MicroRNAs: potential regulators of renal development genes that contribute to CAKUT

Abstract

Congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of childhood chronic kidney disease (CKD). While mutations in several renal development genes have been identified as causes for CAKUT, most cases have not yet been linked to known mutations. Furthermore, the genotype-phenotype correlation is variable, suggesting that there are additional factors that impact the severity of CAKUT. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and are involved in many developmental processes. Although little is known about the function of specific miRNAs in kidney development, several have recently been shown to regulate the expression of, and/or are regulated by, crucial renal development genes present in other organ systems. In this review, we discuss how miRNA regulation of common developmental signaling pathways may be applicable to renal development. We focus on genes that are known to contribute to CAKUT in humans, for which miRNA interactions in other contexts have been identified, with miRNAs that are present in the kidney. We hypothesize that miRNA-mediated processes play a role in kidney development through similar mechanisms, and speculate that genotypic variations in these small RNAs or their targets could be associated with CAKUT.

Introduction

Congenital anomalies of the kidney and urinary tract (CAKUT) are amongst the most frequent birth defects in humans, and are the major cause of childhood chronic kidney disease and end-stage renal disease [1]. The underlying causes of CAKUT remain elusive for the most part, likely because these represent a heterogeneous group of disorders with many underlying etiologies, and a wide range of prognostic outcomes. Thus, molecular tools that may direct prevention strategies, predict functional outcomes or guide interventions would be a helpful addition to the clinical management of these children. This review will address emerging information regarding the role of miRNAs in regulating kidney development.

In humans, kidney development begins around the fifth week of gestation with the outgrowth of the ureteric bud (UB) from the Wolffian duct epithelium. This outgrowth is initiated via cues from a subset of the adjacent intermediate mesoderm, known as the metanephric mesenchyme (MM), which induces the UB to elongate and invade the MM [2]. The UB responds to signals from the MM to undergo iterative branching events to form the collecting duct system [2, 3]. The terminal collecting ducts, whose ampullae are referred to as ureteric buds, induce the formation of new nephrons with each successive round of branching [3]. In response to signals from the UB, MM cells are induced to either condense around the tips of the UB to form nephron progenitors (multipotent, self-renewing renal progenitors) or to become renal stromal cells [4].

Broadly speaking, CAKUTs occur due to abnormalities in kidney and urinary tract development. For example, abnormal UB induction can result in renal agenesis, duplicated collecting systems, vesicoureteral reflux and/or obstruction. Alternatively, if fewer nephrons are induced during kidney development, renal hypoplasia resulting in poor congenital nephron endowment occurs. Children with reduced nephron numbers are thought to have a poorer renal prognosis [5], and are at risk for developing primary hypertension [6]. In addition, impaired nephrogenesis can result in dysplastic kidneys or tumor formation (eg. Wilms tumors), related to abnormal cell fate specification, apoptosis or proliferation. For an in-depth review please refer to [7]. CAKUTs are a highly heterogeneous group of disorders, and the factors that influence the clinical presentations of CAKUTs are not fully understood. For example, the renal phenotype in families with known mutations affecting kidney development can be variable, suggesting that there are additional factors involved [8]. These factors could be mutational (i.e. genetic variation amongst individuals), epigenetic (i.e. DNA methlylation, histone modifications, miRNAs, etc.) and/or environmental (i.e. toxin exposure). Given the heterogeneity, it is likely that the genetic or molecular basis may be different from one subtype to another.

