MS-275. Cells were exposed to the inhibitor for periods ranging between 0 and 36 hours. Lysates were harvested and arrayed in a 10-fold dilution series, in duplicate. Data points were collected and analyzed using a concentration interpolation methodology following normalization. Protein expression profiles revealed up-regulation of ␥-catenin in highly invasive lines, and ␣-catenin in moderate and highly invasive lines following exposure to all HDACis, apicidin and MS-275, respectively. Gelsolin was up-regulated in poorly and moderately invasive lines following exposure to all HDACis. Desmoglein was downregulated in poorly and moderately invasive cell lines with all 4 HDACis, and focal adhesion kinase expression was decreased in moderate and highly invasive lines exposed to valproic acid and MS275. The authors conclude that different classes of HDACis show the potential to modulate the expression of tumor and invasive suppressor genes, identifying HDACis as potential therapeutic agents in bladder cancer. The usefulness of reverse phase protein array in screening the action of different classes of HDACi in bladder model systems presents another avenue for the preclinical screening of targeted therapies. 0022-5347/10/1836-2115/0 THE JOURNAL OF UROLOGY® © 2010 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
AND
RESEARCH, INC.
Diet Sodas and Nephrolithiasis Hypocitraturia is a risk factor for nephrolithiasis. Dietary citrate and alkali intake may have an effect on citraturia, and increasing alkali intake also increases urine pH, which can help prevent uric acid stones. Eisner et al (page 2419) determined the citrate, malate and total alkali concentrations in commonly consumed diet sodas to help direct dietary recommendations for patients with hypocitraturic calcium or uric acid nephrolithaisis. They measured the citrate and malate concentrations in a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis and in 15 diet sodas. The anions were measured by ion chromatography. The pH of each beverage was measured to allow calculation of the unprotonated anion concentrations using the known pKs of citric and malic acid. Total alkali equivalents were calculated for each beverage. Statistical analysis was performed using Pearson’s correlation coefficient. Several sodas contained greater or equal amounts of citrate as alkali and total alkali than a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis. Colas had the lowest amounts of total alkali (less than 1.0 mEq/l). There was no significant correlation between beverage pH and total alkali content. The authors conclude that several commonly consumed diet sodas contain moderate amounts of citrate as alkali and total alkali, and that this information can be helpful for dietary recommendations for patients with calcium nephrolithiasis and specifically those with hypocitraturia. It may also be useful for patients with low urine pH and uric acid stones. The authors emphasize that beverage malate content is important, as malate ingestion will increase the total alkali delivered, which in turn augments citraturia and increases urine pH.
Bladder Regeneration by Collagen Scaffolds Various injuries may result in compromised bladder structure and compliance requiring reconstruction. Currently the major surgical solution is enterocystoplasty by vascularized bowel segments. However, multiple significant complications of this method greatly hamper its clinical application and, thus, it Vol. 183, 2115-2116, June 2010 Printed in U.S.A. DOI:10.1016/j.juro.2010.03.065
www.jurology.com
2115
2116
THIS MONTH IN INVESTIGATIVE UROLOGY
is important to improve the current method or find a better alternative for bladder regeneration. Chen et al (page 2432) from People’s Republic of China investigated a collagen based targeting system for bladder regeneration. A collagen binding domain (CBD) was added to the N-terminal of native basic fibroblast growth factor (bFGF) to allow it bind to collagen. Partial cystectomy was performed in Sprague Dawley rats and the collagen scaffolds loaded with CBD-bFGF, native bFGF or phosphate buffered saline were grafted to the remaining host bladders, respectively. At days 30 and 90 the reconstructed bladders were evaluated for histological analysis and urodynamic examination. This targeting bFGF delivery system induced satisfying histological structures of bladders, promoting more vascularization and ingrowth of smooth muscle cells. Urodynamic examinations revealed well accommodated bladder tissue with volume capacity and compliance. The authors conclude that this targeting delivery system induces better bladder regeneration at the injured site and, thus, could be an effective strategy for bladder regeneration with potential clinical applications.
