Review: The role of microRNAs in kidney disease

Authors


Professor Jonathan Gleadle, Department of Renal Medicine, Level 6, Flinders Medical Centre, Flinders Drive, Bedford Park, SA 5042, Australia. Email: jonathan.gleadle@health.sa.gov.au

ABSTRACT

MicroRNAs (miRNAs) are short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression via blockade of protein translation or by inducing mRNA degradation. These miRNAs potentially regulate the expression of thousands of proteins. As a result, miRNAs have emerged rapidly as a major new area of biomedical research with relevance to kidney disease. MiRNA expression has been shown to differ between the kidney and other organs as well as between different kidney regions. Furthermore, miRNAs have been found to be functionally important in models of podocyte development, diabetic nephropathy and polycystic kidney disease. Of particular interest, podocyte-specific deletion of Dicer, a key enzyme in the biogenesis of miRNA, results in proteinuria and severe renal impairment in mice. One miRNA (miR-192) can also act as an effector of transforming growth factor-β activity in the high-glucose environment of diabetic nephropathy. Differential expression of miRNAs has been reported in kidney allograft rejection. It is anticipated that future studies involving miRNAs will generate new insights into the complex pathophysiology underlying various kidney diseases, generate diagnostic biomarkers and might be of value as therapeutic targets for progressive kidney diseases. The purpose of this review is to highlight key miRNA developments in kidney diseases and how this might influence the diagnosis and management of patients with kidney disease in the future.

INTRODUCTION

MicroRNAs (miRNAs) are endogenous non-coding RNA molecules, 20–22 nucleotides in length. The discovery and characterization of miRNA in the last decade is revolutionizing our understanding of gene regulation, cell differentiation, proliferation, apoptosis, metabolism and pathophysiology of many diseases including kidney diseases. The understanding of miRNA biology and its role in various diseases is still in its early stage but is expanding rapidly. The aim of this review is to highlight miRNA biology in relation to kidney diseases and its importance in understanding disease mechanisms. Future directions in this field will also be discussed.

DISCOVERY AND BIOGENESIS OF MICRORNAs

MiRNAs were first found in the nematode Caenorhabditis elegans in 1993.1 Since then they have also been described widely in plants and mammals.2 MiRNAs are first transcribed in the nucleus as stem-loop primary miRNA, which are then cleaved into shorter precursor miRNA by Drosha, an RNase III, and its essential cofactor called DGCR8 (DiGeorge syndrome critical region 8), a double-stranded RNA-binding protein (Fig. 1).3–6 The precursor miRNAs are transported out of the nucleus via Exportin-5 and once in the cytosol are cleaved into their mature form of 20–22 nucleotides by Dicer, another RNase III.7,8 After cleavage, the miRNA duplex is unwound and the functional strand is loaded onto the RNA-induced silencing complex (RISC) and functions as its guide.9 The mature miRNA guides the RISC complex to a (near) complementary sequence, usually in the 3′ untranslated region (UTR), of a target messenger RNA (mRNA).9 Upon binding, the RISC causes post-transcriptional gene silencing by either cleaving the target mRNA or by inhibiting its translation, so that miRNAs are usually negative regulators of gene expression.10 In addition to their role in such post-transcriptional repression, miRNAs have now been implicated in transcriptional gene silencing by targeting the promoter region but have also been reported to have a positive effect on transcription.11–13

Figure 1.

Current model of biogenesis of microRNAs.

Each miRNA can potentially regulate the translation of a large number of different mRNA and each mRNA can possess multiple binding sites for a single or for many different miRNA because the specificity of miRNA is mainly determined by Watson-Crick base pairing at the 5′ region of the miRNA. Estimates have suggested that the total number of different miRNA sequences in humans may exceed 1000.14 Computational analysis also predicts that over 60% of human genes are potential targets of miRNAs and that there are a large number of other non-coding RNAs of greater nucleotide length than microRNA, which are also likely to have important functions.15 However, direct experimental evidence defining mRNA targets of miRNA regulation has been reported for only a small number of miRNAs and target mRNAs.

