Cell culture
Conditionally immortalized podocytes [11] (SVI; NIPOKA GmbH, Greifswald, Germany) were used for all the cell culture experiments and were handled as previously described [12]. Podocytes were maintained in RPMI 1640 medium (Sigma‒Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Boehringer Mannheim, Mannheim, Germany), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). For expansion, podocytes were cultured at 33°C and 5% CO2. To induce podocyte differentiation, podocytes were cultured at 38°C and 5% CO2 for at least two weeks before the experiments. Prior to exosome isolation, the cells were washed three times with Dulbecco’s PBS (PBS, Sigma‒Aldrich), and the medium was replaced with RPMI 1640 supplemented with 10% exosome-depleted FBS (System Biosciences, Palo Alto, CA, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin.
Exosome isolation
The cells were cultured in exosome-depleted media for 3 days. The conditioned medium was transferred to a 15 mL conical tube and centrifuged for 15 min at 3000×g at room temperature (RT) to remove cells and cell debris. The supernatant was transferred to a fresh 15 mL conical tube and centrifuged again for 15 min at 3000×g at RT to remove residual cell debris. Afterwards, 2 mL of Exoquick TC (System Biosciences) was added to the supernatant. After inverting the tube several times, it was incubated at 4°C overnight. Afterwards, the supernatant was centrifuged for 1 h at 10,000×g at 4°C. The exosome pellet was eluted in RPMI 1640 medium without supplements.
Exosome transfection
Exosome transfection was performed with the Exo-Fect™ siRNA/miRNA Transfection Kit (System Biosciences) following the manufacturer’s instructions with some modifications. We used the Silencer Select Flna siRNA (#s101260, Ambion, Thermo Fisher Scientific), Silencer Select Pxn siRNA (#s72562, Ambion, Thermo Fisher Scientific), pre-miR™ miRNA Precursor miR-21 mimics (#AM17100, Ambion, Thermo Fisher Scientific) and the corresponding negative controls (Silencer™ Cy3™-labeled Negative Control No. 1 siRNA, Silencer™ Negative Control No. 1 siRNA and Cy3™-labeled pre-miR Negative Control #1, Ambion, Thermo Fisher Scientific). The volume of the transfection reaction was 110 µL and included 545 nM of each nucleic acid. After incubation for 15 min at RT, the transfection reaction mixture was applied to 100 µL of the eluted exosomes or PBS as a negative control, respectively. This mixture was incubated for 1 h at 37°C in the dark. Exosome cleanup was performed according to the manufacturer’s instructions.
Exosome treatment
Prior to exosome treatment, differentiated podocytes were washed twice with 10 mL of PBS, and the cells were dissociated with 3 mL of trypsin–EDTA (0.05%, 0.02%, Thermo Fisher Scientific). The trypsin–EDTA reaction was blocked by the addition of 9 mL of RPMI 1640 supplemented with exosome-depleted FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. The cells were subsequently pelleted via centrifugation at RT and 750×g for 5 min. The pellet was subsequently eluted in 1 mL of RPMI 1640 supplemented with exosome-depleted FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. After cell counting, 2.5 × 104 cells per well were seeded in each well of a collagen 4-coated (Thermo Fisher Scientific) 6-well plate in 2 mL of RPMI 1640 supplemented with exosome-depleted FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. The cells were subjected to exosome treatments after 3 days. The cleaned-up exosome elutions from the transfection experiments were applied to the corresponding wells. Cells were treated for 48 h for uptake- and knock down experiments and for different durations (4 h to 1 week) and concentrations (18.75–150 µg) to characterize uptake dynamics.
Protein isolation and Western blot
Protein isolation from cells was performed as previously described [13, 14]. Prior to protein isolation, the cells were washed 3 times with 2 mL of PBS.
For protein isolation from the transfected and untransfected exosomes, 100 µL of exosome mixture was supplemented with 20 µL of Exoquick TC, inverted and incubated on ice for 1 h. Afterwards, the samples were centrifuged for 1 h at 14,000×g and 4 °C. The pellets were eluted in Pierce IP Lysis Buffer (500 mM, Thermo Fisher Scientific). Further protein isolation and concentration determination via the Bradford assay were performed as previously described [13, 14].
