Antibodies
A hybridoma-producing mouse anti-rat Thy 1.1 mAb 1-22-3 (IgG3) was prepared by immunization of BALB/c mice with collagenase-treated fresh rat glomeruli. Ascitic fluid containing mAb 1-22-3 was produced in BALB/c mice primed with 2,6,10,14-tetramethylpentadecane (Sigma Chemical, St. Louis, MO) and injected intraperitoneally with the hybridoma. The obtained fluid was subjected to 50% ammonium sulfate (Kanto Chemical, Tokyo, Japan) precipitation, and the obtained immunoglobulin-rich fraction was dialyzed against phosphate-buffered saline (PBS, Kanto Chemical).
Animals and Ethics
Wistar rats were obtained from (Sankyo Laboratory, Tokyo, Japan).This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, 1996). Euthanasia for rats was performed with carbon dioxide inhalation. The carbon dioxide flow rate displace 30% to 70% of the cage volume per minute.
Preparation of DFAT cells from adipose tissue
Around one g of epidydymal adipose tissue from six male Wistar rats was treated with collagenase and centrifuged. Adipocytes were isolated from the top layer. More than 99% of the isolated cells were mature lipid-filled adipocytes. The mature adipocytes floating on top of the culture medium attached to the upper surface of the culture flasks within a few days. Approximately 10–20% of the adherent cells flattened out by day 3 and changed to a spindle-shaped morphology by day 7. The cells subsequently entered a proliferative log-phase upon inversion of the flasks and changing of the media and reached confluence by day 14. During this stage, the cells lose their lipid droplets completely and exhibit the fibroblast-like morphology of DFAT cells. Six different DFAT cells from six Wistar rats, and combined them as allogenic DFAT cells.
Experimental protocols (Figure 1)
Experiment 1: Distribution of DFAT cells. DFAT cells from Wistar rats were labeled with a Qtracker® Cell Labeling Kit (Molecular probes, Life Technology, Tokyo, Japan). In total, 106 labeled DFAT cells were injected through the tail vein in Wistar rats. At 3 hours and one week after the injection, kidney, aorta, liver, and lungs were removed and fixed in 3% formalin (Kanto Chemical) in PBS and embedded in paraffin.
Experiment 2: Effects of implantation of DFAT cells on mAb1-22-3-induced nephritis. In all male Wistar rats weighing 250 g, the right kidney was nephrectomized. Rats were injected with 1.0 mL of saline containing 0.5 mg of mAb 1-22-3 through the tail vein at 7 days after nephrectomy. Thirty-five days after the nephrectomy, 1.0 mL of saline or 106 DFAT cells in 1.0 mL of saline were injected through the tail vein. Fifty-six days after the nephrectomy, 5-bromo-2-deoxyuridine (BrdU, Sigma Chemical) (0.1 mg/g body weight) was injected through the tail vein. Sixty-three days after the nephrectomy, all rats were killed and the left kidney was removed. BrdU incorporation into kidney was determined at 28 days after implantation of allogenic DFAT cells.
Experiment 3: Effects of implantation of autogenic and allogenic DFAT cells on mAb 1-22-3-induced nephritis. In Wistar rats, the right kidney was nephrectomized. At 7 days after nephrectomy, rats were injected with 1.0 mL of saline containing 0.5 mg of mAb 1-22-3 through the tail vein. Thirty-five days after nephrectomy, 1.0 mL of saline or 106 autogenic DFAT cells prepared from identical Wistar rats or allogenic DFAT cells prepared from other identical Wistar rats in 1.0 mL of saline were injected through the tail vein. Sixty-three days after nephrectomy, all rats were housed in metabolic cages, and urinary protein excretion was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Serum blood urea nitrogen (BUN) and creatinine were measured by SRL, Inc. (Wako, Saitama, Japan). Serum levels of TSG-6, and expression of TSG-6 mRNA in kidney were determined at 28 days after implantation of DFAT cells. All rats were then killed and the left kidney was removed.
Determination of BrdU incorporation into kidney
Removed kidneys were immediately frozen in liquid propane cooled to −196°C by liquid nitrogen. Frozen kidney slices were cut into 4-µm-thick cryostat sections. Sections were rinsed 3×10 min in 50 mM NH4Cl (Kanto Chemical)/PBS to wash out the glutaraldehyde fixative and to reduce background staining. After pretreatment in 5% normal goat serum in PBS, the cryostat sections were incubated overnight in a humidified chamber at 4°C with mouse monoclonal anti-BrdU clone 3D4 (BD Biosciences Pharmingen, San Diego, CA, USA) 1∶300, diluted in PBS-1% BSA. After incubation with primary antibodies, sections were rinsed three times with PBS and covered for 1 h at room temperature in the dark with the appropriate secondary antibodies coupled to fluorescein isothiocyanate (FITC, Sigma Chemical).
Morphological and immunohistological analysis
The 3-mm paraffin sections of removed renal cortex were stained with hematoxylin and eosin. Renal cortical thickness was measured under high magnification (×400). The glomerular injury score (GIS) was obtained by the following formula: [(0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)]/50. To semi-quantify the tubulointerstitial area, 20 areas of renal cortex were randomly selected. The percentage of each area that showed sclerofibrotic change was estimated and assigned a score of 0, normal; 1, involvement of <10% of the area; 2, involvement of 10–30% of the area; 3, involvement of >30–50% of the area; or 4, involvement of >50% of the area. The tubulointerstitial injury score (TIS) was similarly calculated as [(0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)]/20.
Determination of serum TSG-6
Levels of TSG-6 in blood serum were detected with a multi-detection microplate reader using a double-antibody sandwich ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocols. The concentrations of TSG-6 were normalized to the total protein content.
RNA extraction and real-time PCR
Total RNA was extracted from renal cortex and medulla with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed into cDNA with random 9-mers with an RNA PCR Kit (AMV) Ver. 3.0 (Takara Bio, Ohtsu, Japan). Real-time quantitative PCR was performed with diluted cDNA using a FastStart TaqMan Probe Master (Roche Applied Science) and SYBR Select Master Mix (Life Technologies) in an ABI 7500 sequence detector (Life Technologies) according to the manufacturer’s instructions. All assay-on-demand primers and the TSG-6 probe were purchased from Life Technologies. Real-time PCR data were analyzed with standard curves and normalized to 18S ribosomal RNA with its specific primer sets (5′ and 3′ primers: 5′-CGGCTACCACATCCAAGGAA-3′ and 5′-GCTGGAATTACCGCGGCT-3′). Correlation coefficients for the standard curves were all > 0.90.
Statistics
Values are reported as the mean ± SE. Two-way ANOVA with the Bonferroni/Dunn procedure as a post-test was also used. A value of p < 0.05 was considered to be statistically significant.