Non-Coding RNAs in Hereditary Kidney Disorders Part 1

Abstract

Single-gene defects have been revealed to be the etiologies of many kidney diseases with the recent advances in molecular genetics. Autosomal dominant polycystic kidney disease (ADPKD), as one of the most common inherited kidney diseases, is caused by mutations of the PKD1 or PKD2 gene. Due to the complexity of the pathophysiology of cyst formation and progression, limited therapeutic options are available. The roles of noncoding RNAs in development and disease have gained widespread attention in recent years. In particular, microRNAs in promoting PKD progression have been highlighted. The dysregulated microRNAs modulate cyst growth by suppressing the expression of PKD genes and regulating cystic renal epithelial cell proliferation, mitochondrial metabolism, apoptosis, and autophagy. The antagonists of microRNAs have emerged as potential therapeutic drugs for the treatment of ADPKD. In addition, studies have also focused on microRNAs as potential biomarkers for ADPKD and other common hereditary kidney diseases, including HNF1β- associated kidney disease, Alport syndrome, congenital abnormalities of the kidney and urinary tract (CAKUT), von Hippel–Lindau (VHL) disease, and Fabry disease. This review assembles the current understanding of the non-coding RNAs, including microRNAs and long noncoding RNAs, in polycystic kidney disease and these common monogenic kidney diseases. 

Keywords

non-coding RNA; microRNA; Genetic kidney disease; PKD 

1. Introduction 

For many decades, it was initially thought that the majority of transcriptomes are mRNAs, which can translate into proteins based on the code in the mRNAs. However, only approximately 2% of human genes were identified to encode proteins, and the majority of human genes are transcribed into noncoding RNAs (ncRNAs) from the recent findings of the Encyclopedia of DNA Elements (ENCODE) Project Consortium [1]. The ENCODE project also provided evidence that noncoding genes could be pervasively transcribed to regulate protein-coding genes by forming complex regulatory networks [1]. Thus, understanding the role of ncRNAs in human diseases has become one of the most important challenges of science.

In recent years, research into the use of stem cells and a Chinese herbal remedy for the treatment of kidney diseases has gained great attention. The main mechanism of the two therapies is to promote the repair of injured renal tissues and protect the remaining renal functions. 

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The Chinese herbal remedy,cistanche, has been used in traditional Chinese medicine to treat various chronic kidney diseases since ancient times. It is reported that cistanche has the potential to reduce inflammation, reduce kidney fibrosis, and promote the synthesis of extracellular matrix components. It has been revealed that these effects are due to its bioactive components, including many phenolic substances, triterpenoids, and coumarins.

On the other hand, stem cell technology has caused a revolution in medical practice. Research has demonstrated that stem cells can differentiate into various types of renal cells and perform therapeutic activities, including protecting the remaining functional renal tissues, slowing down tissue fibrosis, and repairing damaged renal tissues.

Ultimately, the combination of traditional Chinese medicine with modern science could be the key to treating various kidney diseases. This strategy has gradually been accepted by the medical community and studies have already shown that the combined therapy of cistanche and stem cell treatment may considerably reduce the mortality rate of kidney diseases. 

In conclusion, the use of cistanche and stem cell treatment in the treatment of kidney diseases shows great potential and requires further research. The combined therapy of the two treatments could provide an improved treatment option for those facing kidney diseases.

ncRNAs are mainly classified into long ncRNAs (lncRNA s) and small ncRNAs based on their length, using a cutoff of 200 nucleotides (Figure 1) [2]. LncRNAs are longer than 200 nucleotides and comprise linear lncRNAs (named by default as lncRNAs) and circular RNAs (circRNAs). Small ncRNAs are shorter than 200 nucleotides, which include microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), transfer RNA-derived stress-induced small siRNA (siRNA), small nuclear ribonucleic acid (snRNA), small nucleolar RNA (snoRNA), repeat-associated small interfering RNA (siRNA), small Cajal body-specific RNA (scaRNA) and others. Among them, miRNAs are the most extensively investigated small ncRNAs, in which the first miRNA lin-4 was identified in Caenorhabditis elegans in 1993 [3]. MiRNAs play a critical role in the development and pathogenesis of a variety of diseases, such as cancer, diabetes, cardiovascular disease, and kidney diseases [4]. The dysregulated miRNAs are potential therapeutic targets of human diseases [5]. Given that the miRNAs in body flfluids are stable and easily detectable, and have tissue-enriched expression profiles, they have been reported to be used as potential diagnostic and prognostic biomarkers [5].

