Animal experiments
Animal maintenance and experimental procedures were approved by the Animal Care Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China). Thirty-four MRL/lpr mice aged 11 weeks were introduced and cultivated by the Shanghai Experimental Animal Center of the Chinese Academy of Sciences from the Jackson Laboratory (USA), and 10 male C57BL/6 mice of the same age served as normal controls. For studies involving the role of anti-P-selectin mAb in LN, MRL/lpr mice were randomly divided into the following three groups: MRL/lpr, saline, and anti-P-selectin groups, which consisted of no treatment, treatment with normal saline, and treatment with anti-P-selectin mAb (clone RB40.34, IgG1λ; BD Biosciences, San Diego, California, United States) intraperitoneally at 2 mg/kg body weight twice a week from 12 to 16 weeks of age, respectively. Mice were housed in a specific pathogen-free room at a constant temperature of 22 ± 2 ℃ and a constant humidity of 50 ± 5% under a 12-h day/night cycle. Mice were given free access to chow and water.
Physiologic parameters
Before the mice were euthanized, they were provided water ad libitum, and 24-h urine was collected in metabolic cages. The urinary albumin concentration was measured using a mouse albumin enzyme-linked immunosorbent assay (ELISA) quantitation set (Bethyl Laboratories, Inc., Montgomery, TX, USA). The urinary creatinine concentration in the same sample was measured using the QuantiChrom™ Creatinine Assay Kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s protocol. The serum was collected through the post–glomus venous plexus after anesthetization; anti-dsDNA (FUJIFILM Wako Shibayagi Corporation, Japan) and complement C3 (Icllab, Portland, OR, USA) levels were measured with ELISA kits according to the manufacturer’s protocol.
Kidney histopathology
The kidneys were removed from anesthetized mice and immediately fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm. The sections were stained with periodic acid–Schiff (PAS). PAS micrographs were observed by two renal pathologists independently to estimate semi-quantitative scores for glomerular and tubulointerstitial areas according to the area affected by the lesion: 0 (no), 1 (≤ 25%), 2 (25% to 50%), 3 (≥ 50%), in which glomerular 0–9 points, including mesangial cell proliferation 0–3 points; mesangial matrix hyperplasia 0–3 points; sclerosis 0–3 points; tubulointerstitial 0–9 points, including renal tubular atrophy 0–3 points; interstitial fibrosis 0–3 points; interstitial cells infiltration 0–3 points.
Immunohistochemistry staining
One hour before the sacrifice of mice, the hypoxia probe Hypoxyprobe™-1 (Hypoxyprobe, Inc., Burlington, MA, USA) at 60 mg/kg body weight was injected intraperitoneally, and the intensity and distribution area were analyzed by immunohistochemical (IHC) staining according to the manufacturer’s protocol. IHC staining was performed on paraffin-embedded kidney sections following standard procedures by incubating the sections in a primary antibody against P-selectin (Cell Signaling Technology, Inc., Danvers, MA, USA), hypoxia-inducible factor 1α (HIF-1α; Novus Biologicals, Inc., Littleton, CO, USA), and CD31 (Abcam, Cambridge, MA, USA) at 4 ℃ overnight. After washing, the sections were incubated with biotinylated secondary antibodies, followed by incubation with an avidin–biotin–peroxidase complex for DAB substrate development using the ABC kit (Vector Laboratories, Burlingame, CA, USA) at room temperature, and they were mounted using Aqua PolyMount (Polysciences, Inc., Warrington, PA, USA). Images were acquired using a Leica DM1000 microscope with a digital camera. The results of immunohistochemistry were determined by two renal pathologists independently according to the distribution of light staining in renal tissue with a semi-quantitative score of 0–3, in which 0 indicates negative staining; 1 point, occasional positive staining; 2 points, focal positive staining; and 3 points, diffuse positive staining.
Western blot analysis
Renal cortical tissues were ground and lysed, and cells were collected and lysed in RIPA buffer containing protease inhibitor cocktail. Equal amounts of protein samples were loaded on sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore, MA, USA), probed with antibodies, and visualized with the Luminescent Imaging Workstation (Tanon, China). The band intensities were quantified using ImageJ. Mouse anti–β-actin antibody (Sigma, MA, USA) was used as loading control.
Renal BOLD-MRI examination
Mice were fasted for 4 to 8 h before BOLD-MRI. After anesthetization, mice were placed in a prone position. The limbs were fixed, and the animal-specific eight-channel high-resolution head coil was placed. The T1WI, T2WI, DWI, and BOLD images of kidney cross section were scanned using a GE Signa Excite 3.0T MR scanner. For cross-sectional T1WI and T2WI imaging, sequences included fast spin echo (FSE) T1WI [repetition time (TR)/echo time (TE) 540/12.7 ms], FSE T2WI (TR/TE 4000/46.2 ms), layer thickness 2.4 mm, layer spacing 0.2 mm, field of view (FOV) 6 cm × 6 cm, and matrix 256 × 224. The scope of the scan included both kidneys. For cross-sectional BOLD imaging, sequences included multiecho fast gradient-echo sequence, 8 gradient echoes, TR 110 ms, TE 2.0–27.5 ms, layer thickness 2 mm, and spacing 0.2 mm, with 5 levels of coronal imaging centered on the renal hilum. Parameters were set as flip angle 20°, bandwidth 125, number of excitations (NEX) 1, FOV 15 cm × 15 cm, and matrix 224 × 192 (8 frames per layer). The data were transferred to the ADW4.2 workstation, and the R2* values of the renal cortex and medulla were measured by Functool software on the image near the renal hilum. The R2* value measurement method consisted of the following: the window width/window position of the R2* image was adjusted to about 60.0/5.0, and the R2* color map of the kidney was used to measure the R2* value of the renal cortex. The region of interest (ROI) was posited on the clear boundary of the cortex and medulla manually, and 3 ROIs were placed on each side of the kidney cortex and medulla to calculate their average [11].
Statistical analyses
The group data were expressed as the mean ± standard error of the mean (SEM). Comparisons between the two groups were performed using an unpaired t test after determining the distribution and variance of the data. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used when more than two groups were present. All tests were two-tailed, and P < 0.05 was considered to be a statistically significant result.