miRNAs are regulators of gene expression in diverse biological and pathological courses in life. Their discovery may be considered one of the most important steps in the story of modern biology. miRNAs are packed within exosomes and released by cells for cellular communications; they are present in bodily fluids. Their study opens the way for understanding the pathogenetic mechanisms of many diseases; furthermore, as potential candidate biomarkers, they can be measured in bodily fluids for non-invasive monitoring of disease outcomes. The present review highlights recent advances in the role of miRNAs in the pathogenesis of primary and secondary glomerulonephritides such as IgA nephropathy, focal segmental glomerular sclerosis, lupus nephritis and diabetic nephropathy. The identification of reciprocal expression of miRNAs and their target genes provides the molecular basis for additional information on the pathogenetic mechanisms of kidney diseases. Finally, recent findings demonstrate that miRNAs can be considered as potential targets for novel drugs.

INTRODUCTION

The universe of -omics in kidney diseases is a field of interesting growth. Data from genomics, transcriptomics, proteomics and metabolomics demonstrate the important role of the genome in the pathophysiology of kidney diseases [14]. The genome world is influenced by exogenous factors such as diet, environment, hyperglycaemia and uraemia, and modulated by the epigenome that is represented by methylated DNA, histones and miRNAs. The story of the latter began on 1993 when Lee et al. [5] described the first miRNA in Caenorhabditis elegans. Over the last two decades, it has been demonstrated that miRNAs regulate gene expression at the post-transcriptional level in multiple biological processes of human diseases such as glomerular diseases in nephrology [6].

miRNAs are small non-coding RNAs composed of ~21–25 nucleotides that are produced by genes and, during the multistage cellular process of biogenesis, they move from the nucleus to the cytoplasm where the binding to the 3′-UTR of target mRNAs induces degradation of RNAs or, more frequently, repression of protein translation [7]. Each miRNA regulates hundreds of different mRNAs, thus a large proportion of the transcriptome is modulated by miRNA regulation. This mechanism of miRNA-mediated gene silencing is composed of three phases: translational repression, deadenylation and degradation. More details can be read in the review of Huntzinger and Izaurralde [8]. In vitro studies, in which different types of mammalian cells [HeLa and HEK (human embryonic kidney)-293] have been used, have demonstrated that translation repression precedes mRNA decay [9] and translation inhibition is not triggered by deadenylation. Finally, miRNA-mediated silencing comes in the early step of the translation process [10] that can occur in four distinct ways: inhibition of translation initiation; inhibition of translation elongation; co-translation protein degradation; and premature termination of translation. However, additional information about the mechanistic connections between translational repression and mRNA degradation is necessary.

miRNAs are present in cells and tissues. Their involvement occurs in three different ways: (i) dysregulation of miRNA levels; (ii) a genetic variant of miRNA that alters the binding; and (iii) alteration within 3′-UTRs of mRNA. miRNAs are released by cells into the bodily fluids (i.e. plasma, serum, urine etc.) where they are relatively stable. Circulating miRNAs are resistant to RNase digestion because they are contained in circulating vesicles and others are carried by circulating the Argonaute2 complex that is one mechanism responsible for the stability of circulating miRNAs [11].

Serum, plasma and PBMCs (peripheral blood mononuclear cells) contain a large number of miRNAs; studies have demonstrated that they make up a pool of candidate molecules forming specific classifiers for diagnosis of diseases and for monitoring their outcome. miRNAs have been selected on the basis of their fold changes and potential value in the pathogenesis of disease. For this reason, miRNAs may be considered strategic candidate biomarkers for non-invasive diagnosis and early detection of disease. In the last 5 years, evidence has demonstrated that miRNAs are involved in the development of kidney and are essential for normal renal function [1215]. A cluster of miRNAs (miR-192, miR-194, miR-204 and miR-215) has been found in the human kidney [16] and a differential profile of miRNA expression has been shown by Tian et al. [17] in the renal cortex and medulla of rat kidney. Studies aimed at evaluating the role of miRNAs in the development and progression of renal damage can be considered as potential therapeutic targets in kidney diseases.

The aim of the present review is to highlight the role of miRNAs in the pathophysiology of some glomerular diseases and their implications in the field of therapy.

IgA NEPHROPATHY

IgAN (IgA nephropathy) is the most common worldwide primary glomerulonephritis characterized by the deposition of IgA1–IgG immune complexes in the mesangial area of glomeruli. The first hit of the disease is the abnormal production of galactose-deficient IgA1 (Gd-IgA1) by lymphocytes that move from the mucosa to the bone marrow during the acute phase of upper respiratory tract infections.

Blood

IgA1 is characterized by the presence of the hinge region located between heavy chain 1 and 2. It contains three to five O-linked glycans composed of GalNAc (N-acetylgalactosamine) and galactose. The addition of these glycans is modulated by two enzymes, GALNT2 (N-acetylgalactosaminyltransferase 2) and C1GALT1 (core 1 β1,3-galactosyltransferase 1) respectively. Reduced expression of these two enzymes has been observed in IgAN patients by some investigators [18,19], but the mechanism underlying this abnormal glycosylation is not well known. We have demonstrated that, in PBMCs of IgAN patients, the up-regulation of two miRNAs, let-7b and miR-148b, inhibits the activity of the two enzymes GALNT2 and C1GALT1 respectively [20,21]. These findings were validated in vitro by transfection studies that showed (i) miRNA mimics reduced the endogenous expression of enzymes, and (ii) the loss of the specific miRNAs increased the levels of GALNT2 and C1GALT1 respectively. Moreover, we found that the up-regulation of miR-148b was directly correlated with serum levels of Gd-IgA1. These data suggest a role for miRNAs in the abnormal glycosylation process of IgA1 in IgAN and indicate two potential targets for new therapeutic approaches in patients with IgAN.

High serum levels of let-7b and miR-148b have been recently detected in a multicentre study in which two large cohorts of Caucasian and Asian IgAN patients were enrolled [22]. A logistic regression analysis was performed to develop a predictive model based on these two miRNAs of which the diagnostic accuracy was assessed by the area under the ROC (receiver-operating characteristic) curve (AUC). The combined miRNA biomarker, let 7b and miR-148b, was able to discriminate each cohort of IgAN patients by healthy blood donors and non-IgAN patients. This biomarker seems to be a novel indicator to support the clinicians in the diagnosis of IgAN. Finally, the serum measurement of this biomarker could be used for monitoring the effect of new potential drugs modulating the glycosylation process of IgA1.

Kidney

Wang et al. [23] studied the intrarenal expression of miR-200 family, miR-205 and miR-192, and the gene expression of molecules involved in the EMT (epithelial–mesenchymal transition) process because tubular atrophy and interstitial fibrosis are the most important predictors of poor outcome in the prognosis of IgAN [23]. The decision to study these miRNAs was based on previous in vitro studies that demonstrated some members of the miR-200 family and miR-205 regulate EMT by repressing ZEB (zinc finger E-box-binding) homeobox 1 and ZEB2 that are transcription repressors of E-cadherin (epithelial cadherin) [24]. The intrarenal expression of miRNAs extracted from formalin-fixed and paraffin-embedded renal tissue blocks and quantified by qRT-PCR (real-time quantitative reverse transcription–PCR), showed a down-regulation of miR-200c and high values of miR-141, miR-205 and miR-192. Among genes involved in the EMT process, the intrarenal expression of vimentin was inversely correlated with that of miR-141, whereas the intrarenal expression of E-cadherin was correlated with that of miR-200c. No correlations were found for ZEB1 and SP1 (specificity protein 1). The expression of miRNAs studied was diversely correlated with disease severity and progression of IgAN. Later, the same investigators found elevated levels of miR-146a and miR-155 in kidney biopsies and urine of patients with IgAN [25]. These miRNAs (in tissue and in urine) were inversely correlated with the eGFR (estimated glomerular filtration rate) and positively with proteinuria. All together, these data suggest that intrarenal miRNAs play an important role in the progression of renal damage. These studies have some limitations. First, the investigators did not explore the total spectrum of miRNAs in the renal parenchyma; secondly, data were not validated by functional studies; and, finally, they did not search the cellular expression of miRNAs at the renal level.

