MicroRNAs as Master Regulators of Glomerular Function in Health and Disease

Abstract

MicroRNAs (miRNAs) are important regulators of gene expression, and the dysregulation of miRNAs is a common feature of several diseases. More miRNAs are identified almost daily, revealing the complexity of these transcripts in eukaryotic cellular networks. The study of renal miRNAs, using genetically modified mice or by perturbing endogenous miRNA levels, has revealed the important biologic roles miRNAs have in the major cell lineages that compose the glomerulus. Here, we provide an overview of miRNA biogenesis and function in regulating key genes and cellular pathways in glomerular cells during development and homeostasis. Moreover, we focus on the emerging mechanisms through which miRNAs contribute to different diseases affecting the glomerulus, such as FSGS, IgA nephropathy, lupus nephritis, and diabetic nephropathy. In-depth knowledge of miRNA-based gene regulation has made it possible to unravel pathomechanisms, enabling the design of new therapeutic strategies for glomerular diseases for which available therapies are not fully efficacious.

The kidneys fulfill several key tasks in the human body, from the removal of uremic waste products to the regulation of whole body ion homeostasis, acid-base balance, and BP. All of these functions depend on an adequate filtration rate. The filtering unit of the kidney is the glomerulus, a specialized bundle of capillaries composed of fenestrated endothelial cells surrounded by the glomerular (Bowman’s) capsule. The capillaries are wrapped by podocytes and the entire glomerular tuft is structurally supported by mesangial cells, which secrete growth factors and matrix proteins. Crosstalk between different glomerular cell types is required for the glomerular filtration barrier to be functional, and this crosstalk is tightly orchestrated by controlled gene expression and epigenetic mechanisms. Among epigenetic regulators, microRNAs (miRNAs) have emerged as endogenous molecules that negatively control target genes. miRNAs have been found to be dysregulated in glomerular cells, leading to cell-specific response and damage, and could therefore be exploited as novel targets for glomerular diseases (Figure 1).

Figure 1.

miRNA dysregulation in glomerular diseases. Changes in miRNAs in different populations of glomerular cells (podocytes, PECs, glomerular endothelial cells, and mesangial cells) occurring in FSGS, lupus nephritis (LN), IgA nephropathy (IgAN), and diabetic nephropathy (DN).

miRNA Biogenesis and Function

miRNAs are short (approximately 21 nucleotides) noncoding RNAs that function as guide molecules in RNA silencing by inducing mRNA degradation or blocking protein translation.¹ Since the discovery of the first miRNA in Caenorhabditis elegans, the field has exploded, so much so that current estimates suggest that the human miRNAome is composed of >2500 miRNAs (http://www.mirbase.org).² miRNA genes are individual or clustered and reside in intergenic regions and in the introns of noncoding or coding transcripts, although a minority are in exonic regions or embedded in long–noncoding RNAs (lncRNAs, ≥200 nt).³ miRNAs are transcribed by RNA polymerase II as long capped and polyadenylated hairpin transcripts (pri-miRNA) and their expression is regulated either by epigenetic mechanisms, such as DNA methylation and histone modifications,⁴ or transcription factors including p53^5,6 and ZEB1/2.⁷ Pri-miRNAs—thousands of base pairs in length—are cleaved by the nuclear RNase III Drosha, and release approximately 70-nucleotide stem-looped structures called precursor miRNAs (pre-miRNAs). Drosha processing can be accelerated by different proteins, including p53⁸ and SMADs, the signal transducers of the TGF-β/bone morphogenetic protein,⁹ or slowed down, as occurs upon phosphorylated MeCP2 interacting with the Drosha cofactor DGCR8.¹⁰ Pre-miRNAs are then shuttled by the exportin-5-RanGTP complex to the cytoplasm, where they are cleaved by the RNAse type III endonuclease Dicer, liberating small duplexes of the mature miRNA strand (guide) and a complementary strand of similar length (passenger). It is notable that Dicer action can also be counteracted via interaction with the immune regulator MCPIP1.¹¹ The duplex is finally loaded onto an Argonaute protein (AGO2), forming a pre-RNA–induced silencing complex (pre-RISC) which quickly removes the passenger strand to generate a mature RISC. The passenger strand is rapidly degraded after displacement from the duplex. The mature RISC is an effector complex where miRNAs function as guides by binding through their seed sequence (nucleotides 2–8) to miRNA binding sites usually located in the 3′-untranslated region of target mRNAs, whereas Argonaute proteins function as effectors by recruiting factors which induce translational repression, mRNA deadenylation, and mRNA decay.

