Circulating miRNAs in extracellular vesicles related to treatment response in patients with idiopathic membranous nephropathy

Abstract

Background: Extracellular vesicle (EV)-microRNAs (miRNAs) are potential biomarkers for various renal diseases. This study attempted to identify the circulating EV-miRNA signature not only for discriminating idiopathic membranous nephropathy (IMN) from idiopathic nephrotic syndrome (INS), but also to predict the treatment response of patients with IMN.

Methods: We prospectively enrolled 60 participants, including those with IMN (n = 19) and INS (n = 21) and healthy volunteers (HVs; n = 20) in this study. Using RNA sequencing, we assessed the serum EV-miRNA profiles of all participants. To identify the EV-miRNAs predictive of treatment response in IMN, we also analyzed EV-miRNAs among patients with IMN with and without clinical remission.

Results: The expression levels of 3 miRNAs differed between IMN patients, INS patients and HVs. In addition, compared to HVs, RNA sequencing revealed differential expression of 77 and 44 EV-miRNAs in patients with IMN without and with remission, respectively. We also identified statistically significant (|fold change ≥ 2, p < 0.05) differences in the expression levels of 23 miRNAs in IMN without remission. Biological pathway analysis of miRNAs in IMN without remission indicated that they are likely involved in various pathways, including renal fibrosis.

Conclusion: Our study identified EV-miRNAs associated with IMN as well as those associations with therapeutic response. Therefore, these circulating EV-miRNAs may be used as potential markers for the diagnosis and prediction of treatment response in patients with IMN.

Keywords: Extracellular vesicles; Glomerulonephritis; Treatment outcome; microRNAs.

Fig. 1

Isolated extracellular vesicles (EVs) from the serum samples of patients. A Diagram of the observed peaks of nanoparticles in three patient serum EVs by dynamic light scattering (DLS) using Nano-ZS Zetasizer. B Transmission electron microscopy showing membranous vesicles of purified serum EVs. Scale bar, 200 nm. C Western blot analysis of EVs using positive marker as CD9 and negative marker as GM-130. D Cluster of differentiation 63 (CD63) expression levels in the serum-derived EVs as determined by the enzyme-linked immunosorbent assay (ELISA). Bars represent mean ± SD of three EVs from different samples

Fig. 2

EVs-microRNAs (miRNAs) from patients with idiopathic nephrotic syndrome (INS), idiopathic membranous nephropathy (IMN), and healthy volunteers (HVs). A Heat map showing z-scores of EVs-miRNAs from HVs (n = 20) and patients with INS (n = 21), with 33 upregulated (yellow) and 11 downregulated (blue) miRNAs. B Heat map showing z-scores of EVs-miRNAs from patients with INS (n = 21) and IMN (n = 19), with 3 upregulated (yellow) and 8 downregulated (blue) miRNAs. C Heat map showing z-scores of EVs-miRNAs from HVs (n = 20) and patients with IMN (n = 19) with 31 upregulated (yellow) and 27 downregulated (blue) miRNAs. D Fold change (FC) and p-values of three miRNAs, whose expression levels were specifically up- or down-regulated in patients with IMN compared to HVs and patients with INS

Fig. 3

Extracellular vesicle (EV-miRNAs from patients with idiopathic membranous nephropathy (IMN) with (IMN-W, n = 9) and without (IMN-R, n = 10) clinical remission. A Heat map showing z-scores of EVs-miRNAs from patients with IMN, with or without clinical remission, with 24 upregulated (yellow) and 18 downregulated (blue) miRNAs. B Fold change (FC) and p-values of top 10 miRNAs showed differential expression in patients with IMN-W and IMN-R

Fig. 4

Identification of idiopathic membranous nephropathy without clinical remission (INM-R)-specific EVs-miRNAs. A Venn diagram of overlapping miRNAs among the three datasets shows 23 miRNAs expressed in patients with IMN-R. B Fold change (FC) and p-values of 23 miRNAs, whose expression levels were up- or down-regulated in patients with IMN-R compared to HVs and IMN-W

Fig. 5

Principle component analysis (PCA) of serum-derived EVs. A Results of PCA using serum-derived EVs from HVs, IMN, and INS. B Results of PCA using serum-derived EVs from HVs, IMN-W, and IMN-R