Bao et al. [26] found an up-regulation of miR-21 in both glomerular and tubulointerstitial tissue of IgAN patients with enhanced expression in podocytes and tubular cells. Interestingly, in vitro cultured human MCs (mesangial cells) stimulated with polymeric IgA obtained from serum of IgAN patients produced an abnormal amount of TGFβ1 (transforming growth factor β1) and TNFα (tumour necrosis factor α) that induced up-regulation of miR-21 in podocytes and HK2 (human kidney 2) cells. These cells showed high expression of fibrinogen and Col1 (collagen I). In vitro inhibition of miR-21 prevented PTEN (phosphatase and tensin homologue deleted on chromosome 10)/Akt pathway activation and suppressed the expression of fibrinogen and Col1. Thus it means that inhibition of miR-21 may prevent fibrinogenic activity in IgAN. Recently, the same investigators using the same methodological approach demonstrated that miR-223 was down-regulated when glomerular endothelial cells were incubated in vitro in conditioned medium of human MCs after stimulation with polymeric IgA obtained from serum of IgAN patients [27]. Treatment with MC-derived IL (interleukin)-6 caused the decrease in miR-223 leading to high expression of ICAM-1 and monocyte–endothelial adhesion in glomerular endothelial cells. In addition, treatment with miR-223 mimics inhibits the production of importin α4 and α5 that mediate the translocation of NF-κB (nuclear factor κB) and STAT3 (signal transducer and activator of transcription 3) into the nucleus. In conclusion, glomerular sclerosis indicates the progression of renal damage in IgAN, as shown by repeated biopsies, and glomerular endothelial cells participate in this process. The measurement of the miR-223 level in circulating endothelial cells may be used as non-invasive method for monitoring the progression of renal damage in IgAN.

Recently, Tan et al. [28] studied the miRNA expression profile in a pool of six kidney biopsies from IgAN patients compared with normal renal cortex from six individuals who received nephrectomy for kidney cancer. They observed a total of 85 miRNAs that were differentially expressed in IgAN patients. miR-10b, miR-133b and miR-486 were highly expressed, whereas miR-424, miR-143 and miR-200c were down-regulated. The expression of these miRNAs was correlated with glomerular sclerosis and interstitial fibrosis. Interestingly, they found that miR-148b, previously found to be up-regulated in PBMCs of IgAN patients, was down-regulated in kidney biopsies. They explained this difference on the basis of organ specificity. Fang et al. [29] found a down-regulation of miR-29c and high expression of related TPM1 (tropomyosin 1) and COL2A1 (collagen type II α1) target genes in renal tissue of IgAN patients [29]. These findings were confirmed by additional in vitro studies on cultured HK2 cells in which TGFβ1 significantly reduced miR-29c expression with a related increase in TPM1. The bioinformatics analysis showed that COL2A1 and TPM1 are targets of miR-29c. In the presence of down-regulated miR-29c expression, there was an increased production of collagen and consequent renal fibrosis.

Urine

miR-21, miR-29 family and miR-93 were measured by Wang et al. [30] in urine from IgAN patients. They found low urinary levels of miR-29b and miR-29c that were correlated with proteinuria and renal function, but high levels of miR-93 that was correlated with glomerular scarring. Furthermore, urinary miRNAs (miR-21, miR-29b, miR-29c and miR-93) were correlated significantly with the Smad3 pathway that is involved in fibrosis. The authors concluded that the measurement of these miRNAs may be considered novel biomarkers for monitoring the development of fibrosis. Further study by the same group of investigators observed high values of miR-17 expression in urinary sediment of IgAN patients [31]. It has been demonstrated that the overexpression of this miRNA promotes cell proliferation via post-transcriptionally depression of target genes [32]. Therefore down-regulation of miR-17 in urine may be considered a biomarker of progression of renal damage. Data on low urinary levels of miR-200a, miR-200b and miR-429 that was inversely correlated with vimentin and positively with proteinuria were reported by Wang et al. [33]. They suggested that the measurement of these miRNAs in the urine may be indicative of progression of renal damage and expression of disease severity.

In conclusion, data from PBMCs and serum clearly show that let-7b and miR-148b are involved in the modulation of the glycosylation process of IgA1 which plays an important role in the pathogenesis of IgAN. Data from kidney tissue and urine show that other miRNAs, listed in Table 1, modulate the process of renal fibrosis and progression of renal damage. However, these studies have some limitations. First, data on miRNAs have been obtained only in Asian IgAN patients. Secondly, a limited sample size was used by all investigators. Thirdly, there are no functional studies on these miRNAs. Finally, we believe that, after validation of these findings in different populations, it is necessary to carry out a bioinformatics analysis for studying related associations between all reported miRNAs and target genes.

Table 1
miRNAs and their target genes in IgA nephropathy
miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
PBMCs/serum     
 Serino et al. [20,21let -7b  GALNT2 Glycosylation process of IgA1 
 miR-148b  C1GALT1  
Kidney     
 Wang et al. [23miR-141 miR-200c Vimentin, E-cadherin Interstitial fibrosis 
 miR-192    
 miR-205    
 Wang et al. [25miR-146a    
 miR-155    
 Bao et al. [26,27miR-21  Fibrinogen and Col1 Renal fibrosis 
  miR-223 ICAM1 Monocyte–endothelial adhesion 
 Tan et al. [28miR-10b-5p miR-424-5p  Renal fibrosis 
 miR-133a miR-143-5p   
 miR-133b miR-200c-3p   
 miR-486-5p miR-148b   
 Fang et al. [29 miR-29c TPMI and COL2A1 Renal fibrosis 
Urine     
 Wang et al. [30miR-93 miR-29b Smad3 Renal fibrosis 
  miR-29c   
 Szeto et al. [31miR-17   Cell proliferation 
 Wang et al. [33 miR-200a Vimentin Renal damage 
  miR-200b   
  miR-429   
miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
PBMCs/serum     
 Serino et al. [20,21let -7b  GALNT2 Glycosylation process of IgA1 
 miR-148b  C1GALT1  
Kidney     
 Wang et al. [23miR-141 miR-200c Vimentin, E-cadherin Interstitial fibrosis 
 miR-192    
 miR-205    
 Wang et al. [25miR-146a    
 miR-155    
 Bao et al. [26,27miR-21  Fibrinogen and Col1 Renal fibrosis 
  miR-223 ICAM1 Monocyte–endothelial adhesion 
 Tan et al. [28miR-10b-5p miR-424-5p  Renal fibrosis 
 miR-133a miR-143-5p   
 miR-133b miR-200c-3p   
 miR-486-5p miR-148b   
 Fang et al. [29 miR-29c TPMI and COL2A1 Renal fibrosis 
Urine     
 Wang et al. [30miR-93 miR-29b Smad3 Renal fibrosis 
  miR-29c   
 Szeto et al. [31miR-17   Cell proliferation 
 Wang et al. [33 miR-200a Vimentin Renal damage 
  miR-200b   
  miR-429   

FOCAL SEGMENTAL GLOMERULAR SCLEROSIS

Primary FSGS (focal segmental glomerular sclerosis) is characterized by an extensive flattening of podocyte foot processes, glomerular sclerosis, tubular atrophy and interstitial fibrosis.