The inhibitory activity of an endogenous miRNA is not a function of its absolute expression level in the cells, but depends on its loading into the RISC complex. The miRNAs’ association with RISC varies from cell to cell, due to the relative abundance of mRNA species and their complementarity.¹² It is notable that a given miRNA can regulate several hundred transcripts belonging to different cellular pathways and networks. Because of this, miRNAs are able to switch between cellular programs and are therefore often considered master regulators of the genome.

miRNAs during Glomerular Development

Kidney development depends on reciprocal interaction between two tissues, the ureteric bud, which gives rise to the cells of the collecting duct, and the metanephric mesenchyme. The metanephric mesenchyme contains nephron progenitors that generate glomerular podocytes and renal tubular epithelia, stromal progenitors that differentiate into interstitial cells, mesangial cells, vascular smooth muscle cells and pericytes, and finally vascular progenitors which give rise to glomerular and vascular endothelia.^13,14

miRNAs have been identified in the kidney at all embryonic stages and their sequential expression is crucial during organ development, as demonstrated in mammalian and nonmammalian models with spatial conditional deletion of specific miRNAs and the miRNA processing enzymes Drosha and Dicer. miRNAs are required during early kidney organogenesis as demonstrated by the crucial role played by the miR-30 family during pronephros development in Xenopous. miR-30s target the LIM-class homeobox factor Xlim/Lhx1, a key transcriptional regulator of kidney development.¹⁵ Moreover, miRNA absence in the early metanephric mesenchyme leads to severe renal dysgenesis due to loss of nephron progenitors.^16,17 Similarly, during midgestation the depletion of miRNAs in nephron progenitors results in premature termination of nephrogenesis and nephron apoptosis,¹⁸ whereas the specific inactivation of the miR-17∼92 cluster leads to the defective proliferation of progenitor cells and reduced numbers of developing nephrons which predisposes mice to glomerular dysfunction and proteinuria.¹⁹

Finally, the deletion of Dicer-1 in Foxd1-derived stromal cells during kidney development resulted in hypoplastic kidneys with abnormal differentiation of the nephron tubules and vasculature, leading to kidney failure and perinatal mortality. Notably, the number of glomeruli in mutant kidneys was reduced and abnormalities, including cyst formation and dilated vessels, affected the glomeruli. Podocytes appeared to be immature, with cuboidal epithelial morphology, whereas mesangial cells failed to form in 20% of the glomeruli.²⁰ The role of stromal miRNAs for normal nephrogenesis and glomerular mesangial maintenance was also confirmed in a concomitant study with similar results.²¹

miRNAs in Glomerular Visceral and Parietal Epithelial Cells

Podocytes are terminally differentiated epithelial cells that are located on the visceral side of the Bowman’s capsule. They develop primary and secondary foot processes that wrap around the capillary of the glomerulus.²² miRNAs are critical players in podocyte homeostasis, because targeted deletion of Dicer or Drosha in these cells, both in the developing as well as in the adult kidney, leads to proteinuria and glomerulosclerosis.^23–26 Moreover, several miRNAs have been found to be dysregulated in podocyte injury, a pathologic mechanism causing glomerular injury and sclerosis.

Because mature podocytes must withstand fluctuating pressures and potentially harmful molecules contained in the primary filtrate, they are unlikely to be static structures.²² This remodeling of the actin cytoskeleton plays a primary role in the structural adaptations made by podocytes to preserve their glomerular filtration properties. The podocyte cytoskeleton is finely regulated by a set of miRNAs that are expressed mainly in the adult kidney, such as miR-30, miR-132, miR-134, and miR-29a. miR-30 family members are highly expressed in human podocytes and, in addition to protecting them against apoptosis by directly targeting Notch1 and p53,²⁷ they promote podocyte actin fiber stability by controlling calcium/calcineurin signaling through the inhibition of several components of this pathway (TRPC6, PP3CA, PP3CB, PPP3R1, and NFATC3).^28,29 Dysregulation of calcium/calcineurin signaling leads to podocyte cytoskeletal damage, which is a key feature of various glomerular diseases, such as FSGS, characterized by the early onset of podocyte injury.²⁸ It is notable that miR-30s are significantly downregulated in the podocytes of patients with FSGS and in the rat model of FSGS induced by the podocyte toxin puromycin aminonucleoside. Furthermore, fibrogenic factors, such as TGF-β, reduce miR-30 expression both in vivo as well as in cultured podocytes, possibly due to Smad2-dependent mechanisms.^27,30 On the other hand, the renoprotective effect of glucocorticoids in glomerular disease has been shown to be associated with increased expression of miR-30s.²⁷

