presented on September 3, 2007

Introduction: Interstitial cystitis/Painful Bladder Syndrome (IC/PBS) is of unclear etiology, associated with chronic pelvic pain and urinary frequency and urgency. The current lack of demonstrated biological markers in blood or urine has been a major challenge to treat IC/PBS. We used several proteomic approaches aiming at the identification of new markers related to IC/PBS in a rat model of chronic irritation and in human urine.

Methods: Chronic irritation of rat bladder was caused by a brief (90 secs) intravesical instillation of 0.4N HCl. Whole bladders were collected at different time points after treatment, snap frozen and nuclear and cytosolic protein extracts obtained (NE-PER, Pierce). Samples were resolved in standard 2D-gels stained with an improved Coomasie Stain (Imperial Stain, Pierce) or by Differential Gel Eletrophoresis. Differentially expressed spots were excised and identified by MALDITOF MS/MS. Human 2h urine from six patients (3 IC, 3 Multiple Sclerosis) and five controls were processed in two ways: ice-cold ethanol precipitation, standard desalting and 2D analysis as above, or concentration (Miniplus, Millipore), cold acetone precipitation, 2D-clean up kit (Amersham) and SELDI-TOF analysis.

Results: With two different proteomic strategies, we identified four proteins overexpressed in the irritated bladder compared to normal bladders: calgranulin A and B, released in inflammatory responses; myelin transcription factor 1-like (MyT1L), which may regulate gene expression in the nervous system and pituitary; and R. norvegicus fibrinogen gamma- chain (RNFIBG1 NID), with unclear function so far. Regarding the human urine, ethanol precipitation + desalting yielded cleaner 2D-gel patterns, especially for proteins ≥15 KDa, although no differences were observed compared with control urines. Concentration + acetone precipitation + SELDI yielded several peaks which appear or are present at higher levels in diseased urine compared to normal urine (3572, 5193, 15114, and 20605 Da).

Conclusions:The way of specimen collection, sample preparation and identification method are the key steps that lead to the successful biomarker discovery. These results could help in the accurate diagnosis, early prediction of the disease and also help to elucidate the primary defect and the pathogenesis in chronic cystitis.

Authors: de Miguel F, Chancellor M, Yoshimura N, Tyagi P

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