The expanding roles of microRNAs in kidney pathophysiology

The expanding roles of microRNAs in kidney pathophysiology Abstract MicroRNAs (miRNAs) are short single-stranded RNAs that control gene expression through base pairing with regions within the 3′-untranslated region of target mRNAs. These small non-coding RNAs are now increasingly known to be involved in kidney physiopathology. In this review we will describe how miRNAs were in recent years implicated in cellular and animal models of kidney disease but also in chronic kidney disease, haemodialysed and grafted patients, acute kidney injury patients and so on. At the moment miRNAs are considered as potential biomarkers in nephrology, but larger cohorts as well as the standardization of methods of measurement will be needed to confirm their usefulness. It will further be of the utmost importance to select specific tissues and biofluids to make miRNAs appropriate in day-to-day clinical practice. In addition, up- or down-regulating miRNAs that were described as deregulated in kidney diseases may represent innovative therapeutic methods to cure these disorders. We will enumerate in this review the most recent methods that can be used to deliver miRNAs in a specific and suitable way in kidney and other organs damaged by kidney failure, such as the cardiovascular system. biomarker, chronic kidney disease, microRNA, transplantation, vascular calcification MICRORNAs: THE WATCHMEN OF THE HUMAN GENE PROGRAMME Biogenesis of miRNAs: a story of canonical and non-canonical ways MicroRNAs (miRNAs) are small non-coding RNAs measuring about 20–25 nucleotides (nt) that are involved in the regulation of gene expression. The concept of miRNA was first described in 1993 when the laboratory of Ambros discovered the first gene encoding a miRNA, lin-4 in Caenorhabditis elegans [1]. The lin-4 gene did not code the expected messenger RNA (mRNA), but a small non-coding RNA of approximately 22 nt complementary to a sequence of the 3′-untranslated region (UTR) of the lin-14 mRNA. This binding led to a down-regulation of the expression of the lin-14 gene which was consequently described as the first target gene regulated by a miRNA. During 7 years, lin-4 was one of a kind, as no equivalent was discovered in any living organism. Ruvkun’s laboratory then discovered a second miRNA that was called let-7 [2], expressed in most known species, including humans. More than 3000 miRNAs have since been identified in the human genome, and many efforts have been undertaken to understand how miRNAs are produced and how genes are controlled by their regulatory networks [3]. In the canonical way, miRNA biogenesis starts in much the same way as that of mRNAs, with the transcription of larger RNA species by RNA polymerase II or III (Figure 1). The RNase III Drosha and its protein partner, DiGeorge syndrome critical region 8 (DGCR8), cleave the precursor pri-miRNA near the base of the hairpin stem. This forms a small hairpin (sh) of roughly 60–70 nt called pre-miRNA, which is released in the cytoplasm via the exportin shuttle, where it is recognized and cleaved by the Dicer RNase III, resulting in the double-stranded miRNA/miRNA* duplex, then unwound to single strands by RNA-induced silencing complex (RISC). The RISC complex contains Argonaute 2 (AGO2), another endonuclease complex, and brings the mature miRNA to its target mRNAs in the 3′-UTR region, resulting in gene silencing [4]. FIGURE 1 View largeDownload slide miRNA biogenesis and regulations. miR-223 is transcribed as a single large RNA called pri-miRNA. An enzymatic complex comprising Drosha and DGCR8 processes the pri-miRNA into a hairpin-structured pre-miRNA. It is then exported to the cytoplasm by a nucleocytoplasmic shuttle protein (exportin) where it is cleaved by the Dicer complex into a miRNA duplex. Finally, the miRNA duplex is unwound by the RISC complex to obtain the mature miR-223, which is transported by RISC to bind various mRNA targets. FIGURE 1 View largeDownload slide miRNA biogenesis and regulations. miR-223 is transcribed as a single large RNA called pri-miRNA. An enzymatic complex comprising Drosha and DGCR8 processes the pri-miRNA into a hairpin-structured pre-miRNA. It is then exported to the cytoplasm by a nucleocytoplasmic shuttle protein (exportin) where it is cleaved by the Dicer complex into a miRNA duplex. Finally, the miRNA duplex is unwound by the RISC complex to obtain the mature miR-223, which is transported by RISC to bind various mRNA targets. A subset of miRNAs originates from non-canonical pathways that will typically bypass one or more steps of the canonical process. Instead of originating from their own genes, they are specifically excised from other larger RNAs such as introns, transfer RNAs, small nucleolar RNAs or endogenous short hairpin RNAs [3] (Figure 1). Non-canonical miRNAs resemble canonical miRNAs structurally and functionally and they are mainly involved in stem cell proliferation and immune response. Also, miRNAs can take their origin in the mitochondrial genome and directly regulate mitochondrial transcripts [5]. These mitomiRNAs can impact mitochondria and mitochondria-related pathways with relevance to metabolic regulation, including lipid metabolism, oxidative phosphorylation and electron transport chain components. miRNA modes of action: silencing the mRNA, the protein or both? Once synthesized, miRNAs bind target mRNAs in order to realize the regulation of translation (Figure 1). Bioinformatic analyses indicate that every miRNA has up to 100 potential targets and that conversely multiple miRNAs may target the same protein. The miRNA either inhibits translation mechanisms or triggers destruction of the target mRNA. The consensus is that the predominant mode of action is translational inhibition [6] (Figure 1). Helwak et al. [7] sequenced the miRNA–target RNA duplexes associated with human chaperone protein AGO1. They found that most miRNAs bind mRNAs with their 5′-seed region, but that ∼60% of seed interactions are non-canonical with bulged or mismatched nucleotides. In fact, a study described that ∼20% of miRNA–mRNA interactions involve the miRNA 3′-end, with little evidence for 5′ contacts [7]. This is important as most prediction informatic softwares rely on the 5′-seed region sequence and strongly dictate the way researchers identify miRNAs targets. As our repertoire of experimental tools used to study miRNA mode of action improves, we can bet that our understanding of how, where and why miRNAs function and regulate gene expression will change. We now know who watches the watchmen: long non-coding RNAs regulate miRNAs by sponging and remodelling chromatin The question of the regulation of miRNAs has long remained elusive, and next-generation sequencing has recently provided clues by using systematic sequencing of all RNA species in various cell types. The activity of miRNAs can be regulated by other non-coding RNAs, called long non-coding RNAs (lncRNAs), typically longer than 200 nt, and whose members come from thousands of loci over the genome [3]. They take their origin from exonic, intronic, 3′- and 5′-UTRs, etc. Some are pseudogene transcripts that will inhibit the function of specific miRNAs by acting via ‘sponge-like’ effects; some are able to modulate miRNA expression by chromatin remodelling. The current state of knowledge