A recent work underlines the role of miRNAs in regulating podocyte cytoskeletal dynamics. The study showed that brain-derived neurotrophic factor is able to repair podocyte damage both in vitro and in a mouse model of FSGS by inducing miR-132 and inhibiting miR-134 expression upon binding to its receptor on podocytes. Brain-derived neurotrophic factor induced modulation of miR-132 and miR-134 has been found to be essential for increasing actin polymerization, favoring foot process elongation that contrasts the cell flattening induced by proteinuric conditions.³¹

Adult podocytes also express miR-29 family members that possess potent antifibrotic and proapoptotic activities and have been found to be dysregulated in several diseases affecting both the kidney and the heart.³² An elegant study highlighted the renoprotective effect of miR-29a in diabetes-induced loss of podocyte integrity and renal homeostasis. Diabetic miR-29a transgenic mice had higher nephrin levels and podocyte viability and less glomerular fibrosis compared with diabetic wild-type mice. The overexpression of miR-29a attenuated nephrin ubiquitination and restored proper protein acetylation through the inhibition of histone deacetylase 4. These data demonstrate the importance of post-translational acetylation reactions in podocyte nephrin and suggest that increasing miR-29a action may protect against diabetic podocytopathy.³³

The role of miRNAs has been ascertained in glomerular parietal epithelial cells (PECs) lining the Bowman’s capsule. PECs recently gained attention because they are a heterogeneous population of renal progenitor cells,^34–36 as well as for their contribution to glomerular diseases.³⁴ Robust miR-150 expression in PECs and podocytes of patients with lupus nephritis correlated positively with disease chronicity scores and the expression of profibrotic proteins. Increased miR-150 would foster the production of profibrotic molecules through the downregulation of its predicted target, SOCS1. The latter protein acts as a negative regulator of the JAK/STAT signaling pathway, which promotes the transcription of genes involved in cell proliferation, inflammation, and fibrosis.^37,38 Similarly to miR-150, we identified miR-324–3p increased expression in PECs as well as podocytes in an FSGS rat model. Increased expression of miR-324–3p was associated with the downregulation of its target prolyl endopeptidase (Prep)—involved in the formation of the antifibrotic peptide Ac-SDKP—in fibrotic areas of the kidneys of diseased rats.³⁹ hPECs isolated from naive Bowman’s capsules express significant levels of miR-193a, which works as a suppressor of podocyte differentiation by inhibiting the expression of the transcription factor Wilms tumor protein (WT1).^40,41 WT1 is essential for the development and maintenance of podocytes and glomeruli by inducing the expression of podocalyxin, nephrin, and other genes governing podocyte architecture.⁴² The downregulation of miR-193a is associated with PEC transdifferentiation toward a podocyte phenotype, whereas its over-expression leads to their abnormal activation, a prerequisite for the formation of crescents in proliferative GN.⁴¹ In line with this, extracapillary lesions in humans and mice are mainly constituted by PECs, which over-express miR-193a. Moreover, isolated glomeruli from individuals with FSGS are characterized by increased expression of miR-193a, compared with normal kidneys or kidneys affected by other glomerular diseases.⁴⁰ The involvement of miR-193a in the pathogenesis of FSGS was also supported by data from miR-193a knock-in mice, which develop FSGS with extensive podocyte foot process effacement.⁴⁰ All of the above evidence, together with findings that the number of crescents was reduced by anti–miR-193a in the mouse model of nephrotoxic nephritis,⁴¹ concur to indicate that miR-193 is a promising therapeutic target for FSGS. The mouse model of nephrotoxic nephritis has been also instrumental for identifying the key role of miRNAs in orchestrating T cell–mediated crescentic GN. Mice with miRNA-deficient CD4+ T cells develop less severe GN upon toxin injection. Moreover, the kidneys of patients with ANCA-associated crescentic GN and mice with nephrotoxic nephritis are characterized by the upregulation of miR-155 that drives the Th17 immune response and tissue injury.⁴³

miRNAs in Mesangial and Glomerular Endothelial cells

miRNAs are essential for mesangial cell integrity and function and are involved in their response to injury. Mesangial cells form the structural support of the capillary network and provide a counter force to changes in intraglomerular capillary pressure, contributing to the modulation of GFR. They respond to injury in different ways by producing reactive oxygen species, cytokines, chemokines, and excess matrix proteins, and undergoing apoptosis, abnormal proliferation, and migration.⁴⁴