TECHNIQUES FOR DETECTION OF MIRNA LEVELS

Assaying the levels of specific microRNA sequences was initially cumbersome; however, advances in technology now allow detection with a sensitivity and specificity that can enable monitoring in a clinical setting. Originally, RNA blot analyses provided both quantitative and qualitative information about the various forms of a miRNA within a total RNA sample.1,16 As the number of miRNAs in the miRBase registry17 has increased, microarray technology has been adapted to enable the parallel screening of thousands of miRNAs in one sample.18 More recently, real time reverse transcription-polymerase chain reaction has been adapted to enable relative quantification and quantitative analysis of miRNA levels. Specific amplification of mature miRNAs can be achieved using stem-loop oligonucleotides to prime reverse transcription and enable TaqMan detection,19 while alternate reagents and methods for amplifying precursor and primary transcripts are also commercially available. Microfluidic systems now enable high throughput miRNA PCR profiling with small amounts of input sample RNA, enabling analysis of small biopsies, limited volumes of body fluids, or even formalin-fixed paraffin-embedded archival material.20 The hybridization kinetics of oligonucleotides have been enhanced through the incorporation of locked nucleic acid monomers, which provide an advantage for PCR and in situ hybridization21 and also enhance the potential for employing anti-miRNA strategies in therapeutic roles.22,23

MICRORNAS IN RENAL PHYSIOLOGY

The suggestion of organ-specific roles for miRNAs emerged with the demonstration of tissue-restricted miRNA expression, including clusters of miRNAs that are expressed specifically in the kidney.24 Conversely, the absence or lower levels of particular miRNAs in the kidney compared with other organs may permit renal specific expression of target proteins that are important for kidney function.24,25 Examples of miRNAs that are more abundant in the kidney compared with other organs include miR-192, miR-194, miR-204, miR-215 and miR-216. Tian et al. established the first differential profile of miRNA expression between the renal cortex and medulla of rats indicating a potential role in tissue specification.26 However, cell type-specific miRNAs in the kidney have not yet been reported.

A critical role of miRNA regulation in the progression of glomerular and tubular damage, and the development of proteinuria have been suggested by studies in mice with podocyte-specific deletion of Dicer.27–29 All three reports showed major renal abnormalities in these mice including proteinuria, podocyte foot process effacement, glomerular basement membrane abnormalities, podocyte apoptosis, podocyte depletion and mesangial expansion. There was a rapid progression of renal disease with initial development of albuminuria followed by pathological features of glomerulosclerosis and tubulointerstitial fibrosis. This led to renal failure and death by 6–8 weeks. It is likely that these phenotypes are due to the global loss of miRNAs because of Dicer deletion, but given multiple miRNAs and their myriad targets, the precise pathways responsible require identification.

These investigators also identified specific miRNA changes, for example, the downregulation of the miR-30 family when Dicer was deleted. Of relevance, the miR-30 family was found to target connective tissue growth factor, a profibrotic molecule that is also downstream of transforming growth factor (TGF)-β.30 Thus, the targets of these miRNAs may regulate critical glomerular and podocyte functions. These findings have also been complemented by an elegant study revealing a developmental role for the miR-30 family during pronephric kidney development in Xenopus.31

Recently, another study has shown that deletion of Dicer in the renin secreting cells of mice severely reduced the number of juxtaglomerular cells, decreased expression of the renin genes, lowered plasma renin concentration and decreased blood pressure.32 The kidneys developed striking vascular abnormalities and prominent striped fibrosis. These findings highlight the important roles of Dicer and miRNAs in renal physiology and pathology, although the extent to which such genetic studies reveal an essential and fundamental role of Dicer in cellular function, as opposed to a specific role in renin secreting cells, is arguable. The importance of Dicer in cellular function is further highlighted by Wei's study.33 They established a mouse model with targeted Dicer deletion in renal proximal tubules. These mice had normal renal function and histology despite a global downregulation of miRNAs in the renal cortex. However, these mice were strikingly resistant to renal ischaemia-reperfusion injury, showing significantly better renal function, less tissue damage, lower tubular apoptosis and improved survival compared with their wild-type counterparts.33

