Cell lines and CCR1-expressing cells.
The cell lines 293MR (human CCR1/293 cells) and 293M3X (mouse CCR1/293 cells) have been described previously (12
). The 293M3X/pEAK10-Gqi5 cell line was created by transfecting 293M3X cells with the plasmid pEAK10-Gqi5 using Lipofectamine (Life Technologies Inc., Carlsbad, California, USA) and selecting with 0.5 μg/ml puromycin (Edge BioSystems Inc., Gaithersburg, Maryland, USA). pEAK10-Gqi5 directs the overexpression of the chimeric G protein Gqi5, allowing enhanced cytosolic calcium flux responses from Gi-coupled receptors (12
). pEAK10-Gqi5 was constructed by ligating a HinDIII/NotI fragment from plasmid pLEC-Gqi5-HA (Molecular Devices Corp., Sunnyvale, California, USA) into HinDII/NotI–digested pEAK10 (Edge BioSystems Inc.).
HEK 293 cells stably expressing human (293MR) or murine (293M3X) CCR1 were grown to confluent monolayers in T-225 square-centimeter flasks as described previously (14
). Cells were tested for their ability to bind 125
I-labeled MIP-1α/CCL3 and biological responses by changes in intracellular Ca2+
. The binding assays were performed in transfected cells by oil centrifugation methods as described previously (15
). Nonspecific binding was determined in the presence of either 100 nM or 1 μM unlabeled ligand. The binding data were curve fitted with the computer program IGOR (Wavemetrics, Lake Oswego, Oregon, USA) to determine the affinity and number of sites.
Cytosolic Ca2+ measurements.
Cytosolic Ca2+ measurements in HEK 293 cells overexpressing human and murine (293M3X pEAK10-Gqi5) CCR1 were carried out as follows. Cells were lifted from flasks using an enzyme-free cell dissociation buffer (PBS based; Life Technologies Inc.). The cells were then loaded with fluo-3 by resuspension at 1 to 2 × 106 cells/ml in HBSS, 20 mM HEPES (all Life Technologies Inc.), 3.2 mM CaCl2, 1% heat-inactivated FBS, 2.5 mM probenecid, 0.04% Pluronic F-127, 4 μM fluo-3, pH 7.4, and incubated at 37°C for 1–1.5 hours. The cells were pelleted by centrifugation and resuspended at 106 cells/ml in HBSS, 20 mM HEPES, 1 mM CaCl2, 1% heat-inactivated FBS, 2.5 mM probenecid, pH 7.4, and kept at 37°C for analysis. Chemokine-induced cytosolic calcium transients were collected using a thermostated Deltascan 4000 fluorometer running FeliX version 1.41 (Photon Technologies International Inc., Severna Park, Maryland, USA).
C57BL/6 mice 12 ± 2 weeks of age were obtained from Charles River Deutschland GmbH, Sulzfeld, Germany. All mice were kept in macrolone type III cages under a 12-hour light and dark cycle. Water and standard chow (Sniff, GmbH, Soest, Germany) were available ad libitum.
Determination of pharmacokinetic parameters.
Male mice were subcutaneously dosed with BX471 (20 mg/kg) in a vehicle. The vehicle solution was prepared as follows: 40% cyclodextrin in (#33,260-7; Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) unbuffered saline was mixed, dissolved overnight, and filtered through a 0.45-μm filter. BX471 was added at a concentration of 20 mg/ml, pH 3.3. After complete dissolution, the pH was raised using 1 N KOH to pH 4.5 and filtered as above. Blood samples were withdrawn at various times, and plasma was stored frozen until analyzed. Plasma samples were analyzed either by HPLC using ultraviolet detection methods as described previously (13
Unilateral ureter ligation.
UUO was performed as described previously (8
). Mice were sacrificed 10 days after UUO. Eight mice were sacrificed at day 6. Contralateral kidneys served as intraindividual control. All experimental procedures were performed according to the German animal care and ethics legislation and were approved by the local government authorities.
In group I, control mice were treated subcutaneously with 50 μl of the vehicle for 10 days at 8-hour intervals. The first dose was administered immediately after UUO. Group II mice were treated with 50 mg/kg BX471 in 50 μl vehicle as above from day 1 to 5 at 8-hour intervals (9
). In group III C57BL/6 mice received BX471 in vehicle from day 6 to 10, and in group IV C57BL/6 mice were treated with BX471 in vehicle from day 1 to 10.
