Animal research was approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical University. Naive inbred male rats weighing 200-250 g (Experimental animal center of China, Peking) were used throughout the experiments. Lewis (LEW) rats acted as graft recipients and Fisher (F334) rats as donors. They were housed under standard conditions at controlled temperature, humidity and light/dark cycles, fed a standard diet and had free access to tap water.
Cyclosporine A (Novartis Pharma AG, Basel, Switzerland) was dissolved in cremophor and administered subcutaneously.
Operative procedures were performed under general anesthesia induced by ketamine (100 mg/kg body weight; Ketamin 2%, Huadong-Pharma, Shanghai, China) administered intraperitoneally. The left donor kidney was isolated, removed, cooled and positioned orthotopically into the host whose left renal vessels had been isolated, clamped, and the native kidney removed. Donor and recipient renal artery, vein, and ureter were then anastomosed end-to-end with 10–0 prolene sutures. No ureteral stent was used. Mean ischemia time was 25 minutes (ranges 22–31min). The remaining right kidney of the recipient was removed on day 10, at which time the transplanted kidney was checked for surgical damage. Rats with any overt signs of unsuccessful operation were discarded.
Ten male LEW rats received left renal transplantation orthotopically from ten male F334 donors. To suppress acute rejection, rats were treated with low dose CsA (1.5 mg/kg/day) for the first 10 days. Ten uninephrectomized male F334 rats and ten uninephrectomized male LEW rats were used as control animals.
At week 12, rats were put into metabolic cages and 24 hour urine samples were collected and quantitative proteinuria was determined. At the same time, serum and urine creatinine were measured and creatinine clearance was calculated.
At week 12, recipients and controls were sacrificed and the transplanted kidney removed. Only kidneys without apparent complications of grafting such as pyelonephritis or hydronephrosis were evaluated. Representative portions of the kidneys were snap-frozen in liquid nitrogen and stored at −80°C for PCR analysis or fixed in formalin (4%) for histological and immunohistological evaluation.
Formalin-fixed and paraffin embedded sections were stained with hematoxylin/eosin to assess the grade of cellular infiltration and tubular atrophy. Periodic acid-Schiff (PAS) reaction was used to evaluate the extent of glomerulo- and arterio-sclerosis. Tissue sections were coded and examined in a blinded fashion by light microscopy. At least 200 glomeruli were counted in each section. Renal structural damage was scored semiquantitatively on a scale from 0 to 3+ for interstitial cellular infiltration, tubulopathy, glomerulopathy and arterial intimal fibroplasia using the Banff criteria [16
] and the sum of scores (0–12+) was calculated for each sample.
Goat anti-rat polyclonal antibodies MMP-9 and TGF-beta1 are purchased from Santa Cruz (U.S.A.) and for secondary and tertiary staining from DAKO (Denmark).
The antigen on the formalin-fixed and paraffin embedded sections (2 μm) was restored by microwave, and incubated with primary antibodies as mentioned above using the LSAB techniques. The intensity of tissue staining for MMP-9 and TGF-beta1 was evaluated in a blinded manner by calculating the relative stained area using a computerized pathological image analytical system.
Images were acquired by means of Leica DMR-X microscope coupled to a Leica DC500 digital camera (Leica, Wetzlar, Germany) and the image analysis system Quantimet Q550 (Leica Imaging Systems). Ten randomly selected discontinuous fields (400 ×) per kidney were evaluated, including tubulointerstitial in renal cortex, medulla and the conjunction region. More than 60 tubules in each biopsy section were observed. The positive area was yellow staining and Image-Pro Plus software was used to quantify the integrated optical density. There were four classed intensity of staining (negative vs. pale yellow vs. pale brown vs. russet) and four classed staining area (staining area < 25% vs. staining area 25–50% vs. staining area 50–75% vs. staining area > 75%). product of intensity and relevant staining area were used for statistical analysis.
Total RNA was extracted from the representative portions of the kidneys using Trizol (Gibco/BRL, U.S.A.), which is based on the method described by Chomczynski and Sacchi [17
Representative portions of the kidneys samples were stored in −80°C. 60–100 mg frozen tissue samples were homogenized in 1ml Trizol. 200 μl chloroform was added to each mixture. The mixture was centrifuged at 12,000 g for 15 min at 4°C. The supernatant was treated with 0.5 ml isopropanol. The mixture was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was discarded, and 0.75 ml 75% ethanol was added, and then centrifuged at 7,500g for 5 min at 4°C. The precipitate containing total RNA was stored at −80°C until further processing. RNA concentration was measured spectrophotometrically.
Total RNA was transcribed to complementary DNA(cDNA) by reverse transcription (RT) with hexamer random primers (Promega, U.S.A). 1 μg of total RNA was added to 0.5 μg of primer. A reaction mixture containing MMLV reverse transcriptase (Promega), buffer solution (Promega), dNTP (Genda, Canada) in a concentration of 0.2 mM/L, and 25U recombinant ribonuclease inhibitor (Huamei, China). The reaction allowed to proceed at 37°C for 60 minutes, and was stopped by heating to 95°C for 5 minutes followed by cooling on ice. The cDNA was stored at −30°C for further procedure.
Amplification of specific complementary DNA (cDNA)
Specific cDNA products corresponding to mRNA for rat β-actin, MMP-9 were amplified using the polymerase chain reaction (PCR). 1 μl of cDNA was taken for PCR, which was performed in PCR buffer (Genda), using 0.2 mM/L dNTP (Genda), 1 μM/L of both primers (Genda), and 2.0 U Taq DNA polymerase (Genda). GeneAmp2700 Thermal Cycler (U.S.A) was used for amplification with the following sequence profile: initial denaturation at 95°C for 5 minutes followed by relevant number of cycles of three temperature PCR (denaturing, 94°C for 30 seconds; annealing, 65°C for 30 seconds; and extension, 72°C for 30 seconds) ending with a final extension at 72°C for 7 minutes and cooling to 4°C.
The primers sequence for
up 5′-ATGGTGGGTATGGGTCAGAAGG-3′ (located at exon 2),
down 5′-GTACATGGCTGGGGTGTTGAAGG-3′ (located at exon 4).
The length of the amplified segments was 270 bp;
up 5′-ATCGACTCCAGTAGACAATCC-3′ (located at exon 9)
down 5′-CAGAGAACTCGTTATCCAAGCG-3′ (located at exon 12)
The length of the amplified segments was 443 bp.
The amplified PCR product was identified by electrophoresis of 10 μl sample on 1.5% agarose gel stained with 0.5 μg/ml of ethidium bromide. Sample products were visualized by UV transillumination and the gel was photographed. Specific products were identified by size in relation to a known DNA MARKER (Takara Bio, Dalian, China) run with each gel. MMP-9 cDNA was semiquantitatively analyzed by densitometric comparison to β-actin (internal control) from the same sample after the positive image was digitized by video for computerized densitometry. The results are given as a ratio of intensity of MMP-9 and β-actin mRNA.
All data were expressed as mean ± SD. Statistic analysis was performed with statistical software (SPSS 13.0, Chicago, IL), using One-Way ANOVA and bivariate correlation analysis. Generally p values under 0.05 are considered significant.