MICRORNAS IN RENAL DISEASES

Diabetic nephropathy

Diabetic nephropathy is the leading cause of end-stage kidney disease but our understanding of the disease mechanisms is incomplete. Studies of miRNA expression in diabetic nephropathy have so far emerged predominantly from animal models of diabetes and the effects of hyperglycaemia. In one study, miR-192 levels were shown to be increased in glomeruli isolated from streptozotocin-injected diabetic mice and diabetic mice db/db when compared with non-diabetic mice.34 In this study, miR-192 was shown to regulate E-box repressors that are responsible for controlling the expression of TGF-β-induced extracellular matrix proteins, collagen 1-α 1 and 2 (Col1a1 and 2). Col1a1 and 2 were shown to accumulate during diabetic nephropathy; therefore, these results suggest a potential role of miR-192 in diabetic nephropathy or that miR-192 can be an effector of TGF-β. However, discordantly a recent study demonstrated that miR-192 expression is decreased in proximal tubular epithelial cells in response to TGF-β.35 The loss of miR-192 correlates with tubulointerstitial fibrosis and reduction in eGFR in renal biopsies from patients with established diabetic nephropathy. This suggests that mesangial cell and proximal tubular epithelial cell miRNA expression may exhibit different responses to TGF-β.

Recently, Akt kinase, a key mediator of diabetic nephropathy, was found to be activated through downregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which is targeted by miR-216a and miR-217. In turn, these miRNAs are upregulated by TGF-β, and indirectly by miR-192, in mouse mesangial cells.36,37 In other animal studies, Zhang et al. showed miR-21 expression was downregulated in response to early diabetic nephropathy in vitro and in vivo.38 Overexpression of miR-21 inhibited proliferation of mesangial cells in high-glucose condition. The 24 h urine albumin excretion rate of diabetic db/db mice decreased after exposure to elevated miR-21. The same study also identified PTEN as a target of miR-21.38

Another study has reported overexpression of miR-377 in human and mouse mesangial cells when exposed to high glucose levels.39 MiR-377 has been demonstrated to reduce the expression of p21-activated kinase (PAK1) and manganese superoxide dismutase (mnSOD). This enhances fibronectin production, which is characteristic of mesangial cells in diabetic nephropathy. We anticipate that many other miRNAs expressed in podocytes, tubular and other renal cells will be deregulated under hyperglycaemic conditions.

In diabetic nephropathy, alteration of miRNA expression in response to several pathophysiological states is of interest, notably hypoxic-ischaemic and hyperglycaemic stimuli. The findings by Wang and colleagues have already provided the first glimpse of the effects of hyperglycaemia on miRNA expression in mesangial cells. In addition, hyperglycaemia has been found to affect endothelial dysfunction through miR-221.40

Polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited renal diseases. Genetically, mutations in the polycystic kidney disease-1 gene (PKD1) account for 85% of ADPKD; whereas mutations in the polycystic kidney disease-2 gene (PKD2) are responsible for the remainder.41 PKD2 encodes a protein termed polycystin-2. Aberrant expression of polycystin-2 causes abnormal proliferation of renal tubular and biliary epithelial cells, eventually leading to cystogenesis.42,43

The potential role of microRNAs in control of expression of PKD genes and in mediating functional effects has recently been explored. Two groups have demonstrated that miR-17 directly targets the 3′UTR of PKD2 and post-transcriptionally represses the expression of PKD2.44,45 Moreover, they also showed that overexpression of miR-17 may promote cell proliferation via post-transcriptional repression of PKD2 in HEK293T cells. Finding new miRNAs that target PKD1 is an area of active research.

Using a rat model of PKD, 30 differentially expressed miRNAs have been identified in diseased kidney tissues compared with healthy rat, 29 of which are downregulated.46 Two algorithms: TargetScan and miRanda, predicted targets for significantly deregulated miRNAs in PKD that were correlated with pathways affected in PKD as determined using KEGG, GeneOntology (GO), Biocarta and the Molecular Signature databases.47–50 The deregulated miRNAs in PKD were associated with genes in 24 functional categories, including several pathways important to cyst formation such as mTOR signalling, mitogen-activated protein kinase signalling, Wnt signalling and TGF-β pathway.46 However, these correlations require experimental validation.

