Therapeutic potential of microRNAs for the treatment of renal fibrosis and CKD

Abstract

Chronic kidney disease (CKD), defined as reduced glomerular filtration rate, is increasingly becoming a major public health issue. At the histological level, renal fibrosis is the final common pathway leading to end-stage renal disease, irrespective of the initial injury. According to this view, antifibrotic agents should slow or halt the progression of CKD. However, due to multiple overlapping pathways stimulating fibrosis, it has been difficult to develop antifibrotic drugs that delay or reverse the progression of CKD. MicroRNAs (miRNAs) are small noncoding RNA molecules, 18–22 nucleotides in length, that control many developmental and cellular processes as posttranscriptional regulators of gene expression. Emerging evidence suggests that miRNAs targeted against genes involved in renal fibrosis might be potential candidates for the development of antifibrotic therapies for CKD. This review will discuss some of the miRNAs, such as Let-7, miR-21,-29, -192, -200,-324, -132, -212, -30, -126, -433, -214, and -199a, that are implicated in renal fibrosis and the potential to exploit these molecular targets for the treatment of CKD.

INTRODUCTION

Chronic kidney disease (CKD), defined as reduced glomerular filtration rate, is a major public health issue (45, 84, 99). The economic burden of CKD is substantial, and the Medicaid costs for the treatment of CKD in the US now exceeds $42 billion/year (85, 118). Over the last few years, multipronged interventions have been applied to slow the progression of CKD. These include intensive glycemic control, strict control of blood pressure, correction of dyslipidemia, discontinuation of potentially nephrotoxic drugs and smoking, and blockade of the renin-angiotensin system to lower glomerular capillary pressure (47, 123, 127, 130). However, despite the use of multimodal therapeutic interventions, none of the current strategy reverses the progression to end-stage renal disease. So the need for a more efficacious approach for the treatment of CKD is as pressing as ever (3, 29, 37, 66, 101, 128, 130). At the histological level, renal fibrosis is the final common outcome of CKD, irrespective of the initial injury (57, 58, 74, 129). As discussed here, based on the strong preclinical and clinical data, the use of antifibrotic agents to slow or halt the progression of CKD is a reasonable supposition, although definitive evidence based on clinical trials is needed.

MicroRNAs (miRNAs) are small noncoding RNA molecules, 18–22 nucleotides in length, that constitute a large family of posttranscriptional regulators of gene expression controlling developmental and cellular processes in eukaryotic organisms (61). Analyses of miRNA expression profiles have identified a panel of miRNAs, including microRNA (miR)-215, miR-146a, and miR-886, along with other miRNAs (miR-192, miR-194, miR-21, miR-200a, miR-204, and let-7a–g) whose expression is enriched in the kidney relative to other organs (110). By comparing miRNA levels of normal vs. fibrotic kidneys, Gomez et al. (43) identified 21 miRNAs that were upregulated and three that were downregulated. The signature of upregulated miRNAs included several associated with cell survival in various cancers, including miR-15, -21, -200, and -451. In a comparison of results from human CKD with animal models of kidney injury with fibrosis due to unilateral ureteric obstruction (UUO) or ischemic injury, 24 miRNAs were found to be upregulated in common. These include: let-7i, miR-15b, -21, -25, -132, -199, and -214, suggesting that these miRNAs may play a role in regulating the disease process. Taken together, accumulating evidence have identified a distinct set of miRNAs involved in the process of renal fibrosis that might be a potential target for antifibrotic therapy for CKD. These genes and their targets are summarized in Fig. 1. Below we will discuss the results of recent studies relating specific miRNAs relative to molecular targets participating in renal fibrosis (Table 1). For conciseness, we use the prefix “miR” to designate all microRNAs discussed in this review. Current recommendations for nomenclature call for the use of the prefix “mir”, all lower case, to refer to precursor miRNAs and the mature microRNA to be labeled miR-#, e.g., miR-199a-5p and miR-199a-3p. Unfortunately, many of the published reports do not provide sufficient information to determine which particular strand of mature miRNA was assessed. However, based on the targeted genes reported in the studies that are discussed in our review, we have provided a list of validated mature miRNAs relevant to renal fibrosis (Table 2). In addition, the role of a specific miRNA in fibrosis can be direct, for example by downregulating collagen gene expression. In other cases, the link between a miRNA and renal fibrosis is associative and may involve changes in the expression of profibrotic cytokines or cell death (leading to reparative fibrosis). The reader is encouraged to approach the cited references from that critical perspective.

Fig. 1.

Renal pro- and antifibrotic micro (mi)RNAs for tubular epithelial cells (TECs), mesangial cells (MCs), and podocytes.

Table 1.

Summary of miRNAs involved the process of renal fibrosis and their potential targets

MiRNAs	Potential Targets (genes, gene classes, and signaling networks)
Antifibrotic
Let-7 family: Let-7a, Let-c, Let-7f, Let-7b, Let-7d, Let-7e, Let-7g, Let-7i, and miR-98	TGF-β cell signaling pathway (COL1A2 and Col4a1), EndMT program, fibronectin, FN1, N-cadherin, THBS1, Thbs1, ZEB2, Zeb2, SMAD2, and JAG1
MiR-29 family: miR-29a, -29b, and -29c	ECM genes (Col1a2, LAMC1, LAMC2, FBN1, Fbn1, Eln, and Itgb1), P53, Spry1, and Rho kinase, TGFB1, Tgfb1
MiR-126	EGFL7, VEGFA, DLK1, IRS1, Irs1, SPRED1, Spred1, HOXA11, CXCL12, PI3K/Akt pathway, AKT2
MiR-30 family: miR-30a, -30b, -30c, -30d, -30e	Notch1 and p53 pathways, Ucp2, CTGF, Ctgf, NOTCH1, ZEB2, TP53, Tp53
MiR-146a	IRAK1, Irak1, TLR4; TGF-β1/Smad pathway, TNF receptor-associated factor 6-nuclear factor kappa B pathway, SMAD2, Smad4, TRAF6, Traf6
MiR-34c	Notch/Jag1 pathway, NOTCH1/4
Profibrotic
MiR-21	Fatty acid and lipid oxidation metabolic pathways (PPARA, Mpv17l), ERK/MAPK, MMPs and TIMPs (MMP9, Mmp9, TIMP1, MMP2), PTEN/Akt pathway, PTEN, Pten, AKT2, TGF-β cell signaling pathway (SMAD7, Smad7, Smad3), Wnt-pathway (DDAH1, ADMA), TGFB1
MiR-214 and -199	PTEN; AKT1
MiR-433	TGF-β1/Smad3, AZIN1, JNK1 (MAPK8), ERK/MAPK
MiR-132 and -212 family: miR132, miR132*, miR-212, and miR-212*	TGF-β, STAT3/ERK pathways, MAPK1, cell proliferation (Foxo3/p300); Ep300, Mmp9
MiR-324-3p	Prep, Smad7, Zeb2
MiR-382	Klk5
MiR-216a	Col1a2, Tsc22, Ybx1
Conflicting Findings
MiR-192 family: miR-192, miR-194–2, and miR-215	TGF-β1, miRNA-200b/c, Col1a2 and Col4a1, Zeb1, Zeb2, ZEB2, E-cadherin
MiR-200 family: miR-200a, −200b, -200c, -141, and -429	TGF-β1, Zeb1, ZEB1, Zeb2, ZEB2, TGFB2, Tgfb2, Akt pathway

Shown is a summary of the potential gene targets and signaling pathways of micro (mi)RNAs that are implicated in renal fibrosis and are discussed in this review. Validated genes (miRTarBase (23), http://mirtarbase.mbc.nctu.edu.twphp/index.php, accessed August 5, 2017) are boldfaced and italicized; human genes are in all capital letters; while the mouse/rat genes have initial capital letters.