Inherited kidney diseases, consisting of monogenic and polygenic kidney disorders, have a significant risk for the development of end-stage renal disease (ESRD). Monogenic kidney disease results from a pathogenic mutation of a single causative gene (Table 1) and more monogenic nephropathy genes are continually being identified by whole exome sequencing and whole genome sequencing [6]. Approximately 450 monogenic kidney disorders account for 30% of cases of chronic kidney disease (CKD) in pediatric cohorts and 5–30% in adult cohorts [6]. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 gene and is the most common monogenic kidney disorder [7]. The cyst-lining epithelial cells of PKD are hyperproliferative and hypersecretory, which leads to progressive cyst growth and expansion, and ultimately causes ESRD [7]. Recent evidence suggests that, in addition to the underlying gene mutation, epigenetic regulators modulate cyst growth and act as potential therapeutic targets [8]. One example is the role of miRNA-mediated signaling in promoting cyst formation [9]. This review focuses on the recent advances in the understanding of the role of ncRNAs in the pathogenesis of PKD and discusses the potential application of ncRNA as therapeutic targets of PKD. In addition, this review also summarizes the findings of ncRNAs in other common monogenic kidney diseases, including HNF1β-associated kidney disease, Alport syndrome, congenital abnormalities of the kidney and urinary tract (CAKUT), von Hippel–Lindau (VHL) disease, and Fabry disease.

2. miRNAs

miRNAs, the 20- to 22-nucleotide-long RNA molecules, mainly downregulate gene expression post-transcriptionally [10]. Most miRNAs are processed by the canonical miRNA biogenesis pathway [11]. Primary miRNAs (pri-miRNAs) are transcribed from their genes by RNA polymerase II enzyme, which is then cleaved into pre-miRNAs by the microprocessor complex that consists of an RNA binding protein DiGeorge Syndrome Critical Region 8 (DGCR8) and a ribonuclease III, DROSHA [12]. The pre-miRNAs are exported out of the nucleus by an exportin 5/RanGTP complex [12]. The cytoplasmic pre-miRNAs are processed by the RNase III endonuclease Dicer, resulting in the formation of 22-nucleotide double-stranded mature miRNAs [13]. The mature miRNA duplex is loaded into the Argonaute (AGO) family of proteins in an ATP-dependent manner, to form the miRNA-induced silencing complex (miRISC) [14]. AGO selects a “guide” strand based in part on the thermodynamic stability at the 50 ends of the miRNA duplex [15]. The guide strand has a lower 5 0 stability that is preferentially loaded into AGO [15]. The unloaded strand becomes the passenger strand that is cleaved by AGO2 and degraded by cellular machinery [15]. Multiple non-canonical miRNA biogenesis pathways, including Drosha/DGCR8-independent and Dicer-independent pathways, have been elucidated. In Drosha/DGCR8-independent pathway, the pre-miRNAs resemble Dicer substrates without the cleavage of Drosha [11]. On the other hand, in the Dicer-independent pathway, miRNAs are processed by Drosha and can be loaded into AGO without the cleavage of Dicer [11]. 

The miRISC interacts with 30 UTR of target mRNAs via miRNA response elements (MRE), MREa complementary sequence that is 2–8 nucleotides located at 50 ends of miRNAs. It has been reported that miRNAs also interact with other regions, including 50 UTR, coding sequence, and gene promoter [16]. The binding of miRNA with MRE at 30 UTR of target mRNAs results in mRNA deadenylation and decapping, mRNA cleavage by activation of AGO2 endonuclease, and translation repression [17–19]. The binding of miRNAs to 50 UTR and coding regions of mRNAs downregulates the gene expression [20,21], whereas the binding of miRNAs to the promoter region has been reported to induce transcription [22].

