Synaptopodin

Human frozen kidney sections were fixed in ice-cold acetone and blocked using 5% normal goat serum solution for 1 h at room temperature. Mouse kidneys were fixed with 4% paraformaldehyde and subsequently embedded with paraffin. Paraffin-embedded kidney sections (4 μm) were processed with antibodies to the antigens described below. For immunofluorescent studies, after HIER treatment with Tris–EDTA pH 9 for 20 min at 100 °C, sections were blocked using 5% normal goat serum solution for 1 h at room temperature.
Frozen and/or paraffin sections were incubated with NPR3 (ab97389, abcam, 1:1000), Synaptopodin (61094; Progen, 1:700), CD31 (303105; Biolegend, 1:500), PDGFRβ (MAB1263; R&D System, 1:1000), Nephrin (BP5030; Origene, 1:200), Wilms-Tumor1 (WT1) (ab89901; abcam, 1:100), ⍺-smooth muscle actin (A5228; Merck, 1:1000), tdTomato (TA150128; Origene, 1:50) at 4 °C overnight. Alexa Fluor conjugated secondary antibodies (Invitrogen) were used for visualization. All slides were co-stained with Hoechst 33342 (H3570, Life Technologies: 1:10,000). For paraffin embedded sections an extra step of incubation with sudan black B (0.1% in 70% ethanol, #199664, Sigma-Aldrich) solution was applied before mounting in Dako fluorescent medium (S3023, Agilent).

Paraffin-embedded kidney tissue sections were deparaffinized and subjected to antigen retrieval, which was performed under high pressure in citrate buffer (0.01 mol/L, pH 6.0) for 10 min. After blocking with 5% bovine serum albumin (BSA) for 1 h, the sections were incubated with primary antibodies overnight at 4°C (Nrf2, GeneTex, USA, #GTX103322; synaptopodin, Progen, Germany, #65194), followed by incubation with fluorochrome-conjugated secondary antibodies (Thermo Fisher Scientific) at 37°C for 2 h in the dark. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, Antgene, China). All images were taken with a fluorescence microscope (Olympus, Japan).

Starting with FFPE kidney tissue biopsy specimen slides, antigen retrieval was performed with EDTA solution at pH 8.0 for 56 minutes at 100°C. Primary Abs against APOL1 (5.17D12, Genentech), synaptopodin (Progen Biotechnik, 61094), and CD31 (CST, 3528s) were applied at 1:4000 (final concentration 0.95 μg/mL), 1:100, and 1:1600, respectively, for 60 minutes at 36°C. Ready-to-use 3-hydroxy-2-quinoxaline secondary anti-rabbit multimers (catalog 760-4815, Roche) were incubated for 12 minutes at 36°C. This was followed by addition of anti–3-hydroxy-2-quinoxaline HRP for 12 minutes. DAB (catalog 760-159, Roche) was incubated for 5 minutes at room temperature. Tissue section was counterstained hematoxylin (catalog 760-2021, Roche) for 4 minutes at room temperature. Bluing reagent (catalog 760-2037, Roche) was added for 4 minutes at room temperature.

All procedures that involved animals were performed according to EU guidelines and approved by local Bioethical Commission at University of Gdansk.
Wistar rats, weighing 120–160 g, with free access to the standard diet (Altromin C1324, Germany) and drinking tap water were anesthetized as described before [18 (link)]. Glomeruli isolated from renal cortex by gradual sieving were incubated for 5 days at 37 °C in RPMI1640 (Sigma-Aldrich, Poland) containing 10 % heat-inactivated fetal bovine serum (FBS, Pan-Biotech, Germany), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma-Aldrich, Poland). The outgrowing epithelial cells were trypsinized, passed through a sieve with 33 μm pore size, seeded in 12-well or 6-well plates, and cultivated at 37 °C for next 15–20 days, as described previously [17 (link)]. The seeding density was 50 × 10³ cells/cm². Cell identity and phenotype were confirmed by immunofluorescence, using antibodies to WT-1 (Santa Cruz Biotechnology), podocin (Sigma-Aldrich, Poland), and synaptopodin (Progen, Germany).

The immunofluorescence assay analyses using the frozen renal biopsies from patients, the frozen kidney sections from rats and the cell climbing films were performed as previously described. The following primary antibodies were used in this study: Mfn2 (1:200; Abcam, United Kingdom); WT1 (NB110-60011, 1:50; Novus, GER); Synaptopodin (65194, 1:50; Progen, GER); TOM20 (sc-17764; 1:50; Santa Cruz, United States); Calreticulin (ab92516; 1:100; Abcam, United Kingdom); PERK (1:50; Santa Cruz, United States). The Alex Fluor 488/594 donkey anti-rabbit/Mouse IgG (HL) (ANT023, ANT024, ANT029, ANT030; 1:100; Antgene, Wuhan, China) were used as secondary antibodies. The nuclei were stained with an anti-fluorescence quencher containing DAPI (ANT063; Antgene, Wuhan, China). A confocal microscope (Olympus, Japan) was used to observe all of the microscopic images. The Pearson’s correlation coefficients (PCC) have been calculated in immunofluorescence assay of TOM20 and CRT, which with a value between 1 and −1, was used to quantitative colocalization between two proteins (1 means perfect correlation; −1 means completely excluded; 0 means no relationship) (Schober et al., 2018 (link)).

All experiments were performed in accordance with directive 2010/63/EU for animal experiments, and the protocol was approved by the local ethics committee of the University of Science and Technology, Bydgoszcz, Poland.
Female Wistar rats, weighing 120–140 g, were used for primary podocyte culture as described previously [11 (link)]. All experiments were performed using podocytes that were cultured for 12–20 days. Cell phenotypes were confirmed by podocyte-specific antibodies against Wilms tumor-1 protein (Biotrend, Koeln, Germany) and synaptopodin (Progen, Heidelberg, Germany).