Table 2.

Validated mature miRNAs relevant to renal fibrosis

Family	Target	MiRNA
Antifibrotic
Let-7	COL1A2	hsa-let-7g-5p
	FN1	hsa-let-7g-5p
	JAG1	hsa-let-7b-5p, hsa-miR-98-5p
	SMAD2	hsa-let-7g-5p
	THBS1	hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-let-7e-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-98-5p
	Thbs1	mmu-let-7b-5p
	Zeb2	mmu-let-7b-5p
	ZEB2	hsa-let-7d-5p
MiR-29	Spry1	mmu-miR-29c-3p
	TGFB1	hsa-miR-29b-3p
	Tgfb1	mmu-miR-29b-2-5p
	Col1a2	mmu-miR-29a-3p, mmu-miR-29b-3p
	LAMC1	hsa-miR-29c-3p
	LAMC2	hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p
	Fbn1	mmu-miR-29a-3p, mmu-miR-29b-3p
	FBN1	hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p
	Eln	mmu-miR-29a-3p, mmu-miR-29b-3p,
	Itgb1	rno-miR-29b-3p
MiR-126	EGFL7	hsa-miR-126-3p
	VEGFA	hsa-miR-126-3p
	IRS1	hsa-miR-126-3p
	Irs1	mmu-miR-126a-3p
	SPRED1	hsa-miR-126-3p
	Spred1	mmu-miR-126a-3p
	CXCL12	hsa-miR-126-5p, hsa-miR-126-3p
	AKT2	hsa-miR-126-3p
MiR-30	TP53	hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-miR-30d-5p, hsa-miR-30e-5p
	Tp53	rno-miR-30a-5p, rno-miR-30b-5p, rno-miR-30c-5p, rno-miR-30d-5p, rno-miR-30e-5p
	NOTCH1	hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-miR-30d-5p, hsa-miR-30e-5p
	Ucp2	mmu-miR-30e-5p
	Ctgf	rno-miR-30c-5p
	ZEB2	hsa-miR-30c-5p, hsa-miR-30e-5p
MiR-146a	TRAF6	hsa-miR-146a-5p
	Traf6	mmu-miR-146a-5p, rno-miR-146a-5p
	IRAK1	hsa-miR-146a-5p
	Irak1	mmu-miR-146a-5p, rno-miR-146a-5p
	SMAD2	hsa-miR-146a-5p
	Smad4	rno-miR-146a-5p
	TLR4	hsa-miR-146a-5p, hsa-miR-146b-5p
MiR-34c	NOTCH1	hsa-miR-34c-5p
	NOTCH4	hsa-miR-34c-5p
Profibrotic
MiR-21	PPARA	hsa-miR-21-5p
	MMP9	hsa-miR-21-5p
	Mmp9	mmu-miR-21a-5p
	MMP2	hsa-miR-21-5p
	PTEN	hsa-miR-21-5p
	Pten	mmu-miR-21a-5p, rno-miR-21-5p
	AKT2	hsa-miR-21-5p
	SMAD7	hsa-miR-21-5p
	Smad7	mmu-miR-21a-5p
	DDAH1	hsa-miR-21-5p
	TGFB1	hsa-miR-21-5p
MiR-214 and -199	PTEN	hsa-miR-214-3p
	AKT1	hsa-miR-199a-3p
MiR-433	AZIN1	hsa-miR-433-3p
	MAPK8	hsa-miR-433-3p
MiR-132 and -212	MAPK1	hsa-miR-132-3p
	Foxo3	rno-miR-132-3p, rno-miR-212-3p, mmu-miR-212-3p, mmu-miR-132-3p
	Ep300	mmu-miR-132-3p
	Mmp9	rno-miR-132-3p, mmu-miR-212-3p, mmu-miR-132-3p
MiR-324	Zeb2	mmu-miR-324-3p
MiR-382	Klk5	mmu-miR-382-5p

Source: from miRTarBase (http://mirtarbase.mbc.nctu.edu.twphp/index.php).

ANTIFIBROTIC MiRNAs IN CKD

MiRNA Let-7 family.

Lethal-7 (let-7), one of the first discovered miRNAs, has been implicated in cancer and pluripotency (41, 109). Let-7 and its family members are highly conserved across species and play a significant role in the regulation of cell reprogramming (109). In mice, 12 genes encode members of the let-7 family, which includes nine slightly different miRNAs: let-7a, let-c, let-7f (all encoded by two genes), and let-7b, let-7d, let-7e, let-7g, let-7i, and miR-98 (all encoded by one gene) (41). All let-7 family members are believed to have a similar function because they share a common seed region (nucleotides 2–8), which mediates interaction with the target mRNAs.

Recently, let-7 family members have been suggested to act as negative regulators of profibrotic processes in several disease states, including renal fibrosis (88, 91). Knockdown of let-7b in rat proximal tubular epithelial cells (pTECs) elevated transforming growth factor (TGF)-β1 receptor 1 (TGFBR1) expression and mimicked some of the profibrotic effects of TGF-β1 (113). Similarly, Park et al. (91) found that let-7 family miRNAs target extracellular matrix (ECM) gene products (Col1a2 and Col4a1) of the TGF-β signaling pathway in mouse mesangial cells (MCs) and that the let-7 family negatively modulates ECM accumulation by regulating multiple players in the pathway starting from receptor down to final gene product. Conversely, the negative regulator of let-7 biogenesis, lin28b, was upregulated by TGF-β. Lin28 is a translational enhancer and transcription factor that forms a trimeric complex with OCT4 on DNA (103). It has been widely investigated as an oncogene and was recently demonstrated to suppress the expression of let-7 miRNAs by binding to a motif in the terminal loop of pre-let-7 and recruiting ZCCHC11/TUT4 uridylyltransferase to promote terminal uridylation (10, 103, 107). Of note, in glomeruli of diabetic mice, let-7b levels were decreased, whereas lin28b, Col1a2, and Col4a1 levels were increased. These results suggest that restoring let-7 levels may play a protective role as a natural suppressor of the TGF-β pathway in renal cells during diabetic nephropathy (DN) (91). Park et al. (91) demonstrated that suppression of let-7 miRNAs by lin28 is mediated by TGF-β through the action of Smads. TGF-β-induced enrichment of Smad2/3 showed that the lin28b promoter, which contains a Smad-binding element (SBE), is responsive to TGF-β.

Endothelial-to-mesenchymal transition (EndMT) is an important source of fibroblasts in renal fibrosis. Chen et al. (19) reported that fibroblast growth factor receptor-mediated induction of let-7 miRNA acts as a negative regulator of the EndMT program by inhibiting TGF-β signaling via suppression of TGF-β, TGF-βR1, and Smad2 expression. The endogenous tetrapeptide, N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), has antifibrotic actions in the heart and kidney due to inhibition of cellular proliferation and TGF-β signaling. Nagai et al. (86) demonstrated that the renal antifibrotic and anti-EndMT effects of AcSDKP in Type 1 diabetic CD-1 mice were associated with restoration of let-7 levels. AcSDKP is hydrolyzed almost exclusively by ACE, and administration of an ACE inhibitor plus AcSDKP ameliorated kidney fibrosis and inhibited EndMT compared with therapy with an ACE inhibitor alone. Similar findings were observed in the db/db mouse model of Type 2 DN (88).

