Identification of differentially expressed circulating exosomal lncRNAs in IgA nephropathy patients

In Asian populations, Immunoglobulin A nephropathy (IgAN) is known as the most prevalent of primary chronic glomerular disease. IgAN is mostly characterized by the occurrence of IgA1 deposits in the mesangium of glomeruli; usually occurs in young or middle-aged adults. Among the patients, about 20 ~ 40% of cases eventually progress to end-stage renal disease (ESRD) within 10 ~ 20 years [1]. Although the exact mechanism of IgAN is still largely unknown, it is thought to be an immune related disease with a multi-“hit” pathogenetic process with overproduction of aberrantly glycosylated IgA1(galactose-deficient IgA1, Gd-IgA1) [1]. Till now, renal biopsy is still the standard for IgAN diagnosis, with no effective disease-specific therapies currently available. However, renal biopsy is invasive with limitations in assessing disease activity only at the time of biopsy, which could lead to inconclusive findings and decisions [2].

Although a number of IgAN-specific biomarkers have been discussed, the most reported is Gd-IgA1, it has not been validated in large multiracial cohorts of IgAN patients [3]. There are still other studies reported on serum and urinary biomarkers, such as sCD89 [4], the transferrin receptor (TfR) [5], products of complement system (C3b, C3c, C5b-9) [6]. However, there are limitations of these biomarkers, such as the validation in other populations of IgAN patients or lack sensitivity and specificity towards the disease diagnosis and progression. One study identified circulating sCD89-IgA immune complexes was thought about be associated with IgAN disease progression [7]. However, another study measured sCD89-IgA immune complexes in the 326 IgAN patients and found no association with disease progression. With these contradicting results, it was suggested sCD89-IgA is not a good predictor for assessing the progression of IgAN [8]. To date, there are no IgAN-specific biomarkers yet to replace renal biopsy as a diagnostic or to add valuable information in evaluating progression of IgAN revealing the urgent need to identify effective and non-invasive biomarkers to improve early detection and individualized treatment for IgAN.

Long non-coding RNAs (lncRNAs) are known as a heterogeneous class of transcripts with a of more than 200 bases, with no protein-coding potential [9]. Emerging evidence shows that lncRNAs play important roles in various biological processes, including gene expression, protein folding recruitment during chromatin modifications, X-chromosome inactivation/ and immunoregulation [9]. Recent studies have also reported the link between lncRNAs to various kidney diseases [10‐13]. Li et al. reported that lncRNA MALAT1 expression was substantially increased in diabetic nephropathy (DN) and could reduce pyroptosis of renal tubular epithelial cells, antagonizing the role of miR-23c on the down-regulation of target gene ELAV like RNA binding protein 1 (ELAVL1), which resulted in a better understanding of the pathogenesis of DN and help the development of new therapeutic strategies [10]. lncRNA X-inactive Specific Transcript (Xist) was identified to be up-regulated in membranous nephropathy (MN). Down-regulation of Xist may improve MN by reducing podocytes apoptosis via miR-217/TLR4 pathway [11]. Liao et al. reported that lncRNA RP11-2B6.2 was increased in kidney tissue from lupus nephritis (LN) patients and positively associated with disease activity and the level of type-I interferon (IFN) [12]. In IgAN patients, Zuo et al. reported 167 differentially expressed lncRNAs (55 up-regulated and 112 down-regulated lncRNAs) while compared with controls, which provided new information on the potential role of lncRNAs in IgAN [13]. Subsequently, this explicit change of lncRNA profile indicated the possibility of lncRNAs to be used as a molecular biomarker for IgAN. However, the inherent instability of lncRNAs (easily degraded by RNase in the blood) results in utterly difficult and inconsistent detection of lncRNAs in the blood is, therefore limits their clinical application [14].

Exosomes are small membrane vesicles with diameters less than 150 nm that are present in nearly all biological fluids (e.g., blood, breast milk, saliva and urine) [15]. Exosomes are known to encapsulate certain proteins, lipids, and RNA, and mediate intercellular communication between various types of cells [15]. Increasing researches are showing that not only can exosomes be diagnostic and prognostic markers for various kinds of malignancies [16], but they also play an important role in immunoregulation and the pathogenesis of immune related diseases such as rheumatoid arthritis (RA) [17], systemic lupus erythematosus (SLE) [18] and IgAN [19]. Exosomes could act as a protective barrier that can protect lncRNAs from extracellular degradation [14, 15], hence could be used as an excellent biomarker to detect significant changes in lncRNA profile in IgAN patients. In recent years, high-throughput RNA sequencing (RNA-seq) has become an attractive choice for the identification of differentially expressed genes (DEG) in various diseases as it has covers most of genome and exact detection of even those low-expressing genes. RNA-seq could achieve the resolution of a “single-base” and capture all transcripts [20]. This study utilized RNA-seq to search for differentially-expressed blood exosomal lncRNAs between IgAN patients and their first-degree healthy relatives. In addition, real-time PCR (qRT-PCR) was also conducted to validate the results of RNA-seq, which may provide clues to identify potential novel lncRNA biomarkers for IgAN.

