Study population and biopsy procedure
Patients, 50 years or older and referred to colonoscopy, were enrolled in the study. Medical history was recorded according to case notes. Patients with either a history of or present colorectal neoplasia were included in the neoplasia group, whereas patient with no present or former neoplasia were placed in the control group irrespective of other extra-intestinal diagnoses. Patients were excluded from the study in case of history of any kind of chronic inflammatory condition of the intestine or if they had recently been or were presently undergoing a radiation and/or chemotherapy for CRN. A total of 19 patients were enrolled, with 11 subjects in the neoplasia group and 8 subjects in the control group. There were four women in each group and the mean age was 67 ± 13 yrs in the control group and 69 ± 4 yrs in the neoplasia group.
Six biopsies from each patient were obtained during endoscopy from macroscopically normal appearing mucosa using standard biopsy forceps (Radial Jaw 3) from Boston Scientific with an outside diameter of 2.2 mm. The biopsies were obtained approximately 30 cm from the anus and at least 10 cm from macroscopically abnormal tissue on retraction of the endoscope. Biopsies were immediately transferred to an iced, oxygenized bicarbonate Ringer solution with the following composition (in mM): Na+ (140), Cl- (117), K+ (3.8), PO4
- (2.0), Mg2+ (0.5), Ca2+ (1.0) and HCO3
- (25). pH was adjusted to 7.4 by gassing the media with 95% O2/5% CO2.
The study protocol was approved by the Scientific Ethical Committee of Copenhagen (KA 97161) and Frederiksberg Counties (KF01-232/03) and conducted in accordance with the Helsinki declaration. All patients participating gave written informed consent.
Theophylline, indomethacin, acetazolamide, NPPB, SITS, rifamycin SV, cAMP and cGMP were purchased from Sigma-Aldrich (Seelze, Germany). db-cAMP, db-cGMP and OATP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), OATP1C1 (cat. no.: sc-51350), OATP2B1 (cat. no.: sc-66561), OATP4A1 (cat. no.: sc-51169), OATP4C1 (cat. no.: sc-136775). Bumetanide was received as a gift from Leo Pharmaceuticals (Copenhagen, Denmark). ABC-antibodies were obtained from LifeSpan Biosciences (Seattle, WA, USA), ABCB1 (cat. no.: LS-A9403-50), ABCC3 (cat. no.: LS-C16477-250), ABCC4 (cat. no.: LS-B4-50), and ABCC5 (cat. no.: LS-B2334-50). Oligo primers were obtained from Tag Copenhagen (Copenhagen, Denmark). Normal goat and rabbit serum was purchased from Dako (Glostrup, Denmark). All other chemicals were of analytical grade.
Functional characterization in MUAS-chamber
Biopsies were mounted in MUAS-chambers as previously described . Pretreatment of the biopsies was performed 15 min mounting. Amiloride (100 μM, apically) was added to inhibit ENaC-mediated sodium transport. Theophylline (400 μM, apically and basolaterally) was added to dampen endogenous breakdown of cNTs. Indomethacin (40 μM, basolaterally) was added to inhibit endogenous synthesis of cNTs. Experiments were conducted after additional 20 min equilibration. cAMP, cGMP, dibutyryl-cAMP and dibutyryl-cGMP were used as model substrates of OATP and ABC-transporters and applied either luminally or basolaterally (500 μM). Each experiment was terminated by basolateral addition (in μM) of bumetanide (25), NPPB (500), SITS (1000), and acetazolamide (250), alone or in combination. In a few experiments after pretreatment, rifamycin SV (50–100 μM) was added to either side of the tissue to test for its inhibition of cNT influx.[18, 19]. DMSO was the vehicle for applying lipid soluble drugs and its final concentration always kept below 1 per mille in chambers. A thorough discussion concerning the validity of measured SCC and slope conductance by the MUAS-method is presented earlier .
After completion of the study the biopsies were gently dismounted and preserved in 4% paraformaldehyde for subsequent histological assessment of tissue integrity. Epithelium preservation on biopsies was scored on a scale from 0 to 3, where 0 is overall intact epithelial surface, 1: slightly damaged epithelium, 2: at least 1/3 intact epithelium and 3: practically no intact epithelial surface.
From each patient, one biopsy was frozen on dry ice and saved at −80 °C for rt-qPCR studies. For RNA isolation, biopsies were homogenized using a Polytron (PT 1600 E) for 2 min. RNA isolation was performed using a NucleoSpin® RNA/Protein kit from Macherey-Nagel (Düren, Germany). RNA concentration was determined with NanoDrop® ND-1000 from NanoDrop Technologies (Wilmington, DE, USA), and the purity assessed by means of absorbance ratios (A260/A230 and A260/A280).