MiRNAs are small non-coding RNAs that act as regulators of gene expression through the repression of their target mRNAs (reviewed in [9]). miRNA genes are transcribed by RNA polymerase II, and are subsequently processed to their mature, 22-nucleotide form. They interact primarily with the 3′-untranslated regions (UTRs) of target mRNAs via an 8-nucleotide seed region to direct mRNA degradation or inhibit protein translation. A single miRNA can have hundreds of putative targets, and any given mRNA can be targeted by multiple miRNAs. Individual miRNAs are thought to be capable of targeting multiple proteins in a signaling pathway to maximize their effect. The majority of studies regarding miRNAs in kidney development have thus far blocked the miRNA biogenesis pathway in specific cell lineages. In renal tubules, loss of miRNAs impacted maturation and caused hydroureter, hydronephrosis, and tubular and glomerular cysts [10]. Without miRNAs in juxtaglomerular cells, they no longer expressed renin [11]. Inhibiting mature miRNAs in nephron progenitors increased apoptosis and severely impacted progenitor survival [12, 13]. Loss of miRNAs in podocytes resulted in marked proteinuria and tubular and glomerular injury including tuft collapse and foot process effacement [14]. Finally, miRNA biogenesis disruption in renal tubules along with part of the ureteric bud caused the CAKUT symptoms of low nephron endowment and ureteropelvic junction obstruction [15]. Please refer to several recent reviews for details [16-18].

Despite growing knowledge regarding miRNAs in the kidney, the roles of individual miRNAs in renal development remains largely obscure, and there is limited data regarding the function of miRNAs in human kidney development. Intriguingly, deep RNA-sequencing of murine embryonic kidneys revealed 51 miRNAs that are present in the developing kidney [19]. In this review, we will discuss recently published data on genes in which mutations have been defined in humans with CAKUT, and for which there is evidence of interaction in various experimental systems with miRNA(s) known to be expressed in the kidney (please see schematic 1 for a summary). We speculate that miRNAs may be responsible, at least in part, for the phenotypic variability observed in CAKUTs.

Schematic 1.

MiRNAs associated with renal disease can potentially regulate CAKUT related genes during kidney development. Several of the miRNAs mentioned in this review have already been linked to kidney disease. Presented are the hypothetical pathways in which these miRNAs can potentially interact with CAKUT linked renal development genes (orange text). Arrows indicate a positive interaction, where t-bars indicate inhibition.

A potential Pax2/n-Myc/miR-17~92 interaction in nephrogenesis

The Pax2 gene (paired box 2) codes for a transcription factor critical for the formation of tissues and organs during embryogenesis, including the kidney. Pax2 mutations are associated with Renal Coloboma Syndrome (RCS), which is associated with congenital anomalies of the kidneys [20, 21]. Null mutations in humans have not been described, presumably due to prenatal lethality; however, Pax2^−/− mice completely lack ureters, kidneys and the entire genital tract [22]. In both mice and humans, heterozygous loss of Pax2 results in reduced kidney size [22, 23]. In contrast, transgenic mice that overexpress Pax2 globally had poorly developed podocyte foot processes, proteinaceous dilated tubules, abnormal renal function and died perinatally [24]. This suggests that the gene dosage of Pax2 is essential for normal kidney development, and implies that its levels must be tightly regulated.

One possible mechanism to regulate Pax2 levels is via an interaction with n-Myc and miRNAs. Pax2 and n-Myc can function concertedly to regulate cell proliferation in embryonic renal mesenchymal cells [25]. Pax2 and n-Myc levels appeared to positively regulate each other, where transfected Pax2 cDNA elevated n-Myc levels, and vice versa. In addition, the stimulation of Pax2 transcription by n-Myc was amplified under high glucose conditions, implying that this pathway could be modulated by stress [25]. n-Myc is required for kidney development, and hypomorphic mutations have been associated with fewer developing glomeruli and collecting ducts in the embryonic mouse kidney [26]. n-Myc is thought to transcriptionally activate miR-17~92 via several canonical E-box binding domains located in the 5′UTR of the miR-17~92 loci, and has been shown to do so in primary cerebellar granule neuron precursors [27].

