Circular RNA in renal diseases

Abstract Circular RNA (circRNA) is a newly described type of non‐coding RNA. Active research is greatly enriching the current understanding of the expression and role of circRNA, and a large amount of evidence has implicated circRNA in the pathogenesis of certain renal diseases, such as renal cell carcinoma, acute kidney injury, diabetic nephropathy and lupus nephritis. Studies have found evidence that circRNAs regulate programmed cell death, invasion, and metastasis and serve as biomarkers in renal diseases. Recently, circRNAs were identified in exosomes secreted by the kidneys. Nevertheless, the function of circRNA in renal diseases remains ambiguous. Given that circRNAs are regulators of gene expression, they may be involved in the pathology of multiple renal diseases. Additionally, emerging evidence is showing that circulating circRNAs may serve as novel biomarkers for renal disease. In this review, we have summarized the identification, biogenesis, degradation, and functions of circRNA and have evaluated the roles of circRNA in renal diseases.

inconclusive. In this review, we introduce the relationship between circRNA and renal diseases. Research not only shows that circRNAs play critical roles in the progression of renal diseases but also that they may be used as new diagnostic biomarkers and therapeutic targets.

| CHAR AC TERIS TI C S OF CIRCRNA s 2.1 | Biogenesis of circRNAs
CircRNAs are formed by exon back-splicing and alternative splicing. Precursor messenger RNA (pre-mRNA) is canonically spliced into functional linear RNA via removing introns ( Figure 1). However, back-splicing that is resistant to spliceosomes occurs in a reversed orientation to produce a single exonic or a multi-exonic RNA molecule with a unique exon-exon junction. 12,13 The major mechanisms of back-splicing junction, including intron pairing-derived circularization in which reverse complementary motifs (RCMs) specifically located in the flanking introns lead to direct base pairing to form loops, protein-derived circularization in which a spliceosome functions via RBPs dimerization to bring the splice sites in close proximity, and lariat precursor circularization requiring at least two splicing events, are known to produce circRNAs ( Figure 1). 13,14 In contrast to linear RNA, circRNA is fairly stable and resistant to exonucleases, which enables it to be accumulated in high levels, but the level of circRNA changes during the progression of disease. 15 A study using pluripotent stem cell-derived cardiomyocytes demonstrated that senescence, chronic stress, acute stress, oxidative stress and metabolic stress dynamically up-regulated the expression of cir-cRNAs like circ-Foxo3 and circACC1. 16,17 However, the mechanism of this up-regulation is still ambiguous. These characteristics might be associated with biogenesis and potential functions.

| Detection of circRNAs
It is challenging to detect circRNA because their sequences are almost identical to the sequences of their linear cognate RNAs.
Divergent PCR typically is used to validate circRNAs by amplifying speculative back-spliced junction (BSJ) sites, but it can also amplify the linear RNA containing the same BSJ sequence locus. So far, the abundances and sizes of circRNAs were detected by northern blotting. Typically, Northern blot probes are labelled with the radioisotope 32 P. 18 Radioactive substances are potential health and environmental hazards, and they are relatively difficult to work with; therefore, non-radioactive probes labelled with digoxigenin are suitable alternatives in the use of circRNA detection. 19 However, F I G U R E 1 The biogenesis and functions of circRNAs. A, Pre-mRNA is canonically spliced into linear RNA through the removal of introns. B, CircRNAs are derived from non-canonical splicing events. Three types of circRNA are formed. C, The biogenesis of circRNAs. CircRNAs can be miRNA sponges and modulate miRNA activity. CircRNAs can bind to RBPs and affect their functions and translocations. Certain circRNA regulates transcription and encodes proteins. In addition, circRNA-derived pseudogenes can insert into the genome. D, The degradation of circRNA. CircRNAs are globally degraded by RNase L in early cellular innate immune responses. E, The elimination of circRNA. CircRNAs can be eliminated into the extracellular space by exosomes although assays have been developed to detect circRNAs, further challenges include the understanding of the physicochemical nature and mechanisms of circRNA at multiple levels and the identification of circRNA binding proteins.

| Elimination and degradation of circRNAs
To date, little is known about the elimination and degradation of cir-cRNA in cells. Emerging studies have demonstrated that circRNAs are abundant and stable in exosomes, and can be detected in blood and urine. [20][21][22] CircRNAs are eliminated into the extracellular space by exosomes that are further removed by the reticuloendothelial system or secreted by the liver and the kidney ( Figure 1). 23,24 As cir-cRNA is stable under normal conditions, the degradation of circRNA was examined by subjecting it to several stressors. It was found that the degradation of circRNA upon viral infection simulated by Poly(I:C) was triggered by the essential enzyme RNase L. 25 Based on this finding, we predict that more degradation mechanisms of cir-cRNA will be discovered in diverse cells and tissues.