Blood

Cai et al. [34] measured the serum levels of two kidney-specific miRNAs, miR-192 and miR-205, that are involved in the EMT process. Two categories of young patients with biopsy-proven FSGS and MCD (minimal change disease) were studied [34]. They found increased levels of both miRNAs only in patients with FSGS that were significantly correlated with proteinuria and degree of renal fibrosis. These findings require validation because they were obtained by a single centre in a limited number of serum samples. In addition, there were no data on the effect of drugs such as corticosteroids and calcineurin-inhibitors on the serum levels of these miRNAs. Finally, these data should be implemented by miRNA expression in renal biopsies.

Kidney

Since the miR-30 family, composed of five members from miR-30a to miR-30e, is notably expressed in podocytes, Wu et al. [35] focused their attention on these miRNAs in FSGS considering that they may regulate the gene expression and consequently the function of these glomerular cells. In addition, the miR-30 family controls a series of pathways such as Notch, p53, Wnt/β-catenin and actin cytoskeleton. The Chinese investigators found a down-regulation of all members of the miR-30 family in microdissected glomeruli of 16 renal biopsies from patients with FSGS compared with six controls. In situ hybridization of miR-30a and miR-30d demonstrated an abundant expression in normal podocytes that was reduced in the sclerotic areas of glomeruli. These findings were confirmed in vitro when human podocytes were treated with some growth factors. TGFβ, LPS (lipopolysaccharide) and PAN (puromycin aminonucleoside) down-regulated all five miR-30 family members with consequent severe cytoskeletal damage. The use of a podocyte cell line that stably expressed exogenous miR-30a showed a lower percentage of annexin V-positive apoptotic cells after TGFβ treatment, thus indicating that miR-30a has an anti-apoptotic effect in podocytes. This protective effect of miR-30a was also indirectly demonstrated in podocytes using the miR-30 sponge after TGFβ treatment by direct inhibition of Notch1 and p53 which mediate podocyte injury. Furthermore, the transfer of miR-30a in podocytes of rats treated with PAN improved podocyte injury, reducing Notch1 activation. Glucocorticoids also contributed to improve the miR-30 expression in podocytes. These findings underline the protective role of miR-30 family in the podocyte damage of FSGS.

Kerjaschki's group demonstrated the important role of miR-193a in the destabilization of podocyte foot process [36]. This miRNA was studied following its identification in an miRNA screening in breast tumour genesis and the generation of a miR-193a transgenic mouse that, after treatment with doxycycline, showed a 10-fold increased expression of miR-193a in podocytes of glomeruli and death of mouse from kidney disease characterized by alteration of the glomerular architecture accompanied by increasing albuminuria, tubular atrophy, scarring and cyst formation. Focal sclerosis at the glomerular level increased with time and, after 4 weeks, podocytes were completely flattened and slit diaphragms were fused. The gene expression analysis of isolated glomeruli demonstrated a large number of down-regulated genes, some of which were putative targets of the miR-193 family. Six of them were related to the podocytes and only WT1 (Wilms’ tumour protein 1) was previously demonstrated to be linked to FSGS [37]. The transgenic expression of miR-193a in mice induced development of FSGS with extensive flattening of podocyte foot processes, loss of their architecture and proteinuria. Gebeshuber et al. [36] demonstrated that miR-193a suppressed the transcription factor WT1 that regulates the maturation process of podocytes. The loss of WT1 down-regulates podocyte proteins such as podocalyxin, nephrin and podocin with consequent collapse of the entire podocyte architecture. These combined results obtained from in vitro experiments were validated in mouse model of FSGS and in human tissues of individuals with FSGS. Thus miR-193a initiates a cascade of podocyte-destabilizing molecular events and its blockade by the injection of an 8-meric LNA-193 (locked nucleic acid 193) showed an enhanced expression of WT1, podocalyxin and nephrin in podocytes, and reduced proteinuria. These findings indicate miR-193a as potential therapeutic target of FSGS.

Urine

Wang et al. [38] investigated miRNA expression in the urinary cellular sediment of adult patients with nephrotic syndrome. They studied a panel of 11 miRNAs and two miRNA families (miR-29 and miR-200) and found that miR-200c expression was increased in the urinary cells of patients with MCD and FSGS, but the small number of cases did not give the opportunity to analyse these groups separately. Only miR-184 was correlated with the renal function. The poor results of this study suggest that further research in a large number of patients with long-term follow-up is required.

In conclusion, data summarized in Table 2 show that some miRNAs are involved in the pathogenesis of renal damage in FSGS. Some of them, such as miR-30a and miR-193a participate in the cascade of molecular events occurring in the podocyte which remains the main target of the disease. These miRNAs could be considered for further studies on the treatment of FSGS.

Table 2
miRNAs and their target genes in focal segmental glomerular sclerosis
miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
Blood     
 Cai et al. [34miR-192   Participation in the EMT process 
 miR-205    
Kidney     
 Wu et al. [35 miR-30 family (miR-30aAnnexin V Anti-apoptotic effect in podocytes 
 Gebeshuber et al. [36miR-193a  WT1 Maturation process of podocytes 
Urine     
 Wang et al. [38miR-200c    
miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
Blood     
 Cai et al. [34miR-192   Participation in the EMT process 
 miR-205    
Kidney     
 Wu et al. [35 miR-30 family (miR-30aAnnexin V Anti-apoptotic effect in podocytes 
 Gebeshuber et al. [36miR-193a  WT1 Maturation process of podocytes 
Urine     
 Wang et al. [38miR-200c    

LUPUS NEPHRITIS

SLE (systemic lupus erythaematosus) is an immune complex disease characterized by an aberrant activation of T- and B-lymphocytes that produce an abnormal amount of autoantibodies directed against the nuclear components and other self antigens. There is also the participation of genetic and environmental factors. The local deposition of immune complexes at kidney level causes LN (lupus nephritis). Since miRNAs may be considered potential regulators of innate and adaptive immune cell response and relative inflammatory process, they have been considered key players in this disease.

Blood

Dai et al. [39] for the first time described the miRNA expression profile in PBMCs isolated from Chinese SLE patients and identified 16 miRNAs of which nine were up-regulated and seven were down-regulated. Then, Te et al. [40] investigated on PBMCs and EBV (Epstein–Barr virus)-transformed B-cell lines of African-American and European-American patients with LN. They found 18 miRNAs that were differently expressed in both racial groups; five miRNAs (miR-371-5p, miR-423-5p, miR-638, miR-1224 and miR-663) were specific for SLE disease and three of them, miR-371-5p, miR-423-5p and miR-1224-3p, were associated with LN. Bioinformatics analysis demonstrated that the potential gene targets of these miRNAs are producers of mediators involved in the IFN (interferon) signalling. Unfortunately, this report is lacking functional studies. The different miRNAs identified by Dai et al. [39] and Te et al. [40] may be explained by different populations and races studied and by the different techniques used.

