Proteomic analysis of bladder biopsies from interstitial cystitis/bladder pain syndrome patients with and without Hunner’s lesions reveals differences in expression of inflammatory and structural proteins

This study was approved by the institutional IRB (IRB# 2009-218) and written consent for all participants was obtained. Disease state was determined during cystoscopy by a trained urologist. Bladder samples were obtained from 4 female patients with HIC and 4 female patients with NHIC during routine medical cystoscopy. Two biopsies were collected from each patient: one from a diseased site (glomerulations in NHIC patients, Hunner’s lesions in HIC patients), and another from an adjacent non-disease-apparent (NDA) site as determined by an experienced urologist. 5 mm × 5 mm samples of bladder tissue were snap-frozen and stored at − 80 °C. Patients were asked to complete the validated Interstitial Cystitis Symptom Index and Problem Index (ICSI/ICPI) to measure lower urinary tract symptoms (LUTS) and impact on patient quality of life (QOL) [10]. ICSI scores range from 0 to 19 and ICPI scores range from 0 to 16, with higher scores representing more severe symptoms and impact [3].

Deidentified samples were sent to MSBioworks for processing and analysis. Samples were blinded and randomized to the researchers. Samples were washed in 1 × PBS and mechanically disrupted in a Bullet Blender (NextAdvance) with 1.6 mm stainless steel beads in 100µL of modified RIPA lysis buffer (2% SDS, 150 mM NaCl, 50 mM Tris pH 8, 1X Roche complete protease inhibitor). Samples were incubated at 60 °C for 30 min, then clarified by centrifugation. The protein concentration of each extract was determined by Qubit fluorometry (Invitrogen). 10 μg of each sample was processed by SDS-PAGE using a 10% Bis–Tris NuPage mini-gel (Invitrogen) in the MES buffer system. The migration window (2 cm lane) for each sample was excised into ten equally sized bands, each was processed by in-gel digestion with trypsin using a ProGest robot (DigiLab). Samples were first washed with 25 mM ammonium bicarbonate, followed by acetonitrile. Samples were reduced with 10 mM dithiothreitol at 60 °C, followed by alkylation with 50 mM iodoacetamide at room temperature. Digestion was performed with trypsin at 37 °C for 4 h. Finally, samples were quenched with formic acid and the supernatant was analyzed via nHPLC-MS/MS.

Sample supernatants were analyzed by nHPLC-MS/MS with a NanoAcquity HPLC system (Waters) linked to a Q Exactive mass spectrometer (ThermoFisher). Digested proteins were loaded on a Luna C18 resin (Phenomenex) trapping column and eluted over a Luna C18 resin 75 µm analytical column at a flow rate of 350 nL/min. The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 70,000 FWHM for MS and 17,500 FWHM for MS/MS. The fifteen most abundant ions were selected for MS/MS.

Data was analyzed using Mascot with the following parameters: the database used was SwissProt Human (concatenated forward and reverse plus common contaminants), with the enzyme set to Trypsin/P. Fixed modifications were set to Carbamidomethyl (C), while variable modifications included Acetyl (N-term), Deamidation (N,Q), Oxidation (M), and Pyro-Glu (N-term Q). Mass values were monoisotopic. Peptide and Fragment Mass Tolerances were 10 ppm and 0.02 Da, respectively. 2 max missed cleavages were allowed. After Mascot processing, data was parsed into Scaffold for validation and filtering. Data were filtered using a 1% protein and peptide false discovery rate (FDR) while requiring at least 2 unique peptides per protein. To determine approximate relative abundance of proteins within a given sample and between samples, normalized spectral abundance factor (NSAF) was calculated based on the following equation:where SpC = Spectral Counts, MW = Protein Molecular Weight (in kDa), and N = Total Number of Proteins. Significant proteins were determined by comparing average NSAF values for diseased and adjacent NDA tissues to generate fold change values, as well as a T-test for significance. Proteins were considered significant if they had a fold change difference ≥ 4 and a p value ≤ 0.05. Interactions and relationships between these proteins were mapped using STRING. Proteins were connected by function and interaction, with interactions predicted by experimental data, database searches, text mining, and co-expression.

Significant proteins were uploaded to STRING [11] (string-db.org) and mapped to predict protein–protein interactions, represented by colored lines between protein nodes. These relationships include known interactions determined by experiments (purple) and database mining (teal), predicted interactions via gene neighborhood (green), co-occurrence (blue), or gene fusion (red), or from text mining (yellow), co-expression (black), or protein homology (light blue).

First, we compared diseased areas of HIC tissue to NDA areas of HIC bladders to determine protein differences. 18 proteins were found to be significantly altered when comparing NDA to diseased HIC tissues. Three of these proteins were linked to ubiquitination (Table 2).

Comparing diseased areas of NHIC bladder tissue to NDA areas revealed 13 proteins were significant. These proteins function in various stages of peptide and lipid metabolism, as well as partial function in cytoskeleton organization.

Next, we compared diseased areas of HIC patients with diseased areas of NHIC patients. 34 proteins were found to be significantly increased when comparing diseased HIC tissue to diseased NHIC tissue. STRING’s protein mapping (Fig. 2) revealed that ten of these proteins are involved in immune response and are associated with inflammatory diseases, such as asthma and type I diabetes mellitus, while nine of these proteins are associated with protein processing in the endoplasmic reticulum (ER) (Table 3). In addition, five proteins involved in collagen formation and cellular adhesion were significantly decreased in HIC patients compared to NHIC patients (Table 3).

Finally, we sought to compare NDA tissues of HIC patients with NDA tissue of NHIC patients. 50 proteins were found to be significantly altered when comparing NDA NHIC tissue to NDA HIC tissue. STRING’s protein mapping (Fig. 3) revealed nine proteins involved in protein metabolism and four proteins linked to the Rap1 signaling pathway, which is involved in cell proliferation and adhesion, as well as three proteins involved in the immune response (Table 4).