Lipoxins are endogenously produced lipid mediators that promote resolution of inflammation and inhibit fibrosis, suggesting a possible role in modulating renal disease. Lipoxins attenuate renal fibrosis in a rat UUO model, and this is associated with increased expression of let-7c miRNA (10). Brennan et al. (10) provided evidence that lipoxins suppress TGF-β1-induced renal fibrosis through a mechanism involving upregulation of let-7c and downregulation of its target TGF-βR1. In cultured human pTECs (HK-2 cells), the lipoxin A4 attenuated TGF-β1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1. Moreover, the potential prognostic value of let-7c dysregulation in renal fibrosis is underscored by their finding that several let-7c target genes were upregulated in human CKD biopsies (10).

MiR-29 family.

MiR-29a, -29b, and -29c have a common seed sequence but are encoded by distinct genomic loci. Overall, these antifibrotic miRNAs have been reported to be downregulated in chronic kidney diseases, such as DN, focal segmental glomerulosclerosis (FSGS), membranous nephropathy, and IgA nephropathy (78, 95). Many matrix genes are targets for miR-29, including fibrillin, collagens, elastin, laminins, and integrin-β1. In fact, miR-29 has the potential to reduce the expression of ~20 genes that impact collagen formation. Thus, miR-29 normally provides tonic suppression of matrix production and turnover. Additionally, miR-29 also slows cell growth and may trigger apoptosis in certain cancer cells potentially by suppressing genes that normally silence the p53 transcription factor. Therefore, miR-29 has the potential to both suppress matrix deposition and drive cell death, which may be beneficial if the cells that undergo apoptosis are profibrogenic cells (16).

Antifibrotic effects have been reported for all of the miR-29 family members, miR-29a, miR-29b, and miR-29c. Streptozotocin-induced diabetic miR-29a transgenic mice showed improved renal function with less glomerular fibrosis and inflammation and better podocyte viability compared with diabetic wild-type (WT) mice (71). Knockdown of miR-29a promoted histone deacetylase activity that led to podocyte apoptosis, proteinuria, and subsequent renal dysfunction (71). Reduced expression of miR-29 along with the development of progressive renal fibrosis was demonstrated in obstructive nephropathy (95). In vitro studies confirmed that overexpression of miR-29b inhibited, and knockdown of miR-29 enhanced, TGF-β1-induced expression of collagens I and III by renal tubular cells. In addition, upregulation of miR-29b was implicated in the protection of SS.13^BN consomic rats from renal medullary fibrosis and tubular necrosis following the development of hypertension, as compared with control Dahl salt-sensitive rats, due to the targeting of multiple collagen and ECM-related genes (75). Downregulation of miR-29c was associated with significant increases in interstitial fibrosis, collagen type II α1 (COL2A1) and tropomyosin 1α protein levels in the remnant kidney of rats subjected to 5/6 nephrectomy and in the kidneys of patients with IgA nephropathy (38).

TGF-β1 counteracts the beneficial role of miR-29 family members by downregulating their expression in pTECs, MCs, and podocytes (32). TGF-β-induced downregulation of miR-29 expression is mediated by activation of Smad3 signaling. Binding of Smad3 to an SBE in the promoter region of miR-29 suppresses its transcription in cultured pTECs and fibroblasts, as well as in vivo, in the kidney of mice following UUO (95). In contrast, Smad3 knockout (KO) mice exhibit increased expression of miR-29 along with reduced renal fibrosis in the UUO mouse model (95). Fang et al. (38) reported that cobalt chloride, which increases hypoxia-inducible factor-alpha (HIF-α) protein levels, increased miR-29c expression in human kidney epithelial HK-2 cells. Upregulation of miR-29c was significantly attenuated by knockdown of HIF-1 or HIF-2α (38). These findings suggest that HIF-1 and HIF-2α also contribute to the regulation of miR-29c expression in the kidney.

Qin et al. (95) utilized an ultrasound-microbubble-mediated gene transfer of miR-29 for the treatment of renal fibrosis in a mouse model of UUO. They demonstrated that delivery of miR-29b either before or after induction of UUO reduced renal fibrosis. However, there are some discordant findings regarding the antifibrotic effect miR-29c. Long et al. (76) reported that miR-29c increased accumulation of ECM and induced podocyte apoptosis in a db/db mouse model of Type 2 diabetes. Knockdown of miR-29c reduced albuminuria and mesangial matrix accumulation. They identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c and provided evidence that downregulation of Spry1 promotes Rho kinase activation, resulting in enhanced cell apoptosis and fibronectin synthesis. Treatment with Rho kinase inhibitors attenuates the development of renal damage in different animal models (112). Although data connecting the activation of Rho/Rho kinase pathway and kidney disease in humans are still lacking, these studies have provided compelling evidence for the renoprotective effects of Rho-kinase inhibitors (39, 112, 117).

MiR-126.

MiR-126 is encoded by intron 7 of the epidermal growth factor like-7 (EGFL7) gene and has proangiogenic actions. Microvesicles derived from endothelial progenitor cells were found to protect the kidney from acute ischemic injury due to the generation of miR-126 and miR-296, which inhibited capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis (12). Bijkerk et al. (8) more recently reported that mice overexpressing miR-126 in the hematopoietic compartment displayed a decrease in fibrotic and injury markers after renal ischemia/reperfusion injury. The beneficial effects of miR-126 were attributed to the preservation of vascular integrity due to an increase in the number of circulating hematopoietic stem and progenitor cells and their mobilization to the kidney. Recently, Al-Kafaji et al. (2) found that decreased expression of circulating miR-126 was associated with the development of DN in Type 2 diabetic patients and suggested it may be a promising biomarker for the progression of DN.

MiR-126 regulates the responsiveness of endothelial cells to VEGF. One of the main targets of miR-126 is EGFL7, which is implicated in cell migration and blood vessel maturation and functions by interacting with the Notch receptor and Notch ligand Delta-like 4 (97). By downregulating EGFL7, miR-126 may coordinate angiogenesis and vessel maturation; however, the precise importance of this downregulation is not known (87). MiR-126 also silences Sprouty-related protein (SPRED1) (which negatively regulates VEGF signaling), DLK1, transcription factors (e.g., IRS1 and HOXA11), as well as a regulator of G protein 16 (RGS16), which is an inhibitor of G protein-coupled receptor (GPCR) signaling (40, 97). During tissue repair, delivery of miR-126 to recipient vascular cells suppresses RGS16 and enables CXCR4, a GPCR, to support an autoregulatory feedback loop that increases production of its ligand CXCL12, which in turn promotes recruitment of progenitor cells and vascular protection (131). MiR-126 may also inhibit vascular endothelial cell apoptosis by enhancing PI3K/Akt signaling by directly targeting the negative regulator PIK3R2 (phosphoinositide-3-kinase regulatory subunit 2) (18). Thus, miRNA-126 might protect the kidney via several signal pathways. Further studies are needed to discern the antifibrotic effect and its potential value for the treatment of renal disease.