In this study, the exosomal lncRNA profiles of IgAN patients were measured and compared to their healthy first-degree relative. Through high-throughput RNA seq, we identified lncRNA-G21551 as a potential diagnostic biomarker for IgAN. We also predicted its potential role in IgAN pathogenesis through pathway enrichment analysis and visualization using R package clusterProfiler. The nearest protein-coding gene of lncRNA-G21551 was identified to be FCGR3B, which encodes for the low affinity receptor (FcgR3B receptor) of the Fc segment of immunoglobulin G (IgG). Until the pathogenesis of IgAN is elucidated, renal biopsy will remain the golden standard for the diagnosis of IgAN. Therefore, our finding may bring forward a clue to find the disease biomarkers by exosomal lncRNA profiles in IgAN. The establishment of differentially expressed exosomal lncRNA profiles in IgAN could be important for illustrating the pathogenesis of IgAN.

lncRNAs-based biomarkers have been reported in a variety of diseases including membranous nephropathy, IgAN and hepatocellular carcinoma [11, 13, 14]. Zuo et al. identified 167 differentially expressed lncRNAs (including 55 upregulated lncRNAs and 112 downregulated lncRNAs) in peripheral blood mononuclear cells (PBMCs) of IgAN, which may aid in the elucidation of a basic pathogenic mechanism [13]. However, unprotected lncRNAs are easily degraded by RNAse, and exosomes act as a protective layer that can prevent extracellular degradation of lncRNAs [14]. Recently, emerging studies on exosomal non-coding RNAs (including lncRNAs and miRNAs) in renal disease have been reported [22], but few reports has been focused on IgAN. Min et al. reported a significantly difference in urinary exosomal miRNA profiles (including miR-29c, miR-146a and miR-205) between IgAN and healthy controls, which may serve as novel biomarkers for IgAN [19]. However, the differential expression of lncRNAs in plasma exosomes in IgAN patients was not reported, which may contribute to the discovery of potential new biomarkers or pathogenic factors of IgAN from plasma, as the increased levels of circulatory polymeric IgA1 with aberrant O-glycosylation of its hinge region was reported to be the first-hit as of this disease [1].

It was previously reported that copy number (CN) variation of the FCGR3B gene is associated with susceptibility to systemic lupus nephritis (SLE) and ANCA-associated systemic vasculitis (AASV) [23, 24]. Furthermore, the FcgR3B receptor is primarily expressed on neutrophils; while FCGR3B CNs are correlated with the expression of FcgR3B, functioning for the clearance of immune complex [25]. IgAN is frequently characterized by depositions of IgA (mainly IgA1) or IgA-containing immune complexes in the glomerular mesangial areas or the capillary wall. IgA1 deposits are usually detected along with complement component 3 (C3), and often with IgG or IgM or both in glomeruli [1]. However, a recent study has shown that FCGR3B polymorphisms have significant influence on the incidence and pathological grade of IgAN, suggesting that the impairment of IgG-IC clearance by the FCGR3B gene and subsequent glomerular deposition may also contribute to the glomerular lesions [26].

However, in this current study, we did not carry out mechanistic study on the direct evidence that could elucidate the interaction between lncRNA-G21551 and FCGR3B and the mechanisms involved in IgA nephropathy. The functional research of lncRNA-G21551 in IgAN may be an interesting new research area and is currently the scope of our research group.

In summary, our study demonstrates a significant difference in plasma exosomal lncRNA expression profiles between IgAN patients and their first-degree relatives, providing novel information on the potential role of exosomal lncRNAs in IgAN. Pathway enrichment analysis and visualization reveals that the FCGR3B gene may be closely associated with the pathogenesis of IgAN. Therefore, the expression level of exosomal lncRNA-G21551 could be utilized as a promising biomarker for IgAN diagnosis.