miR-17~92 (homologous to human MIR17HG) codes for six miRNAs that have been shown via numerous studies to be linked to the regulation of cell cycle and proliferation in a variety of cellular contexts [28-30]. The observation that the hypomorphic n-Myc allele resulted in decreased cell division in the developing mouse kidney is interesting given the described role of miR-17~92 in the cell cycle [26, 31, 32]. Additionally, miR-17~92 expression is amplified in several cancers including Wilms’ tumor [27, 33], possibly in response to elevated n-Myc and/or c-Myc levels [34, 35]. In humans, miR-17~92 haploinsufficiency has been linked to Feingold Syndrome [28], a condition that is most frequently ascribed to mutations in n-Myc [36]. Renal defects have been reported in cases of Feingold Syndrome due to n-Myc mutations, including bilateral renal dysplasia and hypoplasia; however it remains unclear what role the miR-17~92 cluster plays during normal kidney development [36, 37]. Together, these data raise the question of whether a Pax2/n-Myc/miR-17~92 pathway plays an important role in kidney development.

miRNAs expressed in polycystic kidney disease can target HNF1β

Recent data showed that miR-17~92 was upregulated in the Ksp/cre; Kif3a^flx/flx model of polycystic kidney disease (PKD) [38]. Interestingly, deletion of the miR-17~92 locus from developing renal tubules and ureters in this model ameliorated cyst growth [38]. Luciferase reporter assays subsequently demonstrated that the PKD1 and PKD2 genes can be targeted by miR-17 [32, 39, 40]. In renal epithelial cells, miR-92a (one of the miRNAs in the miR-17~92 cluster) was found to directly target the 3′UTR of hepatocyte nuclear factor-1β (HNF1β, mutations of which are associated with cystic renal hypodysplasia) [38, 41]. In addition, miR-17~92 expression in mouse kidneys was negatively correlated with HNF1β levels, which is consistent with HNF1β being a direct target of miR-92a [38].

Another miRNA that may target HNF1β is miR-802. In murine liver cells, upregulation of miR-802 was correlated with decreased HNF1β expression, and direct targeting of HNF1β by miR-802 was confirmed using a luciferase assay [42]. Levels of miR-802 may be responsive to stress, since miR-802 was increased in the livers of both obese mice and humans [42], as well as in response to a high potassium diet in the cortical collecting ducts of mice [43]. Interestingly, miRNA microarrays of adult PKD mice revealed that miR-802 is downregulated relative to controls in the kidney [38]. These studies suggest that miR-802 might target HNF1β in the kidney as well.

Potential HDAC/let-7/c-Myc/miR-17~92 pathway in kidney development

Chromatin modifications represent an epigenetic mechanism that allows genes to become more or less transcriptionally active. Examples include histone acetylation and deacetylation, and histone deacetylases (HDACs) have important roles in numerous cellular processes including cell cycle, proliferation, differentiation, and cell death [44]. For example, chromatin immunoprecipitation assays demonstrated that HDAC1 and HDAC2 regulate Pax2 in the developing mouse kidney [45]. Interestingly, about 40% of noncoding RNAs were either upregulated or down-regulated in response to HDAC inhibitors in cancer [46, 47]. In the kidney, HDAC inhibitors expanded the renal progenitor population in zebrafish [48], and enhanced renal recovery after acute kidney injury in both zebrafish and mice [49]. In addition to Pax2, other important kidney development genes regulated by HDACs include: Pax8, WT1, GDNF and Wnt9b [45]. Taken together, these data suggest that HDAC regulation of miRNAs and renal development genes could play an important role during kidney organogenesis.

HDAC inhibitors have been shown to downregulate several members of the let-7 miRNA family in human breast cancer and hepatic stellate cells [47, 50], as well as miR-17~92 levels in colorectal cancer cells [51]. In liver cells, let-7c could directly target the 3′UTR of c-Myc, subsequently altering the expression of miR-17~92 [52], a transcriptional target of c-Myc [27, 53]. Interestingly, recent work showed that c-Myc functions as a direct transcriptional repressor of miRNA genes in B cell lymphomas, including members of the let-7 family [54]. These data suggest that it is possible for c-Myc and let-7 miRNAs to reciprocally regulate each other possibly affecting at least the levels of miR-17~92 miRNAs. Let-7 member miRNAs are expressed fairly ubiquitously in the kidney [12, 55, 56], and their miss-expression was linked with renal cell carcinoma [57]. Since c-Myc, let-7g (at least), and miR-17~92 are all expressed in nephron progenitors [12, 45, 55, 56], it will be interesting to determine if an interaction between these signaling pathways exists during kidney development.