Urinary Nerve Growth Factor Predicts Persistent Detrusor Overactivity After Bladder Outlet Obstruction Relief Detrusor overactivity (DO) is a recognized cause of lower urinary tract symptoms, and is associated with bladder storage symptoms involving urgency and urgency incontinence. In animal models of bladder outlet obstruction (BOO), the mRNA expression of bladder nerve growth factor (NGF) was shown to be increased in conditions associated with DO and continued DO after BOO relief. Lee et al (page 2440) evaluated urinary nerve growth factor (uNGF) as a predictive factor of persistent DO after BOO relief in a rat model. They divided 50 female Sprague-Dawley rats into 10 sham operated controls and 40 with BOO. BOO was induced by partial urethral ligation and was relieved by removal of ligation after 3 weeks. Voided urine was collected before BOO (T1), 3 weeks after onset of BOO (T2) and 3 weeks after BOO relief (T3). Cystometry was performed in awake rats at T2 and T3. Bladder tissue was harvested at T3. uNGF and bladder NGF were measured by enzyme-linked immunosorbent assay and the results were adjusted based on creatinine (Cr) concentration. For 16 rats in which DO disappeared after BOO relief (group 1) uNGF/Cr significantly increased from T1 to T2 (p ⫽ 0.001) and significantly decreased from T2 to T3 (p ⫽ 0.003). In 8 rats with persistent DO despite BOO removal (group 2) uNGF/Cr significantly increased from T1 to T2 (p ⫽ 0.012) but did
not change from T2 to T3 (p ⫽ 0.123). Rats with persistent DO also had significantly higher uNGF/Cr at T1 compared to the control group (p ⫽ 0.015) and group 1 (p ⫽ 0.005). After BOO relief uNGF significantly decreased with resolution of DO but did not change in animals with persistent DO. Since the initial preBOO urinary NGF level was increased in animals with persistent DO, the authors conclude that this increased urinary NGF may be predictive of persistent DO after BOO relief.
Growth Inhibition and Apoptosis Induction by Tumor Necrosis Factor-␣ in Urethral Rhabdosphincter Although detrusor overactivity is considered the primary cause of urgency incontinence in older patients, impairment and decrease of the human urethral rhabdosphincter (HUR) has been reported with aging. This is believed to be due to apoptosis and a cause of urinary incontinence in the elderly. To explore this mechanism Hanada et al (page 2445) from Oita, Japan investigated the effects of tumor necrosis factor (TNF)-␣ on HUR satellite cells. The HUR satellite cells were cultured and selected by magnetic affinity cell sorting. Apoptosis induction was examined by flow cytometry and immunocytochemistry. Caspase cascade activation was determined by Western blot analysis. After TNF receptor expression was confirmed, the TNF signaling pathway was determined. TNF-␣ inhibited the proliferation of HUR satellite cells and caused some positive staining for annexin V-fluorescein isothiocyanate, but not for propidium iodide, suggesting the induction of early phase apoptosis. Increase of sub-G1 fraction was revealed by flow cytometry. Furthermore, activation of caspase-8 and 3, and cleavage of poly (adenosine diphosphate-ribose) polymerase were observed by Western blot analysis. TNF receptor expression at the mRNA and protein level was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively, and phosphorylation of inhibitor of B␣ was observed within 2 to 5 minutes after TNF-␣ treatment. Etanercept, a TNF-␣ antagonist, inhibited activation of inhibitor of B␣ and reversed the effects of TNF-␣ on HUR satellite cells. The authors conclude that since TNF-␣ induces growth inhibition and apoptosis of HUR satellite cells through TNF receptor activation, it might be involved in age related decreases in HUR cell number and a causative factor for urinary incontinence in the elderly. Karl-Erik Andersson Section Editor
AND
RESEARCH, INC.
Diet Sodas and Nephrolithiasis Hypocitraturia is a risk factor for nephrolithiasis. Dietary citrate and alkali intake may have an effect on citraturia, and increasing alkali intake also increases urine pH, which can help prevent uric acid stones. Eisner et al (page 2419) determined the citrate, malate and total alkali concentrations in commonly consumed diet sodas to help direct dietary recommendations for patients with hypocitraturic calcium or uric acid nephrolithaisis. They measured the citrate and malate concentrations in a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis and in 15 diet sodas. The anions were measured by ion chromatography. The pH of each beverage was measured to allow calculation of the unprotonated anion concentrations using the known pKs of citric and malic acid. Total alkali equivalents were calculated for each beverage. Statistical analysis was performed using Pearson’s correlation coefficient. Several sodas contained greater or equal amounts of citrate as alkali and total alkali than a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis. Colas had the lowest amounts of total alkali (less than 1.0 mEq/l). There was no significant correlation between beverage pH and total alkali content. The authors conclude that several commonly consumed diet sodas contain moderate amounts of citrate as alkali and total alkali, and that this information can be helpful for dietary recommendations for patients with calcium nephrolithiasis and specifically those with hypocitraturia. It may also be useful for patients with low urine pH and uric acid stones. The authors emphasize that beverage malate content is important, as malate ingestion will increase the total alkali delivered, which in turn augments citraturia and increases urine pH.