| Function of circRNAs
Given that circRNA is enriched in both cells and exosomes, it is possible that it could be transported outside of cells, especially because it is highly stable in both intracellular and extracellular spaces. 26,27 However, it remains a challenge to demonstrate the functional significance of circRNA because of its sequence similarity with cognate linear RNA. Presently, some functions of circRNA have been explored. CircRNAs may modulate the expression of genes and proteins via multiple pathways ( Figure 1).

| circRNAs as miRNA sponges
Compared to circRNA, miRNA has been extremely well studied.
miRNAs are a class of non-coding RNAs that can bind to their target mRNAs to participate in regulation of their downstream signalling molecules. 28 Recently, increasing evidence is suggesting that cir-cRNAs contain binding sites of miRNA. miRNA bound by circRNA cannot bind to target mRNA and loses its capability to inhibit gene expression, resulting in an increase in mRNA. In contrast to its typical role as a miRNA sponge, circHIAT1 was shown to act as a miRNA storage device to improve the stability of mir-195-5p/29a-3p/29c-3p and partially reverse or block the migration and invasion of cancer cells. 29 Currently, there is no compelling evidence that single circR-NAs act as miRNA sponges; nevertheless, some studies have found that certain circRNAs acted as 'collective sponges' for a few miRNAs such as miR15/16 and let-7. CircRNA has several binding sites for a specific miRNA to mitigate the repression of targeted mRNA. 18 Although it has been reported that single circRNAs could be sponges for miRNA, it is considered rare because circRNAs do not typically show multiple binding sites for miRNA. 30 Therefore, further investigation must be done to elucidate the conditions that prompt circR-NAs to expose binding sites to their corresponding miRNAs.

| circRNA and RNA binding proteins
RBPs are critical for the regulation of RNA during post-transcription, and they participate in the maturity, transport, orientation and translation of RNA. An increasing number of publications have shown that RBPs such as RNA polymerase II can bind to circRNA 4 and impact circRNA splicing, folding, stabilization, processing and localization. 31 CircRNAs interact with multiple proteins and subsequently influence their modes of action. RBP genes could be transcribed to numerous circRNAs that contain binding sites for the corresponding RBP. For instance, circPABPN1 blocks HuR interaction with PABPN1 mRNA by binding to HuR, leading to a low translation rate. 5 Despite these interesting findings, the degree of detectable RBP regulation by cir-cRNA remains an unanswered question.

| Regulating transcription
In humans, some circRNAs derived from back-splicing (with retained introns) or from processed intron lariats remain in the nucleus. 3,4 Cellular localization seems to predict the potential function.

| The translation of circRNA
For many years, circRNAs were considered to be untranslatable because of no 5' 7-methylguanosine (m7G) cap and 3' poly(A) tail.
However, recent studies have indicated that a sub-fraction of endogenous circular RNAs can be translated without the 5' 7-methylguanosine (m7G) cap. For example, circ-ZNF609, which is related to heavy polysomes, was translated in a cap-independent way. 32 In fruit fly brains, some circRNAs are translatable by showing that the UTR structures of ribo-circRNAs (cUTRs) allow for translation without the cap. 33 Modification of N 6 -methyladenosine (m6A) RNA effectively triggers the translation of circRNAs into proteins in human cells. Consensus m6A motifs are enriched in circRNAs, and a single m6A site is sufficient to drive translation initiation. 34 It is controversial whether circRNA can be directly translated. For example, circRNAs that were 126 nucleotides in length were effectively translated into proteins in Escherichia coli cells. 35 Although more evidence in support of circRNA translation is needed, translation may possibly occur. In addition, it remains to be investigated whether the encoded polypeptides derived from circRNAs serve a physiological function.

| circRNA-derived pseudogenes
It is well known that the pseudogenes derived from linear mRNAs can be retrotranscribed and integrated into host genomes. Recently, a study demonstrated that retrotranscription could also occur with cir-cRNAs and that the processed pseudogenes were eventually inserted back into the host genome. 36 The identification of circRNA-derived pseudogenes suggests a novel function of circRNA: altering genomic DNA composition by inserting its retrotranscription product. Despite these findings, the function and mechanism of circRNA-derived pseudogenes remain a mystery.