MiR-30 family.

The miR-30 family consists of five evolutionarily conserved members, miR-30a through -30e (98). Members of the miR-30 family all share a common seed sequence and are encoded by six genes located on human chromosomes 1, 6, and 8 (11). The miR-30 family had been documented to be downregulated in podocytes of patients with FSGS (80, 122). Treatment of cultured human podocytes with TGF-β or puromycin aminonucleoside leads to cytoskeletal damage and apoptosis, which is ameliorated by upregulation of miR-30 expression and aggravated by knockdown of miR-30. The protective role of miR-30 was attributed to inhibition of Notch1 and p53 pathways (122).

The miR-30 family also inhibits epithelial cell ECM production and phenotype changes by downregulating mitochondrial uncoupling protein 2 (Ucp2) (51). UUO in mice was shown to induce UCP2 in tubular epithelial cells, the ablation or inhibition of which attenuated UUO-induced renal fibrosis. In cultured rat kidney epithelial cells, UCP2 expression was induced by TGF-β1, and its knockdown markedly reduced, while overexpression promoted, TGF-β1-induced epithelial-to-mesenchymal transition (EMT). Ucp2 mRNA was demonstrated to be a direct target of miR-30e, which was downregulated in tubular cells of fibrotic kidneys and TGF-β1-treated kidney epithelial cells. Moreover, tubular cell EMT was inhibited by a miR-30e mimic, whereas a miR-30e inhibitor acted like TGF-β1. In addition, connective tissue growth factor (CTGF), a key molecule in the process of renal fibrosis, is regulated by miR-30. Duisters et al. (34) demonstrated that miRNA-30 expression was inversely associated with the amount of CTGF in two animal models of heart disease, as well as in patients with left ventricular hypertrophy. In vitro, knockdown of miR-30 increased CTGF levels in cardiomyocytes and fibroblasts. Overexpression of miR-30c decreased CTGF levels and reduced the production of collagens. These results suggest that the miR-30 family is an attractive therapeutic target for fibrotic diseases including CKD since it influences the fibrotic process by regulating mitochondrial ATP and CTGF production.

PROFIBROTIC MiRNAs IN CKD

MiR-21.

MiR-21 is one of the most abundant miRNAs in human tissues and is upregulated in several animal models of kidney disease and in kidney tissue obtained from patients with CKD. In addition, the kidneys of miR-21 KO mice showed marked protection against the development of renal fibrosis (13). Microarray studies of kidneys from miR-21 WT and KO mice indicate that expression of the predicted target genes of miR-21 are not significantly different under normal conditions (14). However, after expression of miR-21 is increased by UUO or ischemia/reperfusion injury, differences in expression of the target genes of miR-21 were observed between WT and miR-21 KO mice, with miR-21 KO mice having far less renal interstitial fibrosis following injury.

Analysis of gene expression profiles in miR-21 WT and KO mice in response to injury suggested that miR-21 contributes to the development of renal fibrosis via regulation of metabolic pathways involved in fatty acid and lipid oxidation (14). Peroxisome proliferator-activated receptor (PPAR)-α is one of the most potent transcription factors that regulate fatty acid oxidation and is a validated target of miR-21. In normal kidneys, high expression levels of PPAR-α can be detected in interstitial cells, whereas after injury the expression falls secondarily to miR-21 targeting. Upregulation of PPAR-α expression in transgenic mice subjected to UUO decreased ECM accumulation and reduced renal interstitial fibrosis. Those observations confirm the importance of fatty acid oxidation in the prevention of renal fibrosis and the importance of miR-21 silencing of the PPAR-α axis.

Generation of reactive oxygen species (ROS) contributes to renal fibrosis. Mpv17-like protein is a mitochondrial inner membrane protein that regulates the production of ROS and protects against mitochondrial oxidative stress and apoptosis. Mpv17-like protein is downregulated by miR-21 during kidney injury (14). MiR-21 also upregulates extracellular signal-regulated kinases (ERK) signaling in the kidney (14). Both ERK1/2 and TGF-β/Smad signaling pathways seem to play a role in the development of kidney fibrosis in diabetic models (21).

The role of miR-21 in ECM homeostasis may be mediated through the regulation of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Wang et al. (115) described the association between MMP-9/TIMP1 and miR-21 in DN using the kk-ay diabetic mouse. They confirmed computational predictions that MMP-9 is a potential target for miR-21. With the progression of DN, the expression of miR-21 increased in the kidney of kk-ay diabetic mice. They further observed that miR-21 directly downregulates MMP-9 protein expression and indirectly leads to an increase in TIMP1 mRNA expression and protein (115). Notably, treatment of kk-ay DN mice with antagomir-21 improved renal function and decreased renal TIMP1, ColIV, and FN protein levels. Additionally, global transcriptome analysis revealed miR-21 affects the expression of another target involved in ECM regulation, namely RECK (reversion-inducing-cysteine-rich protein with kazal motifs), which is a membrane-anchored glycoprotein inhibitor of MMP-9 and MMP-2 (90).

The expression of several MMPs appear to be regulated by phosphatase and tensin homolog (PTEN), a well-documented tumor-suppressor gene and negative regulator of Akt signaling that was demonstrated to be downregulated by miR-21 during TGF-β1-induced EMT (67, 116). MiR-21 knockdown in kidneys also leads to a decrease in MMP-2 (due to attenuated Akt signaling), suggesting that PTEN is also targeted by miR-21 in the kidney (14, 20). Indeed, the genes for human and mouse/rat PTEN are validated targets for miR-21 (miRTarBase 2016) (23). MMP-2 is implicated in renal interstitial fibrosis possibly via induction of EMT (33). Moreover, Chen et al. (17) demonstrated that cyclosporine A (CsA)-induced nephrotoxicity was associated with increased miR-21 and a rapid increase in phosphorylated Akt and downregulation of PTEN. Inhibition of miR-21 prevented CsA-induced Akt activation and EMT gene expression (17), as well as activation of profibrotic genes in human podocytes and tubular cells in a model of IgA nephropathy (5).

It is well established that TGF-β is a key cytokine in renal fibrosis. TGF-β signaling is regulated by miR-21. TGF-β1, in turn, induces miR-21 expression through Smad signaling (63). McClelland et al. (81) reported that the extent of fibrosis and the decline in renal function in patients with DN correlated with upregulation of miR-21, which they also observed was upregulated in various animal models of fibrotic renal disease and experimental DN. Using rat proximal tubular epithelial cells, these investigators provided evidence that TGF-β1-induced miR-21 expression augments fibrotic gene expression by TGF-β1, by enhancing TGF-β1-induced Smad3 and Akt signaling due to the downregulation of Smad7 and PTEN, respectively. Recent studies demonstrate that Smad3 stimulates the generation of miR-21. Davis et al. (27) reported that the induction of mature miR-21 and pre-miR-21 was not preceded by an increase in pri-miR-21 in response to TGF-β1 stimulation, indicating that upregulation of miR-21 occurs at the posttranscriptional level. Davis et al. (27) suggested that Smad3 associates with the Drosha/DGCR8/p68 microprocessor complex to facilitate cleavage of pri-miRNA to pre-miRNA. In a later study, TGF-β and bone morphogenetic protein (BMP) signaling was found to regulate a group of miRNAs (including miR-21) posttranscriptionally via direct binding of Smad3 to the stem regions of the target pri-miRNAs, which contain a conserved sequence similar to the SBE present in the promoters of Smad target genes (28). Recruitment of Smad3 was postulated to be required for the efficient recruitment of the microprocessor complex. Smad3 may also bind to an SBE of the promoter for the miR-21 gene to modulate its expression in response to TGF-β stimulation (133).