It has been reported that miRNAs play critical roles in kidney development, maintaining homeostasis, acute kidney injury (AKI), and the progression of tubulointerstitial fibrosis [9,23,24]. Deletion of Dicer mediated by Six2-Cre in the progenitors of the nephron epithelium induces apoptosis and premature termination of nephrogenesis [25], in which deletion of Dicer in nephron progenitors induces apoptosis during kidney development that is regulated through increasing the expression of pro-apoptotic protein Bim [26]. Bim is targeted by several miRNAs, including miR-10a, miR-106b, and miR-17-5p, in nephron progenitors [26]. Loss of Dicer mediated by HoxB7-Cre in the ureteric bud epithelium also increases cell proliferation and apoptosis and disrupts ciliogenesis, which leads to the development of cysts [25]. As miRNAs are widely involved in the pathogenesis of AKI and CKD, they should have the promising diagnostic and therapeutic potential [27,28]. 

3. LncRNAs 

Unlike the extensive investigation of miRNAs over the past decade, the information regarding the function of lncRNAs is limited. lncRNAs are categorized as sense, antisense, intronic, intergenic, bidirectional, and enhancer-associated based on the location concerning protein-coding genes [29]. Growing evidence suggests that lncRNAs are central players in the epigenetic regulation of tissue homeostasis during development and disease [30,31]. lncRNAs are enriched in the nucleus and associated with chromatin remodeling complex, thereby regulating the chromatin architecture of genes either in cis (near their transcription sites), or in trans (at sites distant from their transcription site) [32]. lncRNAs also regulate the recruitment of chromatin modifiers and transcription via a variety of mechanisms [32]. IncRNAs can also be exported to the cytoplasm to regulate mRNA stability, modulate translation and interfere with posttranslational modifications [32]. More studies in the past five years have focused on the functional role of lncRNAs in kidney diseases, such as glomerular diseases, tubulointerstitial disease, kidney fibrosis, and acute kidney injury.

4. Noncoding RNA in Polycystic Kidney Disease 

4.1. miRNAs and lncRNAs in ADPKD 

Numerous miRNAs have been investigated in PKD cells and murine models, and human ADPKD. The dysregulated miRNAs modulate cyst growth and interstitial fibrosis through a variety of mechanisms, including directly repressing the expression of PKD genes, regulating cystic cell proliferation, apoptosis, and autophagy, promoting epithelial–mesenchymal transition (EMT) and inflflammation, and causing defects in mitochondrial metabolism and actin cytoskeleton (Figure 2). In the following sections, we discuss the dysregulation of different miRNAs in ADPKD (Table 2). 

4.1.1. mIR-17–92 Cluster 

miR-17–92 cluster is an evolutionarily conserved oncogenic miRNA cluster, which encodes six miRNAs (miR-17, miR-18a, miR-19a, miR-19b-1, miR20a, and miR-92a-1). Deletion of a region of chromosome 13 that includes MIR17HG, encoding human miR-17–92 cluster, causes type 2 Feingold syndrome [49]. Type 2 Feingold syndrome is autosomal dominant, and is characterized by abnormalities of fingers and toes, hearing loss, short stature, or kidney or heart abnormalities [49]. Mice with germline deletion of miR- 17–92 are perinatal lethal with lung hypoplasia and a ventricular septal defect, and a B cell maturation defect [50]. Conditional knockout of miR17–92 in nephron progenitors reduces the number of developing nephrons, which leads to albuminuria, podocyte foot process effacement, and glomerulosclerosis in adult mice [51]. However, inducible deletion of miR- 17–92 in adult mice leads to no obvious abnormalities [52], and kidney-specific knockout of miR-17–92 does not cause any changes in kidney morphology and histology [33].