Regulation of TGF-β/BMP signaling via miRNAs

Bone morphogenic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) super-family of growth factors. BMP signaling is required for normal ureteric budding and nephron induction during kidney development, and BMP4 mutations are associated with renal hypodysplasia [58, 59]. Numerous recent studies have provided evidence of BMP repression by miRNAs, and conversely, TGF-β and BMP signaling have the potential to regulate miRNAs. One intriguing mechanism by which TGF-β and BMP signaling might regulate miRNA levels is via an interaction of their downstream effector proteins, the SMADs, with a component of the DROSHA microprocessor complex (required for processing of mature miRNAs) [60]. A SMAD-DROSHA interaction was recently demonstrated to promote the processing of the miR-21 primary transcript to mature miR-21 in vascular smooth muscle cells [60]. However, it remains unknown exactly how this occurs or how many miRNAs are regulated via this mechanism. Though it was reported that BMP4 treatment increased miR-21 levels in vascular smooth muscle cells [61], it was also shown that BMP4 decreased miR-21 levels in epidermal keratinocytes, suggesting that there are additional factors that regulate the miR-21/BMP4 interaction [62]. Interestingly, upregulation of miR-21 in diabetic nephropathy mouse models correlated with increased expression of proteins that contribute to renal fibrosis [63].

Post-transcriptional regulation of miRNA biogenesis is not the only mechanism by which BMP signaling regulates miRNAs. miRNAs may also participate in regulatory loops for TGF-β/BMP signaling. For example, one member of the miR-17~92 cluster, miR-20a, was reported to target negative regulators of BMP signaling in human mesenchymal stem cells upon osteogenic differentiation [64]. BMP signaling was also associated with miR-17~92 in myocardial differentiation, where Bmp mutant mouse embryos had reduced levels of miR-17~92 miRNAs, and the BMP^−/− phenotype worsened when one copy of miR-17~92 was removed [65].

Another example of the interaction between miRNAs and TGF-β/BMP signaling is miR-302~367. The miR-302~367 cluster was transcriptionally downregulated in human primary pulmonary artery smooth muscle cells in response to BMP4, likely to promote BMP signaling because miR-302 can directly target the type II BMP receptor (BMPRII) [66]. In the kidney, miR-302 is at least expressed in the glomerular mesangium, and its levels were increased in response to connective tissue growth factor (CTGF) [67]. Thus, increased levels of miR-302 might play a role in the progression of diabetic nephropathy as a mediator between CTGF and TGF-β signaling.

Yet another example was demonstrated when BMP4 was found to activate miR-125a transcription in a feedback loop where miR-125a targeted Dies1 (a protein associated with the BMP4 receptor complex), resulting in decreased BMP4 signaling [68]. In the kidney, miR-125a interacts with the RNA binding protein bicaudal-C homolog 1 (Bicc1) to silence at least the protein kinase inhibitor PKIα and the adenylate cyclase AC6 in a PKD mouse model implying a role for miRNAs in regulation of cystic growth [69].

Recent work suggests that a TGF-β1 regulatory loop is miRNA-dependent in diabetic nephropathy [70]. Increased TGF-β1 in mouse models of diabetic nephropathy were shown to result in increased miR-192 expression, which is thought to target the E-box repressor SIP1 [70, 71]. The increase in miR-192 is believed to be mediated by TGF-β1-induced Akt activation, which subsequently results in the release of repression of miR-192 via the transcription factor, Ets-1 [72]. In addition, transfection of mesangial cells with miR-192 and miR-200b/c induced TGF-β1 expression, presumably by down-regulating the E-box repressors, Zeb1/2 [70, 73]. However, the precise mechanism by which TGF-β regulates miR-192 expression may be cell type- or species-dependent. Expression profiling of biopsies from patients with diabetic nephropathy revealed lower miR-192 levels, in association with increased fibrosis and decreased glomerular filtration rate [73]. Additionally, treatment of cultured human proximal tubular cells with TGF-β1 resulted in decreased miR-192 levels [73]. Further studies will be required to reconcile these apparent conflicts. miR-192 also induced the expression of miR-200b/c, implying that miR-192 is upstream of miR-200b/c [70]. Both miR-200b/c and miR-192 enhanced the expression of collagens and are thought to play a role in fibrosis in diabetic nephropathy [17].

