Participants and data collection
We included 40 age- and sex-matched patients with IMN and INS and 20 HVs from a prospective glomerular disease cohort. In the present study, INS included focal segmental glomerular sclerosis and minimal change disease, which were defined by the association of the clinical features of nephrotic syndrome with renal biopsy findings of diffuse foot process effacement using electron microscopy . Patients with glomerulonephritis, who were diagnosed between January 2015 and Jun 2020, were enrolled from Soonchunhyang University Hospital and Presbyterian Medical Center. Patients with secondary causes of MN, such as lupus or malignancy, were excluded. This study was approved by the institutional review board of Soonchunhyang University (IRB No. 2016-01-002-007). Written informed consent was obtained from all the participants.
Remissions were defined according to the 2012 Kidney Disease: Improving Global Outcomes guidelines. Among patients with IMN, complete remission was defined as a reduction in proteinuria to 0.3 g/day. Partial remission was defined as a reduction in proteinuria to between 0.3 and 3.5 g/day (with at least 50% reduction versus baseline). Composite remission included either complete remission within 1 year after renal biopsy or partial remission with less than 2.5 g of proteinuria for 2 years following pathologic diagnosis. A refractory response was defined as the absence of composite remission during the follow-up period. Therefore, a total of 19 patients with IMN were divided into two groups: a well- responding (IMN-W) and refractory (IMN-R) group, based on the achievement of composite remission.
During the follow-up period, treatment decisions for the enrolled patients were made by the treating nephrologist. The most common reasons for initiating immunosuppressive therapy were patient characteristics (proteinuria, renal function, etc.) that were not properly controlled, and nephrologist clinical judgement.
Serum EV RNA isolation and assessment
RNA sequencing was conducted as previously described . Briefly, circulating EVs were isolated from the serum using the ExoQuick isolation agent (System Bioscience, Palo Alto, CA, USA), according to the manufacturer’s guidelines. Supernatants obtained after centrifugation (3,000 × g for 15 min) of the serum samples were mixed with ExoQuick reagent and incubated for 30 min at 4 °C. After another centrifugation at 1500 × g for 30 min, the supernatant was aspirated, and the pellet was retained. After resuspension of the pellet in sterile phosphate-buffered saline, RNA was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). All processes involving the suspension of exosomes were conducted according to the manufacturer’s guidelines. After RNA extraction, purified RNA was eluted in RNase-free water (20 μL). The purified RNA was analyzed using an Agilent Bioanalyzer 2100 with an RNA Pico Chip and Small RNA Chip to examine the size distribution of EV RNAs (Agilent Technologies, Santa Clara, CA, USA).
Characterization of EVs by cluster of differentiation 63 (CD63) detection
CD63 levels in circulating EVs were measured using the Exo-enzyme-linked immunosorbent assay (ELISA)-ULTRA CD63 kit (System Biosciences, Palo Alto, CA, USA), according to the manufacturer’s protocol.
Transmission electron microscopy (TEM)
This protocol was performed as described by Thery et al. and Rikkert et. al. [31,32]. A droplet of exosome solution was placed on Para film, and a Formva-carbon-coated nickel grid (200 meshes, TED PELLA, USA) was floated on the drop to absorb the sample at room temperature. After 10 min, the exosomes were fixed with 2.5% glutaraldehyde and stained with 1% uranyl acetate. The sample was washed with distilled water and dried in the dark. The grid was observed using an electron microscope operating at 75 kV (H-7000B; Hitachi, Tokyo, Japan).
Exosome physicochemical properties
A Nano-ZS Zetasizer (Malvern Inc., UK) was used to estimate the particle size. The samples were diluted ten times with distilled water and particle size was measured three times in a set of 50 repetitions using disposable cuvettes (DTS1070; Malvern Inc., Worcestershire, UK) and analyzed using the Zetasizer software (version 7.11).
cDNA library preparation and small RNA sequencing
The samples were processed to produce exosomal RNA (10 ng) as an input for each library. Small RNA libraries were constructed using a SMARTer smRNA-Seq Kit for Illumina (Takara Bio, Shiga, Japan), according to the manufacturer’s guidelines. Sequencing libraries were constructed by polyadenylation, cDNA synthesis, and polymerase chain reaction (PCR) amplification.