It has been reported that the miR17–92 cluster is upregulated in kidneys of multiple orthologous models of PKD and human ADPKD via c-Myc which directly binds to the conserved Myc binding sites on the promoter of miR17–92 cluster [33,34]. The oncogene c-Myc is upregulated in PKD and to promote cyst progression, and c-Myc transgenic mice develop renal cysts [53,54]. Inhibition of c-Myc through targeting its upstream epigenetic regulator BRD4 via bromodomain inhibitor JQ1 slowed cyst growth in PKD mouse models [54]. Similar to c-Myc transgenic mice, the kidney-specific transgenic overexpression of miR-17–92 develops renal cysts. Conditional knockout of miR-17–92 in PKD models slows cyst growth, preserves renal function, and prolongs the survival of those mice via inhibiting cell proliferation. Furthermore, anti-miR-17 attenuates cyst growth in two PKD animal models and reduces cyst growth in vitro models of human ADPKD. Bioinformatic analysis revealed that the 30 UTR of PKD1 and PKD2 mRNA contains conserved binding sites for miR-17, and the 30 UTR of HNF-1b mRNA contains a miR-92 binding site [33,55]. The transcription factor hepatocyte nuclear factor-1β (HNF-1β) is encoded by HNF-1b, which directly regulates the transcription of PKD2 and PKHD1 [56]. The upregulated miR-17–92 negatively regulates the expression of PKD genes (PKD1, PKD2, and HNF-1b) in a posttranscriptional manner [33]. The disease severity of PKD is associated with the functional PC1 dosage which is suggested by the findings in the hypomorphic PKD1 p.R3277C mouse model [57]. Thus, the potential mechanism by which miR17–92 promotes cyst growth may be through decreasing the expression of PKD genes. 

A recent study has further identified that miR-17 is the primary pathogenic miRNA to promote cyst growth within the miR17–92 family through in vivo screening of anti-miRNAs targeting miR-17, miR-18, miR-19 or miR-25 individually [35]. Anti-miR-17 treatment slowed cyst growth in Pkd1flflox/RC: Ksp-Cre mice, a mouse model that carries a flflox allele and an R3277C mutant allele of Pkd1 gene, through regulating mitochondrial metabolism, mTOR pathway, and inflflammation [35]. Specifically, miR-17 inhibits the expression of peroxisome proliferator-activated receptor-α (PPARα) by binding to the 30 -UTR of its mRNA. miR-17 downregulated the expression of PPARα target genes in cystic kidneys, including Pparg, Ppargc1a, Sod2, Me, Oxct1, Pdk4, Etfa, Etfb, Etfdh, Cd36, Slc27a2, and Cpt2. PPARα is the key regulator of mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO), suggesting that miR-17 promotes cyst growth by affecting the mitochondrial metabolism in renal epithelial cells. These findings also indicate that miR-17-PPARα axis-mediated mitochondrial dysfunction is one of the alterations leading to the pro-proliferative metabolic reprogramming of cyst epithelia, in addition to the defective glucose metabolism and dysregulated lipid and amino acid metabolism [58–60]. Lee et al. have identified RGLS4326 by screening a chemically diverse library of anti-miR-17 oligonucleotides [61]. RGLS4326 is a single-stranded, chemically modified, short oligonucleotide with nine nucleotides that is complementary to the miR-17 seed sequence [61]. The safety of RGLS4326 is supported by the fact that no hematopoietic and renal toxicity is observed in monkeys [61]. RGLS4326 shows preferential kidney distribution and mainly presents in both proximal tubules and collecting ducts in cystic kidneys of the ADPKD mouse model [61]. Treatment with RGLS4326 suppresses cyst growth in human ADPKD models in vitro and slows cyst growth in Pkd2 conditional knockout mice (Pkd2flflox/flflox: Pkhd1-Cre) as well as Pcy/CD1 and Pcy/DBA mice which develop polycystic kidney disease with mutations in NPHP3 (nephronophthisis 3) and are used as mouse models for long-term treatment [61]. By displacing miR-17 from translationally active polysome fractions, RGLS4326 de-represses the expression of miR-17 target genes, including PKD1 and PKD2 [61]. RGLS4326 treatment also normalizes the dysregulated metabolism pathways and inhibits the pro-proliferative pathways in cystic kidneys [61]. RGLS4326 is a potential drug candidate for ADPKD, due to its safety, stability, and therapeutic efficiency in PKD models. A Phase 1b clinical trial is designed to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of RGLS4326 in patients with ADPKD (ClinicalTrials.gov identifier NCT04536688). 

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