Recently, Liu et al. (73) found a new target for miR-21. They suggested that miR-21 interacts with the 3′-untranslated region (UTR) of the mRNA for dimethylarginine dimethylaminohydrolase 1 (DDAH1) in the Wnt pathway in human HK-2 cells. DDAH1 breaks down asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS). Thus, upregulation of miR-21 may contribute to renal fibrosis by reducing DDAH1 activity/expression and increasing ADMA levels, thereby reducing production of NO.

Recent studies using anti-miR-21 oligonucleotides have demonstrated their ability to reduce renal injury and fibrosis in various animal models of acute kidney injury, as well as in Alport syndrome (14). Alport nephropathy is caused by a mutation in a capillary basement membrane protein (COL4A5) resulting in cell stress that secondarily triggers leukocyte recruitment and fibrosis. In primary cell cultures from kidneys obtained from a mouse model of Alport nephropathy, anti-miR-21 oligonucleotides protected cells from mitochondrial dysfunction due to cell stress and inhibited activation of fibroblasts by various stimuli. Weekly delivery of anti-miR-21 oligonucleotides to mice after the onset of the disease profoundly slowed disease progression and improved survival, normalized tubular function, and reduced histological end points, including interstitial fibrosis, glomerulosclerosis, tubular injury, and inflammation (42).

Anti-miR-21 oligonucleotides can be given to animals for many weeks without noticeable deleterious effects (42). Therefore, anti-miR-21 oligonucleotides represent a potential new class of therapeutics to treat fibrotic kidney diseases and have now entered phase II clinical trials (14). Although the safety of delivering modified oligonucleotides to the body has yet to be proven, phase III clinical trials are currently underway to treat a number of diseases. In addition, anti-miR-21 oligonucleotides have proven to be safe in long-term studies in primates and more recently in humans (43). On the other hand, it should be noted that ischemic preconditioning of the kidney induced upregulation of the expression of miR-21 that was shown to protect against subsequent renal ischemia/reperfusion injury by targeting proapoptotic genes (125). This study highlights the complex role of miR-21 in the regulation of fibrosis in the kidney.

MiR-214 and miR-199a.

MiR-214 is expressed at high levels in human and animal models of kidney disease (43). MiR-214 is cotranscribed with the miR-199a family on a single lncRNA strand located on the complementary strand of an intron in the dynamin-3 gene. miR-214 and miR-199a genes are upregulated by activation of the TWIST transcription factor and by hypoxia via HIF-1α. In kidney samples obtained from patients with various kidney diseases, miR-214 was detected in renal tubules, glomeruli, and infiltrating immune cells (30). Denby et al. (30) recently suggested that miR-214 functions by promoting renal fibrosis independently of TGF-β signaling. In the mouse UUO model, decreased expression of miR-214 protected against the development of fibrosis, and treatment of WT mice with anti-miR-214 before UUO resulted in similar antifibrotic effects. MiR-214 antagonism did not block Smad2/3 activation, and TGF-β blockade together with miR-214 deletion provided additional renal protection, indicating that miR-214 induces fibrotic effects independent of Smad2/3. Furthermore, the regulation of endogenous miR-214 was unaffected by inhibition of canonical TGF-β signaling (30).

The endogenous Akt signaling pathway inhibitor PTEN is also a recognized target for miR-214 (69). Wang et al. (120) recently reported that inhibition of miR-214 significantly reduced albuminuria and mesangial expansion in db/db mice, and this was associated with reduced expression of α-smooth muscle actin (SMA), SM22, and collagen IV, as well as partially restored PTEN levels. In vitro studies confirmed that PTEN is a target of miR-214 and that inhibition of miR-214 reduced fibrotic gene expression and partly restored PTEN protein levels in high glucose-stimulated human mesangial cells. Upregulation of miR-214 was specifically linked to hypertrophy in this model, as PTEN overexpression attenuated miR-214-mediated hypertrophy of the mesangial cells exposed to high glucose, whereas PTEN knockdown mimicked hypertrophy of these cells.

As mentioned, the miR-199a family is cotranscribed with miR-214. MiR-199a-5p and miR-199a-3p are produced from a single molecule, pre-miR-199p (15). Several studies in nonrenal tissues have pointed to discrete roles for these miRNAs in modulating cell migration and matrix deposition. However, the significance of the miR-199a family in kidney disease has not been established, nor has the potential differential actions of miR-199a-3p and miR-199a-5p (15). Increased renal expression of miR-199a-5p was reported in the UUO mouse model of kidney fibrosis, and miR-199a-5p was shown to be more effective than miR-199a-3p in reducing caveolin-1 levels in lung fibroblasts, a critical process in the activation of fibroblasts by TGF-β (72). Others have reported that the membrane protein Klotho is a target of miR-199a-5p in rat mesangial cells exposed to high glucose media (121). Suppression of Klotho expression by miR-199a-5p was linked to increased expression of inflammatory and profibrotic genes secondary to enhanced Toll-like receptor 4 (TLR4)/NF-κB p65/NGAL signaling. Given its positive association with inflammation and fibrosis, miR-199a-5p may also represent another therapeutic target for the treatment of renal fibrosis associated with diabetes.

MiR-433.

Early studies of miR-433 focused on its role in cancer (25). Recently, miR-433 was reported to be upregulated in renal and cardiac fibrosis (70, 106). Human miR-433, located at chromosomal region 14q32.2, belongs to the DLK1-DIO3 miRNA cluster and functions as a tumor suppressor in several human cancers. Epigenetic programming has an important role in the regulation of miR-433 (126). CpG islands surrounding the miR-433 gene are hypermethylated, and treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) induces miR-433 expression.

MiR-433 was upregulated in the UUO mouse model, while knockdown of miR-433 attenuated the induction and progression of renal fibrosis in this model (70). In vitro, overexpression of miR-433 in rat tubular epithelial cells enhanced TGF-β1-induced fibrosis, while its knockdown attenuated fibrosis. Smad3 (but not Smad2) was shown to bind the miR-433 promoter to induce its expression. Thus, miR-433 is an important component of TGF-β/Smad3-induced renal fibrosis through induction of a positive feedback loop that amplifies TGF-β/Smad3 signaling. Antizyme inhibitor 1 (AZIN1), a regulator of polyamine synthesis, was identified as a target gene of miR-433 linked to TGF-β signaling in renal fibrosis (70). Overexpression of miR-433 suppressed AZIN1 expression, while AZIN1 upregulation suppressed TGF-β signaling and fibrosis. As an important component of TGF-β/Smad3-induced renal fibrosis, miR-433 may be a potential therapeutic target in renal fibrosis disease.

MiR-132 and miR-212 family.

