The preferred treatment for upper urinary tract renal calculi is extracorporeal shock wave lithotripsy (ESWL®). However, this treatment can result in renal epithelial cell injury, an important factor in calculi formation, which may explain why ESWL is associated with a high rate of recurrent renal calculi. Previous studies of stone recurrence rates after ESWL have principally been clinical followup studies, limited by patient variability in terms of stone size, location and composition, and by the number and energy of shock waves that patients received. Therefore, definitive conclusions regarding the influence of ESWL on stone formation cannot be drawn from these studies.
Xue et al (page 762) from Shaanxi, China investigated the influence of kidney damage secondary to shock waves on calcium oxalate crystal retention in the kidney in 4 groups of rats. Group 1 served as controls, group 2 (sham) was given 25 ml 0.75% ethylene glycol daily for 14 days, group 3 received 15 kv, 1 Hz shock waves 500 times to the left kidney followed by 25 ml 0.75% ethylene glycol daily for 14 days, and group 4 received the same treatment as group 3 except the number of impacts was increased to 1,000. Both kidneys were removed at the end of the experiment. Kidney sections revealed that the kidneys that did not receive shock waves had fewer crystals than those that received a shock wave impact, which had crystals in major areas. The density of calcium oxalate increased with the number of shock wave impacts. The authors conclude that the kidney damage caused by shock wave therapy in their study was sufficient to trigger new stone formation in the presence of urinary calcium oxalate supersaturation, and that the quantity of stone formation was related to kidney injury severity. Their findings support previous studies demonstrating that renal tubular epithelial cell injury is a key factor in the stone formation process.
Topoisomerase II α and Renal Medullary Carcinoma
Renal medullary carcinoma (RMC) is a rare renal malignancy that primarily affects young patients with sickle cell hemoglobinopathy. RMC is an aggressive renal neoplasm without currently available effective therapy. Topoisomerase II α (TOP2A) is a gyrase involved in cell proliferation and DNA maintenance and repair, and it is a target of inhibiting agents such as anthracyclines. Prior studies demonstrated a correlation between TOP2A protein expression and clinical response to anti-TOP2A agents in cases of breast carcinoma and in tumor cell lines including renal cell carcinoma (RCC).
Albadine et al (page 735) from Baltimore, Maryland evaluated TOP2A expression in relation to the proliferation index and TOP2A gene copy number status in a series of 14 RMCs. Archival tissues obtained from 3 institutions were analyzed using monoclonal antibodies for TOP2A and Ki67. TOP2A gene copy number was evaluated using fluorescence in situ hybridization. Locus specific TOP2A gene and chromosome 17 centromere probes were used. Percentages of tumor cells with polysomic, eusomic or monosomic chromosome 17 status were also determined. TOP2A was over expressed in 11 of 13 (85%) RMCs (median 50%, range 1% to 80%). A high Ki67 proliferation index was noted in 13 of 14 tumors (median 87.5%, range 2% to 100%). Ki67 expression was greater than TOP2A expression in all 13 informative tumors. A strong, statistically significant correlation was found between TOP2A and Ki67 expression (pairwise CC 0.9, p = 0.0000). TOP2A over expression was associated with shorter survival. No TOP2A amplification was detected in any of the 14 RMCs including the 11 tumors with TOP2A over expression. TOP2A gene deletions were noted in 4 tumors, and of these 4 deletions 2 were associated with chromosome 17 monosomy and 2 were in eusomic chromosome 17 tumors. The authors conclude that TOP2A is over expressed in 85% of RMCs, potentially lending support for using TOP2A inhibitor agents to treat this aggressive renal tumor.
Bcl2 -938CC Genotype and Poor Renal Cancer Prognosis
Renal cell carcinoma is the third leading cause of death among urological tumors, accounting for about 2% of adult malignancies. B cell lymphoma (Bcl2) family proteins, which are well-known as mitochondrial membrane proteins, are important regulators of programmed cell death or apoptosis in normal tissues and cancer cells. In RCC cases previous studies have shown an association between increased Bcl2 expression and better prognosis. A single nucleotide polymorphism (SNP) (-938C/A, rs2279115) was found in the bcl2 gene, whose -938A allele is significantly associated with increased Bcl2 expression compared with that of the C allele. Since there is currently no information about the role of the bcl2 -938C/A SNP in RCC, Hirata et al (page 721) from San Francisco, California investigated the polymorphism at the bcl2 -938C/A site and its effects on clinical characteristics in patients with RCC. They genotyped bcl2 -938C/A SNP in 216 patients with RCC, and in 209 age and gender matched healthy subjects. They also investigated the relationship between the bcl2 -938C/A polymorphism, Bcl2 expression, proliferation and apoptosis status in RCC tissues using immunohistochemistry and TUNEL assay. The association of the bcl2 -938C/A SNP with survival of patients with RCC was also analyzed using Kaplan-Meier curves. Survival in Bcl2 positive cases was significantly longer than in negative cases. On univariate and multivariate analyses the bcl2 -938CC genotype was independently associated with poor prognosis. Kaplan-Meier analysis showed that survival of patients with CC genotypes was significantly worse than that of those with CA+AA genotypes. CC genotype carriers had significantly lower Bcl2 expression and higher proliferative activity in renal cancer tissues than CA+AA genotype carriers. The authors conclude that for the first time it is possible to show that the bcl2 -938C/C genotype has a worse prognosis and lower survival in patients with RCC. In addition, the bcl2 -938C/A SNP was shown to be an independent adverse prognostic factor for RCC.