Additional miRNAs involved in TGF-β/BMP signaling are the highly conserved, non-homologous miR-143 and miR-145 [74]. TGF-β and BMP4 induced expression of miR-143 and miR-145 in pulmonary artery smooth muscle cells, presumably through transcriptional activation of an upstream CArG box [75]. This subsequently led to a decrease in their direct target, Krüppel-like factor-4 (KLF4) [75]; a transcription factor that defines pluripotency [76]. Both miR-143 and miR-145 are expressed in E15.5 mouse kidney by RNA-sequencing [19], and KLF4 is expressed in smooth muscle cells of the kidney where TGF-β signaling is active [77]. Interestingly, miR-145 overexpression decreased levels of BMP4 mRNA in esophageal adenocarcinoma cells [78]. Together, although multiple miRNAs have been implicated in TGF-β/BMP signaling, the function of these miRNAs remains largely undefined in the developing kidney.

Potential miRNA links to Eya1/GDNF

The invasion of the UB into the neighboring MM depends on glial-cell-line-derived neurotrophic factor (GDNF) signaling by the MM to its receptor tyrosine kinase, Ret, in the ureteric bud [79]. Mutations in both GDNF and Ret were demonstrated in stillborn fetuses with bilateral or unilateral renal agenesis [80]. The transcription factors, Pax2 and Eya1, function as part of a complex to activate expression of GDNF [81], and Eya1 is associated with Branchiootorenal (BOR) syndrome [82, 83]. Interestingly, miR-562 is expressed in the kidney and was shown to directly target Eya1 in Wilms’ tumors via luciferase reporter assays [84]. In addition, miR-562 is harbored in the genetic locus 2q37, whose deletion has been associated with Wilms’ tumor [84].

Notably, GDNF could regulate the expression of miR-21 and miR-24-2 precursors via an ERK1/2 MAPK signaling pathway in human BE(2)-C cells [85]. Another study showed GDNF induction of miR-2, −21, −218, and let-7f, −7g, −7i in human glioblastoma cells [86]. It is not clear to what extent miR-21, miR-24-2 or miR-2 are expressed in the kidney, but miR-218 and let-7 member miRNAs are [12, 55, 56]. Furthermore, the miss-expression of miR-218 has been linked with renal cell carcinoma [57, 87]. Whether these miRNAs play an important role in kidney development remains unknown.

Six1 and miRNAs

The transcription factor Six1 is required for UB invasion in renal development, and is also associated with BOR syndrome [88-90]. Six1 upregulated miR-106b~25, which was shown to activate TGF-β signaling via targeting Smad7 in human breast cancer [91]. The miR-106b~25 cluster is expressed in the developing kidney [12], and is paralogous to the miR-17~92 cluster; thus, having the potential to target similar mRNAs. Six1 was also shown to be a miRNA target of miR-185 in various cancers, including Wilms’ tumor [92]. Interestingly, Six1 has been suggested to regulate c-Myc, which can also be regulated by miR-185 [92, 93].