The deposition of immune complexes or Igs in the mesangium activates mesangial cells, causing their proliferation and excess matrix production, which lead to glomerular injury.⁴⁴ The activation of mesangial cells is a typical hallmark of immune-mediated glomerular diseases, of which lupus nephritis and IgA nephropathy are the main examples. In patients and mice with lupus nephritis, mesangial expansion is mediated by the upregulation of miR-148a-3p through the post-transcriptional inhibition of PTEN (phosphatase and tensin homolog), the major negative regulator of PI3K/Akt signaling, with consequent Akt activation and cell proliferation.⁴⁵ The activation of human mesangial cells after the deposition of immune complexes leads to the exuberant production of chemokines and cytokines, which contribute to cell proliferation and cause intense inflammation. In this context, the activation of the type I IFN signaling pathway in kidney tissues was recently shown to be key to the pathogenesis of lupus nephritis, because the expression level of IFN-inducible genes correlates with disease activity and severity.⁴⁶ miR-744 acts as a feed-forward regulator of IFN signaling in human mesangial cells. Type I IFN promotes the transcription of proinflammatory genes through miR-744, which targets phosphatase PTP1B, leading to the activation of downstream signal components of the type I IFN pathway.⁴⁷ Moreover, in mesangial cells, type I IFN, via the transcription factor IRF-1, induces the expression of human EGF 2 (HER-2), which switches off protective miRNAs including miR-26a and miR-30b, which directly regulate the cell cycle in mesangial cells.⁴⁸ More recent studies provide another clue to the complex activation of the type I IFN pathway in the kidney, showing that this pathway might be negatively regulated by miR-130b. Downregulated miR-130b expression was observed in kidney tissues from patients and lupus-prone mice, which correlated negatively with abnormal activation of the IFN response. Interestingly, the administration of miR-130b agomir reduced the IFNα-accelerated progression of experimental lupus nephritis and ameliorated glomerular lesions.⁴⁹

Mesangial cells are also strongly affected in IgA nephropathy, due to the massive deposition of aberrantly O-glycosilated polymeric IgA1. IgA1 O-glycosilation occurs in immune cells due to two enzymes, GALNT2 and C1GALT1, whose expression is controlled by let-7b and miR-148b, respectively.^50,51 Of interest, miR-148b and let-7b were found upregulated in PBMCs of patients with IgA nephropathy.^50–52 Several pieces of evidence indicate that IgA nephropathy is often aggravated by mucosal infection, with secretory IgA being the most important antibody in mucosal immunity. A recent study demonstrated that secretory IgA isolated from patients with IgA nephropathy induces mesangial cell proliferation and fosters the production of proinflammatory cytokines such as IL-8 and IL-1β, through the downregulation of miR-100–3p and miR-877–3p, respectively.⁵³

It goes without saying that miRNAs also play a key role in glomerular endothelial cells, highly flattened and fenestrated cells. This unique feature allows them to selectively filter large volumes of plasma at a prodigious rate.⁵⁴ Endocapillary hypercellularity contributes to renal injury in IgA nephropathy,⁵⁵ a process in which miR-223 plays an active role.⁵⁶ Patients with IgA nephropathy are characterized by low levels of miR-223, which induces glomerular endothelial cell activation by upregulating importin α4 and α5, which are responsible for NF-κB and STAT3-mediated cell proliferation and monocyte adhesion.⁵⁶ Moreover, glomerular endothelial cell injury is a critical pathologic process at the early stage of diabetic nephropathy⁵⁷ and it has been suggested that it is mediated by miR-29c⁵⁸ and miR-93.⁵⁹