considers that the crosstalk between the two types of non-coding RNAs contributes to pathogenesis and progression of various diseases [3]. miRNA polymorphism may explain at least partly phenotypic divergences in humans Single-nucleotide polymorphisms (SNPs) have been described in miRNA sequences and may also modify their synthesis and activity. For example, SNPs in miR-146a and miR-499 are associated with coronary artery disease in various Asian populations [8]. Others have been linked to renal cancer [9]. Several studies have shown that some SNP variants lead to increased levels of the mature miRNA sequence, some to reduced levels of the mature miRNA sequence relative to wild type or even to the generation of novel miRNAs due to alterations in Drosha and/or Dicer processing sites [10]. miRNAs are novel biomarkers in the cardiovascular field Cardiovascular disease (CVD) is the leading cause of mortality and morbidity in chronic kidney disease (CKD). The latest research shows numerous dysregulations of miRNA in cardiovascular pathologies, making miRNAs possible new biomarkers of CVD associated with CKD [11]. For instance, they are deregulated in rapid aortic valve calcification [12] and in carotid-related stroke [13]. As will be discussed below, knowledge is sparser in the nephrology field. MIRNAs AND THE NEPHROLOGY FIELD: AN EMERGING STORY The expression of numerous miRNAs is altered in cellular and pre-clinical models of CKD and related vascular calcification Patients afflicted with later stages of CKD exhibit a higher cardiovascular morbimortality, which is associated with the presence of atherosclerosis and/or vascular calcifications. The calcification process is a common consequence of uraemia and is due to a complex, active process of osteogenesis resembling bone formation in blood vessels [14]. Our team has focused on the roles of uraemic toxins (including inorganic phosphate) in in vitro models of vascular calcification and osteoclastogenesis [15] (Figure 2). We have pointed out in vitro and in vivo the roles of miR-126, miR-143, miR-145 and miR-223 in the acquisition of a pro-calcifying phenotype in human primary cultures of vascular smooth muscle cells (VSMCs) [16, 17]. Magnesium treatment was able to revert at least partly the effect of Pi on miRNA expression in VSMCs [18]. These results were confirmed in vivo in tissue biopsies from CKD and/or atherosclerotic mice displaying vascular calcification [17]. miR-126 and miR-223 levels were increased in aortas of these mice, whereas miR-143 and miR-145 levels were decreased [17]. Sevelamer administration reduced the aortic expression of miR-223 and miR-126 in this model. We thus suggested a direct link between the observed changes in miRNA levels and uraemic vascular toxicity (Figure 2). Importantly, at high uraemia values we found low seric miR-223 levels (contrary to variations in aortic wall), hinting at a similar regulation in human patients [17]. In another model, we also showed that, in vitro, Pi is responsible for a marked decrease in osteoclastogenesis in RAW cells, in conjunction with a lower cellular miR-223 levels [19]. An anti-miR specific for miR-223 inhibited osteoclastogenesis in the same way as Pi. In contrast, the overexpression of miR-223 led to an increased differentiation, as reflected by tartrate-resistant acid phosphatase activity. miR-223 affected the expression of not only its target genes NFIA and RhoB but also of osteoclast marker genes and of the Akt signalling pathway that induces osteoclastogenesis [19]. These results were confirmed by measuring bone resorption activity in human VSMCs differentiated into osteoclasts—suggesting that the modulation of miR-223 in CKD patients might be of value in managing the complications related to CKD (Figure 2). Also, another team found that expression levels of miR-223 in renal biopsies were greater in patients with progressive chronic renal failure than in patients with stable CKD; this suggests that miR-223 may have a role in aggravating renal dysfunction [20]. FIGURE 2 View largeDownload slide Implication of miR-223 in the vascular calcification process. miR-223 expression is modulated during vascular calcification in CKD. Hyperphosphataemia increases miR-223 expression in VSMCs, which transdifferentiate into ‘osteoblast-like’ synthetic cells with higher mobility. The same high Pi concentration decreases miR-223 levels in vascular precursors of monocytes/macrophages and inhibits their receptor activator of nuclear factor κB ligand–induced differentiation into osteoclast-like cells. FIGURE 2 View largeDownload slide Implication of miR-223 in the vascular calcification process. miR-223 expression is modulated during vascular calcification in CKD. Hyperphosphataemia increases miR-223 expression in VSMCs, which transdifferentiate into ‘osteoblast-like’ synthetic cells with higher mobility. The same high Pi concentration decreases miR-223 levels in vascular precursors of monocytes/macrophages and inhibits their receptor activator of nuclear factor κB ligand–induced differentiation into osteoclast-like cells. miRNAs are potential biomarkers in the various stages of CKD At the moment, glomerular filtration rate is the best marker of renal function, but its measurement in routine practice is tricky, so it is usually estimated from creatinine serum levels. The serum urea level and the albuminuria and proteinuria values (divided by the creatininuria value) are also used as markers of renal function. Nevertheless, a variety of studies have stressed that these markers lack sensitivity for early diagnosis of CKD [21]. Over the past decade, several potential promising biomarkers have emerged: urinary liver fatty acid-binding protein, urinary N-acetyl-β-d-glucosaminidase, urinary connective tissue growth factor, serum apolipoprotein A-IV, urinary and serum neutrophil gelatinase-associated lipocalin, serum and urinary kidney injury molecule 1 or serum fibroblast growth factor 23 (recently reviewed in Rysz et al. [21]). Most of these markers are very sensitive and specific but have not yet been validated in clinical practice. In recent years, miRNAs have attracted increasing interest as potential biomarkers to assess the severity of the disease or to identify a group of patients with comorbidity [22]. Mitchell et al. [23] have demonstrated for the first time the presence of circulating miRNAs in human plasma. This discovery was surprising, as plasma is known to have high RNase activity. These circulating microRNAs have many origins and ways of transport conferring the stability and the protection needed against RNase activity [24]. miRNAs could be also released by active secretion via cell-derived membrane vesicles or released by a protein miRNA complex comprising AGO2 and/or high-density lipoproteins (HDLs). The main advantage of miRNAs relates to their stability in serum. Furthermore, miRNAs might meet the sensitivity, specificity and reproducibility criteria required for an ideal, non-invasive biomarker [22] (Figure 3). However, the possible involvement of a given miRNA in several different diseases remains a problem. FIGURE 3 View largeDownload slide The potential of miRNAs in renal disorders, a bench to bedside approach: cellular and preclinical models are used to screen the most useful miRNAs in a nephrological pathology. Alternatively, patient