Glomerular endothelial derived vesicles mediate podocyte dysfunction: A potential role for miRNA

MicroRNAs (miRNA) are shown to be involved in the progression of several types of kidney diseases. Podocytes maintain the integrity of the glomerular basement membrane. Extracellular vesicles (EV) are important in cell-to-cell communication as they can transfer cellular content between cells, including miRNA. However, little is known about how extracellular signals from the glomerular microenvironment regulate podocyte activity. Using a non-contact transwell system, communication between glomerular endothelial cells (GEnC) and podocytes was characterised in-vitro. Identification of transferred EV-miRNAs from GEnC to podocytes was performed using fluorescence cell tracking and miRNA mimetics. To represent kidney disease, podocyte molecular profiling and functions were analysed after EV treatments derived from steady state or activated GEnC. Our data shows activation of GEnC alters EV-miRNA loading, but activation was not found to alter EV secretion. EV delivery of miRNA to recipient podocytes altered cellular miRNA abundance and effector functions in podocytes, including decreased secretion of VEGF and increased mitochondrial stress which lead to altered cellular metabolism and cytoskeletal rearrangement. Finally, results support our hypothesis that miRNA-200c-3p is transfered by EVs from GEnC to podocytes in response to activation, ultimately leading to podocyte dysfunction.

Primary human podocytes were used for all other studies. Human podocyte primary cells were purchased from Celprogen and cultured following manufacturer's instructions. Primary podocytes were maintained and proliferated in a 37°C, 5% CO2, 95% humidified incubator. Podocytes were seeded onto Human Podocyte Primary Cell Culture Extra-Cellular Expansion Matrix precoated flasks (Celprogen). Podocytes were grown and maintained in Human Podocyte Primary Cell Culture Complete Media with Serum (Celprogen). Media were renewed every 2-3 days and cells were subcultured at 60-70% confluency, at a 1:2 cell density. Cells were used in experiments up to passage 12, as cells retain morphological features of podocytes at least up to this passage (3).

FM1-43 staining
GEnC were harvested and resuspended in 5μg FM1-43FX (Thermo Fisher Scientific) and incubated in the dark for 5 minutes. GEnC were washed to remove any remaining (free-floating) dye. GEnC were seeded onto transwell inserts for co-culture. In some experiments, cells were simulated with 1μg/ml LPS, as described above. Podocytes were collected, and backdrop suppressor reagent (Thermo Fisher Scientific) was added to quench any extracellular dye. FM1-43FX positive cells were counted by flow cytometry (BD AccuriC6) or imaged on a Leica SP5 inverted confocal microscope to detect FM1-43FX expression.

RNA isolation and quantification
Total RNA was extracted from the cells using a miRNeasy kit (QIAGEN) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). For RNA sequencing, RNA was purified from podocytes using the Total RNA Purification Kit (Norgen Biotec Corp) (see below).

BCA protein assay
EVs were lysed in NP40 + PI. A set of protein standards were prepared from a 2mg/ml albumin standard (BSA) ampule. The 2 set of dilutions ranging from 2000μg/ml to 25μg/ml. Reagent A and Reagent B were mixed at a 50:1 ratio to prepare a WR. A sufficient volume to pipette each sample in duplicates plus standards plus unknown. For the microplate procedure, 25μl of each standard or unknown sample plus 200μl of WR were added. The plate was incubated at 37 o C for 30 minutes and absorbance was read at 650nm on a plate reader. The average absorbance of the blank standard was subtracted from the samples and unknown replicates. A standard curve was created to determine the protein concentration of EVs in μg/ml.

