Ethical statement
All the enrolled subjects provided informed written consent. The study protocol was approved by the Bioethics Committee of the A.O.U. Città della Salute e della Scienza Hospital (protocol no. 0021671). The study was conducted according to the principles expressed by the Declaration of Helsinki of 1975, as revised in 2013.
Patients
A total of three patients diagnosed with steroid-resistant nephrotic syndrome were recruited in this study. Genetic and clinical features of patients are described in Table 1. During follow up patient 1 and 2 did not respond to all the treatments attempted, progressed to CKD5 and were successfully transplanted without recurrence. Patient 3, which initially proved to be steroid resistant, had a late satisfactory response to steroids combined with angiotensin-converting enzyme inhibitors and one single dose of Rituximab, and after 48 months of follow up has a normal kidney function and minimal proteinuria.
Generation and characterization of podocyte cell lines
The urines of the three patients with steroid-resistant nephrotic syndrome and of a healthy pregnant woman (Table 1) were freshly collected and centrifuged at 200 × g for 10 min. The pellet was resuspended in DMEM/F-12 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% foetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA), 50 IU/ml penicillin, 50 g/ml streptomycin, 5 mM glutamine, 5 g/ml insulin, 5 g/ml transferrin, and 5 mg/ml selenium (all from Sigma-Aldrich, St Louis, MO, USA). Subsequently, primary cells were grown at 37 °C up to the third passage and characterized as podocytes (NS-POD1, NS-POD2, NS-POD3 and CTL-uPOD, respectively). In addition, a conditionally immortalized podocyte cell line (AS-POD) from an Alport syndrome patient was used (10). Finally, tissue‐derived podocytes, kindly gifted by MA Saleem, were used as an additional control for selected experiments (CTL-tPOD) (20).
Generation and characterization of nKPC cell line
Urine sample (500 mL) was collected from a newborn (born at 32 gestational weeks) at day 1 after birth from a catheter bag, as described (18). The sample was centrifugated at 1’500 rpm for 10 min and the cell pellet was resuspended in α‐MEM Medium (Gibco/BRR ThermoFisher, MA, USA) supplemented with 20% Chang Medium B (Irvine Scientific, Santa Ana, California, USA) and 2% Chang Medium C (Irvine Scientific), 20% FCS (Invitrogen, Carlsbad, CA, USA), 50 IU/mL penicillin, 50 g/mL streptomycin, 5 mM glutamine (all from Sigma‐Aldrich, St. Louis, MO, USA). At passage 2, 1 x 104 primary nKPCs were infected in DMEM F12 20% FCS with a retrovirus containing a pBABE-puro-hTERT plasmid (Addgene plasmid #1771) (21). The day after the infection, the medium was replaced with their growth medium. From passage 3, cells were selected in their growth medium containing 1 µg/mL puromycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for four weeks, until passage 7.
Isolation of nKPC-EVs
EVs were obtained from nKPC supernatant. The cells were cultured overnight in RPMI deprived of FCS, then the supernatant was centrifuged for the removal of cell debris and apoptotic bodies at 3,000 g for 20 min. EVs were purified by a 2 h-ultracentrifugation at 100,000 g at 4 °C (Beckman Coulter, Brea, CA, USA) and used fresh or stored at −80 °C after resuspension in RPMI supplemented with 1% dimethyl sulfoxide. Analysis of the size distribution and particle quantification were performed using NanoSight NS300 (NanoSight Ltd, Malvern, UK) equipped with a 405 nm laser and the Nanoparticle Tracking Analysis (NTA) 2.3 software (NanoSight Ltd., Malvern, UK). MSC and serum-derived EVs were isolated as previously described (16, 22).