MiR-132 and miR-212 are closely clustered in the genome and are transcribed together under the regulation by the cAMP response element binding protein (CREB), the expression of which is regulated by angiotensin II (ANG II) (35). The miR212/132 gene has an equivalent structure in all species; the two miRNAs genes are organized in tandem on chromosome 10 in the rat, 11 in mouse, and 17 in humans (96). The miR212/132 gene cluster produces four mature miRNAs: miR132, miR132*, miR212, and miR212*. MiRNA and miRNA* sequences represent the two strands of the duplex-derived from the precursor miRNA. Although they are produced in equal amounts by transcription, their accumulation differs and depends on differences in the thermodynamic stability of the 5′-end of each strand (96).

Recently, miR-132 and miR-212 were reported to be increased in the heart, aorta, and kidney of ANG II-induced hypertensive rats and decreased in human arteries obtained from patients undergoing bypass surgery, in response to ANG II type 1 receptor (AT1) blockade (35). In renal fibrosis, most of the α-SMA-positive myofibroblasts arise from perivascular cells. Bijkerk and colleagues (7) found that miR-132 increased 21-fold during pericyte-to-myofibroblast transformation in mice subjected to UUO and that miR-132 silencing decreased collagen deposition and tubular apoptosis. Further study identified a rate-limiting role for miR-132 in myofibroblast proliferation. This role of miR-132 in regulating cell proliferation was selective for the interstitial compartment. MiR-132 was found to coordinately regulate the expression of several genes involved in STAT3/ERK pathways, TGF-β signaling, and cell proliferation (Foxo3/p300) (7). In light of evidence that MMP-9 is a target of miR-132 (111), the role of MMP-9 in mediating the effect of miR-132 on renal fibrosis warrants additional consideration. Overall, the available data support miR-132 as a potential target for antifibrotic therapy in CKD (7).

MiR-324-3p.

Macconi and colleagues (79) analyzed the miRNA expression profile of glomeruli from the Munich Wistar Frömter (MWF) rat, which spontaneously develops progressive nephropathy. They found that miR-324-3p was the most upregulated miRNA. Given the remarkable antifibrotic effects of ACE inhibitors, they focused on potential targets of miR-324-3p involved in the metabolism of pro- or antifibrogenic peptides, including ANG II. Bioinformatic analysis indicated that prolyl endopeptidase (Prep) was a miR-324-3p target involved in the formation of ANG (1–7), which has renoprotective actions, and the antifibrotic peptide AcSDKP. Transient transfection of cultured tubular cells with a miR-324-3p mimic reduced Prep protein and activity. Overexpression of miR-324-3p in MWF rats reduced expression of Prep in glomeruli and tubules and the excretion of AcSDKP and increased deposition of collagen. Conversely, treatment with an ACE inhibitor downregulated glomerular and tubular miR-324-p, promoted renal Prep expression, increased plasma and urine AcSDKP levels, and attenuated renal fibrosis. These results suggest that the renoprotective effects of ACE inhibitors partially result from modulation of the miR-324-3p pathway, suggesting that it may be a potential therapeutic target for renal fibrosis (79). Recently, Xu and colleagues (124) found Smad7 is a direct target of miR-324-3p. Considering the important role of Smad7 as a negative regulator of TGF-β1/Smad3 signaling in renal fibrosis, the role of miR-324-3p in opposing Smad7 should be further investigated (119).

MiRNAs WITH CONFLICTING RESULTS

MiR-192 family.

The gene for miR-192, which is highly expressed in the normal kidney, is located on chromosome 11q13.1, 109 bp upstream of the region encoding miR-194-2 (22, 104). It is likely that miR-192 and miR-194-2 are regulated as a common transcriptional unit (104). Several studies from rodent models of kidney diseases and cell lines demonstrate a profibrotic role of miR-192 in both renal tubular and mesangial cells (24, 31, 56, 94). In this regard, Sun and collaborators (102) reported that administration of the anticancer drug Taxol interferes with cell proliferation by altering the cytoskeleton ameliorates fibrosis in the remnant kidney model by lowering miR-192 levels.

For the most part, a functional link between increased miR-192 levels and TGF-β1-driven renal fibrosis has been well documented (24, 56, 62). Like miR-21, miR-192 promotes renal fibrosis by amplifying TGF-β signaling. Glomeruli from diabetic mice show increased miR-192 expression and either TGF-β (via Smad3) or high glucose-induced miR-192 expression in TECs (24, 56). In MCs, miR-192 mediates TGF-β-induced collagen expression by downregulating zinc finger E-box binding homeobox 1/2 (Zeb1/2) expression (56). In TECs, miR-192 overexpression enhances, and its inhibition opposes, TGF-β1-induced collagen expression (24). Kato et al. (53) provided evidence that an miRNA circuit involving miR-129 mediates regulation of TGF-β1 levels in renal MCs. TGF-β1 levels were upregulated by miR-192 or miR-200b/c. Levels of miR-200b/c were also upregulated by miR-192, suggesting that miR-200b/c is a downstream target of miR-192. The activity of the TGF-β1 promoter was upregulated by TGF-β1 or miR-192, demonstrating that the miR-192/miR-200 cascade induces TGF-β1 expression. TGF-β1 increased occupancy of the activators, upstream stimulatory factors and Tfe3, and decreased that of the repressor Zeb1 on the TGF-β1 promoter E-box binding sites. Moreover, inhibitors of miR-192 decreased the expression of miR-200b/c, Col1a2, Col4a1, and TGF-β1 in mouse MCs and mouse renal cortex. Thus, miRNA-regulated circuits may amplify TGF-β1 signaling, thereby accelerating the progression of CKD (53). There is also a TGF-β-induced feedback amplification of the circuit between p53 and miR-192 in mouse MCs, via the miR-192 target Zeb2, which was implicated in the pathogenesis of DN. MiR-192-mediated reduction in Zeb2 enhanced p53 levels, which in turn drove miR-192 expression.

In contrast to these reports, Krupa et al. (62) reported that loss of miR-192 promotes fibrogenesis in DN. However, the stage of the disease may have an impact on the role of miR-192. For example, Kato and colleagues (56) observed upregulation of the renal expression of miR-192 and collagen α-2(I) in Type 1 and 2 in the early stage of mouse models of diabetes. Krupa and colleagues (62) found decreased miR-192 expression in advanced-stage human DN renal biopsy samples accompanied by tubulointerstitial fibrosis. Jenkins et al. (50) reported that TGF-β1 downregulates miR-192 and miR-194, which are cotranscribed in the shared precursor pri-miR-192/194. They identified binding sites for hepatocyte nuclear factor (HNF) and p53 within the miR-194-2/192 promoter region required for constitutive activity, which was decreased by TGF-β1 through an Alk5-dependent mechanism. TGF-β1 treatment decreased binding of HNF to the promoter, whereas knockdown of HNF-1 inhibited mature miR-192 and miR-194 expression (50). In cultured human proximal tubular cells, Krupa and colleagues (62) also reported downregulation of miR-192 expression by TGF-β1, while increased expression of this transcript repressed Zeb1 and Zeb2 expression, thereby de-repressing E-cadherin and exerting an antifibrotic effect.