samples can be screened using high-throughput techniques. miRNAs can further be validated as prognostic biomarkers in large human cohorts, then used as innovative therapeutic approaches to improve kidney function in the patients. FIGURE 3 View largeDownload slide The potential of miRNAs in renal disorders, a bench to bedside approach: cellular and preclinical models are used to screen the most useful miRNAs in a nephrological pathology. Alternatively, patient samples can be screened using high-throughput techniques. miRNAs can further be validated as prognostic biomarkers in large human cohorts, then used as innovative therapeutic approaches to improve kidney function in the patients. The expression of miRNAs in CKD has been documented; in most cases, levels decrease as CKD progresses [25]. Neal et al. [26] used real-time quantitative PCR (qPCR) and found that the levels of most miRNAs decreased as the CKD stages increased. Others used a cohort of 90 patients at CKD stages 3, 4 and 5D to show a decrease in serum expression levels of miR-125b, miR-145 and miR-155 [27]. The serum concentration of certain heart-specific miRNAs additionally seemed to be inversely related to renal function [11]. We have shown in a murine model that serum miR-223 levels decrease in CKD mice. Conversely, Ulbing et al. [28] reported that miR-223 expression was lower in CKD stage 4 and 5 patients than in healthy controls. This down-regulation disappeared after renal transplantation. The results wait to be confirmed in larger cohorts. The evidence for an effect of renal function on circulating miRNA levels in plasma and urine is limited and sometimes contradictory. Pharmacokinetics studies showed that the liver, the reticuloendothelial system and the kidney are responsible for excreting a proportion of circulating miRNAs [29]. However, small interfering RNAs (siRNAs) conjugated to diethylenetriaminepentaacetic acid (DTPA) accumulate in the kidney three times more than DTPA alone when administered to animals, suggesting that small RNAs might be actively reabsorbed after renal filtration through the proximal tubule [29]. In vivo imaging experiments have clearly demonstrated that siRNAs are freely filtered in the glomerulus and are reabsorbed within 10 s by proximal tubular epithelial cells [30]. However, the renal excretion system does not seem to be involved since the decrease in circulating miRNA levels in CKD patients in Neal et al.’s study was not associated with an increase in excreted miRNAs in urine [26]. Some researchers have looked at the known accumulation of RNases in renal failure [31], as this might increase the degradation of miRNAs in the plasma. That being said, circulating miRNAs are usually protected by exosomes, HDL and AGO proteins, and no consistent differences in exosome abundance or stability have been observed in uraemic vs. normal serum [32]. miRNA levels remain stable when plasma is subjected to prolonged room temperature incubation [31]. Some in vitro studies indicate that miRNA synthesis is low in CKD but that the miRNA-processing enzymes Drosha and Dicer are normally expressed [33]. With regard to the biogenesis of urinary miRNAs, the RNAs are released by cells in the nephron and downstreamed in the urinary tract [31]. The miRNAs in the urinary tract may be associated with membrane-bound extracellular vesicles, such as microvesicles (formed by outward budding of the plasma membrane) and exosomes (secreted from multivesicular endosomes formed in the endocytic tract) [24]. Can seric miRNA measurement be useful in acute kidney disease? Acute kidney injury (AKI) leads to tubular injury and the development of fibrosis. miR-146a is transcriptionally upregulated by ligands of interleukin-1 receptor/Toll-like receptor family members via the activation of nuclear factor κB in renal proximal tubular cells. miR-146a knockout mice submitted to renal ischaemia–reperfusion injury exhibited tubular injury [34]. So miR-146a could be a key mediator of the development of AKI and CKD. Other miRNAs have been shown to be instrumental in various pre-clinical models. For example, miR-223 in AKI is associated with experimental sepsis [35] and miR-140 in cisplatin-induced AKI [36]. What if miRNAs could help to predict the outcome of kidney graft? Rejection is the main consequence of allograft failure and its monitoring by the use of invasive biopsies is risky and burdensome to transplant patients. Circulating miRNA levels could be of tremendous help to the clinician as novel and easily accessible biomarkers of allograft rejection and transplant failure. A significant number of miRNA and transplant studies have been performed in renal transplantations. A pioneering study investigated whether acute rejection (AR) is associated with alterations in miRNA levels in renal biopsies [20]. The authors found an overexpression of miR-142, miR-155 and miR-223 in AR biopsies, suggesting that miRNA expression levels may serve as biomarkers of human renal allograft status. The kidney-specific miRNA-146a seems to be the most interesting risk factor in the development of rejection as it is part of an miRNA signature of renal ischaemia–reperfusion injury [37], an increase of its expression in dendritic cells promotes allogeneic kidney graft survival [38] and SNPs in miR-146a are associated with renal cancer [9]. The various miRNAs implicated in nephropathies are summarized in Table 1. Table 1 miRNAs implicated in nephropathies miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] Table 1 miRNAs implicated in nephropathies miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] We now have to reliably and reproducibly measure serum miRNAs in routine clinical practice miRNAs are promising innovative biomarkers in the nephrology field, but we are far from the identification of a precise and useful miRNA signature in a given kidney pathology. At present, miRNAs’ value as biomarkers is at least partly limited by difficulties in normalizing their expression levels, which may lead to poor reproducibility between different laboratories [22]. Real-time PCR is suitable for analysing a small number of miRNAs because it is accurate, rapid and easily transferable into routine practice. Several endogenous circulating miRNAs (such as U6 and miR-1249) were initially used to normalize expression levels of circulating miRNAs; however, the validity of this approach was recently challenged because of high variability [22]. Due to the low concentration of miRNAs in the circulation, standardization is of the utmost importance. Henceforth, a specific amount of exogenous miRNA (e.g. synthetic C.elegans miR-39) is added to avoid experimental bias [22]. The time required to quantify circulating levels of miRNAs by real-time qPCR is also an obstacle to their use in clinic. Several new approaches are in development. For instance, a new method of nanobiophotonic detection is being evaluated. It involves Förster resonance energy transfer after the excitation of two fluorophores, without the need for sample preparation or signal amplification [42]. Alternatively, Smith et al. [32] have developed a simple method for the electrochemical detection of miRNAs in urine samples. MIRNAS ARE POTENTIAL NEW THERAPEUTIC SOLUTIONS IN NEPHROLOGY Lessons from pre-clinical models Regulation of gene expression is critical for all