Protein extraction and Western Blot
For protein analysis, podocytes, nKPCs and nKPC-EVs were lysed at 4 °C for 20 min in RIPA buffer (20 nM Tris·HCl, 150 nM NaCl, 1% deoxycholate, 0.1% SDS 1% Triton X-100, pH 7.8) supplemented with protease and phosphatase inhibitor cocktail and PMSF (Sigma-Aldrich, St. Louis, MO, USA). To determine total protein concentration of podocyte lysate, Bradford solution was used, according to the manufacturer’s procedures (Bio-Rad Inc, Berkeley, CA, USA). At variance, total protein concentration of nKPCs and nKPC-EVs was determined spectrophotometrically using a micro-BCA™ Protein Assay Kit, as previously described (23). Either 30 µg (for podocytes) or 8 µg (for nKPCs and nKPC-EVs) of proteins were electrophoresed through 4-12% Mini-Protean TGX Stain-Free Gels (Bio-Rad). Using the iBLOT2 system (Life Technologies, Carlsbad, CA, USA), gels were blotted onto PVDF membrane filters according to the manufacturer’s procedures. Each membrane was immersed in blocking solution, consisting in 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) in PBS, for 1 h before overnight incubation with primary antibodies at the indicated dilutions. After rinsing in wash buffer (0.1% Tween in PBS), horseradish peroxidase-conjugated secondary antibodies were used for 1 h at 1:3000–1:5000 dilutions. After final washings, membranes were incubated with ECL chemiluminescence reagent (Bio-Rad). Images were acquired using a ChemiDoc ™ XRS+ System (BioRad, Milan, Italy). For podocytes analysis, the following antibodies were used: rabbit monoclonal anti-Podocin (Cat. No. sc-21009; Santa-Cruz, Dallas, TX, USA), and mouse monoclonal anti-CD2AP (Cat. No sc-25272, Santa-Cruz). Goat monoclonal anti-Vinculin (Cat. No. sc-7648 Santa-Cruz) was used as housekeeping. In the case of nKPCs and nKPC-EVs protein analysis, mouse monoclonal anti-CD63 (Cat. No sc-5275, Santa-Cruz) and rabbit monoclonal anti-Calreticulin (Cat. No 2891, Cell signalling, Milan, Italy) were used. The protein bands were detected using either rabbit, mouse, or goat peroxidase-labeled secondary antibody.
Immunofluorescence
Immunofluorescence on podocytes was performed as follows: cells were fixed in 4% paraformaldehyde for 20 min at room temperature and permeabilized with PBS 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 4 °C. PBS 1.5% BSA (Sigma-Aldrich, St. Louis, MO, USA) was used to block non-specific sites for 20 min at room temperature. Subsequently, Texas Red-X Phalloidin (Cat. No. T7471; Thermo Fisher Scientific) was incubated for 1 h. Fixed cells were washed with PBS 0.1% BSA before nuclear staining with 4.6-diamidine-2-phenylindole (DAPI, Sigma-Aldrich) for 8 min. After the final wash, coverslips were mounted with Fluoromount. Images were acquired by the videoconfocal system ViCo microscope Nikon Eclipse 80i (Nikon, Japan).
Transmission Electron Microscopy
The transmission electron microscopy (TEM) was performed on EVs placed on 200-mesh nickel formvar carbon-coated grids (Electron Microscopy Science) for 20 min to promote adhesion. The grids were then incubated with 2.5% glutaraldehyde plus 2% sucrose. EVs were negatively stained with NanoVan (Nanoprobes, Yaphank, NY, USA) and observed using a Jeol JEM 1400 Flash electron microscope (Jeol, Tokyo, Japan) (24).
Super resolution microscopy
Super-resolution microscopy was performed with Nanoimager S Mark II microscope from ONI (Oxford Nano- imaging, Oxford, UK) equipped with a 100x, 1.4NA oil immersion objective, an XYZ closed-loop piezo 736 stage, and triple emission channels split at 640, 555 and 488 nm on nKPC-EV. EV profiler Kit (EV-MAN- 1.0, ONI) was used for the experiments following manufacturer’s protocol. The Kit contains fluorescent antibodies, anti CD9-488, CD63-568 and CD81-647, washing buffer and the imaging buffer. Images were acquired sequentially in dSTORM mode in total reflection fluorescence (TIRF). Single-molecule data was filtered using NimOS software (v.1.18.3, ONI). Data analysis was conducted using Collaborative Discovery (CODI) online analysis platform www.alto.codi.bio from ONI and the drift correction pipeline version 0.2.3 was used (25).
RNA Isolation and Real Time PCR
Total RNA of podocytes untreated or treated for 24 h with nKPC-EVs (5×104 EV/cell) was isolated using Trizol Reagent (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. At variance, RNA of nKPCs and of nKPC-EVs was extracted using miRNeasy mini kit (Qiagen GmbH, Qiagen Strasse 1, 40724 Hilden, Germany) according to the manufacturer’s protocol. RNA was then quantified spectrophotometrically (Nanodrop ND-1’000, Wilmington, NC, USA). For the gene expression analysis, quantitative real-time PCR (RT-PCR) was performed. Briefly, one-strand cDNA was produced from 200 ng of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). RT-PCR experiments were performed in a 20 μL-reaction mixture containing 5 ng of cDNA template, the sequence-specific oligonucleotide primers (purchased from MWG-Biotech, Eurofins Scientific, Brussels, Belgium), and the Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA). GAPDH mRNA was used to normalize the RNA inputs. The fold change expression with respect to the control was calculated for all the samples. Primer list can be found in supplementary data (Supplementary table I).