miRNAs during Glomerular Obsolescence

Diabetic nephropathy has been instrumental for the identification of various miRNAs and their roles in different phases of damage leading to glomerular obsolescence. The development and progression of diabetic nephropathy involve complex interplay between metabolic, hemodynamic, growth, and inflammatory factors. Hyperglycemia is associated with the excessive production of reactive oxygen species generated by NADPH oxidase, together with the overactivation of the aldose reductase/polyol pathway. NADPH oxidase threshold activity is settled by miR-25 levels that directly silence NOX4, one of the major NADPH oxidase subunits.⁶⁰ miR-25 expression is suppressed under hyperglycemic conditions leading to increased NADPH oxidase activity and ROS production.⁶⁰ Moreover, aldose reductase favors oxidative stress by downregulating miR-200a-3p and miR-141–3p, which are able to suppress Keap1-Nrf2, TGF-β1/2, and Zeb1/2 signaling.⁶¹ Little is known about miRNA regulation of inflammation during diabetic nephropathy. During early diabetic nephropathy, miR-146a was upregulated in the kidneys and macrophages of diabetic mice.⁶² The induction of diabetes in miR-146a−/− mice resulted in greater proteinuria, higher renal macrophage infiltration, and more fibrosis than in wild-type diabetic mice. Furthermore, a lack of miR-146a was associated with the upregulation of proinflammatory and profibrotic genes and the expression of markers of inflammasome activation. This evidence pointed to miR-146a acting as a protective epigenetic regulator in diabetic nephropathy by tilting the balance toward anti-inflammatory signaling.⁶²

Podocyte loss, mesangial hypertrophy, matrix accumulation, and basement membrane thickening are among the earliest pathologic features of diabetic nephropathy, with TGF-β playing a key role in their development.⁶³ Podocyte loss, due to both detachment from the glomerular basement membrane and apoptosis, appears to be a prerequisite early event that leads to glomerular obsolescence. Podocyte apoptosis is controlled by miR-29c and miR-21. miR-29c expression is increased in db/db mice and activates Rho kinase by targeting Sprouty homolog 1, thus favoring fibronectin assembly and apoptosis.⁵⁸ In contrast, there are data that suggest that miR-21 has an antiapoptotic role in podocytes because it ameliorates TGF-β and hyperglycemia-induced glomerular injury through the repression of proapoptotic signals.⁶⁴

The hypertrophic response of mesangial cells results either from the activation of the PI3K/Akt pathway or the modulation of cell cycle–regulated proteins. Different miRNAs govern mesangial hypertrophy by serving as endogenous silencers of PTEN expression, as occurs for miR-216, miR-217,^65,66 and miR-21,^67–69 which are increased in diabetic mice glomeruli and in cultured mesangial cells exposed to TGF-β or high glucose. PI3K/Akt signaling is also controlled by miR-200b/c, which repress the PI3K inhibitor FOG2.⁷⁰ Finally, under hyperglycemic conditions, miR-34a upregulation induces mesangial proliferation and glomerular hypertrophy by targeting Growth arrest specific 1 (GAS1),⁷¹ whereas miR-196a downregulation contributes to mesangial hypertrophy by switching on the expression of p27kip1, a cyclin-dependent kinase inhibitor, and therefore arresting mesangial cells in the G1 phase.⁷²