cellular processes, with dysregulation often resulting in the development of disease. miRNAs are instrumental players in the fine-tuning of gene regulation networks and have thus potential therapeutic impacts [43]. Several recent studies have shown that endothelial miRNAs have important roles during vasculogenesis and in the response to inflammation and haemodynamic stress [44, 45]. It has been shown for example that miR-126–based therapy maintains a proliferative reserve in endothelial cells (ECs) and can prevent atherosclerosis [41]. Indeed, the pro-angiogenic miR-126 is abundant in ECs, plays an important role in vascular dysfunction and modulates the expression of vascular cell adhesion molecule-1 and chemokine CXCL-12. We have recently shown that miR-126 is involved in the vascular remodelling under laminar shear stress in human endothelial cells and is implicated in the modulation of syndecan expression [40]. miRNAs could have a role(s) in CKD vascular remodelling and may therefore represent useful targets to prevent or treat vascular complications of CKD [15]. To further confirm this, it was very recently shown that administration of miR-126 rescued EC proliferation at predilection sites and limited atherosclerosis [41]. This suggests a possible direct link between the observed miRNA alterations and the vascular damage caused by CKD [17]. Concerning kidney graft models, Xu et al. [39] showed in a murine model a role for miR-21 up-regulation in the protective effect of delayed ischaemic preconditioning against subsequent renal ischaemia–reperfusion. The timely and effective delivery of miRNAs and miRNAs inhibitors. CRIPSR/cas9 gene editing, gold or iron nanotechnologies: what they can bring to the table Chemically modified miRNA precursors or inhibitors (such as sponges) can be injected directly into the bloodstream (Figure 4) [46]. Modern viral vectors can also be used (Figure 4) [46]. Lentiviruses seem to be a good solution for kidney delivery since they proved capable of infecting kidney podocyte cells and delivering the therapeutic gene product, they escape the immune system and they are efficient vectors suited to deliver miRNA therapeutic sequences [47]. Different approaches of injured arterial wall treatment have used systemic, polymer-based particles or local, modified stents to induce endothelial regeneration in CKD [46, 48]. One can use naturally occurring nanomaterials already present in the human body, namely exosomes or HDL [32], to deliver functional miRNAs in a therapeutic goal, as has already been done with miR-21 [49]. Also, another team showed that HDL particles are able to deliver miR-223 to ECs in order to suppress expression of its mRNA targets [32]. Efforts are under way to assess nanotechnology, with novel gold nanoparticles of 13 ± 1 nm, each functionalized with a monolayer of double-stranded alkylthiol-modified RNA molecules (Figure 4) [50]. These new vehicles can enter cells without the aid of co-carriers, reside there for up to 24 h after transfection and deliver endogenous miRNAs in situ [50]. The same approach could be used to deliver miR-126 or miR-223 in diseased vessels in the CKD context. It was shown that 3-nm glutathione-protected gold nanoclusters accumulate preferably in the kidney [51], and they would thus be prime candidates. It is difficult to prioritize among all these promising solutions, but in our opinion lentiviral vectors and gold nanoparticles specifically accumulating in the kidney are the most promising techniques in the foreseeable future for treating kidney disorders. FIGURE 4 View largeDownload slide New therapeutic approaches in kidney diseases. miRNAs levels can be either enhanced or decreased according to the clinical context in kidney and blood vessels. Various strategies are currently being developed to modify their expression. Recent gene therapy vectors, for example, adeno-associated virus or lentivirus, can be used to deliver miRNA precursors to diseased tissues. One can also use chemically modified RNAs, inhibitory sponge complementary sequences or miRNAs chemically bound to nanoparticles. The CRIPSR/Cas 9 system can also be used to alter the miRNA sequence and improve its function. FIGURE 4 View largeDownload slide New therapeutic approaches in kidney diseases. miRNAs levels can be either enhanced or decreased according to the clinical context in kidney and blood vessels. Various strategies are currently being developed to modify their expression. Recent gene therapy vectors, for example, adeno-associated virus or lentivirus, can be used to deliver miRNA precursors to diseased tissues. One can also use chemically modified RNAs, inhibitory sponge complementary sequences or miRNAs chemically bound to nanoparticles. The CRIPSR/Cas 9 system can also be used to alter the miRNA sequence and improve its function. That being said, another system has arisen recently that could prove equally useful. The CRISPR/cas9 system is an emerging genome editing tool that is now increasingly used in the miRNA research field. For example, CRISPR/cas9 constructs can be constructed with single-guide RNAs targeting selected sites of miRNA precursors to almost completely obliterate the expression of these miRNAs in the long term (Figure 4) [52]. Additionally, the technique can be used to target miRNAs of the same family or with highly conserved sequences. CONCLUDING REMARKS We need new solutions to improve diagnosis and monitoring in modern nephrology and miRNAs are prime candidates to be developed as innovative biomarkers in the early diagnosis and prognosis of patients afflicted with kidney disorders. The development of new pharmacologic therapies enabling the modulation of miRNA levels should be considered to provide possible therapies of most common kidney diseases in an expanding world population of ageing and diabetic patients. FUNDING Part of the work described in this review was funded by a grant from the Picardie Regional Council. CONFLICT OF INTEREST STATEMENT Z.A.M. reports grants and other funding from Amgen, Sanofi-Genzyme, Bayer, the French government, MSD, GlaxoSmithKline, Eli Lilly, FMC, Baxter and Outsuka. The other authors have declared no conflicts of interest. The results presented in this paper have not been published previously in whole or part, except in abstract format. REFERENCES 1 Lee RC , Feinbaum RL , Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 . Cell 1993 ; 75 : 843 – 854 Google Scholar CrossRef Search ADS PubMed 2 Reinhart BJ , Slack FJ , Basson M et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans . Nature 2000 ; 403 : 901 – 906 Google Scholar CrossRef Search ADS PubMed 3 Rong D , Sun H , Li Z et al. An emerging function of circRNA–miRNAs–mRNA axis in human diseases . Oncotarget 2017 ; 8 : 73271 – 73281 Google Scholar PubMed 4 Guo H , Ingolia NT , Weissman JS et al. 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Sci Rep 2016 ; 6 : 22312 Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nephrology Dialysis Transplantation Oxford University Press

The expanding roles of microRNAs in kidney pathophysiology

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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0931-0509
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1460-2385
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10.1093/ndt/gfy140
