Proteomics and Clinical Translation in Urological Disease
Proteomics is broadly defined as a collection of scientific approaches and technologies to characterize the protein content, and modifications within cells, tissues, body fluids and whole organisms at a particular stage or condition. It is a rapidly growing new discipline that has the potential to increase and improve the understanding of protein function and interaction in the context of systems biology. It is a translational science with the potential to identify many new therapeutic targets as well as diagnostic and prognostic biomarkers of disease. As discussed by Vaezzadeh et al (page 835) from Boston, Massachusetts, proteomics approaches combine powerful technologies such as protein/peptide separation, identification and bioinformatic detection, and quantitation based on powerful computational data processing tools. Based on a literature search they review current proteomics technologies, discuss applications in urology and provide a perspective on the future of proteomics in clinical medicine. Advantages, challenges and limitations of current proteomics approaches are also discussed. The authors conclude that proteomics offers new approaches to the study of diseases of the genitourinary tract, and the potential to identify clinically relevant biomarkers and new therapeutic targets.
TRAIL and Oxidative Stress
TRAIL, an endogenous protein involved in immunosurveillance and a novel drug in clinical trials, is of particular interest as a cancer therapy because it can induce apoptosis in cancer cells but not in normal cells. Because some cancers develop resistance to TRAIL, safe and effective methods of TRAIL sensitization are of great clinical interest. White-Gilbertson et al (page 1178) from Charleston, South Carolina explored how chemotherapy and oxidative stress (OS) affect TRAIL sensitivity and the expression of proteins in the apoptotic pathway. They assessed sensitivity to TRAIL in viability assays, and measured apoptosis by caspase-3/7 activity and/or nuclear condensation using Hoechst staining. Western blotting was used to determine cleavage, phosphorylation or alterations in protein expression. TRAIL reduced the viability of 5637 but not of J82 or T24 bladder carcinoma cells. Chemotherapy with doxorubicin or cisplatin decreased the expression of the anti-apoptotic protein cFLIPS and increased caspase-8 cleavage, reversing TRAIL resistance in T24 cells. Chemotherapy mediated TRAIL sensitization was mimicked by low concentrations of hydrogen peroxide, which resulted in the phosphorylation of translation elongation factor 2, and reduced the expression of several short half-life, anti-apoptotic proteins including FLIPS, XIAP and survivin. The authors conclude that induction of OS by low concentrations of hydrogen peroxide may reverse TRAIL resistance. This warrants the further exploration of hydrogen peroxide as an adjuvant intravesical treatment to lower the apoptotic threshold of bladder cancer cells.
Uropathogen Interaction with Urological Stent Surfaces
Bacterial adherence to indwelling urological devices is a common problem with more than 90% of stents removed from patients containing bacterial biofilms. Ureteral stents also commonly become encrusted. Various coatings have been developed to reduce bacterial adherence. To date there has been no in vitro testing of coating with heparin. Lange et al (page 1194) from Minneapolis, Minnesota determined the effects of heparin coating on the bacterial adherence of common uropathogens and physical stent properties. Heparin coated ureteral stents (Radiance®) and noncoated controls (Endo-Sof™) were tested against triclosan eluting stents (Triumph®) and noneluting controls (Polaris™) for the adherence of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus and Pseudomonas aeruginosa for 7 days. Adherent bacteria were determined and biofilms were visualized using fluorescent dyes. The radial, tensile and coil strengths of the Radiance and Polaris stents were compared to determine the effect of heparin coating on physical stent characteristics.
Heparin coating did not decrease bacterial adhesion compared to its control. The adhesion of E. coli was limited by all stents tested. The Polaris stent showed significantly greater resistance to bacterial adherence for Klebsiella, Pseudomonas and Enterococcus than the Endo-Sof and Radiance stents, but was more susceptible to S. aureus adherence. The Triumph stent resisted all bacteria except Pseudomonas and Enterococcus. Mature biofilms were observed on all stents with lower viability on the Triumph stent. The Radiance stent demonstrated higher tensile and lower compression strength than the control. The authors conclude that heparin coating does not reduce bacterial adherence to ureteral stents. Drug eluting antimicrobials have an inhibitory effect on bacterial adherence and the Polaris stent showed the least bacterial adherence of the nondrug eluting ureteral stents tested.
