Ifn γ

According to previous methods [58 (link)], ileum tissue from Gn pigs were fixed in buffered formalin, embedded in paraffin, and cut into serial sections (4 μm). Deparaffinized and rehydrated sections were boiled in 10 mM sodium citrate buffer (pH = 6.0) for 10 min. After washing twice with Tris-buffered saline with Tween-20 (TBST), sections were blocked with 10 % normal goat serum in TBST for 1 h at room temperature. Sections were incubated with primary IFN-γ (1:300 v/v, Cell Sciences, Canton, MA, USA), CD80 (1:200 v/v, Ancell, Bayport, MN, USA), p38, p-p38, ERK or p-ERK antibodies (1:300 v/v, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing three times with TBST, the HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) was added and the sections were incubated for 1 h at room temperature. The Diaminobenzidine-HRP detection system was added and sections were incubated at room temperature. All incubation steps were conducted in a humidified chamber. Sections were then counterstained with hematoxylin, dehydrated, and cover-slipped. Assessment of positivity of IHC staining [59 (link)] were conducted under a microscope (ECLIPSE Ti, Nikon Corp., Tokyo, Japan).

The immortalized mouse podocytes were developed by Prof. Dr. Karlhans Endlich, University of Heidelberg, Germany and kindly provided by Prof. Dr. Niels Olsen Saraiva Camara, Institute of Biomedical Sciences, University of Sao Paulo. As previously described^{42 (link)}, the cell culture was grown in 75-cm² flasks (Corning, New York, NY, USA) coated with type I collagen and maintained in RPMI-1640 medium (Thermo Fisher Scientific INC, St Peters, MO, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 30 IU/mL IFN-γ (Cell Sciences, Newburyport, MA, USA), 100 IU/mL penicillin, 100 μg/mL streptomycin and 2 mmol/L L-glutamine (pH 7.4 ) at 33 °C in a 5% CO₂ atmosphere. The culture medium was changed every two days until 85% confluence. Cells at passages 8 to 10 were detached by incubation with 5 mL trypsin-EDTA solution (Thermo Fisher Scientific, 0.05%) for 5 min at 37 °C. The cells were seeded for differentiation at specific cell densities into cell culture dishes with a diameter of 100 mm × 20 mm (Corning) in the same conditions, except for the absence of IFN-γ. The cells were differentiated for 10–15 days. Podocyte differentiation was confirmed by the identification of podocin and synaptopodin on protein expression. Phalloidin staining was used to identify the podocyte cytoskeleton.

Polarization of primary KCs and ImKCs was achieved by culturing cells for 24 h in media containing 20 ng/mL IFN-γ (Cell Sciences, Newburyport, MA, USA, #CRI001B) or 20 ng/mL IL-4 (BioLegend, San Diego, CA, USA, #574302) + 20 ng/mL of IL-13 (BioLegend, San Diego, CA, USA, #575902). Following polarization, media were removed and replaced with culturing media without cytokines and harvested for RNA or infected with the virus. Macrophage polarization was validated by qRT-PCR.