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Abstract

Abstract MicroRNAs (miRNAs) are short single-stranded RNAs that control gene expression through base pairing with regions within the 3′-untranslated region of target mRNAs. These small non-coding RNAs are now increasingly known to be involved in kidney physiopathology. In this review we will describe how miRNAs were in recent years implicated in cellular and animal models of kidney disease but also in chronic kidney disease, haemodialysed and grafted patients, acute kidney injury patients and so on. At the moment miRNAs are considered as potential biomarkers in nephrology, but larger cohorts as well as the standardization of methods of measurement will be needed to confirm their usefulness. It will further be of the utmost importance to select specific tissues and biofluids to make miRNAs appropriate in day-to-day clinical practice. In addition, up- or down-regulating miRNAs that were described as deregulated in kidney diseases may represent innovative therapeutic methods to cure these disorders. We will enumerate in this review the most recent methods that can be used to deliver miRNAs in a specific and suitable way in kidney and other organs damaged by kidney failure, such as the cardiovascular system. biomarker, chronic kidney disease, microRNA, transplantation, vascular calcification MICRORNAs: THE WATCHMEN OF THE HUMAN GENE PROGRAMME Biogenesis of miRNAs: a story of canonical and non-canonical ways MicroRNAs (miRNAs) are small non-coding RNAs measuring about 20–25 nucleotides (nt) that are involved in the regulation of gene expression. The concept of miRNA was first described in 1993 when the laboratory of Ambros discovered the first gene encoding a miRNA, lin-4 in Caenorhabditis elegans [1]. The lin-4 gene did not code the expected messenger RNA (mRNA), but a small non-coding RNA of approximately 22 nt complementary to a sequence of the 3′-untranslated region (UTR) of the lin-14 mRNA. This binding led to a down-regulation of the expression of the lin-14 gene which was consequently described as the first target gene regulated by a miRNA. During 7 years, lin-4 was one of a kind, as no equivalent was discovered in any living organism. Ruvkun’s laboratory then discovered a second miRNA that was called let-7 [2], expressed in most known species, including humans. More than 3000 miRNAs have since been identified in the human genome, and many efforts have been undertaken to understand how miRNAs are produced and how genes are controlled by their regulatory networks [3]. In the canonical way, miRNA biogenesis starts in much the same way as that of mRNAs, with the transcription of larger RNA species by RNA polymerase II or III (Figure 1). The RNase III Drosha and its protein partner, DiGeorge syndrome critical region 8 (DGCR8), cleave the precursor pri-miRNA near the base of the hairpin stem. This forms a small hairpin (sh) of roughly 60–70 nt called pre-miRNA, which is released in the cytoplasm via the exportin shuttle, where it is recognized and cleaved by the Dicer RNase III, resulting in the double-stranded miRNA/miRNA* duplex, then unwound to single strands by RNA-induced silencing complex (RISC). The RISC complex contains Argonaute 2 (AGO2), another endonuclease complex, and brings the mature miRNA to its target mRNAs in the 3′-UTR region, resulting in gene silencing [4]. FIGURE 1 View largeDownload slide miRNA biogenesis and regulations. miR-223 is transcribed as a single large RNA called pri-miRNA. An enzymatic complex comprising Drosha and DGCR8 processes the pri-miRNA into a hairpin-structured pre-miRNA. It is then exported to the cytoplasm by a nucleocytoplasmic shuttle protein (exportin) where it is cleaved by the Dicer complex into a miRNA duplex. Finally, the miRNA duplex is unwound by the RISC complex to obtain the mature miR-223, which is transported by RISC to bind various mRNA targets. FIGURE 1 View largeDownload slide miRNA biogenesis and regulations. miR-223 is transcribed as a single large RNA called pri-miRNA. An enzymatic complex comprising Drosha and DGCR8 processes the pri-miRNA into a hairpin-structured pre-miRNA. It is then exported to the cytoplasm by a nucleocytoplasmic shuttle protein (exportin) where it is cleaved by the Dicer complex into a miRNA duplex. Finally, the miRNA duplex is unwound by the RISC complex to obtain the mature miR-223, which is transported by RISC to bind various mRNA targets. A subset of miRNAs originates from non-canonical pathways that will typically bypass one or more steps of the canonical process. Instead of originating from their own genes, they are specifically excised from other larger RNAs such as introns, transfer RNAs, small nucleolar RNAs or endogenous short hairpin RNAs [3] (Figure 1). Non-canonical miRNAs resemble canonical miRNAs structurally and functionally and they are mainly involved in stem cell proliferation and immune response. Also, miRNAs can take their origin in the mitochondrial genome and directly regulate mitochondrial transcripts [5]. These mitomiRNAs can impact mitochondria and mitochondria-related pathways with relevance to metabolic regulation, including lipid metabolism, oxidative phosphorylation and electron transport chain components. miRNA modes of action: silencing the mRNA, the protein or both? Once synthesized, miRNAs bind target mRNAs in order to realize the regulation of translation (Figure 1). Bioinformatic analyses indicate that every miRNA has up to 100 potential targets and that conversely multiple miRNAs may target the same protein. The miRNA either inhibits translation mechanisms or triggers destruction of the target mRNA. The consensus is that the predominant mode of action is translational inhibition [6] (Figure 1). Helwak et al. [7] sequenced the miRNA–target RNA duplexes associated with human chaperone protein AGO1. They found that most miRNAs bind mRNAs with their 5′-seed region, but that ∼60% of seed interactions are non-canonical with bulged or mismatched nucleotides. In fact, a study described that ∼20% of miRNA–mRNA interactions involve the miRNA 3′-end, with little evidence for 5′ contacts [7]. This is important as most prediction informatic softwares rely on the 5′-seed region sequence and strongly dictate the way researchers identify miRNAs targets. As our repertoire of experimental tools used to study miRNA mode of action improves, we can bet that our understanding of how, where and why miRNAs function and regulate gene expression will change. We now know who watches the watchmen: long non-coding RNAs regulate miRNAs by sponging and remodelling chromatin The question of the regulation of miRNAs has long remained elusive, and next-generation sequencing has recently provided clues by using systematic sequencing of all RNA species in various cell types. The activity of miRNAs can be regulated by other non-coding RNAs, called long non-coding RNAs (lncRNAs), typically longer than 200 nt, and whose members come from thousands of loci over the genome [3]. They take their origin from exonic, intronic, 3′- and 5′-UTRs, etc. Some are pseudogene transcripts that will inhibit the function of specific miRNAs by acting via ‘sponge-like’ effects; some are able to modulate miRNA expression by chromatin remodelling. The current state of knowledge considers that the crosstalk between the two types of non-coding