Use of intravoxel incoherent motion imaging to monitor a rat kidney chronic allograft damage model

This study was approved by the Ethics Committee of Capital Medical University (approval: AEEI-2017-013). The experimental animals were 8–12 weeks old, specific pathogen-free (SPF) grade, male, inbred Fisher and Lewis rats with body weights of 200–250 g. The rats were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (permit number: SCKX Jing 2012–0001). The animals were housed at 22 ± 1 °C at a relative humidity of 55 ± 5% under 12 h light-dark cycles and were fed a standard rat diet. The animals were under the protection of the U.K. Animals (Scientific Procedures) Act 1986 and associated guidelines (EU Directive 2010/63/EU) for animal experiments. The Guide for the Care and Use of Laboratory Animals (NRC 2011) was also obeyed. The hematoxylin and eosin (HE) staining reagent kit, periodic Schiff-methenamine silver (periodic acid-silver methenamine, PASM) staining solution, and Masson’s trichrome staining solution were purchased from Techlab Biotechnology Co., Ltd. (Beijing, China). The anti-alpha smooth muscle actin (α-SMA) and anti-vimentin antibodies were purchased from Abcam Limited Corporation (Cambridge, UK).

A total of 20 Fisher and 20 Lewis rats were used to establish the CAD model in the experimental group. The following animal experiments were carried out in animal laboratory at night. The rats were anesthetized with isoflurane in oxygen. General anesthesia was induced with 3% and maintained with 1.5% isoflurane. The left kidney of healthy Fisher rats was used as the donor kidney. The left kidney of Lewis rats was resected, and orthotopic transplantation of the donor kidney was performed on the left side. The rats received subcutaneous injections of low-dose cyclosporine at a dose of 1.5 mg/kg/day on the day of surgery and days 1–10 after surgery. The right original kidney was resected 7 days after surgery. In the control group, 40 Lewis rats were used to establish the orthotopic syngeneic kidney transplantation model. The same anesthesia procedure was adopted, and male Lewis rats in the same litter were used as the donors and recipients. After surgery, the same treatment was performed as described for the experimental group. The donor rats were euthanized by adjusting the CO₂ flow rate in the cage to 5 L/min until one minute after breathing stopped.

Tail vein blood samples were extracted from all rats for assessment of transplanted kidney function. The measurement was performed once every 2 weeks. The blood creatinine concentration (mmol/L) was measured using the picric acid method with the Dimension Rxl Max automatic biochemical analyzer (Siemens, Newark, DE, USA).

After the MRI examinations, 5 rats from each group were randomly selected using the random number table method at postoperative weeks 4 and 12. All rats were euthanized using the same method described for the donor rats. After the rats were sacrificed, the kidneys were removed immediately, the kidney capsule was removed, and the kidneys were thoroughly washed with ice-cold normal saline. The transplanted kidneys were longitudinally dissected from the coronal plane of the renal hilum and placed in a 10% neutral-buffered formalin solution to fix for 24 h. All acquired kidneys were embedded in paraffin, sectioned (3-μm thickness) and stained with HE, Masson’s stain and PASM. Immunohistochemistry-stained sections were also fixed, embedded and sectioned using the same methods. The sections were placed in 3% H₂O₂ to block endogenous peroxidase, followed by pH 6 citric acid buffer, heated at 98 °C in a microwave oven for 15 min, and then cooled for antigen retrieval. The primary antibody was dropped onto the sections (1100 antibody dilution), incubated at 4 °C overnight and washed with phosphate-buffered saline (PBS) for 5 min × 3 times. Each section was incubated with 50 μL of horseradish peroxidase (HRP)-labeled avidin delivered in a dropwise manner, incubated at 37 °C for 30 min and washed 3 times with PBS. Each section received freshly prepared diaminobenzidine (DAB) solution in a dropwise manner, and the reaction was observed and controlled under a microscope. After the nuclei were stained, the sections were mounted using neutral balsam.

Routine pathological sections and immunohistochemistry-stained sections were independently observed under a microscope by 2 senior pathologists. Routine pathological sections of kidney specimens were scored using the chronic allograft damage index (CADI). The CADI scoring system includes 6 sections of CAD pathology (renal interstitial inflammation, renal interstitial fibrosis, renal tubular atrophy, increased basement membrane matrix, glomerular sclerosis and arterial intimal hyperplasia). The sections are scored according to the degree of pathological changes as follows: none, 0; mild, 1; moderate, 2; and severe, 3. The CADI is sum of the scores of all 6 sections [10]. For analysis of the immunohistochemistry-stained sections, a known positive section was used as the positive control, and α-SMA and vimentin were quantitated according to the immunohistochemistry staining range. Five fields per specimen were observed under a 100× microscope using the double-blind random method.

No deaths occurred among the rats in the experimental and control groups. All rats completed all experiments without any adverse event. The differences in blood creatinine between the rats in the experimental and control groups were not significant until 8 weeks (P<0.05). Compared to that of the control group, the creatinine level in the experimental group gradually increased (Fig. 1).

MRI examinations were successfully performed in all animals. The locations of the transplanted kidneys were more fixed than those of the original kidney, which might be due to the surgical procedure (Fig. 2).

Kidney tissue staining and immunohistochemistry sections from the different groups were observed under a light microscope. Compared with those of the control group, the experimental group already exhibited pathological changes of CAD at 4 weeks after modeling, which included mild proliferation of small arterial intima combined with a small amount of monocyte infiltration, glomerular focal sclerosis, renal tubular atrophy and interstitial fibrosis in the transplanted kidneys. The CAD pathological changes were gradually aggravated over time. Small arterial intimal hyperplasia was gradually aggravated until lumen occlusion developed, monocyte infiltration gradually increased, glomerular sclerosis, tubular atrophy, and interstitial fibrosis were gradually aggravated, and the CADI gradually increased (Fig. 5). The above findings were consistent with the pathological features of CAD and indicated that the CAD rat model was successfully established.

The graft interstitial α-SMA and vimentin expression levels in the experimental group gradually increased over time. The quantitative analysis showed that the differences were statistically significant (P<0.05) (Fig. 6).

The linear correlation analysis showed that the DWI parameter ADC (r = − 0.849, P < 0.01; r = − 0.874, P < 0.01; and r = − 0.875, P < 0.01) and IVIM parameters D (r = − 0.665, P < 0.01; r = − 0.702, P < 0.01; and r = − 0.764, P < 0.01) and f (r = − 0.941, P < 0.01; r = − 0.905, P < 0.01; r = − 0.853, P < 0.01) were negatively correlated with the CADI and the α-SMA and vimentin expression levels, respectively (Table 2).