Interesting is the role of miRNAs in DNA methylation process in SLE. Pan et al [41] demonstrated that miR-21 and miR-148a were highly expressed in CD4+ T-cells of SLE patients and MRL/lpr mouse strain; these miRNA abnormalities contributed to DNA hypomethylation in these cells. miR-21 indirectly down-regulated DNMT1 (DNA methyltransferase 1) expression by targeting the RASGRP1 (Ras guanyl-releasing protein 1) gene that mediates the Ras/MAPK (mitogen-activated protein kinase) pathway upstream of DNMT1. miR-148a directly inhibited DNMT1 expression by targeting the protein coding region of its transcript, thus modulating the post-transcriptional control mechanism of DNA methylation. These results provide evidence for a dysregulation of the DNA methylation process caused by an increased expression of the two miRNAs in SLE patients. Another miRNA regulating the DNA methylation in CD4+ T-cells was studied by Zhao et al. [42] in 30 SLE patients. They found that up-regulated miR-126 directly inhibited the DNMT1 translation via interaction with its 3′-UTR, thus reducing the relative expression of protein. In conclusion, three overexpressed miRNAs are responsible for the DNA hypomethylation in CD4+ T-cells that contributes to T-cell autoreactivity in SLE.

The role of miRNAs has also been explored in immunity and some studies have identified miR-146a and miR-181a as regulators of innate and adaptive immunity respectively. Tang et al. [43] demonstrated that among 52 miRNAs differentially expressed in SLE patients compared with normal controls, seven of them were more than 6-fold less expressed. Among these, miR-146a targeted some genes such as IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6) that are involved in the innate immunity because they are two signal transducers in the NF-κB activation pathways. Therefore, miR-146a reduces the inflammatory response by down-regulating IRAK1 and TRAF6. miR-146a expression levels were negatively correlated with SLE disease activity [SLEDAI (SLE disease activity index) score] and IFN-inducible gene scores demonstrating its important role in the innate immunity involved in the progression of the disease. These findings were confirmed in vitro on PBMCs in which the overexpression of miR-146a reduced the induction of the type I IFN pathway by targeting the key components of the signalling cascade (IRAK1 and TRAF6). In addition, miR-146a reduced the protein expression of IRF5 (interferon-regulatory factor 5) and STAT1 that are key signalling proteins of the type I IFN pathway. Later, the same group demonstrated that the genetic variants (rs57095329) in the promoter region of the miR-146 gene was responsible for a reduced expression of miR-146a and was associated with SLE susceptibility [44]. This variant in individuals with GG genotype had the lowest miR-146a expression and conferred reduced binding of Ets1, a transcription factor involved in the process of miR-146a expression. This genetic variant is an independent risk factor for SLE in Asians.

Considering that miR-146a dysregulation is responsible for hyperactivation of type I IFN pathway, the manipulation of this miRNA could be a probable therapeutic intervention in SLE. Pan et al. [45] investigated this hypothesis by administering VLPs (virus-like particles) containing miR-146a into lupus-prone BXSB mice. These particles transferred the packaged pre-miR-146a RNA into cells and tissues leading to the overexpression of miR-146a. The 20-week-old BXSB mice had a low expression of miR-146a in PBMCs, lung, spleen and kidney. Notably, after miR-146a-VLP administration the expression of this miRNA increased in cells and tissues and a significant reduction of serum anti-dsDNA antibodies and ANA (autoantibody to nuclear antigen) was observed. Furthermore, miR-146a-VLPs reduced the expression of SLE-related inflammatory cytokines (IFNα, IL-1β and IL-6), and suppressed both IRAK1 and TRAF6 in PBMCs. These findings suggest that miR-146a-VLPs may be considered a therapeutic option for murine lupus. The benefit of this treatment in SLE patients remains to be demonstrated.

Zhao et al. [46] demonstrated that a down-regulation of miR-125a in T-cells of SLE patients was responsible for an up-regulation of KLF13 (Krüppel-like factor 13), leading to an elevation of inflammatory chemokines such as RANTES (regulated upon activation, normal T-cell expressed and secreted) regulating the activation of T-cells. This miRNA could be considered a therapeutic target for reducing the inflammatory cytokine production in SLE. In the context of immune tolerance loss in SLE, Divekar et al. [47] reported interesting data on Dicer insufficiency and miR-155 overexpression in Tregs (regulatory T-cells) from two mouse models (MRL/lpr and MRL+/+). They demonstrated that the miRNA signature in Tregs plays a role in the development and function of these cells, especially miR-155 overexpression which reduces CD62L expression causing an altered function of Tregs in the MRL/lpr mice.

Circulating miRNAs have been measured in serum and plasma samples of patients with SLE. Wang et al. [48] found significant low serum levels of the miR-200 family, miR-205 and miR-192 in SLE patients that was correlated with disease activity. They studied these miRNAs because they inhibit the expression of ZEB1 and ZEB2 which are two molecules involved in the process of EMT and fibrosis. Unfortunately, the investigators did not study the correlations between miRNAs and their protein targets; in addition, the data obtained were not validated by in vitro functional studies and in other cohorts of SLE patients. Then, the same group investigated two additional miRNAs (miR-146a and miR-155) that were found to be significantly lower in the serum samples of 40 SLE patients and positively correlated with renal function [49]. These data confirmed the results of Tang et al. [43] observed previously in PBMCs. Furthermore, they found an improvement of miRNAs after administration of calcitriol [49]. These data were not confirmed by functional studies and by long-term follow-up. However, these results may indicate that calcitriol modulates the immune system by increasing the serum levels of miR-146a.

Recently, Dai's group described miRNA profiles in a pool of plasma samples from SLE patients compared with RA (rheumatoid arthritis) patients, who had never received non-steroidal anti-inflammatory and immunosuppressive drugs, and healthy controls [50]. They identified an miRNA profile constituted by up-regulated miRNAs (miR-126, miR-21, miR-451, miR-223 and miR-16), and down-regulated miRNAs (miR-125a-3p, miR-155 and miR-146a). The KEGG map individualized the gene targets of these miRNAs and the most significant pathways were MAPK and ERK (extracellular-signal-regulated kinase) pathways. MAPK is an important regulator of inflammatory and immune responses; ERK is defective in T-cells of SLE because there is a reduced activation of PKCδ (protein kinase Cδ) activation. Several up-regulated circulating miRNAs (miR-126, miR-21, miR-223 and miR-16) are highly expressed in CD4+ T-cells and in PBMCs as reported in previous papers [41,42]. Thus it means that circulating plasma miRNAs are mainly derived from circulating blood cells; however, there is a selective release of miRNAs from cells. These findings have not been confirmed in another cohort of SLE patients.

Szeto's group measured miR-146a and miR-155 in serum and urine samples of patients with SLE [49] because these miRNAs are regulators of the immune system. They found low levels of these miRNAs in serum that were correlated positively with renal function and negatively with SLEDAI and proteinuria. These findings are consistent with data reported by Tang et al. [43] who showed a down-regulated expression of miR-146a in PBMCs of SLE patients that negatively modulate the type I IFN pathway.