MiR-15a has been reported to modulate the expression of cell cycle regulator Cdc25A and affect hepatic cystogenesis in a rat model of PKD.51In situ hybridization suggested that miR-15a was downregulated in liver tissues of patients with ADPKD, autosomal recessive PKD or congenital hepatic fibrosis, as well as rats with PKD. Conversely, overexpression of miR-15a in cells derived from the PKD rat led to a decrease in Cdc25A protein, small decreases in G1-S phase transition and cellular proliferation, and a larger drop in cyst growth in vitro. This disproportionate effect on cyst growth suggests that decreased miR-15a may promote cystogenesis through alternate mechanisms in addition to increased cell proliferation.

Other kidney diseases

In trying to understand the role of microRNAs in renal diseases an obvious approach has been to compare microRNA expression between samples from normal and affected patients. In renal disease, such studies have included patients with IgA nephropathy, lupus nephritis, hypertension and renal cancer. A study by Dai and colleagues compared miRNA expression of IgA nephropathy biopsy samples from 11 patients with three control patients.52 They were able to identify 132 miRNA in both patients with IgA nephropathy and normal control renal tissue samples, of which 31 miRNAs were downregulated and 35 upregulated in diseased tissues. More recently, another study has reported differential intrarenal expression of miR-200c, miR-141, miR-205 and miR-192 in IgA nephropathy and findings correlated with disease severity and progression.53 The deregulated expression of miR-200c and miR-205 is of particular interest given their link with epithelial-to-mesenchymal transition (EMT).

Sixty-six miRNAs have also been found to be differentially expressed in a small number of human kidney tissues from patients with Class II lupus nephritis as compared with healthy control subjects.54 Differential expression of miRNAs (16 miRNA, 7 downregulated and 9 upregulated) in peripheral blood mononuclear cells (PBMC) has also been reported in patients with systemic lupus erythematosus when compared with normal healthy subjects.55

Elevated levels of angiotension receptor 1 (AGTR1) have been shown to lead to hypertension. MiR-155 has been reported to downregulate the expression of AGTR1.56 The miR-155 target site in the 3′-UTR of human AGTR1 contains a single nucleotide polymorphism rs5186, which is associated with hypertension in some subpopulations.57 In a recent study, several other miRNA, miR-200a, miR-200b, miR-141, miR-429, miR-205 and miR-192, were increased in kidney biopsy samples from patients with hypertensive glomerulosclerosis.58 However, miR-155 was not evaluated in this study.

Differential miRNA expression has also been linked to both renal and transitional cell carcinomas.59–61 Hypoxia-regulated miRNAs, such as miR-210, have been found to be expressed differentially in renal cell carcinomas and may have implications for tumour pathogenesis.61 Similarly, an oncogenic cluster of miRNAs has been implicated in Wilms tumour.62 The role of miRNAs in cancers is a rapidly evolving area of research and a detailed review is beyond the scope of this article.

While these differences in tissue microRNA expression are interesting, defining whether changes are disease-specific or fundamental to disease pathogenesis remains a major challenge.

PROGRESSION OF CHRONIC KIDNEY DISEASE AND KIDNEY FIBROSIS

Transition of epithelial to mesenchymal cells is recognized as a substantial contributor to the development of kidney fibrosis.63 Epithelial mesenchymal transition (EMT) describes a reversible series of events during which epithelial cells undergo morphological changes and acquire mesenchymal characteristics. These events involve epithelial cells losing cell–cell contacts, apical-basal polarity and epithelial-specific junctional proteins such as E-cadherin while acquiring mesenchymal markers including vimentin and N-cadherin.64 The end result is that immobile epithelial cells revert to an immature undifferentiated phenotype with enhanced migratory ability reminiscent of an earlier development stage and can embed in interstitium. EMT is known to be involved in implantation, embryogenesis and organ development. It also has been shown to associate with cancer progression and metastasis.65 EMT has been suggested to contribute to kidney fibrosis, which is defined as an excessive deposition of extracellular matrix, mediated predominantly by fibroblasts and mesenchymal cells, leading to structural destruction and renal failure. The possible sources of fibroblasts and mesenchymal cells in kidney fibrosis include de novo proliferation of resident tissue fibroblasts, circulating fibrocytes from bone marrow or perivascular smooth muscle cell expansion (myofibroblasts). It has been demonstrated recently that a large proportion of interstitial fibroblasts actually originate from tubular epithelial cells via EMT in diseased kidney.66–68