Sox17, Wnt signaling, and miRNAs

Mutations in the HMG-box transcription factor, Sox17, are associated with CAKUT, primarily vesicoureteric reflux (VUR) [94, 95]. Sox17 functions as an antagonist of the Wnt/β-Catenin pathway via a Wnt signaling repression domain [96-99]. In embryonic stem cells, Wnt signaling increased the expression of certain miRNAs (miR-181c/338-5p/222/196a/196b/let-7e) [100]. Furthermore, overexpression of a pool of these miRNAs recapitulated the effects of Wnt activation, including increased global histone acetylation, directly affecting the Sox17 promoter [100]. These data suggest a possible Sox17-Wnt signaling feedback loop regulated by miRNAs, mediated globally through chromatin modifications.

Sox17 has also been shown to be a direct miRNA target of miR-151 and miR-141 [101, 102]. miR-141 is expressed in the developing kidney, and its downregulation was reported in nephroblastomas along with miR-200c and miR-192 [103]. Interestingly, inhibition of Sox17 by miR-141 in cell culture resulted in upregulation of downstream genes of the Wnt signaling pathway including c-Myc [102]. MiR-151 was reported to be elevated in the serum of children with nephrotic syndrome, although the significance of this observation remains unclear [104].

The Renin-Angiotensin-Aldosterone system and miRNAs

The Renin-Angiotensin-Aldosterone system plays an important role in responding to physiological cues in regulating blood pressure, and is also causally linked to CAKUT. Evidence for this first emerged when ACE inhibitors or Angiotensin Receptor Type I (AGTR1) antagonists were found to cause fetal anuria [105, 106]. Subsequently mutations in AGT, REN, ACE, and AGTR1 are linked to renal tubular dysgenesis [107, 108]. Signaling through the Renin-Angiotensin-Aldosterone system can be altered upon stress, and in such instances, results in miss-expression of miRNAs [109-111].

In a spontaneous progressive nephropathy model in rats, miR-324-3p levels were increased in association with reduced levels of prolyl endopeptidase (Prep), an enzyme involved in angiotensin metabolism [112]. Accordingly, ACE inhibition down-regulated miR-324-3p, increased Prep levels, and alleviated renal fibrosis in that model [112]. Additionally, HEK293N cells over-expressing AGTR1 had increased levels of miR-29b, -129-3p, −132, -132-3p and −212 [113]. The RAAS can also be targeted by miRNAs, where miR-802 targeted AGTR1 in the gastrointestinal tract[114].

Conclusions

The precise molecular mechanisms behind many cases of CAKUT still remain elusive. Recent work on the functional roles of miRNAs suggests that miRNAs play a role in kidney development, and that their misexpression could contribute to kidney disease and progression. In that context, miRNAs are likely to be important in fine-tuning the expression of important renal development genes, and could be downstream effectors of developmental programs themselves.

While genetic mutations have clearly been linked to a subset of patients with CAKUT, there is significant phenotypic variation amongst family members. In these instances, one possibility is that there is an as yet undetermined gene interacting with the known mutated renal development gene. Another possibility that we would propose is that mutations in genes that code for miRNAs are potential candidates for disease modification. In addition, it is possible that single nucleotide polymorphisms (SNPs) in the 3′UTR of important renal development genes alter visibility to miRNA targeting. The idea of disease-related SNPs (dSNPs) in the 3′UTRs of crucial renal development genes provides a promising mechanism linking genetics, epigenetics, and disease phenotypes [115]. For example, a polymorphism in the miR-155 binding site of AGTR1 hinders the ability of miR-155 to downregulate AGTR1 in humans, and this polymorphic allele has been associated with hypertension [116]. Alternatively, the lack of genotype-phenotype correlation could be due to interactions with environmental or stress factors. Other work has shown that miss-expression of miRNAs in response to stress can surprisingly mimic the effects due to genetic mutations [117, 118]. Accordingly, some of the studies described in this review also integrate environmental stressors as a component of these regulatory pathways.

Ultimately, the emerging information regarding how miRNAs regulate developmental pathways (and are regulated by these pathways themselves) will provide a more comprehensive view of renal development, and has the potential to provide unique insights into the underlying causes of CAKUT. This may then, in turn, lead to novel molecular tools that may direct prevention strategies, predict functional outcomes or guide interventions would be a helpful addition to the clinical management of these children.