Conflicting findings regarding the role of miR-194-2/192 in renal fibrosis may be attributed to intrinsic differences in the animal models used, the cell types, the disease stage studied, and/or experimental conditions used for cultured cells. One study described a biphasic induction of miR-192 by TGF-β1 in murine MCs that initially involves the Smad pathway followed by a mechanism that sustains miRNA expression by relaxing the chromatin structure of the miR-192 gene through Ets1 and histone H3 acetylation by Akt-activated p300 (54). This mechanism may contribute to DN since activation of Akt and p300 and acetylation of Ets-1 and histone H3 were found to be increased in glomeruli from diabetic db/db mice. In the kidney, HNF expression is restricted to the tubular compartment, whereas it is absent in MCs and podocytes, thus partly explaining the cell-specific regulation of miR-192 (56). Further investigation on the potential role of miR-192 and the mechanisms that regulate miR-192 expression during renal fibrosis in different species is necessary (26, 49).

MiR-215, which is located at chromosome 1q41, has the same 8-mer seed sequence as miR-192. It only differs by two nucleotides and thus is another member of the miR-192 family (52, 104). Recently, Lan et al. (64) reported that exogenous miR-215 decreased fibroblast cell proliferation, without significant apoptosis compared with controls. They concluded that miR-215 has a role in inhibiting fibroblast proliferation in the ocular surface conjunctiva. This suggests that miR-215 may have benefit in reducing renal fibrosis as well.

MiR-200 family.

The miR-200 family has been ascribed a role in maintaining epithelial cell differentiation, suggesting it might have antifibrotic actions in DN (114). This family consists of five members (miR-200a, -200b, -200c, -141, and -429), encoded by two separate genomic loci on chromosome 1 (miR-200b∼200a∼429 and miR-200c∼141) (9). Members of this family have very similar seed sequences that recognize the 3′-UTR sequence in the target transcript of the regulated genes. On the basis of their seed sequence, the family members can be classified into two functional groups. Group 1 comprises miR-200b, miR-200c, and miR-429, and group 2 comprises miR-200a and miR-141. Because of the differences in seed sequence, the two groups have different mRNA targets (93).

The mechanism for the antifibrotic effects of miR-200 may involve prevention of EMT in pTECs, which is a common pathway in renal fibrosis (93). Tang et al. (105) demonstrated that miR-200b suppresses TGF-β1-induced EMT in HK-2 cells via increased E-cadherin and reduced fibronectin mRNA and protein expression. The former was due to decreased expression of Zeb1 and Zeb2, while the latter resulted from the direct targeting of the fibronectin mRNA. Suppression of TGF-β1-induced EMT occurred independently of TGF-β1-induced phospho-Smad2/3 and phospho-p38 and p42/44 signaling. In vivo, expression of Zeb-1 and Zeb-2 increased after UUO, and administration of the miR-200b precursor blocked this effect. Despite elevated miR-200 levels following induction of UUO, delivery of miR-200b-precursor ameliorated fibrosis in these mice (89). MiR-200s are also are predicted to repress the expression of TGF-β2, which may inhibit EMT and fibrosis (6). Gregory et al. (44) also reported that members of the miR-200 family and miR-205 are downregulated during TGF-β1-mediated EMT in MDCK renal epithelial cells, and forced expression of the miR-200 family alone was sufficient to prevent EMT.

In contrast, Kato et al. (53) found that the amount of miR-200b/c increased in mouse MCs treated with TGF-β1 in vitro and in glomeruli obtained from diabetic mice. Increased miR-200b/c was linked to increased collagen expression via downregulation of Zeb1/2. Further study demonstrated that TGF-β1 levels were upregulated by miR-200b/c, creating an autoregulatory loop to amplify TGF-β1 signaling in chronic renal fibrotic diseases (53). Additionally, previous reports indicated that Smad3 directly binds to a SBE in the promoter region of miR-200b/a and functions as a transcriptional activator in a TGF-β-independent manner (1, 83). In TGF-β1-treated MCs and in glomeruli of diabetic db/db mice, miR-200b and miR-200c repressed the PI3K inhibitor FOG2, thereby enhancing PI3K/Akt signaling (92). Thus, TGF-β1-induced expression of miR-192 activates the miR-200 family (along with miR-216a and miR-217), leading to Akt activation, hypertrophy, and fibrosis in MCs.

OTHER MiRNAs AND RENAL FIBROSIS

MiR-146a was reported to inhibit inflammation by suppressing NF-κB signaling by targeting mRNAs such as tumor necrosis factor receptor-associated factor 6 (TRAF6), interleukin-1 receptor-associated kinase 1 (IRAK1), and TLR4 (68). Overexpression of miR-146a inhibited renal fibrosis, α-SMA expression, and macrophage infiltration in a UUO mouse model (82). In addition, miR-146a inhibited TGF-β1–Smad and TNF receptor-associated factor 6–NF-κB signaling pathways (82, 132). These results suggest that miR-146a attenuates renal fibrosis by inhibiting profibrotic and inflammatory signaling pathways and might be another therapeutic target for the treatment of renal fibrosis (82). On the other hand, increased miR-146a expression was observed in kidney samples obtained from patients with DN (46). Moreover, upregulation of miR-146a in the kidney of streptozotocin-induced diabetic rats was not accompanied by downregulation of inflammatory mediators, suggesting a possible defect in the miR-146a-mediated inhibitory loop in diabetes that allows for sustained activation of NF-κB (4).

Recently, Morizane et al. (83) demonstrated that overexpression of miR-34c decreased renal fibrosis and expression of α-SMA, CTGF, collagen types I and III, and fibronectin in mice with UUO. Additionally, they found that miR-34c overexpression attenuates EMT induced by TGF-β in a mouse tubular cell line. They speculated that miR-34c ameliorated renal fibrosis through suppression of the Notch/Jag1 pathway, although a direct relationship between miR-34c and Jag1 was not demonstrated (83). MiR-34c might be a novel target for the treatment of renal fibrosis as well.

In mouse MCs, TGF-β1 increased expression of Col1a2 associated with the upregulation of miR-216a (55). This effect was mediated by posttranscriptional upregulation of Tsc22 due to the alleviation of the inhibitory actions of the RNA-binding protein Ybx1 in P-bodies. Ybx1 was found to be a specific target of miR-216a. Together with the concurrent removal of the inhibitory actions of Zeb1 on the Col1a2 promoter, the interaction of Tsc22 with transcription factor E3 (Tfe3) increases Col1a2 expression in diabetes. In accordance with this model, an increase in Tsc22 was observed in the glomeruli of diabetic db/db mice (55).

UUO-induced renal interstitial fibrosis in mice has been shown to be associated with the upregulation of miR-382, which targets kallikrein 5, an enzyme involved in the degradation of several ECM proteins (60). Intravenous delivery of an anti-miR-382 prevented the decrease of kallikrein 5 protein expression and attenuated inner medullary fibrosis. Moreover, the protective effects of anti-miR-382 treatment were eliminated by knockdown of renal kallikrein 5. Previous work provided evidence that miR-382 is upregulated by TGF-β1 in human renal epithelial cells and contributes to EMT (59). This action of miR-382 was linked to downregulation of superoxide dismutase 2 (SOD2). Thus, more than one process underlies the profibrotic actions of miR-382 in the kidney.

POTENTIAL OF NOVEL ANTIFIBROTIC STRATEGIES USING MiRNAs

The growing knowledge of the role of miRNAs in the regulation of profibrotic pathways in various renal diseases has driven interest in developing agents that block pathogenic miRNAs or restore levels of renoprotective miRNAs. Nearly all aspects of TGF-β signaling that are implicated in renal fibrosis are regulated by miRNAs as presented in Fig. 2. Growing evidence illustrates that miRNAs are both downstream effectors of TGF-β-dependent renal fibrosis and upstream regulators of TGF-β-dependent signaling (32). Feedback loops between TGF-β and miRNAs have heightened the focus on the use of miRNA-based therapies for the treatment of renal disease.