Rt-qPCR for eight transporter genes was performed on biopsies from 6 patients in both groups using β-actin as reference gene. The following eight transporters were selected based on previous reports on presence in human colonic mucosa: OATP1C1, OATP2B1, OATP4A1, OATP4C1, ABCB1, ABCC3, ABCC4, and ABCC5 . Gene sequences were retrieved via http://www.ensemble.org and the primers were designed using Primer 3 software. NetPrimer software was used to identify and exclude primers with a tendency to dimer and/or hairpin formation.
Selected primers were: β-actin (forward: ACCCAGCACAATGAAGATCA, reverse: CGTCATACTCCTGCTTGCTG), OATP1C1 (forward: CCTCAGAAGAAAAGCAACCAT reverse: ACCATCAATAACTCCCACCAG), OATP2B1 (forward: TGATCTGCTTCGCCTTAGTTT, reverse: CTGGATCTGCTCTCTTTGGTC), OATP4A1 (forward: TTCCTGATGACAGAACAGTGC, reverse: ACAGCCGACTTTAAACCACAG), OATP4C1 (forward: GGCTTTCTGCTTCACTACTGC, reverse: CATAACGCTTCTCAACAGTGG), ABCB1 (forward: TGGATTCATCAGCTGCATTT, reverse: TGATGGAGTCATTGTGGAGAA), ABCC3 (forward: TACTCTCTGCCCTCATCTTGG, reverse: AAAACAGGCGGGAGAGAAA), ABCC4 (forward: CCCTCACTGAAACAGCAAAA, reverse: TAGTTAAGGTCGAGGGCTGTC), ABCC5 (forward: GCTCTTCTTGCCACAGTCTCT, reverse: TTTTCGTGGCTTTCTTCTCTG).
cDNA synthesis was conducted using an iScript™ cDNA Synthesis Kit from Bio-Rad (Copenhagen, Denmark) according to manufactures instructions as previously described .
For rt-qPCR, cDNA was amplified using a Fast SYBR® Green Master Mix (Applied Biosystems). Samples were run in four dilutions (0.05, 0.5, 5.0, and 10% cDNA v/v) on 384 well plates in volumes of 10 μL and primer and master mix concentrations of 0.6 μM and 50%v/v, respectively. All samples were run in triplicate with β-actin on all plates. Amplification was performed using a 7900HT Fast Real-Time PCR System from Applied Biosystems (Foster City, CA, USA) in accordance with the supplier’s manual. Standard and dissociation curves confirmed acceptable amplification efficiencies and specificities of the primer sets. The expression profiles were calculated using the deltaCT-method in which the amplification calculation is adjusted for primer-set efficiencies . Results were analyzed using adjuvant SDS 2.3 software. Results are based on the 10% cDNA dilution.
One colon biopsy from each patient was put aside in 4% neutral buffered formaldehyde after the endoscopic procedure. Biopsies were subsequently embedded in paraffin and cut in 10 μm thin slices. Immunohistochemical localization of transporters was performed on biopsies from two patients in each group. The sections were deparaffinated and rehydrated, followed by heat treatment in a microwave oven in order to unmask epitopes. The sections were blocked with a 2% bovine serum albumin solution for 10 minutes, to rule out unspecific antibody adhesion, followed by incubation with a primary antibody at 4 C overnight in the following concentrations: OATP1C1 (1:100), OATP2B1 (1:100), OATP4A1 (1:100), OATP4C1 (1:400), ABCB1 (1:400), ABCC3 (1:400), ABCC4 (1:400), and ABCC5 (1:400). Biotionylated secondary antibodies were detected using a streptavidin-bioptin-complex coupled to horse radish peroxidase and a diaminobenzidin stain, color-enhanced with CuSO4. Finally, the biopsies were periodic acid-Shiff-hematoxylin stained. For negative controls primary antibody was replaced by either normal goat (Dako X0902) or rabbit (Dako X0907) serum (1:100). Images were recorded using an Ortoplan microscope (Wetzlar, Germany) fitted with an Evolution MP camera (Silverspring, MD, USA) and analysis was performed using Image-Pro 5.0 software.
Hypothesis testing was done by unpaired student’s t-test or Mann–Whitney rank sum test. P-values below 0.05 were considered significant. All SCC-signals obtained were included with the same weight regardless of the number of biopsies from each patient. This procedure was chosen in order to reduce the importance of outliers in the single patient. All statistics were performed using SigmaPlot 11.0 software.