RNAs contributes to pathogenesis and progression of various diseases [3]. miRNA polymorphism may explain at least partly phenotypic divergences in humans Single-nucleotide polymorphisms (SNPs) have been described in miRNA sequences and may also modify their synthesis and activity. For example, SNPs in miR-146a and miR-499 are associated with coronary artery disease in various Asian populations [8]. Others have been linked to renal cancer [9]. Several studies have shown that some SNP variants lead to increased levels of the mature miRNA sequence, some to reduced levels of the mature miRNA sequence relative to wild type or even to the generation of novel miRNAs due to alterations in Drosha and/or Dicer processing sites [10]. miRNAs are novel biomarkers in the cardiovascular field Cardiovascular disease (CVD) is the leading cause of mortality and morbidity in chronic kidney disease (CKD). The latest research shows numerous dysregulations of miRNA in cardiovascular pathologies, making miRNAs possible new biomarkers of CVD associated with CKD [11]. For instance, they are deregulated in rapid aortic valve calcification [12] and in carotid-related stroke [13]. As will be discussed below, knowledge is sparser in the nephrology field. MIRNAs AND THE NEPHROLOGY FIELD: AN EMERGING STORY The expression of numerous miRNAs is altered in cellular and pre-clinical models of CKD and related vascular calcification Patients afflicted with later stages of CKD exhibit a higher cardiovascular morbimortality, which is associated with the presence of atherosclerosis and/or vascular calcifications. The calcification process is a common consequence of uraemia and is due to a complex, active process of osteogenesis resembling bone formation in blood vessels [14]. Our team has focused on the roles of uraemic toxins (including inorganic phosphate) in in vitro models of vascular calcification and osteoclastogenesis [15] (Figure 2). We have pointed out in vitro and in vivo the roles of miR-126, miR-143, miR-145 and miR-223 in the acquisition of a pro-calcifying phenotype in human primary cultures of vascular smooth muscle cells (VSMCs) [16, 17]. Magnesium treatment was able to revert at least partly the effect of Pi on miRNA expression in VSMCs [18]. These results were confirmed in vivo in tissue biopsies from CKD and/or atherosclerotic mice displaying vascular calcification [17]. miR-126 and miR-223 levels were increased in aortas of these mice, whereas miR-143 and miR-145 levels were decreased [17]. Sevelamer administration reduced the aortic expression of miR-223 and miR-126 in this model. We thus suggested a direct link between the observed changes in miRNA levels and uraemic vascular toxicity (Figure 2). Importantly, at high uraemia values we found low seric miR-223 levels (contrary to variations in aortic wall), hinting at a similar regulation in human patients [17]. In another model, we also showed that, in vitro, Pi is responsible for a marked decrease in osteoclastogenesis in RAW cells, in conjunction with a lower cellular miR-223 levels [19]. An anti-miR specific for miR-223 inhibited osteoclastogenesis in the same way as Pi. In contrast, the overexpression of miR-223 led to an increased differentiation, as reflected by tartrate-resistant acid phosphatase activity. miR-223 affected the expression of not only its target genes NFIA and RhoB but also of osteoclast marker genes and of the Akt signalling pathway that induces osteoclastogenesis [19]. These results were confirmed by measuring bone resorption activity in human VSMCs differentiated into osteoclasts—suggesting that the modulation of miR-223 in CKD patients might be of value in managing the complications related to CKD (Figure 2). Also, another team found that expression levels of miR-223 in renal biopsies were greater in patients with progressive chronic renal failure than in patients with stable CKD; this suggests that miR-223 may have a role in aggravating renal dysfunction [20]. FIGURE 2 View largeDownload slide Implication of miR-223 in the vascular calcification process. miR-223 expression is modulated during vascular calcification in CKD. Hyperphosphataemia increases miR-223 expression in VSMCs, which transdifferentiate into ‘osteoblast-like’ synthetic cells with higher mobility. The same high Pi concentration decreases miR-223 levels in vascular precursors of monocytes/macrophages and inhibits their receptor activator of nuclear factor κB ligand–induced differentiation into osteoclast-like cells. FIGURE 2 View largeDownload slide Implication of miR-223 in the vascular calcification process. miR-223 expression is modulated during vascular calcification in CKD. Hyperphosphataemia increases miR-223 expression in VSMCs, which transdifferentiate into ‘osteoblast-like’ synthetic cells with higher mobility. The same high Pi concentration decreases miR-223 levels in vascular precursors of monocytes/macrophages and inhibits their receptor activator of nuclear factor κB ligand–induced differentiation into osteoclast-like cells. miRNAs are potential biomarkers in the various stages of CKD At the moment, glomerular filtration rate is the best marker of renal function, but its measurement in routine practice is tricky, so it is usually estimated from creatinine serum levels. The serum urea level and the albuminuria and proteinuria values (divided by the creatininuria value) are also used as markers of renal function. Nevertheless, a variety of studies have stressed that these markers lack sensitivity for early diagnosis of CKD [21]. Over the past decade, several potential promising biomarkers have emerged: urinary liver fatty acid-binding protein, urinary N-acetyl-β-d-glucosaminidase, urinary connective tissue growth factor, serum apolipoprotein A-IV, urinary and serum neutrophil gelatinase-associated lipocalin, serum and urinary kidney injury molecule 1 or serum fibroblast growth factor 23 (recently reviewed in Rysz et al. [21]). Most of these markers are very sensitive and specific but have not yet been validated in clinical practice. In recent years, miRNAs have attracted increasing interest as potential biomarkers to assess the severity of the disease or to identify a group of patients with comorbidity [22]. Mitchell et al. [23] have demonstrated for the first time the presence of circulating miRNAs in human plasma. This discovery was surprising, as plasma is known to have high RNase activity. These circulating microRNAs have many origins and ways of transport conferring the stability and the protection needed against RNase activity [24]. miRNAs could be also released by active secretion via cell-derived membrane vesicles or released by a protein miRNA complex comprising AGO2 and/or high-density lipoproteins (HDLs). The main advantage of miRNAs relates to their stability in serum. Furthermore, miRNAs might meet the sensitivity, specificity and reproducibility criteria required for an ideal, non-invasive biomarker [22] (Figure 3). However, the possible involvement of a given miRNA in several different diseases remains a problem. FIGURE 3 View largeDownload slide The potential of miRNAs in renal disorders, a bench to bedside approach: cellular and preclinical models are used to screen the most useful miRNAs in a nephrological pathology. Alternatively, patient samples can be screened using high-throughput techniques. miRNAs