Fig. 2.

Key pro- and antifibrotic miRNAs implicated in transforming growth factor (TGF)-β signaling in the kidney. TGF-β is the central player in the initiation and progression of renal fibrosis by driving endothelial-to-mesangial transition (EndMT) and epithelial-to-mesenchymal transition (EMT), as well as extracellular matrix (ECM) remodeling and protein accumulation. Both canonical (Smad-dependent) and noncanonical (MAPKs and AKT) signaling pathways are involved and targeted by various miRNAs (boldface) that broadly favor or oppose fibrosis; however, given their broad range of gene targets, it is unlikely that a particular miRNA is strictly anti- or profibrotic. Plus, the same gene target may have conflicting actions. Although Zeb is implicated in the EMT program via in part the repression of E-cadherin, Zeb is also involved in repressing TGF-β expression. Conflicting actions are also reported for the ERK signaling cascade, which is involved in EndMT/EMT but also inhibits receptor Smads. In the text box, the human gene is shown where validated in human or in both human and rodent. Antifibrotic miRNAs are colored red; profibrotic miRNAs are colored green. See the text for additional details.

Due to their distinct features, such as short sequence and homology across species, mature miRNAs are attractive candidates as potential therapeutic agents. In vivo manipulation of the activity of specific miRNAs in the kidney has been achieved by the delivery of inhibitors to block miRNA function or mimics to restore miRNA levels. Many miRNAs are activated by stress and appear to be inactive in healthy cells, which provides a therapeutic advantage for developing safe therapies that target miRNAs. RNA oligonucleotides distribute widely throughout the body and access intracellular compartments. However, anti-miRNA oligonucleotides accumulate in the kidney and liver following systemic delivery, making them ideal for treating renal fibrotic disease. Interestingly, healthy glomeruli do not take up anti-miRNA oligonucleotides, but podocytes and other cell types in injured glomeruli do, indicating that antioligonucleotide therapy may benefit glomerular as well as tubulointerstitial diseases of the kidney (77).

Many obstacles, including safety issues and delivery methods, must be overcome before miRNA-based therapies for CKD can be translated into clinical practice. Ideally, the target miRNA should be kidney specific to avoid adverse effects in other tissues and organs. To minimize side effects, the treatment should affect only one cellular target (or targets acting in the same pathway). Recent studies have identified the targets of particular miRNAs in cells and tissues by a variety of techniques, such as precipitating RNA-induced silencing complexes (RISCs) that incorporate a specific miRNA have shown that one miRNA frequently has >100 targets (43). Commonly, many of these other targets are modestly silenced by the miRNA, although many target genes are functionally related.

Parallel with the discovery of RNA-based gene silencing was the development of anti-miRNA oligonucleotides capable of entering cells after systemic administration to suppress translation. Similar efforts have been made to generate miRNA mimics, which are double-stranded RNA molecules that separate intracellularly, with one strand loading into the RISC and functioning as a miRNA. Though miRNAs are generally stable with a long half-life, individual miRNAs can undergo rapid decay in specific cellular contexts (110). Several advances have been made to enhance RNA stability in vivo by molecular modification of the backbone (43). These modifications include replacing the phosphodiester backbone with a phosphorothioate backbone and the use of 2′-O-methyl RNA molecules and locked bicyclic nucleic acids. Also, 2′-O-methoxyethyl (MOE) and 2-oxy-methyl (OMe) modifications enhance the affinity for complementary nucleotides. Another strategy is to use three strands of RNA with two having locked nucleic acid (LNA) technology for enhanced stability. Modification of the native RNA backbone markedly reduces the type I interferon-mediated immune response cells have to extracellular RNA or DNA (77).

Although such mimics have been widely used in cultured cells, their utilization as drug candidates has been hindered by delivery and selectivity for the target organ. These limitations have been partially overcome as summarized in Fig. 3 by targeted administration or by using vectors containing kidney-specific and inducible promoters (110). Delivery of antisense or nucleic acid-based therapeutics might also benefit from nanocarrier-based delivery systems, as hydrophilic agents typically cannot cross cellular or organelle membranes. Ultrasound-microbubble-mediated gene transfer has also been used to deliver plasmids that produce various miRNAs to mammalian kidneys. High-intensity ultrasound causes microbubble oscillation, resulting in the release of the therapeutic agent(s) attached to or contained within microbubbles (36, 100).

Fig. 3.

Delivery strategies for intrarenal targeting of miRNAs involving direct renal interstitial infusion, pelvic perfusion, and systemic delivery. (Kidney image adapted and reproduced with permission from the copyright holder http://servier.com).

In addition to anti-miRNAs, miRNA inhibition can be attained by expressing miRNA-target sequences able to capture pathogenic miRNAs (miRNA sponges) or by short hairpin RNA plasmids that nullify miRNA expression by RNA interference (110). Effective knockdown can also be achieved using oligonucleotides complementary to the 3′-UTR of the target mRNAs (masking approach) or to the sequence of the miRNA themselves (erasers). Many studies are now investigating the potential of miRNA targets selected on the basis of their known function in fibrosis or identification as targets genome-wide association studies of kidney disease to intervene in disease progression.

Miravirsen is an antimiR-122 for hepatitis C virus infection and the first miRNA-targeted drug to enter a phase II clinical trial (48, 65, 108). It is an LNA-modified DNA phosphorothioate antisense oligonucleotide, which acts by sequestering mature miR-122 in a stable heteroduplex. However, so far the potential for targeting miRNAs as effective antifibrotic therapy has been demonstrated only in experimental models of CKD. As most miRNAs are highly pleiotropic and act differently depending on cell type, a single miRNA may not be able to reverse the process of renal fibrosis. Rather, a complex of miRNAs or anti-miRNAs targeting different key points in the fibrotic process may prove more effective.

In conclusion, in vivo delivery of miRNA mimics or inhibitors has emerged as a promising therapeutic strategy for the treatment of CKD. Successful kidney transfection has been achieved in animal studies by intraperitoneal, intravenous, or subcutaneous injection of either mimics or inhibitors or, more frequently, by intravenous injection of plasmids expressing miRNAs or short-hairpin RNAs (110). It is likely that several agents targeting various miRNAs will enter clinical trials over the next few years and new renoprotective therapeutic agents based on this approach will enter clinical practice in the next decade.

GRANTS

This work was supported in part by National Institutes of Health grants HL-36279 (R. J. Roman) and DK-104184 (R. J. Roman), AG-050049 (F. Fan), P20GM-104357 (cores B and C, R. J. Roman; Pilot, F. Fan); American Heart Association Grant 16GRNT31200036 (F. Fan); and National Natural Science Foundation of China Fellowship Grants 81270939, 81472983, and 81571625 (W. Lv).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

W.L. drafted manuscript; W.L., F.F., Y.W., E.G.-F., C.W., L.Y., G.W.B., and R.J.R. approved final version of manuscript; F.F., Y.W., E.G.-F., C.W., L.Y., G.W.B., and R.J.R. edited and revised manuscript.