Recently, Carlsen et al. [51] developed a well-designed study in which the results were validated in two independent cohorts (Danish and Swedish SLE patients). The up-regulation of miR-142-3p and miR-181a, and the down-regulation of miR-106a, miR-17, miR-20a, miR-203 and miR-92 represented the miRNA pattern of patients with SLE. Interestingly, the most targeted genes of these miRNAs led to TGFβ signalling, actin cytoskeleton, MAPK signalling and cytokine/cytokine receptor pathways that are involved in the inflammatory process of SLE. Then, the authors using the four top-performing miRNAs (miR-142-3p, miR-106a, miR-17 and miR-20a) created a predictive model by unsupervised hierarchical clustering. This top model was validated in the second cohort of patients; however, no difference was observed between patients with or without treatment, demonstrating that the dysregulation of these miRNAs in SLE is independent of immunosuppressive therapy. The presence of active nephritis was associated with decreased levels of miR-342-3p, miR-223 and miR-20a. The difference between the values of the two most significant miRNAs (miR-142-3p and miR-106a) was used to classify samples and the ROC curves of this combined biomarker were 0.95 and 0.89 in the Danish and Swedish cohort respectively. Thus this study suggests the use of a classifier based on four miRNAs for analysing the SLE risk.

In conclusion, as shown in Table 3, some miRNAs, such as miR-148a and miR-126, play an important role in the DNA methylation process; others, such as miR-21, miR-146a, miR-125 and miR-155, are involved in the dysregulated innate and adaptive immunity of SLE/LN; finally many other miRNAs are responsible for abnormal production of inflammatory molecules/cytokines.

Table 3
miRNAs and their target genes in systemic lupus erythaematosus/lupus nephritis

G, glomeruli; TI, tubulointerstitium.

miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
PBMCs     
 Dai et al. [39miR-189 miR-196a Hox3a  
 miR-61 miR-17-5p Transcription factor E2F1  
 miR-78 miR-409-3p   
 miR-21 miR-141   
 miR-142-3p miR-383   
 miR-342 miR-142   
 miR-299 miR-184   
 miR-198    
 miR-298 (mouse)    
 Te et al. [40miR-371-5p  IL32, IFIT3, IFIT2, FGR, IRF5, CD40, PTTG1 IFN pathway 
 miR-423 -5p  SLC2A4, VGF, SOX12  
 miR-638  CD79B, LY6E, ZNF330  
 miR-663  IL32, IFI35, CENTA1, LY6E, ZNF330  
  miR-1224-3p GPDH, PMVH, BSG  
 Pan et al. [41miR-21  RASGRP1 DNA methylation 
 miR-148a  Coding sequence of DNMT1  
 Zhao et al. [42miR-126  DNMT1  
 Tang et al. [43 miR-31   
  miR-95   
  miR-99a   
  miR-130b   
  miR-10a   
  miR-134   
  miR-146a IRAK1, TRAF6, IRF5, STAT1 IFN pathway 
 Zhao et al. [46 miR-125a KLF13 Activation of T-cells 
 Divekar et al. [47miR-155 (mouse)  CD62L Function of Tregs 
Serum/plasma     
 Wang et al. [48 miR-200a/b/c   
  miR-429   
  miR-205 ZEB1 and ZEB2 EMT process and fibrosis 
  miR-192 ZEB2  
 Wang et al. [49 miR-146a   
  miR-155   
 Wang et al. [50miR-126 miR-125a-3p MAPK pathway/ERK pathway Regulator of inflammatory response 
 miR-21 miR-155   
 miR-451 miR-146a   
 miR-223    
 miR-16    
 Carlsen et al. [51miR-142-3p miR-106a  TGF signalling/MAPK signalling 
 miR-181a miR-17   
  miR-20a   
  miR-203   
  miR-92   
Kidney     
 Dai et al. [52miR-516-5p miR-637   
  miR-223  Granulopoiesis 
 Lu et al. [53miR-638 (TI) miR-638 (G)   
 miR-198 (G, TI)    
 miR-146a (G, TI)  Fn14  
 Zhou et al. [54miR-150  COL1, SOCS1 Renal fibrosis 
Urine     
 Wang et al. [48 miR-200a/c   
  miR-141   
  miR-429   
 Wang et al. [49miR-146a   Regulators of immune system 
 miR-155    
miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
PBMCs     
 Dai et al. [39miR-189 miR-196a Hox3a  
 miR-61 miR-17-5p Transcription factor E2F1  
 miR-78 miR-409-3p   
 miR-21 miR-141   
 miR-142-3p miR-383   
 miR-342 miR-142   
 miR-299 miR-184   
 miR-198    
 miR-298 (mouse)    
 Te et al. [40miR-371-5p  IL32, IFIT3, IFIT2, FGR, IRF5, CD40, PTTG1 IFN pathway 
 miR-423 -5p  SLC2A4, VGF, SOX12  
 miR-638  CD79B, LY6E, ZNF330  
 miR-663  IL32, IFI35, CENTA1, LY6E, ZNF330  
  miR-1224-3p GPDH, PMVH, BSG  
 Pan et al. [41miR-21  RASGRP1 DNA methylation 
 miR-148a  Coding sequence of DNMT1  
 Zhao et al. [42miR-126  DNMT1  
 Tang et al. [43 miR-31   
  miR-95   
  miR-99a   
  miR-130b   
  miR-10a   
  miR-134   
  miR-146a IRAK1, TRAF6, IRF5, STAT1 IFN pathway 
 Zhao et al. [46 miR-125a KLF13 Activation of T-cells 
 Divekar et al. [47miR-155 (mouse)  CD62L Function of Tregs 
Serum/plasma     
 Wang et al. [48 miR-200a/b/c   
  miR-429   
  miR-205 ZEB1 and ZEB2 EMT process and fibrosis 
  miR-192 ZEB2  
 Wang et al. [49 miR-146a   
  miR-155   
 Wang et al. [50miR-126 miR-125a-3p MAPK pathway/ERK pathway Regulator of inflammatory response 
 miR-21 miR-155   
 miR-451 miR-146a   
 miR-223    
 miR-16    
 Carlsen et al. [51miR-142-3p miR-106a  TGF signalling/MAPK signalling 
 miR-181a miR-17   
  miR-20a   
  miR-203   
  miR-92   
Kidney     
 Dai et al. [52miR-516-5p miR-637   
  miR-223  Granulopoiesis 
 Lu et al. [53miR-638 (TI) miR-638 (G)   
 miR-198 (G, TI)    
 miR-146a (G, TI)  Fn14  
 Zhou et al. [54miR-150  COL1, SOCS1 Renal fibrosis 
Urine     
 Wang et al. [48 miR-200a/c   
  miR-141   
  miR-429   
 Wang et al. [49miR-146a   Regulators of immune system 
 miR-155    

Kidney

A few studies of miRNA transcriptomics on renal biopsies of patients with SLE have been published, whereas many results have been obtained from kidneys of lupus-prone mice. In the present review, we have focused only on studies from human beings (Table 3). Dai et al. [52] applied renal transcriptomics in five patients with class II LN and identified 66 miRNAs of which 30 were down-regulated and 36 up-regulated. Only miR-516-5p and miR-637 were validated by qRT-PCR. Finally, miR-223 that regulates the granulopoiesis was found down-regulated in SLE patients. None of these miRNAs was included in the list of miRNAs described by the same group of investigators in PBMCs [39]. This confirms that miRNAs are organ-specific.