can further be validated as prognostic biomarkers in large human cohorts, then used as innovative therapeutic approaches to improve kidney function in the patients. FIGURE 3 View largeDownload slide The potential of miRNAs in renal disorders, a bench to bedside approach: cellular and preclinical models are used to screen the most useful miRNAs in a nephrological pathology. Alternatively, patient samples can be screened using high-throughput techniques. miRNAs can further be validated as prognostic biomarkers in large human cohorts, then used as innovative therapeutic approaches to improve kidney function in the patients. The expression of miRNAs in CKD has been documented; in most cases, levels decrease as CKD progresses [25]. Neal et al. [26] used real-time quantitative PCR (qPCR) and found that the levels of most miRNAs decreased as the CKD stages increased. Others used a cohort of 90 patients at CKD stages 3, 4 and 5D to show a decrease in serum expression levels of miR-125b, miR-145 and miR-155 [27]. The serum concentration of certain heart-specific miRNAs additionally seemed to be inversely related to renal function [11]. We have shown in a murine model that serum miR-223 levels decrease in CKD mice. Conversely, Ulbing et al. [28] reported that miR-223 expression was lower in CKD stage 4 and 5 patients than in healthy controls. This down-regulation disappeared after renal transplantation. The results wait to be confirmed in larger cohorts. The evidence for an effect of renal function on circulating miRNA levels in plasma and urine is limited and sometimes contradictory. Pharmacokinetics studies showed that the liver, the reticuloendothelial system and the kidney are responsible for excreting a proportion of circulating miRNAs [29]. However, small interfering RNAs (siRNAs) conjugated to diethylenetriaminepentaacetic acid (DTPA) accumulate in the kidney three times more than DTPA alone when administered to animals, suggesting that small RNAs might be actively reabsorbed after renal filtration through the proximal tubule [29]. In vivo imaging experiments have clearly demonstrated that siRNAs are freely filtered in the glomerulus and are reabsorbed within 10 s by proximal tubular epithelial cells [30]. However, the renal excretion system does not seem to be involved since the decrease in circulating miRNA levels in CKD patients in Neal et al.’s study was not associated with an increase in excreted miRNAs in urine [26]. Some researchers have looked at the known accumulation of RNases in renal failure [31], as this might increase the degradation of miRNAs in the plasma. That being said, circulating miRNAs are usually protected by exosomes, HDL and AGO proteins, and no consistent differences in exosome abundance or stability have been observed in uraemic vs. normal serum [32]. miRNA levels remain stable when plasma is subjected to prolonged room temperature incubation [31]. Some in vitro studies indicate that miRNA synthesis is low in CKD but that the miRNA-processing enzymes Drosha and Dicer are normally expressed [33]. With regard to the biogenesis of urinary miRNAs, the RNAs are released by cells in the nephron and downstreamed in the urinary tract [31]. The miRNAs in the urinary tract may be associated with membrane-bound extracellular vesicles, such as microvesicles (formed by outward budding of the plasma membrane) and exosomes (secreted from multivesicular endosomes formed in the endocytic tract) [24]. Can seric miRNA measurement be useful in acute kidney disease? Acute kidney injury (AKI) leads to tubular injury and the development of fibrosis. miR-146a is transcriptionally upregulated by ligands of interleukin-1 receptor/Toll-like receptor family members via the activation of nuclear factor κB in renal proximal tubular cells. miR-146a knockout mice submitted to renal ischaemia–reperfusion injury exhibited tubular injury [34]. So miR-146a could be a key mediator of the development of AKI and CKD. Other miRNAs have been shown to be instrumental in various pre-clinical models. For example, miR-223 in AKI is associated with experimental sepsis [35] and miR-140 in cisplatin-induced AKI [36]. What if miRNAs could help to predict the outcome of kidney graft? Rejection is the main consequence of allograft failure and its monitoring by the use of invasive biopsies is risky and burdensome to transplant patients. Circulating miRNA levels could be of tremendous help to the clinician as novel and easily accessible biomarkers of allograft rejection and transplant failure. A significant number of miRNA and transplant studies have been performed in renal transplantations. A pioneering study investigated whether acute rejection (AR) is associated with alterations in miRNA levels in renal biopsies [20]. The authors found an overexpression of miR-142, miR-155 and miR-223 in AR biopsies, suggesting that miRNA expression levels may serve as biomarkers of human renal allograft status. The kidney-specific miRNA-146a seems to be the most interesting risk factor in the development of rejection as it is part of an miRNA signature of renal ischaemia–reperfusion injury [37], an increase of its expression in dendritic cells promotes allogeneic kidney graft survival [38] and SNPs in miR-146a are associated with renal cancer [9]. The various miRNAs implicated in nephropathies are summarized in Table 1. Table 1 miRNAs implicated in nephropathies miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] Table 1 miRNAs implicated in nephropathies miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] miRNA Nephropathy(ies) and associated disorders Relevant gene targets References miR-21 Renal ischemia–reperfusion PTEN, NF-κB, TGFBR2 Xu et al. [39] miR-125b CKD, vascular calcification EPO, EPOR, BCL2, c-Jun, c-RAF Metzinger-Le Meuth et al. [15], Chen et al. [27] miR-126 CKD, endothelial dysfunction, atherosclerosis VCAM, CXCL12 Taibi et al. [17], Schober et al. [40], Mondadori Dos Santos et al. [41] miR-140 Cisplatin-induced AKI HDAC4, CXCL12 Liao et al. [36] miR-142 Kidney graft AR COX-2, SOCS1, TGFBR1 Anglicheau et al. [20] miR-143 CKD, vascular calcification KLF1, HK2, VCAN Taibi et al. [17] miR-145 CKD, vascular calcification MYOCD, ERK-5, MUC1 Taibi et al. [17], Louvet et al. [18], Chen et al. [27] miR-146a AKI, kidney graft AR, renal cancer NF-κB Du et al. [9], Amrouche et al. [34], Godwin et al. [37], Wu et al. [38] miR-155 CKD, vascular calcification, kidney graft AR SMAD family, PU.1, HDAC Anglicheau et al. [20], Chen et al. [27] miR-223 AKI, CKD, kidney graft AR, osteoclastogenesis, vascular calcification NF-κB, NFIA, RhoB Metzinger-Le Meuth et al. [15], Rangrez et al. [16], Taibi et al. [17], M’Baya-Moutoula et al. [19], Anglicheau et al. [20], Colbert et al. [35] We now have to reliably and reproducibly measure serum miRNAs in routine clinical practice miRNAs are promising innovative biomarkers in the nephrology field, but we are far from the identification of a precise and useful miRNA signature in a given kidney pathology. At present, miRNAs’ value as biomarkers is at least partly limited by difficulties in normalizing their expression levels, which may lead to poor reproducibility between different laboratories [22]. Real-time PCR is suitable for analysing a small number of miRNAs because it is accurate, rapid and easily transferable into routine practice. Several