From each mouse the obstructed and contralateral kidneys were fixed in 4% formalin in PBS and embedded in paraffin. Two-micrometer sections were stained with periodic acid-Schiff reagent and silver following the instructions of the supplier (Bio-Optica, s.p.a., Milano, Italy). To count interstitial cells 12 high-power fields (hpf’s; ×400) were analyzed by a blinded observer. Positive cells were counted per hpf omitting positive cells in glomerular fields. Quantitation of the interstitial volume was performed as described previously (8
). In brief, the interstitial volume index (IInt
) was determined by superposing a grid containing 100 (10 × 10) sampling points on photographs of 12 nonoverlapping cortical fields of silver-stained tissue (×400) of each kidney. The number of points overlying interstitial space were counted. The indices of interstitial collagen deposition (Icol
), tubular cell damage (ITCD
), and tubular dilatation (ITdil
) were assessed accordingly (8
All immunohistological studies were performed on paraffin-embedded sections as described (8
). The following rat and rabbit Ab’s were used as primary Ab’s: rat anti-mCD45 (leukocytes, 1:100; PharMingen, San Diego, California, USA), rat anti-F4/80 (macrophages, 1:50; Serotec Ltd., Oxford, United Kingdom), rat anti-CD3 (lymphocytes, 1:50, Serotec Ltd.), rat anti-FSP1 (activated fibroblasts, 1:500; from F. Strutz) (16
Isolation of renal cells for FACS analysis.
A preparation of isolated renal cells including infiltrating leukocytes was obtained from obstructed and contralateral kidneys, as described previously (8
). The resulting supernatant and blood samples taken from anesthetized mice by retrobulbar puncture were then labeled for flow cytometry. Samples were incubated with 5 μg/ml of mAb’s against murine CCR2, murine CCR5, or the isotype control rat IgG2b (PharMingen), as described (17
). To identify leukocyte subsets samples were incubated with the following directly conjugated cell-specific Ab’s: CD11b fluorescein-isothiocyanate (clone M1/70), CD4 allophycocyanin, and CD8 CyChrome (all PharMingen). Approximately 100,000 gated events were collected in each analysis.
Real-time quantitative RT-PCR.
From each animal aliquots of both kidneys were snap-frozen in liquid nitrogen and stored at –80°C. RNA preparation and real-time RT-PCR on a TaqMan ABI 7700 Sequence Detection System (PE Biosystems, Weiterstadt, Germany) was performed as described (18
). Controls consisting of ddH2
O were negative for target and the housekeeper, GAPDH. The following oligonucleotide primers (300 nM) and probes (100 nM) were used: murine collagen I α1 (gb X 54876; bp 1984–2102): sense, 5′-TGCTTTCTGCCCGGAAGA-3′, antisense,5′-GGGATGCCATCTCGTCCA-3′, internal fluorescence-labeled probe (FAM), 5′-CCAGGGTCTCCCTTGGGTCCTACATCT-3′; murine GAPDH (gb M32599; bp 730–836): sense, 5′-CATGGCCTTCCGTGTTCCTA-3′, antisense 5′-ATGCCTGCTTCACCACCTTCT-3′, internal fluorescence-labeled probe (VIC), 5′-CCCAATGTGTCCGTCGTGGATCTGA-3′. All probes were obtained from PE Biosystems.
Kidneys were homogenized in RIPA buffer (Roche Molecular Biochemicals, Mannheim, Germany). Extracted proteins were boiled in loading buffer for 10 minutes, resolved by 8% SDS-PAGE, and transferred to an Immobilon-P membrane (Millipore, Eschborn, Germany). After blocking, the filter was incubated with the rabbit polyclonal anti-collagen I Ab (1:1,000; Chemicon International, Temecula, California, USA), and immune complexes were visualized using a peroxidase-conjugated Ab (1:5,000 in blocking solution; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA) and then processed for detection by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech Europe, Freiburg, Germany).
Data were expressed as mean plus or minus SD. Comparison of groups was performed using univariate ANOVA, and post hoc Bonferroni’s correction was used for multiple comparisons. Paired Student t test was used for the comparison of single groups (RT-PCR and FACS data). A P value of less than 0.05 was considered to indicate statistical significance.