| CIRCRNA AND RENAL D IS E A S E S
CircRNA expression profiles in renal diseases such as renal cell carcinoma (RCC), diabetic nephropathy (DN), acute kidney injury (AKI) and lupus nephritis (LN) have been reported. 9,11,37,38 In addition, accumulating evidence has shown that circRNAs are involved in modulating inflammation, cell death and fibrosis. [39][40][41] Based on these findings, circRNAs are promising therapeutic targets in renal diseases and should be intensely further investigated.

| Dysfunction of circRNA in renal cell carcinoma
Increasing evidence indicates that circRNAs participate in carcinogenesis, including RCC. Substantial progress has been made in the identification of differential expression of circRNAs in RCC. The interactions between circRNAs, miRNAs and genes are complex.
A previous study obtained RNA microarray data from clear cell RCC (ccRCC) tissues and used control sample data from the Gene Expression Omnibus and The Cancer Genome Atlas to analyse the relationship between circRNAs, miRNAs and genes in RCC. The results revealed that 324 circRNAs were down-regulated, whereas 218 circRNAs were up-regulated in RCC. In addition, a circRNA-miRNA-mRNA interaction network was constructed. 42

| Promising biomarkers for the diagnosis of renal cell carcinoma
CircRNAs are involved in controlling cell proliferation and have also been implicated in cancer development; they play critical roles in carcinoma progression including metastasis, invasion and drug tolerance. 6,21 RCC originating in renal tubular epithelial cells is common and causes a large number of deaths worldwide. Given the stability of circRNA, researchers are focusing on these molecules as potential biomarkers for the diagnosis and pathology of renal cancer. It was found that HHLA2 and circ-ABCB10 were significantly up-regulated in ccRCC tissues, and correlated with short overall survival and a poor prognosis. 43,44 Downregulation of Hsa-circ-0001451, which conspicuously increased RCC proliferation, indicated that its levels were highly correlated with RCC differentiation. 45 CircRNA microarray analysis in ccRCC tissues and cells has indicated that significantly up-regulated levels of circPCNXL2 are associated with poor overall survival of ccRCC patients. 8 This evidence suggests that circRNAs may be considered robust biomarkers.

| Potential therapeutic targets of renal cell carcinoma
The functions of other circRNAs have been recently investigated.
The possibility of an association of DEmRNA with RCC onset and progression was indicated by GO and KEGG pathway analysis. 47 One study focused on the effects of ionizing radiation on human embryonic kidney (HEK) 293T cells and verified that 158 circRNAs were significantly differentially expressed after ionizing radiation exposure. 48 Furthermore, cancer-specific alternative splicing of circRNA in ccRCC has been identified; 4498 circRNA alternative splicing events were detected in the study. 49 The function of circ-AKT3 was confirmed to suppress ccRCC metastasis by increasing E-cadherin expression via competitively binding miR-296-3p. 50 CircPCNXL2, acting as an miRNA sponge, bound to miR-153 to modulate ZEB2 expression in ccRCC. 8 Furthermore, circ-ZNF609 was significantly upregulated in RCC, which regulated FOXP4 expression by sponging miR-138-5p to promote RCC cell growth and invasion. 37 CircC3P1 was shown to restrain RCC activity by regulating the miR-21/PTEN axis. 51 Based on these findings, circRNAs could have potential applications as modulators or prognostic biomarkers for RCC (Table 1).

| CircRNA and diabetic nephropathy
Diabetic nephropathy (DN) is pathologically characterized by albuminuria, glomerular hypertrophy, autophagy, hypertrophy of mesangial cells and glomerular basement membrane thickening. 52,53 The prominent inflammation, observed in both the beginning and ongoing stages of kidney injury, contributes to the pathogenesis of DN. 54,55 High glucose, AGEs and oxidative stress could simultaneously induce the activation of NF-κB to cause inflammation. 56 Glomerular hypertrophy is associated with increases in extracellular matrix (ECM) proteins, leading to later irreversible deterioration, such as interstitial fibrosis. 57 Recent evidence suggests that activation of Wnt/β-catenin signalling in DN plays a pivotal role in driving renal fibrosis. 58 Besides the non-enzymatic glycation of proteins to form advanced glycation end products (AGEs), growth factors, such as TGF-β1, could induce persistent inflammation and fibrosis to accelerate the progression of DN by activating downstream Smaddependent or Smad-independent pathways. 7,58 Taken together, these results indicate that renal fibrosis is closely connected to renal tissue inflammation, and an excessive inflammatory response and fibrogenesis are typical features in DN (Figure 2A).
Emerging evidence indicates the role of circRNAs in DN. For instance, a recent study confirmed that circRNA-15698 is highly expressed in both DN mice and mouse mesangial cells. When exposed to high glucose levels, the levels of fibrosis-related proteins, such as collagen type I (Col. I), and Col. IV were significantly increased.
The knockdown of circRNA-15698 by small interfering RNAs (siR-NAs) led to a significant decrease in these fibrosis-related proteins.
Mechanistically, circRNA-15698 acted as an miR-185 sponge, increasing TGF-β1 protein levels and stimulating ECM-related protein synthesis in DN. 11 Furthermore, one recent study suggested that up-regulated circLRP6 modulated the mesangial cell injury by  (Table 1).
Although the roles of circRNA in inflammation and fibrosis in DN remain to be discovered, their roles in regulating inflammation and fibrosis in other organs or tissues, including the liver, lungs, heart and blood vessels, have been demonstrated ( Figure 2B). Considering their targets, such as TGF-β1, also function in DN, these studies may act as references for the future study of DN. [60][61][62][63][64] For example, cir-cHIPK3 expression was significantly up-regulated in diabetic retinas and, acting as an endogenous miR-30a-3p sponge, led to increased vascular endothelial growth factor-C and Wnt2 expression, which contributed to inflammation and vascular dysfunction. 65 TGF-βmediated induction of circActa2 promotes α-SMA protein expression via sponging of miR-548f-5p in vascular smooth muscle cells. 66 CircRNA CDR1 was found to act as an miR-7 sponge to up-regulate TGFBR2, which primarily functions during pulmonary fibrosis progression by stimulating the process of epithelial-mesenchymal  CircRNA microarray analysis showed that 113 up-regulated and 94