The samples were subsequently adjusted to 20 μg/lane (TSG101) and 10 μg/lane (CD9) for qualitative analysis. For quantitative analysis, we used 20 µL of each exosome sample per lane. For the analysis of cultured podocytes after treatment with FlnA-siRNA exosomes, we adjusted all the samples to 2 µg per lane. For the comparison of cultured podocytes and isolated exosomes we used 30 µg per lane. All samples were mixed with 6× sample buffer (0.35 M Tris [pH 6.8], 0.35 M SDS, 30% v/v glycerol, 0.175 mM bromophenol blue) and boiled at 95°C for 5 min. The protein samples were separated on a 4–20% gradient Mini-Protean TGX gel stain-free (Bio-Rad, Hercules, CA, USA). The separated proteins were blotted on nitrocellulose membranes via the Trans-Blot Turbo RTA Transfer Kit (Bio-Rad) and the Trans-Blot Turbo Transfer System (Bio-Rad) at 2.5 A/25 V for 5 min. Membranes were washed in 1× TBS + T wash buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, and 1 mM MgCl2 supplemented with 0.1% Tween-20; AppliChem, Darmstadt, Germany) and blocked in wash buffer supplemented with 5% milk powder (blocking solution) for 1 h at room temperature. The primary antibodies were diluted in blocking solution and incubated with the membranes overnight. After being washed 3 × 5 min with wash buffer, the membranes were incubated with secondary antibodies for 45 min, washed again for 4 × 5 min with wash buffer, developed with the ECL Prime Western Blotting Detection Reagent (Cytiva Europe GmbH, Freiburg, Germany) and visualized on X-ray films (Cytiva Europe GmbH) by using Carestream Kodak autoradiography GBX developer/fixer solutions. For normalization and usage of alternative antibodies on the same blot, the blots were stripped. The following antibodies were used at the final concentrations: anti-TSG101 (Sigma‒Aldrich; 1:1000), anti-CD9 (Thermo Fisher Scientific, 1:2000), anti-FlnA (Sigma‒Aldrich; 1:4000), anti-Gapdh (Santa Cruz Biotechnology, Santa Cruz, USA; 1:2000), and secondary anti-rabbit HRP (Santa Cruz Biotechnology; 1:6000 for TSG101; 1:5000 for CD9; 1:15,000 for FlnA and Gapdh).
Transmission electron microscopy
For transmission electron microscopy (TEM), 10 mL of cell- and debris-free cell culture media were treated with ExoQuick-TC (System Biosciences) for exosome isolation according to the manufacturer’s instructions with minor modifications. To 10 mL of cell culture media, 3.3 mL of ExoQuick-TC was added, and the mixture was stored overnight at 4°C. Then, the samples were centrifuged at 10,000×g for 60 min, and the supernatant was discarded. After treatment the pellets were prepared for TEM according to Asadi and coworkers [15] via the flotation method for the staining procedure. Briefly, isolated exosomes were fixed with 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.5) and then allowed to adsorb onto a glow-discharged carbon-coated porous Pioloform film on a 400-mesh grid (Plano GmbH) for 20 min on ice. The grid was then transferred onto four droplets of deionized water on ice for 2 min each and finally onto a drop of staining mixture for 10 min on ice. To prepare the staining mixture, 100 µL of 3% aqueous uranyl acetate was added to 900 µL of 2% methylcellulose, followed by the addition of 75 µL of deionized water and 25 µL of 1% phosphotungstic acid (pH 7). After blotting with filter paper, the grids were air-dried. All the samples were examined with a LEO 906 transmission electron microscope (Carl Zeiss Microscopy Deutschland GmbH, Oberkochen, Germany) at an acceleration voltage of 80 kV. For acquisition of the images at 100,000× magnification, a wide-angle dualspeed CCD camera Sharpeye (Tröndle, Moorenweis, Germany) was used, operated by the ImageSP software. All micrographs were edited by using Adobe Photoshop CS6.
RNA isolation
For the miR-loading experiments, the cells were cultured for 24 h in the presence of transfected or untransfected exosomes or control exosomes as indicated. For the siRNA-loading experiments, the cells were treated for 48 h with the transfected exosomes or control exosomes as indicated. RNA isolation was performed as previously described [13]. Prior to the administration of Tri-reagent, the cell layer was washed twice with 2 mL of PBS.
Taqman reverse transcription
cDNA synthesis was performed starting with 10 ng of total RNA via Taqman™ miRNA Assays and the Taqman™ miRNA Reverse Transcription Kit (Thermo Fisher Scientific). The following Taqman™ miRNA assays were used: Hsa-miR-21-5p: ID #000397 and U6 snRNA: ID #715680. The RT-reactions were performed according to the manufacturer’s instructions. The negative controls included no template- and no reverse transcriptase controls.
Taqman qPCR
qPCR was performed with the Taqman™ miRNA Assays mentioned above and the Taqman™ Universal Master Mix II without UNG (Thermo Fisher Scientific) following the manufacturer’s instructions. The reaction mixtures contained 1.33 μL of undiluted cDNA solution and 18.67 μL of Master Mix. qPCR was performed on a Bio-Rad iQ5 thermal cycler (Bio-Rad) with the following cycling scheme: 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 60 s at 60°C. All samples were run in triplicate. The negative controls included the no template controls from cDNA synthesis and an extra no template control for the qPCR. Ct values were calculated with automatically set thresholds and baselines via cycler software (Bio-Rad). Raw Ct values ≥ 38 were excluded from the analysis. All Ct values were normalized against U6 and the Cy3 w/o exosome Ctrl.