Identification of Bladder Distinct Myocardin Splice Variants
The importance of myocardin in the development of smooth muscle tissues has been well established. However, previous observations have suggested that the myocardin signaling pathway may be critical for the normal differentiation of bladder smooth muscle in a manner distinct from that observed in other smooth muscle tissues. Saha et al (page 766) from Oxford, Ohio characterized myocardin expression in the developing and adult bladder to identify potential tissue specific differences that may have a role in detrusor smooth muscle development. Reverse transcriptase and quantitative polymerase chain reaction (PCR) were used to characterize and determine the precise levels of myocardin expression in the mouse and human bladder vs various other tissues. Sequence analysis was used to confirm the genomic location of the various PCR products identified by this analysis. Exonic profiling of the mouse myocardin gene identified a series of unique myocardin splice variants derived from a novel 305 bp exon located between exons 2 and 3 of the previously identified myocardin gene. Each variant showed differential patterns of expression in the mouse and primary protein sequences suggested unique functionalities for each of the myocardin variants identified. Identical myocardin splice variants were also observed in the human bladder as well as a unique human specific exon 12 myocardin splice variant that was not observed in the mouse. The authors conclude that the identification of a series of unique myocardin splice variants that are differentially expressed in the bladder as well as in other muscle and nonmuscle tissues provides a potential molecular platform for mediating many unique tissue specific functions associated with the myocardin transcriptional program.
DNMT1 Silencing and Prostate Cancer PC3 Cell Invasiveness
In somatic cells the DNA methylase DNMT1 maintains genomic DNA methylation at 5′-CpG-3′ residues. DNMT1 has emerged as a promising target for therapeutic intervention since its activity appears to augment the activity of de novo DNA methylases. Molecules that specifically inhibit DNMT1 activity or expression have shown promise in clinical trials as effective anticancer agents. DNMT1 depletion causes distinct phenotypic changes in colon and gastric cancer cell lines, suggesting that the extent to which DNMT1 influences the expression of its target genes is cell type specific. Yaqinuddin et al (page 756) from Karachi, Pakistan determined the impact of DNMT1 depletion from prostate cancer derived cells on their gene expression profiles and cellular phenotype. Small interfering RNA was used to silence DNMT1 expression in prostate cancer derived PC3 cells, and the resulting cell line was validated by reverse transcriptase-PCR and Western blotting. Proliferation, migration and invasion assays were performed in engineered cells to assess the effect of DNMT1 silencing on cellular phenotype. DNA microarrays were used to monitor changes in gene expression. Loss of DNMT1 dramatically decreased cell proliferation but significantly increased cell migratory and invasive potential. Additionally, in the limited set of genes whose expression and DNA methylation status were determined, DNMT1 loss was not only associated with increased CDKN3 and claudin-3 expression, but also culminated in specific demethylation of Rb1 and RAR-β promoters. The authors showed for the first time that the lack of DNMT1 in prostate cancer cells may enhance their invasiveness and migration. The genetic and phenotypic consequences of silencing DNMT1 in PC3 cells are markedly different from those in colon and gastric cancers, indicating that DNMT1 preferentially targets certain gene promoters.
Transient receptor potential vanilloid subfamily 1 (TRPV1) is a member of the transient receptor potential superfamily of nonselective cation channels. It is activated by capsaicin, heat, protons and various endogenous mediators generated during tissue injury or inflammation. TRPV1 is highly expressed on primary sensory afferents, where it functions as an integrator of signals involved in somatosensory and visceral pain, and hypersensitivity. In the bladder TRPV1 is largely expressed on C and Aδ afferents, but may also be present on urothelial cells, myofibroblasts and smooth muscle cells, suggesting a broader role for TRPV1 in bladder mechanosensory transduction.
Cefalu et al (page 776) from Palo Alto, California investigated the pharmacological effect of 2 potent, selective, competitive TRPV1 antagonists, JNJ17203212 and JYL1421, in anesthetized rodent models of bladder function. Both drugs were evaluated in the anesthetized rat volume induced micturition reflex (Refill) model. JNJ17203212 was further evaluated in the Refill model in capsaicin desensitized rats, and in rat capsaicin and mouse citric acid models of irritant induced detrusor overactivity. When the drugs were given systemically in the Refill model they caused an increase in micturition threshold volume. JNJ17203212 also decreased bladder contraction amplitude but JYL1421 was without effect. Capsaicin desensitization significantly increased baseline micturition threshold volume and decreased bladder contraction amplitude in the Refill model compared to sham treated controls and JNJ17203212 produced no further effect after capsaicin desensitization. JNJ17203212 was also effective in 2 models of irritant induced detrusor overactivity, preventing the decrease in micturition threshold volume and the increase in bladder contraction amplitude observed with intravesical instillation of 10 μM capsaicin, and the decreased voiding interval induced by intravesical citric acid. The authors conclude that by using the selective TRPV1 antagonists they found a significant role for TRPV1 on capsaicin sensitive afferents in bladder filling in naïve anesthetized rats and in models of irritant induced detrusor overactivity. JNJ17203212 also inhibited detrusor overactivity induced by intravesical capsaicin and intravesical citric acid. These data increase our understanding of the role of TRPV1 in sensory modulation of the micturition reflex under nonirritant and inflammatory conditions.
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