Western blot
EVs and cells were lysed in NP40 supplemented with protease inhibitors (Sigma). 1/5 -mercaptoethanol was added to the loading buffer as a reducing agent. Samples were incubated at 95oC for 3 minutes and then run on 15% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) (Invitrogen) at 100 volts, until the dye had reached the bottom of the gel. Proteins were then transferred to a nitrocellulose membrane (GE healthcare). Membranes were blocked in 10% milk for at least 30 minutes, washed 3x in TBST and incubated overnight in primary antibody. Membranes were then washed 3x in TBST. Protein was detected with horseradish peroxidase-conjugated secondary antibodies in combination with Amersham ECL Western Blotting Reagents (GE Healthcare). Gene enrichment analysis was used to determine the biological function of the upregulated mRNAs. Using GO, (http://geneontology.org/page/go-enrichmentanalysis) an online resource for analysis, data enrichment analysis was performed on the sets of genes that were significantly upregulated in the treated podocytes. This determined which gene ontology terms were over represented in the treated podocytes. PANTHER groups are annotated subfamilies of related genes that are likely to share function. The core module is a large protein library that contains all protein coding genes from 82 organisms, organised first into subfamilies on the basis of their shared functions. The results obtained from the analysis are therefore based on enrichment relative to the set of all protein coding genes in the human genome. The statistical overrepresentation test compares the uploaded gene list and it determines whether a particular class of genes is overrepresented. For each function category e.g. cell proliferation for biological processes, the binomial test is applied (from the PANTHER reference genome data set) to determine whether there is a statistical overrepresentation or underrepresentation of the genes in the test list relative to the reference list. Prep was then implemented. 3% Agarose Gel Cassettes were used to collect fragments of 100-250bp. The cassette was prepared for loading as follows. Buffer was removed from the elution modules and replaced with 41μl of fresh electrophoresis buffer. The wells were sealed with an adhesive tape strip and continuity tested to calibrate the cassette. 50μl of buffer was removed from each sample well and 40μl of sample (30μl of sample plus 10μl of marker plus 5μl buffer) was loaded into the cassette. The library size distribution and integrity and concentration were assessed using the Agilent 2100 BioAnalyzer and Qubit fluorimeter, as previously described. Equal concentrations of all individual libraries were pooled for multiplex sequencing runs and concentrated using DNA Clean and Concentrator 5 kit (Zymo). Libraries were run independently on 3 individual sequencing lanes. Single end sequencing of multiplexed libraries was performed on an Illumina NextSEQ 500 as above. The raw (not normalised) mRNA counts of 3 separate experiments for RNA sequencing reads were loaded into DESeq2, these counts were then normalised by variance-stabilising transformation.

Primary antibodies
Data was organised with a cut off of minimum P value of 0.05 and fold change of >1.5. The miRNA enrichment analysis and annotation (MIEAA) tool (https://ccb-compute2.cs.uni-saarland.de/mieaa_tool/) was used as an overrepresentation analysis predictor. MIEAA calculates the significance of categories for a test set and determines if the specific category is over-represented or under-represented for the test set with respect to a reference set. Over 14,000 categories are available for analysis on the site. From the input data, a results table is created containing the category, subcategory, P-value (computed by applying the fishers exact test), expected and observed number of miRNAs, and the respective miRNAs per subcategory. http://bioinformatics.psb.ugent.be/webtools/Venn/ was used to create Venn diagrams. Statistically upregulated genes were inputted into the site to create Venn diagrams.

Seahorse Assays
Seahorse XFe96 analyser (Agilent) was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR

ROS staining
MitoSOX Red reagent is a mitochondrial superoxide indicator fluorogenic dye. MitoSOX produces red fluorescence when oxidised by superoxide. Podocytes were treated and harvested using cell dissociation buffer (Thermo Fisher Scientific) and stained with 5µm MitoSOX Red (Thermo Fisher Scientific). 10 minutes after staining, cells were analysed by flow cytometry on a BD LSRFortessa X-20.

VEGF measurement.
Concentrations of VEGFA, were measured using Duoset ELISA kits (R&D systems, Abingdon, UK), according to the manufacturers protocol. Absorbance was measured at 450nm using a Bio-Tek ELX 800 microplate reader (Bio-Tek Instruments).

Podocyte actin staining
Coverslips with adhered, EV or control treated podocytes were washed 2x in PBS, fixed in 4% PFA for 15 minutes and washed with 0.1M glycine. Cells were permeabilised with Triton X-100 and actin was stained using fluorescently conjugated phalloidin (Life technologies). Cells were mounted in DAPIvectashield (Vector labs) and imaged using a Leica SP5 confocal microscope.
miRNA mimic transfection 1x10 5 podocytes in 24 well plates were transfected with Lipofectamine 2000 (Invitrogen) and miRNA mimic miR-200C-3p or miR-29C-3p or negative control miRNA mimic (Life technologies) according to manufacturer's instructions. Cells were incubated for 24 hours. Supernatant was then removed, and cells were washed. Fresh media was then added to the podocytes and supernatant was collected 24-hours later. VEGF ELISA was performed as described above.