Lu et al. [53] examined the miRNA expression in the glomerular and tubulointerstitial compartments of kidney from LN patients. miR-638 had lower expression at the glomerular level and higher expression in the tubulointerstitial compartment, whereas miR-198 and miR-146a were highly expressed in both compartments. Glomerular expression of miR-638 was correlated with the histological activity index and the tubulointerstitial expression was correlated with proteinuria and the SLEDAI score. Finally, the glomerular expression of miR-146a was correlated with both eGFR and the histological activity index. The glomerular and tubulointerstitial expression of the target gene Fn14 was correlated with glomerular expression of miR-146a and miR-155 respectively. The tubulointerstitial expression of CXCR3 (CXC chemokine receptor 3) was correlated with that of miR-146a.

Zhou et al. [54] identified 16 miRNAs with >2-fold increases in 14 FFPE renal biopsies from patients with LN. Of these, miR-150 was up-regulated 4-fold and this increase was validated by qRT-PCR in seven renal biopsies from an independent cohort of LN patients. There was a strong correlation of this miRNA with changes in CI (chronicity index); the ROC analysis showed that renal miR-150 predicted CI≥4 with high diagnostic accuracy. This miRNA was predominantly expressed in PTCs (proximal tubular cells) and was not correlated with the activity index, whereas miR-150 expression correlated with renal Col1 and targeted SOCS1 (suppressor of cytokine signalling 1). Therefore miR-150 overexpression down-regulated SOCS1 protein and this positive correlation on renal fibrosis was documented in co-transfected PTCs and MCs in which the increase in miR-150 down-regulated SOCS1. Consequently, the investigators concluded that this miRNAs modulates some proteins of renal fibrosis in LN patients. In addition, they demonstrated that TGFβ, which is an important regulator of fibrosis, induced a 6-fold increase in miR-150 in podocytes that was reversed by transfection of an miR-150 inhibitor in PTCs. These findings were confirmed in renal biopsies of LN patients by low expression of SOCS1 in the presence of high miR-150. In conclusion, chronic inflammation leads to an increase in TGFβ which induces high expression of miR-150 directly in PTCs and podocytes, whereas this miRNA is increased by other factors in MCs. Thus miR-150 may be considered a potential biomarker for evaluating the renal outcome of patients with LN. However, these findings need validation in a large cohort of patients.

Urine

Two studies of urinary miRNAs reported by Szeto's group found low significant levels of miR-200a, miR-200c, miR-141 and miR-429 in patients with SLE [48,49]. There was no correlation between serum and urinary levels of these miRNAs and finally urinary levels did not correlate with any clinical parameter. In the second article, they focused on the levels of miR-146a and miR-155 because they are regulators of the immune system [49]. Only the urinary values of miR-146a were significantly higher in SLE patients compared with healthy subjects, but were not correlated with clinical findings.

In conclusion, data from the aforementioned studies, listed in Table 3, indicate that miRNAs dysregulated in their expression contribute to altered DNA methylation, innate and adaptive immune cell responsiveness and production of inflammatory mediators in SLE/LN. The easy measurement of specific circulating miRNAs, described above, by qRT-PCR suggests that they can be used for detecting phases of the disease at the blood, kidney and urine level. Thus miRNAs can be considered potential biomarkers for monitoring the outcome of the disease and response to immune suppressive therapy. Finally, the possibility to improve the disease outcome by miRNA-based therapies has been shown by Pan et al. [45] who ameliorated the serum levels of miR-146a after the administration of miR-146a-VLPs.

DIABETIC NEPHROPATHY

DN (diabetic nephropathy) is the most common microvascular complication of Type 1 and Type 2 diabetes mellitus. Since it is the leading cause of ESKD (end-stage kidney disease), prevention today is a challenging task in the world. Several studies have evaluated the role of miRNAs in Type 2 DN considering that the first hit is represented by hyperglycaemia and the second one by the increased production of TGFβ in renal cells during the onset and progression of renal lesions. This leads to the development of mesangial hypertrophy, glomerular basement membrane thickening and tubulointerstitial fibrosis associated with declining eGFR. In this scenario, it has been demonstrated that an altered expression of miRNAs documented previously in animal models of DN and then in renal biopsies from patients with DN is present. Kato et al. [55] first reported an intrarenal overexpression of miR-192 in mouse MCs and then in glomeruli of STZ (streptozotocin)-induced diabetic mice (Type 1 DN) and diabetic db/db mice (Type 2 DN). TGFβ is the growth factor inducing the overexpression of miR-192 in mouse MCs with sequential repression of the target gene SIP1 (Smad-interacting protein 1) and abnormal production of Col1α2. The mechanism by which TGFβ up-regulates miR-192 is based on the Ets1-binding site of this miRNA. Later, Putta et al. [56] confirmed that miR-192 increases collagen expression by targeting E-box repressor ZEB1 and ZEB2. They evaluated the efficacy of an LNA–anti-miR-192 in C57BL/6 diabetic mice and demonstrated a decreased renal expression of extracellular matrix associated pro-fibrotic genes such as Col1α2, Col4α1, TGFβ, connective tissue growth factor and fibronectin. Thus the accumulation of matrix proteins in the kidney was significantly suppressed after LNA–anti-miR-192 treatment and, furthermore, a decrease in proteinuria and albuminuria was observed in the diabetic mice. Therefore miR-192 may be considered a target for treatment of DN and for antagomiRs as potential agents that inhibit miRNAs. Wang et al. [57] demonstrated that the role of miR-192 in human kidney disease is more complex than previously reported. They investigated the role of miR-192 and miR-215 in combination on rat PTCs, rat primary MCs and human immortalized podocytes as translational regulators of the ZEB2 transcription factor and ECM (extracellular matrix) production. They also studied the miRNA expression in kidney of STZ-induced diabetic apoE (apolipoprotein E)-knockout mice [57]. They demonstrated that the combination of the miRNAs studied was down-regulated in its expression by TGFβ and this decrease was associated with high production of ECM, high levels of ZEB2 and decreased transcription of E-cadherin. These results have been confirmed in humans, because Krupa et al. [58], in a study carried out on pooled RNA obtained from formalin-fixed paraffin-embedded renal biopsies, demonstrated a lower expression of miR-192 in patients with severe DN that was correlated with tubulointerstitial fibrosis and low estimated eGFR. These findings were validated in vitro by treating PTCs with TGFβ1 that showed decreased miR-192 expression. The absence of a follow-up represents the major limitation of this study. However, the contrasting results obtained by the investigators in vitro and in vivo may be explained by different sources of material (mouse compared with human kidney tissue) and diverse experimental models (conditions and time points).

Other miRNAs are involved in the development of renal injury in DN. Wang et al. [59] demonstrated an up-regulation of miR-377 in cultured human and mouse MCs exposed to high concentration of glucose and TGFβ that represent the classic diabetic milieu in in vitro experiments. These results were confirmed in renal cortex of kidneys from non-obese diabetic (NOD/Lt) mice and STZ-induced Type 1 diabetes models after 22 and 10 weeks of diabetes respectively. In addition, in both primary and immortalized MCs, cultured in vitro in the presence of high glucose concentration, the overexpression of miR-377 was associated with increased production of fibronectin. Among 1092 potential target genes of miR-377, 67 were confirmed to be targets of human MCs. Three of them, PAK1 (p21-activating kinase 1), SOD1 (superoxide dismutase 1) and SOD2 were reduced as protein expression by exogenous miR-377 in diabetic MCs, thus inducing an increased production of fibronectin. These results suggest that miR-377 participates indirectly to increase the matrix proteins in glomeruli of DN.