F I G U R E 2
CircRNA-mediated fibrosis and inflammation in other organs. A, Inflammation promotes progressive renal fibrosis. When renal injury occurs, circulating immune cells are recruited to the kidney and activate intrinsic kidney cells such as podocytes, which produces tissue damage factors such as cytokines, growth factors, and reactive oxygen species leading to myofibroblast accumulation and ECM production. B, CircRNA-mediated fibrosis and inflammation in other organs or tissues. CircRNA-mediated fibrosis has been identified in several organs such as the liver, heart, lungs, retina and vascular intima down-regulated circRNAs were detected in plasma samples derived from SLE patients. 67 Emerging evidence indicates that circRNAs are involved in the pathophysiology of SLE. 68,69 Collectively, these data suggest that circRNAs could be used as potential biomarkers or therapeutic targets for SLE.
Although SLE is an autoimmune disease that could damage any organ, it commonly attacks the kidney. LN is a primary cause of morbidity and mortality in SLE. 70  A positive correlation between miR-150 and renal chronicity index was found in LN patients, while circHLA-C seems to be negatively correlated with miR-150. 10 In another study, miR-150 attenuated expression of the antifibrotic protein suppressor of cytokine signalling 1 (SOCS1) and promoted renal fibrosis in LN. 72 In addition, the cir-cRNA-002453 level was highly elevated in plasma derived from LN patients compared to SLE patients without LN, rheumatoid arthritis patients and healthy controls. In particular, its expression showed a positive correlation with 24-hour proteinuria and renal SLE disease activity index scores (Table 1). 38 Recently, a bioinformatic analysis showed that high hsa_circ_0000479 levels were correlated with low albumin levels, positive urine protein and low haemoglobin, which indicated that it might be involved in SLE-associated renal injury. 73 Although evidence of the molecular mechanisms of circRNAs in LN is currently lacking, future investigations will identify and illuminate the functions of circRNAs in LN and eventually contribute to its diagnosis and treatment.