Soon after hyperglycemia-induced mesangial hypertrophy, extracellular matrix proteins accumulate in the glomeruli. Excessive matrix production in glomerular cells is orchestrated by several miRNAs, the majority of which are induced by TGF-β. miR-26a, miR-93, and miR-29b/c are antifibrotic miRNAs whose expression is suppressed in podocytes in the presence of a diabetic milieu. miR-26a inhibition leads to increased levels of its target CTGF, a major downstream profibrotic factor of TGF-β. It is notable that miR-26a was found to be downregulated in patients with diabetic nephropathy,⁷³ autoimmune lupus nephritis, and IgA nephropathy.⁷⁴ miR-93 expression was found to be significantly reduced in the kidneys of patients with diabetic nephropathy and in experimental models of diabetes. It is associated with the upregulation of vascular endothelial growth factor A, together with its targets fibronectin and Col4a3 in db/db mice,⁵⁹ and of Msk2 (mitogen and stress-activated kinase 2), a histone kinase that phosphorylates H3S10 (Histone H3 Ser10), which in turn mediates chromatin remodeling and gene activation.⁷⁵ It is notable that miR-93 over-expression reverses high glucose–induced changes in chromatin structure and the global gene transcription of podocytes. The knock-down of Msk2 through the pharmacologic restoration of miR-93 or by a kidney-targeted shRNA system in diabetic mice results in significant improvements in biochemical and histologic features of diabetic nephropathy. Moreover, Msk2 expression is elevated in human patients with diabetic nephropathy in the face of reduced miR-93.⁷⁵ Finally, miR-29s suppression results in the increased expression of type I, II, and IV collagens in rodent models of renal fibrosis.⁷⁶ Increased matrix production in mesangial cells is induced by a fine balance between the upregulation of miR-192, miR-200b/c, miR-216a, and miR-377 and the downregulation of miR-29s and let-7 in experimental diabetes. Specifically, miR-192^77,78 and miR-200b/c⁷⁹ target the E-box repressors Zeb1/2, with the subsequent activation of Col1a2, Col4a1, miR-200b/c, miR-217, and miR-216a. The latter increases Col1a2 expression through a mechanism that favors the interaction of Tsc22 with the transcription factor E3 on the far upstream E-box region of Col1a2.⁸⁰ High glucose, via TGF-β1, upregulates miR-377, which suppresses the expression of p21-activated kinase and superoxide dismutases leading to enhanced susceptibility to oxidant stress and the accumulation of fibronectin.⁸¹ In mesangial cells, TGF-β1 lowers the expression of miR-29s⁷⁶ and let-7,⁸² antifibrotic miRNAs targeting different isoforms of collagen. Moreover, decreased miR-29a expression attenuates the Dickkopf-1/Wnt/β-catenin signaling, contributing to apoptosis and extracellular matrix deposition in STZ-treated mice.⁸³ Notably, in mesangial cells TGF-β induces signaling loops that amplify and create a chronic state of profibrotic pathway activation, modulating the expression of miR-192, miR-200s, miR-21, and miR-130b.^79,84,85

Recent work has demonstrated that diabetic conditions upregulate a megacluster of approximately 40 miRNAs (miR-379 megacluster) and their host lnc-MGC, an lncRNA that is regulated by an endoplasmic reticulum stress–related transcription factor (CHOP).³ miRNAs in the megacluster have several common target genes involved in protein synthesis and endoplasmic reticulum stress and their dysregulation leads to the hypertrophy and fibrosis typical of diabetic nephropathy. In diabetic mice, the inhibition of the cluster miRNAs by an antisense oligonucleotide targeting lnc-MGC results in reduced expression of profibrotic genes, reduced mesangial expansion, and decreased glomerular size in the kidneys. An increase in miR-379 megacluster expression was also observed in human mesangial cells treated with high glucose or TGF-β1. Moreover, most of the cluster miRNAs were expressed in the glomeruli of patients with diabetes, and the expression of some of their precursors increased with glomerular damage, suggesting relevance for human glomerular diseases.³

miRNAs as Potential Therapeutic Agents

Because of the role of miRNAs in regulating several pathways, miRNAs hold the promise of being able to yield a new class of therapeutics. Basically, miRNA-based therapies comprise two approaches: miRNA inhibition or replacement. Several intriguing methods have been developed to inhibit endogenous miRNAs that show a gain-of-function in diseased tissues, from miRNA sponges (target sequences able to capture miRNAs) to the masking approach (oligonucleotides complementary to the target mRNA binding site sequence), but the most widely adopted tool is antisense oligonucleotides (antagomir), designed against the mature sequence of miRNAs. Instead, the replacement of miRNAs is pursued by oligonucleotides, agomirs that “mimic” native miRNAs. Both antagomirs and agomirs need to be chemically modified (primarily with phosphorothioates or 2’O-methoxyethyl or locked nucleic acid backbones) to increase stability (otherwise they are degraded by serum nucleases in the bloodstream) and entrance into the cell through the negatively charged cell membrane.⁸⁶ Intravenously injected antisense oligonucleotides yielded significant inhibition of related targets in both the livers and kidneys of mice⁸⁷ and nonhuman primates.⁸⁸

No matter what strategy is used, the major concerns of miRNA-based therapies include target delivery and safety. In the context of glomerular diseases, the target miRNA should be delivered specifically to the kidney in order to avoid any potential adverse effects in other tissues and organs, and should affect only one target (or targets acting in the same pathway) to avoid effects on unintended templates.