Several studies have now found that EMT is regulated by miRNAs, notably the miR-200 family and miR-205.69–72 These miRNAs have been implicated in the EMT process occurring in cancer development.72 The miR-200 family and miR-205 are downregulated in Madin Darby canine kidney cells undergoing TGFβ-induced EMT.69 Their decrease with TGF-β exposure is linked to the EMT response. Evidence has recently emerged that the miR-200 family and miR-205 are elevated in patients with hypertensive nephrosclerosis.58

Recently, Yamaguchi et al. have proposed an important mechanism for podocyte dehiscence and loss through EMT.73 In other disease processes, particular miRNAs were found to be substantially altered during EMT.65 Future work is required to determine the significance of miRNA involvement in EMT during the development of diabetic nephropathy.

ALLOGRAFT REJECTION

Renal transplantation is the treatment of choice for patients with end-stage kidney disease because of superior survival and quality of life when compared with patients on maintenance dialysis. Despite improvements in immunosuppression, acute rejection (AR) and chronic allograft nephropathy remain major challenges. There is a need to better understand the mechanism of rejection and develop novel biomarkers for diagnosis and management of rejection.

Two studies have found differential expression of miRNAs during AR of kidney allografts. One study characterized the association between intrarenal miRNAs and clinicohistological status of renal allografts.74 A subset of 17 miRNAs, out of 365, was found to be differentially expressed in AR biopsies compared with normal allograft biopsies. The altered expression of 6 of the 17 miRNAs identified was validated with quantitative analysis. Impressively, they reported that AR can be predicted accurately using intragraft levels of miR-142-5p (100% sensitivity and 95% specificity) or miR-155 (100% sensitivity and 95% specificity). In addition, miRNA levels were evaluated in isolated PBMC and human renal tubular epithelial cells. Some of the miRNAs found to be increased in AR were also expressed in PBMC. This indicates that cellular infiltration of immunological cells may explain the changes in miRNA expression.

Using a similar approach, Sui et al. reported 20 miRNAs that were differentially expressed, of which 12 were downregulated and 8 upregulated in AR, when compared with normal allograft biopsies.75 The next challenge in this research is to determine if changes in miRNA expression are due to AR alone, or due to other factors such as renal function, viral infection status and time since transplantation.

A growing number of studies have found several human viruses such as cytomegalovirus, Epstein-Barr virus (EBV) and BK virus that encode viral miRNAs and their specific expression can be associated with different phases of viral infection. Furthermore, there is differential expression of EBV-encoded miRNAs in peripheral blood cells of EBV carriers (latent infection) and patients with acute EBV infection.76 This might provide a diagnostic test to differentiate active viral infection from carriage that is important in the management of renal transplant patients.76–79 Further research is needed to examine the role and function of these miRNAs in the pathophysiology of the infection.

MICRORNAS AS DIAGNOSTIC BIOMARKERS

Recent progress in miRNA research presents opportunities for understanding kidney diseases, including identification of new diagnostic biomarkers. The potential value of miRNAs as biomarkers for human cancer research has been demonstrated and may provide more accurate tumour classification than mRNA analysis.80 MiRNA profiles offer some important potential advantages over standard mRNA or other protein-based profiles. MiRNAs appear to be very stable in tissues and biological fluids, including serum and are protected from endogenous RNase by virtue of their small size and perhaps by packaging within exosomes.81 In addition, the tissue-specific nature of miRNA expression makes them ideal candidates for biomarkers.82 The total number of human miRNAs, estimated to be between 700 and 1000, is considerably smaller than the number of protein-coding mRNAs (about 22 000).14,83

