Proteomic investigation on chronic bladder irritation in the rat

Abstract

Objectives: Interstitial cystitis (IC) is a painful bladder syndrome associated with urinary frequency and urgency. Elusive cause of IC makes its diagnosis only possible by exclusion in many cases. In this study, we used proteomics for identifying disease-associated proteins in a rat model of chronic bladder irritation.

Methods: Chronic irritation of the rat bladder was caused by a brief (90 seconds) intravesical instillation of 0.2 mL of 0.4 N HCl. Whole bladders were collected at different time points after treatment, snap frozen, and nuclear and cytosolic protein extracts were obtained. Samples were resolved in standard 2-dimensional (2D) gels stained with an improved Coomasie stain or by differential gel electrophoresis (DIGE). Differentially expressed spots were excised and identified by MALDI-TOF MS/MS. Histologic and Western blot analyses were also performed.

Results: Bladder morphology and histologic appearance of bladder sections after HCl treatment reflected hemorrhage, edema, epithelial denudation, detrusor mastocytosis, and eosinophilia. Proteomic analysis of irritated rat bladder revealed marked overexpression of 4 nuclear proteins and marked underexpression of 1 nuclear protein compared with normal rat bladders. Among these proteins, inflammation-associated calgranulin A (over) and smooth muscle protein-22/transgelin (under) showed opposed expression patterns after bladder irritation.

Conclusions: Presence of mast cells and eosinophils and overexpression of calgranulin A confirm the inflammatory component of HCl-irritated bladder. Altered expression of nuclear proteins is of particular interest because of their possible role as a prognostic marker in inflammatory bladder disorders. However, more studies are needed before clinical application of these findings can be established.

Figure 1

Time-course of the gross pathological changes in rat bladder after HCl-induced chronic irritation. Severe hemorrhagic changes induced by HCl were seen 24 hrs later that seem to persist till 4^th day and only partially resolve by the 8^th day.

Figure 2

Histological analysis of rat bladder after HCl irritation. (A, D) Sham, (B, E) 1 day, and (C, F) 8 days after treatment. Severe edema in the lamina propria (*) and epithelial denudation (arrow) at day 1, and massive cellular infiltration in the lamina propria (#) and re-epithelization (arrow head) at day 8 after HCl irritation are shown. Hematoxilin and eosin staining. Magnification 20x (A–C), 100x (D–F).

Figure 3

Coomasie-stained 2D-electrophoresis pattern of nuclear extract from rat bladder tissue following chronic bladder irritation. Spots of interest (48 marked) were selected by DeCyder software, isolated from the gels, and submitted to MALDI-TOF analysis, yielding 33 positive identifications from 25 different proteins.

Figure 4

Merged images of DIGE analysis after reciprocal labeling of nuclear extracts from normal and from HCl-irritated rat bladder (day 8). Normal extract was labeled with Cy5 (left) and Cy3 (right) and HCl-treated extract was labeled with Cy3 (left) and Cy5 (right). Computer image analysis selected four spots (A1–A4) as overexpressed and one (A5) as underexpressed in HCl-irritated bladder compared to normal rat bladder. See text for protein ID details.

Figure 5

Temporal changes in Calgranulin A and transgelin levels in nuclear extracts of HCl-irritated bladder analyzed by Western blotting. β-actin served as internal loading control.

Figure 6

Time-course of the number of mast cell (top) and eosinophil (bottom) infiltration in rat bladder after HCl-induced chronic irritation. *p<0.05,**p<0.01 vs. control group at each point with Wilcoxon test; n=4–9 per group.