Flow cytometry
After puromycin selection, nKPCs were detached using a nonenzymatic cell dissociation solution, resuspended in PBS 0.1% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with antibodies. Cells were incubated with either phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, or allophycocyanin (APC)-conjugated antibodies against CD90 (Cat. No. 130-114-859, Miltenyi Biotec, Bergisch Gladbach, Germany) CD73 (Cat. No. 550257, BD Bioscience, Franklin Lakes, NJ, USA), CD146 (Cat. No. 550315, BD Bioscience) and CD29 (Cat. No. 130-101-256, Miltenyi Biotec) CD133-1 (Cat. No. 130-090-826, Miltenyi Biotec) and appropriate isotype control. Stained cells were then analyzed using FACSCalibur machine using CellQuest software (Becton Dickinson Bioscience Pharmingen, Franklyn Lake, NJ, USA).
MACSPlex Analysis
nKPC-EVs were subjected to bead-based multiplex EV analysis by flow cytometry (MACSPlex Exosome Kit, human, Miltenyi Biotec, Auburn, CA, USA), as previously described (25). Briefly, 5´109 EVs were diluted with a MACSPlex buffer (MPB) to a final volume of 120 μL and 15 μL of MACSPlex Exosome Capture Beads (containing 39 different antibody-coated bead subsets) were added to each sample. The samples were then incubated on an orbital shaker overnight (14–16 h) at 450 rpm at 4 °C protected from light, followed by several washings with MPB using centrifugations (3’000 g, 5 min). For EV counterstaining, 5 mL of each APC-conjugated anti-CD9, anti-CD63, and anti-CD81 detection antibodies were added to each sample and incubated on an orbital shaker at 450 rpm for 1 h at room temperature. After additional washings, samples were subjected to flow cytometric analysis using FACS Celesta (BD Biosciences, Franklin Lakes, NJ, USA).
Permeability assay
Permeability assays were performed by measuring BSA filtration from the lower to the upper compartment, after 24-hour treatments. Complete medium (500 mL) containing or not FITC-BSA (1 mg/mL, Sigma Aldrich) was placed in the lower compartment and upper podocyte compartments, respectively. To measure the podocyte filtration ability in basal to apical direction, 100 μl of medium was taken after 6 h from the upper compartment and the passage of FITC-BSA was determined by fluorimetry in triplicate. FITC signal was measured in triplicates using Promega™ GloMax® Plate Reader (Promega Italia S.r.l., Milano, Italy). Data are expressed as the mean amount of filtered BSA-FITC of four different experiments using at least three inserts for each condition in each experiment. Podocytes were treated for 24 h with nKPC-EVs (2-5 x104 EV/cell), methylprednisolone (Urbason, Sanofi 40 mg/mL), cyclosporin (Sandimmun, Novartis 25 mg/mL), tacrolimus (Prograf, Panacea Biotec, 1 ng/mL) and rituximab (Mabthera, Roche, 12 mg/mL).
RNA sequencing analysis
Total RNA from untreated or treated podocytes was isolated using Trizol Reagent (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. RNA was then quantified spectrophotometrically (Nanodrop ND-1’000, Wilmington, NC, USA). Libraries for RNA-seq were generated using a TruSeq RNA stranded sample preparation kit v2 (Illumina Inc, San Diego, CA, USA) following the manufacturer’s instructions, using 1 µg of total RNA as input material. Libraries were pooled and sequenced with a NextSeq 500 sequencer (Illumina Inc) generating 75-bp paired-end sequences. Further analyses were performed using transcript per million (TPM) tables and genes with an average |log2 Fold Change| ³1 were considered for further analysis using Expression Suite and Funrich V3 Software (Bundora, Australia).
Statistical analysis
Data are shown as mean ± SD. Statistical analysis was carried out on Graph Pad Prism (GraphPad Software, Inc., San Diego, CA, USA) by using one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparisons test, or by unpaired t-test, where appropriate. A p value < 0.05 was considered significant.