Bladder Regeneration by Collagen Scaffolds Various injuries may result in compromised bladder structure and compliance requiring reconstruction. Currently the major surgical solution is enterocystoplasty by vascularized bowel segments. However, multiple significant complications of this method greatly hamper its clinical application and, thus, it Vol. 183, 2115-2116, June 2010 Printed in U.S.A. DOI:10.1016/j.juro.2010.03.065
www.jurology.com
2115
2116
THIS MONTH IN INVESTIGATIVE UROLOGY
is important to improve the current method or find a better alternative for bladder regeneration. Chen et al (page 2432) from People’s Republic of China investigated a collagen based targeting system for bladder regeneration. A collagen binding domain (CBD) was added to the N-terminal of native basic fibroblast growth factor (bFGF) to allow it bind to collagen. Partial cystectomy was performed in Sprague Dawley rats and the collagen scaffolds loaded with CBD-bFGF, native bFGF or phosphate buffered saline were grafted to the remaining host bladders, respectively. At days 30 and 90 the reconstructed bladders were evaluated for histological analysis and urodynamic examination. This targeting bFGF delivery system induced satisfying histological structures of bladders, promoting more vascularization and ingrowth of smooth muscle cells. Urodynamic examinations revealed well accommodated bladder tissue with volume capacity and compliance. The authors conclude that this targeting delivery system induces better bladder regeneration at the injured site and, thus, could be an effective strategy for bladder regeneration with potential clinical applications.
Urinary Nerve Growth Factor Predicts Persistent Detrusor Overactivity After Bladder Outlet Obstruction Relief Detrusor overactivity (DO) is a recognized cause of lower urinary tract symptoms, and is associated with bladder storage symptoms involving urgency and urgency incontinence. In animal models of bladder outlet obstruction (BOO), the mRNA expression of bladder nerve growth factor (NGF) was shown to be increased in conditions associated with DO and continued DO after BOO relief. Lee et al (page 2440) evaluated urinary nerve growth factor (uNGF) as a predictive factor of persistent DO after BOO relief in a rat model. They divided 50 female Sprague-Dawley rats into 10 sham operated controls and 40 with BOO. BOO was induced by partial urethral ligation and was relieved by removal of ligation after 3 weeks. Voided urine was collected before BOO (T1), 3 weeks after onset of BOO (T2) and 3 weeks after BOO relief (T3). Cystometry was performed in awake rats at T2 and T3. Bladder tissue was harvested at T3. uNGF and bladder NGF were measured by enzyme-linked immunosorbent assay and the results were adjusted based on creatinine (Cr) concentration. For 16 rats in which DO disappeared after BOO relief (group 1) uNGF/Cr significantly increased from T1 to T2 (p ⫽ 0.001) and significantly decreased from T2 to T3 (p ⫽ 0.003). In 8 rats with persistent DO despite BOO removal (group 2) uNGF/Cr significantly increased from T1 to T2 (p ⫽ 0.012) but did
not change from T2 to T3 (p ⫽ 0.123). Rats with persistent DO also had significantly higher uNGF/Cr at T1 compared to the control group (p ⫽ 0.015) and group 1 (p ⫽ 0.005). After BOO relief uNGF significantly decreased with resolution of DO but did not change in animals with persistent DO. Since the initial preBOO urinary NGF level was increased in animals with persistent DO, the authors conclude that this increased urinary NGF may be predictive of persistent DO after BOO relief.
Growth Inhibition and Apoptosis Induction by Tumor Necrosis Factor-␣ in Urethral Rhabdosphincter Although detrusor overactivity is considered the primary cause of urgency incontinence in older patients, impairment and decrease of the human urethral rhabdosphincter (HUR) has been reported with aging. This is believed to be due to apoptosis and a cause of urinary incontinence in the elderly. To explore this mechanism Hanada et al (page 2445) from Oita, Japan investigated the effects of tumor necrosis factor (TNF)-␣ on HUR satellite cells. The HUR satellite cells were cultured and selected by magnetic affinity cell sorting. Apoptosis induction was examined by flow cytometry and immunocytochemistry. Caspase cascade activation was determined by Western blot analysis. After TNF receptor expression was confirmed, the TNF signaling pathway was determined. TNF-␣ inhibited the proliferation of HUR satellite cells and caused some positive staining for annexin V-fluorescein isothiocyanate, but not for propidium iodide, suggesting the induction of early phase apoptosis. Increase of sub-G1 fraction was revealed by flow cytometry. Furthermore, activation of caspase-8 and 3, and cleavage of poly (adenosine diphosphate-ribose) polymerase were observed by Western blot analysis. TNF receptor expression at the mRNA and protein level was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively, and phosphorylation of inhibitor of B␣ was observed within 2 to 5 minutes after TNF-␣ treatment. Etanercept, a TNF-␣ antagonist, inhibited activation of inhibitor of B␣ and reversed the effects of TNF-␣ on HUR satellite cells. The authors conclude that since TNF-␣ induces growth inhibition and apoptosis of HUR satellite cells through TNF receptor activation, it might be involved in age related decreases in HUR cell number and a causative factor for urinary incontinence in the elderly. Karl-Erik Andersson Section Editor