endogenous circulating miRNAs (such as U6 and miR-1249) were initially used to normalize expression levels of circulating miRNAs; however, the validity of this approach was recently challenged because of high variability [22]. Due to the low concentration of miRNAs in the circulation, standardization is of the utmost importance. Henceforth, a specific amount of exogenous miRNA (e.g. synthetic C.elegans miR-39) is added to avoid experimental bias [22]. The time required to quantify circulating levels of miRNAs by real-time qPCR is also an obstacle to their use in clinic. Several new approaches are in development. For instance, a new method of nanobiophotonic detection is being evaluated. It involves Förster resonance energy transfer after the excitation of two fluorophores, without the need for sample preparation or signal amplification [42]. Alternatively, Smith et al. [32] have developed a simple method for the electrochemical detection of miRNAs in urine samples. MIRNAS ARE POTENTIAL NEW THERAPEUTIC SOLUTIONS IN NEPHROLOGY Lessons from pre-clinical models Regulation of gene expression is critical for all cellular processes, with dysregulation often resulting in the development of disease. miRNAs are instrumental players in the fine-tuning of gene regulation networks and have thus potential therapeutic impacts [43]. Several recent studies have shown that endothelial miRNAs have important roles during vasculogenesis and in the response to inflammation and haemodynamic stress [44, 45]. It has been shown for example that miR-126–based therapy maintains a proliferative reserve in endothelial cells (ECs) and can prevent atherosclerosis [41]. Indeed, the pro-angiogenic miR-126 is abundant in ECs, plays an important role in vascular dysfunction and modulates the expression of vascular cell adhesion molecule-1 and chemokine CXCL-12. We have recently shown that miR-126 is involved in the vascular remodelling under laminar shear stress in human endothelial cells and is implicated in the modulation of syndecan expression [40]. miRNAs could have a role(s) in CKD vascular remodelling and may therefore represent useful targets to prevent or treat vascular complications of CKD [15]. To further confirm this, it was very recently shown that administration of miR-126 rescued EC proliferation at predilection sites and limited atherosclerosis [41]. This suggests a possible direct link between the observed miRNA alterations and the vascular damage caused by CKD [17]. Concerning kidney graft models, Xu et al. [39] showed in a murine model a role for miR-21 up-regulation in the protective effect of delayed ischaemic preconditioning against subsequent renal ischaemia–reperfusion. The timely and effective delivery of miRNAs and miRNAs inhibitors. CRIPSR/cas9 gene editing, gold or iron nanotechnologies: what they can bring to the table Chemically modified miRNA precursors or inhibitors (such as sponges) can be injected directly into the bloodstream (Figure 4) [46]. Modern viral vectors can also be used (Figure 4) [46]. Lentiviruses seem to be a good solution for kidney delivery since they proved capable of infecting kidney podocyte cells and delivering the therapeutic gene product, they escape the immune system and they are efficient vectors suited to deliver miRNA therapeutic sequences [47]. Different approaches of injured arterial wall treatment have used systemic, polymer-based particles or local, modified stents to induce endothelial regeneration in CKD [46, 48]. One can use naturally occurring nanomaterials already present in the human body, namely exosomes or HDL [32], to deliver functional miRNAs in a therapeutic goal, as has already been done with miR-21 [49]. Also, another team showed that HDL particles are able to deliver miR-223 to ECs in order to suppress expression of its mRNA targets [32]. Efforts are under way to assess nanotechnology, with novel gold nanoparticles of 13 ± 1 nm, each functionalized with a monolayer of double-stranded alkylthiol-modified RNA molecules (Figure 4) [50]. These new vehicles can enter cells without the aid of co-carriers, reside there for up to 24 h after transfection and deliver endogenous miRNAs in situ [50]. The same approach could be used to deliver miR-126 or miR-223 in diseased vessels in the CKD context. It was shown that 3-nm glutathione-protected gold nanoclusters accumulate preferably in the kidney [51], and they would thus be prime candidates. It is difficult to prioritize among all these promising solutions, but in our opinion lentiviral vectors and gold nanoparticles specifically accumulating in the kidney are the most promising techniques in the foreseeable future for treating kidney disorders. FIGURE 4 View largeDownload slide New therapeutic approaches in kidney diseases. miRNAs levels can be either enhanced or decreased according to the clinical context in kidney and blood vessels. Various strategies are currently being developed to modify their expression. Recent gene therapy vectors, for example, adeno-associated virus or lentivirus, can be used to deliver miRNA precursors to diseased tissues. One can also use chemically modified RNAs, inhibitory sponge complementary sequences or miRNAs chemically bound to nanoparticles. The CRIPSR/Cas 9 system can also be used to alter the miRNA sequence and improve its function. FIGURE 4 View largeDownload slide New therapeutic approaches in kidney diseases. miRNAs levels can be either enhanced or decreased according to the clinical context in kidney and blood vessels. Various strategies are currently being developed to modify their expression. Recent gene therapy vectors, for example, adeno-associated virus or lentivirus, can be used to deliver miRNA precursors to diseased tissues. One can also use chemically modified RNAs, inhibitory sponge complementary sequences or miRNAs chemically bound to nanoparticles. The CRIPSR/Cas 9 system can also be used to alter the miRNA sequence and improve its function. That being said, another system has arisen recently that could prove equally useful. The CRISPR/cas9 system is an emerging genome editing tool that is now increasingly used in the miRNA research field. For example, CRISPR/cas9 constructs can be constructed with single-guide RNAs targeting selected sites of miRNA precursors to almost completely obliterate the expression of these miRNAs in the long term (Figure 4) [52]. Additionally, the technique can be used to target miRNAs of the same family or with highly conserved sequences. CONCLUDING REMARKS We need new solutions to improve diagnosis and monitoring in modern nephrology and miRNAs are prime candidates to be developed as innovative biomarkers in the early diagnosis and prognosis of patients afflicted with kidney disorders. The development of new pharmacologic therapies enabling the modulation of miRNA levels should be considered to provide possible therapies of most common kidney diseases in an expanding world population of ageing and diabetic patients. FUNDING Part of the work described in this review was funded by a grant from the Picardie Regional Council. 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Sci Rep 2016 ; 6 : 22312 Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Nephrology Dialysis TransplantationOxford University Press

Published: May 25, 2018

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