MiRNA profiling might have relevance to the early detection of acute kidney injury and predict progression in chronic kidney disease (CKD). Indeed, several miRNAs have been associated with tissue hypoxia,84–87 which is recognized as an important contributor to the development of acute kidney injury (AKI) as well as progression of CKD, particularly in predisposing conditions such as diabetes and hypertension. Further studies are needed to examine if hypoxia-regulated miRNAs can serve as early biomarkers for AKI or progression of CKD. MiRNAs with roles, or differential expression, in EMT, inflammation, fibrosis and activation of renal stem cells may also be relevant biomarkers in these conditions.63,66,88

MICRORNA IN BLOOD AND EXOSOMES

The discovery of plasma- or serum-derived miRNAs and free circulating exosomes that contain miRNAs has opened up a new frontier in understanding their physiological or pathophysiological roles.81,89–92 Many of the most highly expressed miRNAs in microvesicles are thought to have roles in cellular differentiation. This has led to speculation that miRNAs in microvesicles circulate to target tissues and have an endocrine function.93 It has also been hypothesized that the circulating miRNAs play a part in cell-to-cell communication.81 Thus far, plasma- or serum-derived miRNA expression has yet to be reported in association with kidney diseases.

MiRNA expression and clearance may be altered in renal failure but this area has not been studied. One study performed miRNA array analysis in cultured human proximal tubular (HK-2) cells exposed to control versus uraemic dialysate. Forty-eight miRNAs were deregulated of which 15 were upregulated and 33 downregulated, respectively. It is possible that the uraemic environment can alter miRNA expression.94 These new insights potentially may have broad ranging implications for the role of microRNAs in the pathogenesis of uraemia.

Exosomes are 40–100 nm diameter membrane vesicles of endocytic origin that are released by most cell types under both physiological and pathological conditions. They are taken up by surrounding host cells and therefore function to promote intercellular communication.95 Exosomes have now been identified in blood, urine and other body fluids.96 Tumours also release exosomes into peripheral circulation and exosomes can be isolated from the blood by differential centrifugation or enriched using cell surface markers such as epithelial cell adhesion molecule.91,92 Exosomes seem to be particularly rich in miRNAs.90 MiRNA expression profiling in exosomes of ovarian cancer patients revealed a high correlation to that of its tumour counterpart.91 These data suggest that miRNA expression profiles from circulating exosomes can be used as a surrogate marker for diagnostic or prognostic purposes. For a number of kidney diseases, miRNAs in peripheral circulation may serve as a measure of disease stage or for monitoring therapeutic response or disease recurrence.

MICRORNAS IN URINE

MicroRNAs have been detected in urine.97,98 Theoretically, these urinary miRNAs may be filtered and excreted by, or directly from, the kidney and/or urinary tract. Melkonyan et al. detected 22 different urinary miRNAs, but none was kidney-specific.97 Analysis of miRNA expression in single urine samples revealed the miRNA ratios miR-126 : miR-152 and miR-182 : miR-152 were significantly elevated in urine of urothelial bladder cancer patients compared with urine of healthy donors and patients with urinary tract infections, enabling a separation of tumour patients from the control groups.98 The ratio miR-126 : miR-152 showed an average 9.9-fold increase in urine samples from patients with bladder cancer in comparison with healthy donors. These studies have revealed a new possibility in the development of non-invasive investigation of kidney diseases by using specific urinary miRNAs as biomarkers for disease diagnosis or progression.

Exosomes have also been detected in urine.99–101 Urinary exosomes are a rich source of intracellular kidney injury biomarkers because they are released from every segment of the nephron, including podocytes.99 Urinary exosomal transcription factors have already been proposed as renal tubular cell biomarkers for acute kidney injury.102 Zhou et al. demonstrated that levels of miR-27b and miR-192 in urinary exosomes could differentiate lupus patients with or without nephritis.103 It is expected that miRNA-containing exosomes in the urine can provide both valuable diagnostic and prognostic information for patients with kidney diseases.