By using an integrated in vitro and in vivo approach, Long et al. [60] demonstrated an increased expression of miR-29c in cultured podocytes and renal endothelial cells after stimulation with a high concentration of glucose and in diabetic kidney of db/db mice. This miRNA inhibited the Spry1 (Sprouty homologue 1) target gene and indirectly stimulated Rho kinase [60]. Thus miR-29c promoted in vitro cell apoptosis and increased fibronectin synthesis, and in vivo caused accumulation of mesangial matrix in glomeruli. The intraperitoneal administration of anti-miR-29c in aged db/db diabetic mice restored Spry1 and improved the mesangial matrix expansion in glomeruli and albuminuria. In conclusion, these findings indicate another target for DN therapy. The same group of investigators identified miR-93 as a novel regulator of VEGF (vascular endothelial growth factor) in in vitro and in vivo experimental models of hyperglycaemic conditions [61]. VEGFα was the putative target of miR-93 because the use of inhibitor anti-miR-93 increased the release of VEGF in vitro and in vivo in diabetic db/db mice. Thus miR-93 is another target of DN.

Kato's group reported an up-regulation of other miRNAs (miR-200b/c, miR-216a and miR-217) in mouse MCs stimulated by TGFβ and in renal glomeruli of STZ-induced diabetic mice (Type 1) and db/db mice (Type 2) [62,63]. The investigators demonstrated that a cascade of miRNAs (miR-192 and miR-200b/c) modulated TGFβ1 production and accelerated renal fibrosis because miR-192 up-regulates miR-200b/c that target ZEB1 and ZEB2. Thus TGFβ1 is both autoregulated and regulated by the above miRNAs [62]. In addition, miR-192 up-regulates miR-216a that targets Ybx1, PTEN and Akt, thus causing glomerular hypertrophy, a similar effect to that induced by TGFβ [63]. Another miRNA orchestrates the hypertrophic effect of high glucose concentration on kidney cells. Dey et al. [64] found miR-21 to be the modulatory link between glucose and PTEN and Akt/TORC1 (target of rapamycin complex 1) activity. They demonstrated the important role of this miRNA in in vitro experiments using rat and human kidney glomerular MCs and in vivo in OVED26T1D mice that showed overexpression of miR-21 in hypertrophic glomeruli. This is an additional target for DN therapy. A recent study by Wang et al. [65] supported this important role of miR-21 because they observed that the overexpression of this miRNA enhanced the TGFβ1-induced EMT by inhibiting Smad7 and down-regulating Smad3. In fact, the administration of miR-21 inhibitor in diabetic mice reduced renal interstitial fibrosis. These results were not confirmed by Zhang et al. [66] who demonstrated a down-regulation of miR-21 in glomeruli of db/db mice in the early phase of DN. Furthermore, the overexpression of this miRNA inhibited the proliferation of MCs in vitro and reduced albuminuria in vivo.

miR-1207-5p was found to be abundantly expressed in kidney cells and up-regulated by glucose and TGFβ1 in a dose- and tissue-dependent manner. PVT1 (plasmacytoma variant translocation 1), which increases PAI-1 (plasminogen activator inhibitor 1) and TGFβ1 in MCs, is associated with DN. Alvarez et al. [67] demonstrated that the levels of miR-1207-5p and PVT1 increased in response to high glucose levels: both miR-1207-5p and PVT1 increased the expression of TGFβ1, PAI-1 and fibronectin and contributed to ECM accumulation in the kidney.

Fu et al. [68] demonstrated a significant reduction of endogenous miR-25 in rat MCs treated with high glucose concentrations and in kidneys of diabetic rats associated with increased NOX (NADPH oxidase) activity characterized by high NOX4 expression levels. These results were confirmed by administration of exogenous miR-25 precursor that reduced endogenous NOX4 miRNA expression and its half-life via mRNA degradation.

The role of miR-200a and miR-141 was investigated by Wang et al. [69] in two models of renal fibrosis (apoE-knockout mice affected by STZ-induced DN and C57BL/6 mice) and in rat PTCs treated with TGFβ1 and TGFβ2 for 3 days. miR-200a and miR-141 are direct translational repressors of TGFβ2 by targeting the 3′-UTR of this gene. The down-regulation of miR-200a and miR-141 caused the development and progression of TGFβ-induced EMT and fibrosis in in vitro and in vivo animal models.

A down-regulation of miR-451 was observed in early phase of DN by Zhang et al. [70]. This miRNA normally down-regulates the expression of YwhaZ and p38 MAPK signalling in in vivo and in vitro models (db/db mice). Therefore the occurrence of miR-451 down-regulation in diabetic mice causes mesangial hypertrophy.

In conclusion, many miRNAs illustrated in Figure 1 and Table 4 have been found to be deregulated in animal models of DN and in a few cases of renal biopsies. Numerous studies have explained in which way miRNAs may modulate the ECM accumulation, fibronectin, collagen deposition, glomerular hypertrophy and the ECM process. Further studies with large number of patients such as multicentre studies are necessary to improve our knowledge on the role of the above quoted miRNAs in the pathogenesis of human DN.

Dysregulation of miRNAs induced by hyperglycaemia and TGFβ in mice with diabetic nephropathy

Figure 1
Dysregulation of miRNAs induced by hyperglycaemia and TGFβ in mice with diabetic nephropathy
Figure 1
Dysregulation of miRNAs induced by hyperglycaemia and TGFβ in mice with diabetic nephropathy
Table 4
miRNAs and their target genes in diabetic nephropathy
miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
Kidney     
 Kato et al. [55miR-192 (STZ-treated mouse) miR-196a SIP1 Abnormal production of Col1α2 
 Wang et al. [57 miR-192/215 ZEB2 ECM accumulation 
 Krupa et al. [58 miR-192 (humans)   
 Wang et al. [59miR-377 (NOD and STZ-treated mouse)  PAK1, SOD1, SOD2 Increased production of fibronectin 
 Long et al. [60miR-29c (db/db mouse)  Spry1 Renal fibrosis 
 Long et al. [61 miR-93 VEGFA Angiogenesis 
 Kato et al. [62miR-200b/c (db/db mouse)  ZEB1, ZEB2 Collagen production 
 Kato et al. [63miR-216a  Ybx1, PTEN, Akt Collagen production 
 Dey et al. [64miR-21 (mice)  PTEN Renal fibrosis 
 Wang et al. [65miR-21 (mice)   EMT process 
 Zhang et al. [66 miR-21 PTEN, PI3K, Akt Prevention of mesangial hypertrophy 
 Alvarez et al. [67miR-1207-5p  PVT1 ECM accumulation 
 Fu et al. [68 miR-25 3′-UTR NOX4  
 Wang et al. [69 miR-141 3′-UTR TGFβ1  
  miR-200a   
 Zhang et al. [70 miR-451 (db/db mice) YwhaZ, p38MAPK Mesangial hypertrophy 
miRNAs
ReferenceUp-regulationDown-regulationTarget gene(s)Biological effect(s)
Kidney     
 Kato et al. [55miR-192 (STZ-treated mouse) miR-196a SIP1 Abnormal production of Col1α2 
 Wang et al. [57 miR-192/215 ZEB2 ECM accumulation 
 Krupa et al. [58 miR-192 (humans)   
 Wang et al. [59miR-377 (NOD and STZ-treated mouse)  PAK1, SOD1, SOD2 Increased production of fibronectin 
 Long et al. [60miR-29c (db/db mouse)  Spry1 Renal fibrosis 
 Long et al. [61 miR-93 VEGFA Angiogenesis 
 Kato et al. [62miR-200b/c (db/db mouse)  ZEB1, ZEB2 Collagen production 
 Kato et al. [63miR-216a  Ybx1, PTEN, Akt Collagen production 
 Dey et al. [64miR-21 (mice)  PTEN Renal fibrosis 
 Wang et al. [65miR-21 (mice)   EMT process 
 Zhang et al. [66 miR-21 PTEN, PI3K, Akt Prevention of mesangial hypertrophy 
 Alvarez et al. [67miR-1207-5p  PVT1 ECM accumulation 
 Fu et al. [68 miR-25 3′-UTR NOX4  
 Wang et al. [69 miR-141 3′-UTR TGFβ1  
  miR-200a   
 Zhang et al. [70 miR-451 (db/db mice) YwhaZ, p38MAPK Mesangial hypertrophy 