Immunofluorescence staining
Prior to immunofluorescence staining, the cells were fixed with 2% paraformaldehyde (PFA) for 10 min and permeabilized with 0.3% Triton-X (Sigma‒Aldrich) for 3 min. Then, the cells were blocked for 1 h with blocking solution (2% FBS, 2% BSA and 0.2% fish gelatin in PBS). The primary antibodies were diluted 1:100 in blocking solution and incubated for 1 h at RT. We used the following primary antibodies: anti-FlnA (Sigma‒Aldrich), anti-CD9 (BD Biosciences, San Jose, CA), anti-Rab5 (Cell Signaling Technology, Leiden, Netherlands) and anti-Rab7 (Cell Signaling Technology). The following secondary antibodies were diluted 1:300 in blocking solution and incubated with the cells for 1 h: anti-mouse-Cy2 (Jackson ImmunoResearch Laboratories, Ely, UK) and anti-rabbit-Cy2 (Jackson ImmunoResearch Laboratories). The actin cytoskeleton was visualized by staining with Alexa Fluor 647 phalloidin (1:100; Thermo Fisher Scientific) for 1 h, which was subsequently added to the secondary antibody solution. For nuclear staining, DAPI diluted 1:100 in PBS (Sigma‒Aldrich) was used for 2 min. All the samples were mounted in Mowiol (Carl Roth, Karlsruhe, Germany).
Confocal laser scanning microscopy (cLSM)
Confocal laser scanning microscopy (cLSM) was performed on a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) with 20×, 40× and 63× oil immersion objectives. For image acquisition, Leica Application Suite software (Leica Microsystems, Version 2.6.0) was used. All z-stacks were acquired throughout the whole z-plane (20 µm) in 0.5 µm steps. Pre-miR-21 experiments were performed in 20 × 1 µm steps. The xzy-projection was acquired in 20 × 1.73 µm steps. For 3D-SIM, a Zeiss Elyra PS.1 A system (Zeiss Microscopy, Oberkochen, Germany) equipped with a 63 × oil immersion objective was used. Z-Stacks with a size of 1280 µm × 1280 µm and a slice-to-slice distance of 0.43 µm were acquired over approximately 2 µm using a 561 nm laser (3.5% laser power, exposure time: 150 ms), a 488 nm laser (3.5% laser power, exposure time: 150 ms) and a 405 nm laser (7% laser power, exposure time: 100 ms). The grating was shifted and rotated five times on every frame while widefield images were acquired. Z-stacks were acquired in 4 planes of 0.43 µm. 3D-SIM reconstruction was performed with Zeiss ZEN Black. ZEN blue software was used for maximum intensity projections.
Flow cytometry and imaging flow cytometry
For flow cytometry, three independent replicates were processed including untreated podocytes, podocytes treated with exosomes without Cy3-miRCtrl that went through the transfection process as controls and Cy3-miRCtrlExos. Untreated podocytes were used as reference. Prior to flow cytometry, 300,000 cells of each treatment group were treated as described above for 48 h. Afterwards cells were washed twice with PBS and detached with Trypsin–EDTA for 5 min. The detached cells were diluted in RPMI 1640 supplemented with exosome-depleted FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. Subsequently, the cells were centrifuged for 3 min at 750×g at RT. The pellet was washed with PBS and centrifuged again at the same conditions. After discarding the supernatant, the cells were fixed by the addition of PFA (2%) for 15 min. Finally, the cells were washed twice with PBS and resuspended in 500 µL PBS, stored on ice and transferred to flow cytometry. Flow cytometry was performed as previously described on an Aria III, Canto II, LSR II (BD Biosciences, Franklin Lakes, NJ, USA) equipped with a 561 nm laser (PE) [16]. 10,000 cells were analyzed in each treatment group. Imaging flow cytometry was performed using an Amnis ImageStreamX MK I (Cytek Biosciences, Fremont, CA, USA) with a 561 nm laser and a 40× magnification as described before [16]. For imaging flow cytometry 1000 cells of each treatment group have been analyzed.
Statistical analysis
Statistical analysis was performed via GraphPad Prism V5.01 (GraphPad Software, CA, USA; https://www.graphpad.com). The data were checked for a Gaussian distribution via the Kolmogorov–Smirnov test. All groups were tested for statistically significant differences via two-way ANOVA and the Bonferroni post hoc correction. All values are displayed as the means with standard deviations. P values ≤ 0.05 were considered statistically significant.