Nephrin

Immunohistochemical analysis was performed on 4-mm-thick paraffin-embedded kidney sections. After dewaxing and rehydrating, endogenous peroxidase was quenched for 10 min with 0.3% H₂O₂ in methanol. Heat-mediated antigen retrieval and enzymatic techniques were performed according to recommendations for the specific antibodies. A blocking step was performed using 10% normal goat serum. The kidney sections were incubated overnight at 4 °C with anti-collagen IV (Abcam, Cambridge, MA, USA), nephrin (Santa Cruz, CA, USA), TGF-β1 (Santa Cruz) or WT-1 (Abcam) primary antibodies. After washing with TBS and incubation with biotinylated secondary antibodies and Vectastain ABC reagent (Vector Laboratories Inc., Burlingame, CA, USA), the samples were visualized using diaminobenzidine staining and counterstaining with hematoxylin. Negative controls had the primary antibodies omitted. Sections were examined using a Nikon Eclipse 80i microscope (magnification, ×400). Immunostaining signals were highlighted and quantified using Image-Pro Plus 6.0 software. Areas with positive staining were quantified and expressed as a percentage of the entire area (n = 20–30).

Slides were immunostained by Invitrogen’s Histostain-SP kits using the Labeled-Strept-avidin-Biotin (LAB-SA) method. Slides were immersed in 3% hydrogen peroxide to block endogenous peroxidase activity. Slides were incubated with primary antibodies of collagen IV (Abcam), TGF-β1, ICAM-1, and Nephrin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All slides were incubated with biotinylated secondary antibodies and horseradish peroxidase-conjugated streptavidin. The detection was visualized using chromogen and counterstaining with 3-6-ethylcarbazole (AEC) followed with hematoxylin (Zymed, Camarillo, CA, USA).

Immunofluorescent staining was performed as previously described [Alcendor 2017] in chamber slide cultures containing mock and BKV-infected GVU cells (podocytes, mesangial cells, and glomerular endothelial cells). Briefly, cells were washed twice with PBS, pH 7.4, air- dried, and fixed in absolute methanol for 20 min at −20 °C. Next, cells were air-dried for 10 min, hydrated in Tris-buffered saline (TBS) (pH 7.6) for 10 minutes, and incubated separately for 1 h with monoclonal antibodies to the BKV major capsid protein VP1 (Santa Cruz Biotech, Temecula, CA, USA), von Willebrand factor (Santa Cruz Biotech), nephrin (Santa Cruz Biotech), and SV40 Large T-Antigen (Abcam), all at a dilution 1:50 in PBS pH 7.4 [30 (link)].

Murine renal tissue from each group was fixed for immunohistochemical staining in 10% neutral formalin, processed in the standard manner, and cut into 3 μm sections. Slides were deparaffinized, hydrated in ethyl alcohol, and washed in tap water. Then, the slides underwent antigen retrieval and blocking with 5% BSA. To assess RANKL, RANK, and nephrin in renal tissues, the sections were incubated with an anti-RANKL polyclonal antibody (RANKL, 1 : 50, Santa Cruz, CA, USA), an anti-RANK polyclonal antibody (RANK, 1 : 50, Santa Cruz, CA, USA), or an anti-nephrin polyclonal antibody (nephrin, 1 : 50, Santa Cruz, CA, USA) overnight at 4°C. After washing, a secondary donkey anti-rabbit antibody was added for 30 minutes (min) at 37°C; the slides were then washed and incubated with DAB for 2 min before counterstaining with hematoxylin. Sections were viewed and imaged with a light microscope (Ni-U, Nikon Corporation, Tokyo, Japan). Images were analyzed quantitatively by Image-Pro Plus 6.0 (IPP, Media Cybernetics, Inc., USA).

FK506 was purchased from Fujisawa Pharmaceutical Co., Ltd (Osaka, Japan), STZ was purchased from Sigma Chemical Co (St. Louis, Mo, USA), microalbumin assay kit was purchased from Exocell Inc (Philadelphia, Pa., USA), and blood glucose assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-CaN, nephrin, podocin, phosphorylated NF-κB p65 (p-p65) and iNOS polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-macrophage monoclonal (ED-1) antibody was from Boster Biotechnology (Wuhan, China). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG as well as fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG was from Boster Biotechnology (Wuhan, China). Chemiluminescence kit was from Amersham Life Science (Little Chalfont, UK).