CLINICAL UTILITY OF miRNAs AS DIAGNOSTIC BIOMARKERS

miRNAs are stable in serum/plasma and urine because they are packed within exosomes that have a key role in cell communications [71]; moreover, they can be detected free in urine and in cellular sediment. Their measurement in bodily fluids opens the way for using miRNAs as potential candidate biomarkers for non-invasive diagnosis of kidney diseases. In addition, miRNA expression in bodily fluids may serve for staging the disease or for monitoring the therapeutic response. Finally, the measurement of miRNAs in urine may be used as a non-invasive tool for investigating the progression of kidney disease.

miRNAs are present in renal tissue; they are stable due to their size and protected from endogenous RNase. The pattern of up- and down-regulated miRNAs obtained by renal biopsies (fresh frozen tissue or formalin-fixed paraffin-embedded tissue) can serve for obtaining new information on the pathogenesis of the disease or as drug targets for new therapeutic strategies. Probably in the near future accurate diagnosis and classification of the biopsy-proven kidney disease using an miRNA expression profile in association with histological findings could improve diagnosis and personalize therapy.

POTENTIAL THERAPEUTIC FUNCTION OF miRNAs IN HUMAN GLOMERULONEPHRITIS

Individual miRNAs regulate the expression of multiple target genes and their related functions. Two main strategies have been investigated in animal models. First, inhibition in vivo of miRNA activity by the administration of (i) sponge miRNAs that have RNA transcripts containing multiple copies of complementary binding sites to an miRNA of interest; (ii) antagomiRs with high binding affinity for specific miRNAs such as LNA. Treatment with antagomiRs has been carried out in mouse models of DN by blocking miR-192 and miR-29c [55,60]. Another approach to interfere with the binding of an miRNA to a specific target mRNA can be achieved through the introduction of oligonucleotides with perfect complementarity in the 3′-UTR of the target mRNA-binding site sequence. This approach is referred to as ‘masking’ or ‘target occupation’. Secondly, in the presence of low miRNA activity, it is necessary to administer: (i) miRNA vectors such as shRNA in LN for improving miR-146a [44]; and (ii) double-stranded miRNA mimics such as miR-29b mimics to block renal fibrosis in a rat model of obstructive nephropathy [72].

Recently, some miRNA-targeted drugs have entered into Phase II clinical trials or preclinical phase: (i) LNA–anti-miR-122, miravasen, has been used with success (good efficacy, tolerability and safety) for treatment of hepatitis C because miR-122 is involved as a host factor of the virus; (ii) miR-195 antagonist for post-myocardial infarction; and (iii) miR-34a and let-7 administration for prostate and lung cancer respectively.

CONCLUSIONS

The presence of differentially expressed groups of miRNAs in blood, kidney and urine demonstrate that they are tissue-specific. In silico analysis has shown that an miRNA may regulate the expression of hundreds of target genes; this means that miRNAs may have an important role in biological regulation. Studies discussed in the present review suggest that various miRNAs might have specific roles in different kidney diseases. In some limited conditions, miRNAs may be used as candidate biomarkers for early diagnosis of disease. Early detection of these miRNAs can enhance clinical management, improve long-term outcomes and greatly increase quality of life.

Abbreviations

     
  • apoE

    apolipoprotein E

  •  
  • C1GALT1

    core 1 β1,3-galactosyltransferase 1

  •  
  • CI

    chronicity index

  •  
  • Col

    collagen

  •  
  • DN

    diabetic nephropathy

  •  
  • DNMT1

    DNA methyltransferase 1

  •  
  • E-cadherin

    epithelial cadherin

  •  
  • ECM

    extracellular matrix

  •  
  • eGFR

    estimated glomerular filtration rate

  •  
  • EMT

    epithelial–mesenchymal transition

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • FSGS

    focal segmental glomerular sclerosis

  •  
  • GALNT2

    N-acetylgalactosaminyltransferase 2

  •  
  • Gd-IgA1

    galactose-deficient IgA1

  •  
  • HK2

    human kidney 2

  •  
  • IFN

    interferon

  •  
  • IgAN

    IgA nephropathy

  •  
  • IL

    interleukin

  •  
  • IRAK1

    IL-1 receptor-associated kinase 1

  •  
  • IRF5

    interferon-regulatory factor 5

  •  
  • LN

    lupus nephritis

  •  
  • LNA

    locked nucleic acid

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MC

    mesangial cell

  •  
  • MCD

    minimal change disease

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NOX

    NADPH oxidase

  •  
  • PAI-1

    plasminogen activator inhibitor 1

  •  
  • PAN

    puromycin aminonucleoside

  •  
  • PBMC

    peripheral blood mononuclear cell

  •  
  • PTC

    proximal tubular cell

  •  
  • PTEN

    phosphatase and tensin homologue deleted on chromosome 10

  •  
  • PVT1

    plasmacytoma variant translocation 1

  •  
  • qRT-PCR

    real-time quantitative reverse transcription–PCR

  •  
  • ROC

    receiver-operating characteristic

  •  
  • SLE

    systemic lupus erythaematosus

  •  
  • SLEDAI

    SLE disease activity index

  •  
  • SOCS1

    suppressor of cytokine signalling 1

  •  
  • SOD

    superoxide dismutase

  •  
  • Spry1

    Sprouty homologue 1

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • STZ

    streptozotocin

  •  
  • TGFβ1

    transforming growth factor β1

  •  
  • TNF

    tumour necrosis factor

  •  
  • TPM1

    tropomyosin 1

  •  
  • TRAF6

    TNF receptor-associated factor 6

  •  
  • Treg

    regulatory T-cell

  •  
  • VEGF

    vascular endothelial growth factor

  •  
  • VLP

    virus-like particle

  •  
  • WT1

    Wilms’ tumour protein 1

  •  
  • ZEB

    zinc finger E-box-binding homeobox

FUNDING

This review has been supported by grants from Regione Puglia (BISIMANE project) [grant number CP44/2009] and Ministero dell’Università e della Ricerca [grant numbers PONa3_00134 and FIRB RBAP11B2SX]. We are grateful to the Schena Foundation for the scientific and financial support.

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