Of the miRNA-based clinical studies, only one relates to kidney disease. RG-012, an antagomir against miR-21, was recently developed to treat Alport nephropathy, a disease for which there is yet no effective therapy. Alport nephropathy is caused by defects in the genes encoding α3, α4, and α5 chains of type IV collagen, a key component of the glomerular basement membrane,⁸⁹ resulting in the disruption of the glomerular filtration barrier, podocyte damage, and glomerular obsolescence. Preclinical studies proving the efficacy of anti–miR-21 were conducted in a mouse model of Alport nephropathy with a null mutation in the α3 chain of type IV collagen.⁹⁰ Renal cells of knock-out mice respond to stress caused by defects in the glomerular basement membrane by increasing miR-21 expression that induces inflammatory and profibrotic signaling.^90,91 Anti–miR-21–treated mice had better kidney function, with less glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation, and an increase in lifespan compared with vehicle-treated mice.

In December of 2015, a phase 1 clinical trial to evaluate the safety, tolerability, and pharmacokinetics of subcutaneous anti–miR-21, RG-012, in healthy volunteers was successfully completed. RG-012 was well tolerated and no serious adverse effects occurred. A phase 2 therapeutic intervention study of RG-012 in patients with Alport syndrome (NCT02855268) will start as soon as the ATHENA study provides data on the natural history of the disease (NCT02136862). Anti–miR-21 treatment developed for the cure of Alport nephropathy seems to be a very promising antifibrotic therapy that is translatable to other renal diseases, such as diabetic nephropathy and IgA nephropathy, where miR-21 has been shown to be actively involved in mediating kidney fibrosis.^92,93 To date, anti–miR-21–based therapies have been successful at curing different CKD in experimental animal models.^91,94,95 miR-21 knockdown ameliorates microalbuminuria and renal fibrosis in a model of type 2 diabetes,⁹⁵ but a recent work has demonstrated miR-21 may have a potential protective role in glomerular injury, indicating that more studies are needed to fully understand the role of mir-21–targeting in diabetes.⁶⁴

Cells have an endogenous machinery that can control miRNA expression, consisting of lncRNAs, which, besides hosting miRNAs, might act as miRNA sponges.⁹⁶ An example is offered by lncRNA H19, which contains several binding sites for the miRNA let-7. The study of lncRNA involvement in glomerular disease is still in its infancy. The few available findings indicate that certain lncRNAs interfere with inflammatory/developmental processes related to diabetic nephropathy. There are data indicating that increased expression of lncRNA MALAT1 in endothelial cells exposed to high glucose, as well as in the kidneys of diabetic mice, is associated with robust expression of inflammatory genes.⁹⁷ Moreover, H19 expression is reduced in the kidneys of E13 embryos carried by hyperglycemic mothers and E13 embryo explants exposed to high glucose. Such a decrease may be responsible for the perturbed epithelial/mesenchymal interactions in the developing kidney, leading to epigenetic changes during the prenatal period that could contribute to disease susceptibility in adult life.⁹⁸

lncRNAs could also block the transcription of miRNAs, and more in general of genes, by recruiting the Polycomb repressive complex 2 (PRC2), an enzyme able to add methyl groups to histone proteins thus silencing transcription of the surrounding genes, to certain areas in the genome.⁹⁶ Interestingly, a biotechnology company has recently developed an innovative RNA-targeted tool comprising short, single-stranded RNA oligonucleotides that interfere with the binding of the lncRNA to PRC2 protein leading to the transcription of specific genes (http://ranarx.com/our-science/).

The Role of miRNAs as Biomarkers

Scientists are motivated to look for novel biomarkers that provide earlier warning signs before the kidney is irreversibly damaged, more specific information on disease progression, and, ideally, response to treatment. Several studies have looked for circulating and urinary miRNAs as biomarkers in patients with glomerular diseases. Urine miRNAs are passively filtered from plasma or possibly shed from the urinary tract. They are stable in both plasma and urine thanks to the fact that they circulate either in association with Argonaute 2 complexes and lipoproteins or packaged into microvesicles, exosomes, and apoptotic bodies.^99,100 Stability, together with the fact that urine can be collected noninvasively and repeatedly, and the fact that the miRNA profile changes according to the type of renal disease, makes urine miRNAs ideal candidates for glomerular diseases.¹⁰¹ Moreover, urine miRNAs are quantifiable by real-time quantitative PCR, microarrays, and next-generation sequencing. In order to monitor kidney diseases, urinary exosome miRNA content would be preferable to the assessment of urinary miRNAs. Exosomes are actively secreted from live renal cells, whereas the urinary miRNA population might also contain plasma miRNAs.