OPPORTUNITIES – NEW THERAPEUTIC STRATEGIES

The evidence implicating miRNAs in the pathophysiology of human diseases has triggered great interest in developing modalities to inhibit miRNAs and their functions. Manipulations of miRNAs can coordinately affect many components of a pathway as opposed to the gene-specific suppression achieved by siRNA targeting. Specific miRNA activity can be inhibited by several methods, which involve antisense strategies and include chemically modified antisense oligonucleotide inhibitors (antagomirs) or the transgenic introduction of tandem miRNA-binding site repeats (known as Decoy or Sponge technologies).23,104,105 One particularly useful form of oligo inhibitor is the antisense locked nucleic acid-modified oligonucleotide, which shows enhanced therapeutic potential.106,107 This strategy has been successfully used in vivo to inhibit hepatic miR-122 activity and thereby reduce serum cholesterol levels in primates,106 as well as reduce Hepatitis C viral load.108

As described above, several miRNAs such as miR-192 and miR-377 lead to extracellular matrix accumulation, podocyte dysfunction, albuminuria and EMT in diabetic nephropathy. It is plausible to suggest that silencing such miRNAs with ‘antagomirs’ may represent a potential therapeutic strategy. Conversely, in kidney diseases in which miRNAs are downregulated, restoring miRNA function by the administration of miRNA mimics may have therapeutic potential.

MicroRNAs have also been reported to modulate replication of viral RNA. One example is miR-122, which is abundant in the liver, facilitates replication of hepatitis C viral RNA suggesting that this miRNA is a target for antiviral intervention.22,108 This interesting model raises the possibility of using similar approaches, possibly also exploiting viral miRNAs, to limit the replication of BK virus in renal allograft and cytomegalovirus, EBV viruses in transplant recipients.

There are currently sparse data on the pharmacokinetics of these oligonucleotides obtained from animal studies. Observations so far have suggested that these inhibitors are eliminated mainly through the renal route and as a consequence, it will be essential to learn the effect of human renal impairment on the clearance of these molecules.109,110 Silencing miRNAs with ‘antagomirs’ in kidney disease may take advantage of higher renal concentration after systemic administration compared with other organs or tissues.

FUTURE CHALLENGES

There are several major challenges in exploring the role of miRNAs in kidney diseases. Most importantly many fundamental questions remain regarding miRNA biology. The mechanism of regulation of miRNA production is not completely clear. While many miRNAs are located within introns of host genes, their expression does not always correlate perfectly with that of host genes suggesting further, post-transcriptional, regulation.23,111,112 Examples of such regulation are the influence of Lin28 proteins on Let-7 production and p53 on the processing of several miRNAs.113,114 Initially, miRNAs were thought to suppress translational inhibition by interfering with the binding of essential translational initiation factors.115 However, other translational repression mechanisms and translational activation and transcriptional effects have been reported.11,115–118 Specific targets for most miRNAs remain unclear. Bioinformatic analyses have predicted many thousands of miRNA-target pairs but only a small proportion of these has been validated experimentally (Table 1). Furthermore, the use of miRNAs as therapeutic agents is attractive but faces considerable challenges, including development of safe and reliable organ and cell-specific delivery systems, avoidance of toxicity derived from off-target effects and from activation of the innate and adaptive immune response. Given these challenges, the most immediate clinical benefits are likely to emerge from identification of miRNAs that can be used as reliable biomarkers for diagnosis, prognosis and response to therapy, in both kidney and allograft disease.

Table 1.  Validated miRNA targets in kidney diseases
miRNAExperiment modelValidated targetsRelation to kidney diseaseReference
miR-15aRatCdc25AProgression of cell cycle leading to growth of cysts51
miR-17HEK 293T cell linePKD2Cell proliferation, cystogenesis44
miR-17-92 cluster PTENEnlarged kidney glomeruli, mesangial expansion, hypercellularity, proteinuria37
miR-21Mesangial cell & db/db micePTENMesangial cells proliferation38
miR-30 familyMouseCTGFHigh expression in kidney glomeruli and loss in podocyte-specific Dicer knockout29
miR-192Mouse & humanZEB1 and ZEB2Enhanced expression in diabetic mouse mesangial cell by TGF-β; enhanced expression by high glucose in human mesangial cells34
miR-216a, miR-217 clusterMousePTENEnhanced expression in diabetic mouse mesangial cells by TGF-β37
miR-377HumanPAK1 and MnSODEnhanced expression by high glucose in human mesangial cells39

Ancillary