The libraries were gel-purified and validated by assessing their size, purity, and concentration using an Agilent Bioanalyzer. The libraries were quantified using quantitative PCR (qPCR), according to the qPCR Quantification Protocol Guide (KAPA Library Quantification Kits for Illumina® Sequencing Platforms). We assessed the quality of the libraries using TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Equimolar amounts of libraries were pooled and sequenced on an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, USA) to generate 51 base reads. Image decomposition and quality value calculations were performed using modules in the Illumina pipeline. All procedures for next-generation sequencing (NGS) analysis were performed at Macrogen (Seoul, Korea).
Analysis of RNA sequencing data and proportions of miRNAs
Following sequence alignment, known and novel miRNAs were identified using the miRDeep2 algorithm. Prior to sequence alignment, we retrieved the Homo sapiens reference genome release hg19 from the UCSC Genome Browser, which was indexed using Bowtie (1.1.2), a program for aligning experimental and reference sequences. The reads were then aligned to the mature and precursor H. sapiens miRNAs obtained using miRBase 21. Uniquely clustered reads were sequentially aligned to the reference genome using miRBase 21 and the non-coding RNA database Rfam 9.1 to identify known miRNAs and other types of RNAs, respectively.
Analysis of miRNA expression levels
The raw data (reads for each miRNA) were normalized to the relative log expression using DESeq2. For preprocessing, miRNAs absent from more than 50% of all samples were excluded, leaving only mature miRNAs for analysis. We added 1 to the normalized read count of the filtered miRNAs to facilitate log2 transformation and draw a correlation plot. For each miRNA, the base mean and log-fold changes were calculated between the groups. We conducted a statistical hypothesis test to compare the groups using the negative binomial Wald test in DESeq2. miRNAs differentially expressed between the two groups were defined as having a |fold change| ≥ 2 and a false discovery rate (FDR)-adjusted p-value of < 0.05. We also performed hierarchical clustering analysis using complete linkage and Euclidean distance as measures of similarity to display the expression patterns of the differentially expressed miRNAs that satisfied the criteria of a |fold change| ≥ 2 and an FDR-adjusted p-value of < 0.05. All data analyses and visualization of the differentially expressed genes were performed using R 3.3.1 (www.r-project.org).
Identification of miRNA target genes and their molecular pathways
We uploaded miRNAs that were differentially expresed in the HVs and patients with IMN-W and IMN-R into commonly used analysis programs, such as DIANA-miRPath and miRSystem, for further analyses. The DIANA-miRPath v.3.0 database used DIANA-microT-CDS and TargetScan 6.2 to analyze miRNA-gene interactions. The database schema incorporated the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Gene Ontology (GO), and GO slim annotations. Gene and miRNA annotations were derived from the Ensembl and miRBase databases, respectively.
Detection of anti-PLA2R antibodies
Serum samples collected from each patient at the time of kidney biopsy were stored at –80 °C and thawed simultaneously for the measurement of anti-PLA2R antibodies. The serum levels of anti-PLA2R antibodies were determined using commercially available ELISA kits (EUROIMMUN AG, Lubeck, Germany). Briefly, PLA2R-coated microplates were incubated with human sera diluted 1:101 in the sample buffer for 30 min and visualized after incubation with anti-human IgG horseradish peroxidase (HRP) conjugate for 30 min. Optical density was measured at 450 nm using a microplate absorbance reader (Model 550; Bio-Rad, Hercules, CA, USA). According to the manufacturer’s guidelines, values ≥ 20 RU/mL were considered positive.
Continuous variables with normal distributions are expressed as the mean ± standard deviation; variables without a normal distribution are expressed as medians with interquartile ranges. The t-test was used to analyze the statistical significance of the differences between continuous variables, and the chi-square test was used for categorical variables to compare the baseline characteristics between HVs and patients with IMN. The IMN group was further divided into two groups, and continuous variables were compared among the three subgroups (HVs vs. IMN-W vs. IMN-R) using the Kruskal-Wallis multiple comparison test. Statistical significance was set at P < 0.05. Statistical analysis was performed using SPSS (version 22.0; IBM Corp., Armonk, NY, USA).