To date, several small clinical studies have found a correlation between the urinary expression of miRNAs and clinical and histologic parameters in diabetic nephropathy, IgA nephropathy, lupus nephritis, and FSGS (Table 1). To strengthen miRNA diagnostic power, future work in this area should focus on large-scale studies and should identify their causal relevance.

Table 1.

miRNAs as biomarkers of glomerular diseases

Disease	Body Fluid	Candidate miRNAs	Notes	Ref
FSGS
	Blood	↑miR-186, ↑miR-192, ↑miR-205	Correlated with proteinuria	104, 105
	Urine	↑miR-196a, ↑miR-490, ↑miR-30a-5p	Correlated with disease activity	102
	Urine	↓miR-1915, ↓miR-663, ↑miR-155		103
	Urine	↑miR-200c	Correlated with eGFR	106
LN
	Blood	↓miR-342-3p, ↓miR-223, ↓miR-20a	Correlated with the onset of LN	108
	Blood	↓miR-200b/c, ↓miR-429, ↓miR-205, ↓miR-192	Correlated with eGFR	110
	PBMCs	↑miR-371-5p, ↑miR-423-5p, ↓miR-1224-3p	Correlated with the onset of LN	107
	Urine	↓miR-26a, ↓miR-30b		48
	Urine	↑miR-221, ↑miR-222	Correlated with disease activity	109
	Urine	↑miR-146a	Correlated with eGFR	111
	Urine	↑miR-155	Correlated with proteinuria	111
IgAN
	Blood	↑miR-148b, ↑let-7b	The combined miRNA levels have diagnostic accuracy	52
	CECs	↓miR-223	Correlated with proteinuria	56
	Urine	↑miR-146a, ↑miR-155, ↓miR-3613–3p, ↓miR-4668–5p ↓miR-29b/c, ↓miR-200a/b, ↓miR-429	Correlated with proteinuria	112, 113, 114, 115
	Urine	↑miR-93	Correlated with glomerular scarring	114
	Urine	↑miR-17		101
DN
	Blood	↑let-7b-5p, ↑miR-21-5-p, ↓let-7c-5p, ↓miR-29a-3p	Correlated with risk of ESRD	117
	Urine		miRNA signature specific for each stage	116
	Urine	↓miR-15, ↓miR-192	Correlated with proteinuria	101, 106
	Urine	↑miR-126		121
	Urinary Ex	↑miR-130a, ↑miR-145, ↓miR-155, ↓miR-424		118
	Urinary Ex	↑miR-15b, ↑miR-34a, ↑miR-636	Correlated with proteinuria	120
	Urinary EV	↑miR-192	Correlated with albuminuria	119

Ref, references; ↑, increased; ↓, decreased; LN, lupus nephritis; IgAN, IgA nephropathy; DN, diabetic nephropathy; CEC, circulating endothelial cell; Ex, exosomes; EV, extracellular vescicles.

In conclusion, significant progress has been attained in our knowledge of how miRNA biology is essential for the dynamic control of gene expression in the kidney and is a crucial facet of glomerular cell function. It turns out that miRNAs are at the forefront of glomerular kidney disease, behaving either as the pathogenic agent or as a stress responder. Because miRNAs induce potentially reversible epigenetic changes, they could serve as potential therapeutic targets for glomerular diseases. An example of miRNA-based therapeutics is provided by the use of anti–miR-21 to cure Alport syndrome, which has recently successfully completed a phase 1 clinical trial. The most recent, promising therapeutic strategy for activating endogenous miRNA expression contemplates interfering lncRNA-mediated silencing of target miRNAs, although a lot of work should be done to achieve this. miRNAs have gained a great deal of attention in the field of the biomarker search for kidney diseases, with those derived from urinary exosomes likely providing a snapshot of the kidney’s status. A single miRNA is unlikely to offer diagnoses, instead a clear miRNA signature is needed for glomerular diseases on the basis of studies confirmed in appropriate cohorts of patients of different ethnicity and disease severity.

Disclosures

None.