Mice, study design, and follow-up
Five-month-old NZB/WF1 mice (JAX®; Charles River Laboratories, Barcelona, Spain) were randomly assigned to four groups. At 6 months of age, treatment was initiated as follows, by group: intraperitoneal cyclophosphamide (CYP) 50 mg/kg/10 days (n = 14), subcutaneous CP-690,550 (CP, XELJANZ® [tofacitinib]; Pfizer, New York, NY, USA) 48 mg/kg/day (n = 8), oral mycophenolate mofetil (MMF, CellCept®; ROCHE FARMA, Madrid, Spain) 30 mg/kg/daily (n = 8), and subcutaneous PBS treatment (n = 14) as a control group. Mice were treated for 12 weeks. Their body weight was determined twice monthly from the beginning to the end of follow-up. Mice were placed in metabolic cages to collect 24-h urine specimens before the onset of treatment, and monthly thereafter. Blood was obtained from the tail vein at monthly intervals. Kidneys were processed for histological and biochemical studies at the end of the study or at death.
The experiments were carried out in accordance with current European Union legislation on animal experimentation and were approved by the animal experimentation ethics committee, the University of Barcelona Institutional Ethics Committee for Animal Research, and the Animal Experimentation Commission of the Generalitat de Catalunya (Catalonian government). The mice were housed in a room at constant temperature with a 12-h dark/12-h light cycle, were given free access to water, and were fed a standard laboratory diet.
Proteinuria, albuminuria, and renal function
Twenty-four-hour urinary protein was determined by Pyrogallol red reaction (AU400 Clinical Chemistry System; Beckman Coulter, Brea, CA, USA) in the Veterinary Clinical Biochemistry Laboratory of Universitat Autonoma de Barcelona. Twenty-four-hour urinary albumin was determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions.
RNA extraction, reverse transcription, and gene expression analysis using quantitative real-time polymerase chain reactions
For molecular studies, the kidney was immediately snap-frozen in liquid nitrogen and stored at −80 °C. RNA was extracted from kidneys using the PureLink™ RNA Mini Kit (Ambion/Thermo Fisher Scientific, Barcelona, Spain) according to the manufacturer’s instructions. RNA purity was analyzed using a NanoDrop ND-1000 V3.3 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA was stored at −80 °C. A total of 500 ng of RNA was used for reverse transcription with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Warrington, UK) in accordance with the manufacturer’s instructions. Tissue expression levels of the following mediators of immunity and inflammation were quantified by TaqMan real-time polymerase chain reaction (ABI Prism® 7700, Applied Biosystems, Spain) using the comparative Ct method: complement component 3 (C3), chemokine (C-C motif) ligand 2 (CCL2), CCL5, CD40L, IL-2, IL-6, Toll-like receptor 9 (TLR9), vascular cell adhesion molecule (VCAM)-1, STAT1, STAT2, STAT3, STAT4, and STAT5a.
Plasma ELISA for anti-DNA antibodies and serum cytokine analysis
Levels of anti-DNA antibodies were measured using a commercially available ELISA kit (Alpha Diagnostic International, San Antonio, TX, USA) according to the manufacturer’s instructions. Serum IL-12, TNF-α, IFN-γ, and monocyte chemoattractant protein (MCP)-1 cytokines were measured using the BD FACSCanto flow cytometer with a cytometric bead array kit (BD CBA mouse inflammation kit) according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA). Data were acquired and analyzed using BD FCAP software and CBA software (BD Biosciences). Serum IL-17 (R&D Systems, Minneapolis, MN, USA) and IFN-α (PBL Assay Science, Piscataway, NJ, USA) cytokine levels were quantified using commercially available ELISA kits according to the manufacturers’ instructions.
Renal lupus histopathology
For histological analyses, 1- to 2-mm-thick coronal slices of kidney tissue were fixed in 4 % formaldehyde and embedded in paraffin. For light microscopy, 3- to 4-μm-thick tissue sections were stained with hematoxylin and eosin stain and periodic acid-Schiff stain. To determine the extent of renal damage, two blinded pathologists analyzed all kidney biopsies. Typical glomerular active lesions of LN were evaluated: mesangial expansion, endocapillary proliferation, glomerular deposits, extracapillary proliferation, and interstitial infiltrates, as well as tubulointerstitial chronic lesions, tubular atrophy, and interstitial fibrosis. Lesions were graded semiquantitatively using a scoring system from 0 to 3 (0 = no changes, 1 = mild, 2 = moderate, and 3 = severe). Finally, a total histological score was derived from the sum of all the described items.
Paraffin-embedded tissue sections were also stained for CD3 (Abcam, Cambridge, UK). The sections were blocked and labeled with immunoperoxidase using a VECTASTAIN ABC kit and an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s protocol. Peroxidase-conjugated antibody staining was followed by diaminobenzidine substrate development (Sigma-Aldrich, Madrid, Spain).
Renal immunofluorescence studies
Slices of kidney were fixed in 4 % paraformaldehyde, embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek, Alphen aan Den Rijn, the Netherlands), and stored at −80 °C. Fluorescent staining of 5-μm cryostat sections was used for confocal microscopy to quantify glomerular immunoglobulin G (IgG) and C3 deposition. Sections were directly stained with fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (Sigma-Aldrich) and FITC-conjugated C3 (Nordic-MUbio, Susteren, the Netherlands). For analysis of C3 and IgG deposition, at least ten glomeruli were visualized and photographed with an immunofluorescence confocal microscope (Leica TCS SL spectral confocal microscope; Leica Microsystems, Mannheim, Germany). Fluorescence was quantified and normalized with Simulator-Leica confocal software (Leica Microsystems) and expressed as mean fluorescence intensity.
To quantify kidney macrophages, F4/80 immunohistochemistry was used. Briefly, 3-μm-thick kidney tissue sections embedded in paraffin were incubated with primary antibody antimouse F4/80 (eBioscience, San Diego, CA, USA) and incubated overnight at 4 °C. Staining was visualized using a secondary Alexa Fluor 546 dye (Molecular Probes/Thermo Fisher Scientific, Eugene, OR, USA). Nuclei were stained blue with DRAQ5 (eBioscience). To determine macrophage infiltration, at least 15 high-power fields were counted, and the number of positive cells was determined and expressed as a mean value.
Statistical analysis
Overall survival was analyzed with the Kaplan-Meier method. One-way analysis of variance with post hoc tests were performed to compare proteinuria and anti-dsDNA antibodies throughout the follow-up, and gene expression and circulating cytokines were evaluated at the time the mice were killed. To compare histological data, a nonparametric Kruskal-Wallis test was used. A p value <0.05 was considered significant. Data are expressed as mean ± SEM.