An Investigational Prostate Cancer Methylation Assay
Carcinoma of the prostate is the leading cause of newly diagnosed cancer in males and the second leading cause of male cancer related deaths in the United States. Early detection of prostate cancer may be made more effective and efficient by implementing an assay offering greater specificity and predictive value when used in conjunction with risk factors or predictive models to determine which patients should undergo biopsy. Prostate specific antigen (PSA) tests have low specificity, which frequently results in unnecessary biopsy and typically limits screening to patients with PSAs greater than 4.0 ng/ml. Baden et al (page 1186) from Raritan, New Jersey evaluated an investigational prostate cancer methylation specific polymerase chain reaction assay that detects aberrant methylation in 3 markers (GSTP1, RARβ2 and APC) that indicate the presence of prostate cancer. The assay was evaluated in post-digital rectal examination urine samples (178 cancer/159 noncancer) collected prospectively at 9 clinical sites. Subject PSAs ranged from 2.0 to 10.0 ng/ml and all subjects underwent transrectal ultrasound guided needle biopsy with 6 or more cores sampled. Detection of 1 or more markers indicated positivity. Methylation specific polymerase chain reaction assay performance was better in whole vs divided urine cohorts (p = 0.035). Assay AUC was 0.72 in the whole urine cohort and 0.67 in the combined population. These values were higher than those of PSA alone using 4.0 ng/ml as the cutoff (p = 0.00 and 0.01, respectively). Moreover, the assay together with the Prostate Cancer Prevention Trial risk calculator or a standard nomogram significantly improved AUC for the whole urine cohort and the combined population (p <0.05) vs predictive algorithms alone. The assay positive predictive value was 54% in the whole urine cohort with PSA 2.0 to 4.0 ng/ml and the negative predictive value was 87% with PSA 4.1 to 10.0 ng/ml. The authors conclude that this investigational assay used with current screening algorithms may potentially add value to the biopsy decision making process.
Mechanisms Underlying the Low Prevalence of Pediatric Calcium Oxalate Urolithiasis
Pediatric nephrolithiasis is relatively uncommon and the majority of urinary stones in children are composed of calcium oxalate (CaOx). It is known that pediatric urinary macromolecules (UMMs) show stronger inhibition of CaOx crystal growth, aggregation and adhesion than those of adults. To investigate the mechanisms underlying the development of CaOx urolithiasis, Momohara et al (page 1201) from Suita, Japan evaluated the differences between pediatric and adult UMMs in inhibitory activity against oxalate induced renal tubular cell injury. Urine samples were collected from healthy men and their sons. The protective effects of UMMs against oxalate induced injury to Madin-Darby canine kidney (MDCK) cells were examined by lactate dehydrogenase assay and immunostaining. Variations in the relative abundance of proteins involved in stone formation such as osteopontin and calgranulin B were analyzed. The investigators found that pediatric urine had significantly higher UMM and glycosaminoglycan concentrations than that of adults (p <0.01). UMMs inhibited oxalate induced MDCK cell injury in a concentration dependent manner and stronger activity was observed in children (p <0.05). TUNEL staining and 8-OHdG immunostaining indicated stronger inhibition of apoptosis and OS in MDCK cells pretreated with pediatric UMMs (p <0.01). Osteopontin and calgranulin B expression were positively correlated (p = 0.03), and these proteins showed greater down-regulation in children (p <0.01). The authors conclude that a reason for the low prevalence of pediatric urolithiasis is that UMMs have stronger inhibitory effects against oxalate induced renal cell injury and OS induced apoptosis in children than in adults.
Sildenafil as a Protecting Drug for Warm Ischemic Kidney Transplants
Normothermic ischemia produces microvascular endothelial injury in organs for transplantation. Previous studies have shown that the phosphodiesterase type 5 inhibitor sildenafil induces an excellent preconditioning protecting effect in the heart. In an experimental minipig model Lledó-García et al (page 1222) from Madrid, Spain studied the protective effects of sildenafil on kidney grafts. Minipigs in group 1 (7) were given an oral dose of 100 mg sildenafil 1.5 hours before surgery. The 7 minipigs in group 2 received no sildenafil. Right single nephrectomy was completed after 45 minutes of warm ischemia by complete vascular clamping. Before autotransplantation all kidneys were submitted to 60 minutes of hypothermic pulsatile perfusion. Renal flow, arterial pressure and renal vascular resistance were recorded in real time for 60 minutes after autotransplantation. Nitric oxide levels were determined in blood samples from the renal vein at predefined intervals. Optical and electronic microscopy was done in all organs at the end of the procedure. In group 1 vs 2 renal vascular flow was significantly higher (153.41 vs 32.07 ml per minute per 100 gm, p <0.01) and renal vascular resistance was significantly lower (0.61 vs 3.08 mm Hg/ml per minute, p <0.01). No significant differences were observed in systemic arterial pressure values between groups 1 and 2 (84.79 vs 83.65 mm Hg, p >0.05). Nitric oxide levels were significantly higher for all periods in group 1 (49.04 vs 17.16 μM, p <0.01). No significant differences were observed in histological studies, although endothelial cell structure was better preserved in the sildenafil group. The authors conclude that for the first time in the literature it is possible to demonstrate a positive effect of sildenafil in the immediate posttransplantation outcome of warm ischemic kidneys without systemic secondary effects.
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