| CircRNA and acute kidney injury
AKI, characterized as a rapid decline in renal function, is a severe complication in critically ill patients and has been identified as an independent risk factor concerning survival. 74,75 Accumulating evidence shows that the major pathological features of AKI include oxidative stress, inflammation and programmed cell death of renal tubular epithelial cells. 76,77 Many key pathways or modulators, such as the NF-κB-mediated pro-inflammatory pathway, the caspase-correlated apoptosis pathway and TGF-β/Smad signalling, are involved in the pathophysiological process of AKI. 78,79 Unfortunately, specific therapy for AKI is still lacking. 74 Recently, circRNAs have become the focus of research, as they might have potential in the diagnosis, as well as the treatment, of AKI.
CircRNAs were dysregulated in types of AKI models induced by nephrotoxic agents and LPS. Differentially expressed circRNAs have been associated with various biological processes, including cellular component organization and biogenesis, localization, and other processes during AKI. RNA-Seq results demonstrated that 1664 circRNAs were significantly highly expressed and 474 cir-cRNAs were uniquely expressed in the kidney. 80 Thirty-four up-regulated and 22 down-regulated circRNAs were verified in mouse kidneys of IR-induced AKI models. 9 Three hundred and sixty-eight circRNAs were found to be differentially expressed in response to cisplatin-induced AKI. 81 Additionally, the study revealed that the expression of 38 circRNAs was significantly dysregulated in contrast-induced AKI. 82 The expression of circ-Dnmt3a, circ-Akt3, circ-Plekha7 and circ-Me1 was in the top 20 most significantly differentially expressed circRNAs, and was predicted to be associated with PI3K-Akt signalling in AKI. 40 Serum circR-126, which may act as a biomarker for predicting the mortality of AKI via miR-126-5p sponging, was elevated in AKI patients. 83 CircANRIL promoted inflammation and apoptosis of HK2 cells treated with LPS, via miR-9/NF-κB pathways. 84 Moreover, the functions of circRNAs in modulating programmed cell death and cell cycle progression indicate that they could be novel potential regulators of AKI. 21 For instance, mmu-circRNA015947 interacted with miRNAs to induce downstream gene expression and, as a result, participated in apoptosis-correlated pathways, which are involved in the pathogenesis of IR injury. 85 Furthermore, by modulating miR-671, circRar1 induced the transcriptional activity of apoptosis-linked factors, such as caspase-8, in lead-induced neurotoxicity. 86 CircRNAs demonstrate great promise as disease biomarkers due to high resistance to exonucleases, and they might even be highly accumulated. Their putative function as miRNA sponges makes them particularly interesting therapeutic targets for future research.
Despite these findings, the understanding of the function of circRNA in AKI is at a rather early stage, and further investigation is required to explore the modulation of circRNAs during AKI progression, including during injury, repair and AKI-to-chronic kidney disease (CKD) transition, and to verify circRNAs that are specifically expressed in the kidney. Such circRNAs could be used as biomarkers for the diagnosis of AKI (Table 1). Whether they might serve as therapeutic targets will no doubt be the patient of future research.

| CircRNA and kidney calculi
Kidney calculi are common chronic renal disease, and approximately 1 in 17 Chinese adults suffer from this illness. 87 Patients with kidney calculi generally present with pain and urinary tract infections that can result in the gradual loss of kidney function. 88 A recent study found that 145 circRNAs, including 58 up-regulated circRNAs, were differentially expressed in urolithiatic rat kidneys. 89 Rno-miR-138-5p and rno-miR-672-5p, which are co-expressed with miRNAs, have been proven to be involved in kidney calculi. 89 Calcium phosphate crystals are abnormally deposited in the vessel walls, leading to vascular calcification, a common complication of CKD. This study showed that circSamd4a exerts an anti-calcification role, via functioning as an miRNA sponge in a vascular calcification model, to reduce the complications of CKD. 90 To date, although information regarding the roles of circRNAs in kidney calculi is scarce, these findings present a novel perspective on the potential action of circRNA in kidney calculi progression.

| CircRNA and hypertensive nephropathy
Hypertension is a common chronic disease that presents with high arterial pressure and may be accompanied by pathological changes in vital organs such as the heart, kidney and blood vessels. Evidence shows that certain circRNAs play a role in the pathological develop- were elevated in both hypertensive nephropathy patients and angiotensin II-infused mice, and resulted in renal inflammation and fibrosis by acting on NF-κB/p65. 94 So far, no reference to circRNA sponging of miR-103a-3p in kidney diseases has been reported, but the down-regulation of miR-103a-3p by circTCF25, and the promotion of proliferation and migration have been reported in bladder cancer. 95 Circulating miR-103a-3p levels are tightly linked to hypertensive nephropathy 94 ; therefore, focusing on its corresponding circRNA might be a novel therapy target for hypertensive nephropathy.

| CircRNA and idiopathic membranous nephropathy
Idiopathic membranous nephropathy (IMN), regarded as an organ-specific autoimmune disease, is one of the major causes of nephritic syndrome in adults and is an important factor in the recurrence of nephritic syndrome in patients after renal transplantation. 96 As the pathogenesis of the disease is not fully understood, and because few sensitive biomarkers have been found that reflect disease activity, an effective treatment for IMN is lacking in modern medicine. 97 It has recently been found that circRNAs may play a function in the occurrence and development of IMN.

| CON CLUS ION
The exploration of abundant circRNAs has expanded our understanding of the diversity of gene expression. Differently expressed circRNAs have been observed in a variety of normal tissues and diseases, which indicates that circRNAs may have vital physiological and pathological function. 99 CircRNAs play key roles in gene expression regulation and biological processes, mainly through the regulation of miRNA and target gene expression. 2 In addition, circRNA-derived pseudogenes have been found to integrate into genomic DNA and alter its composition. 36 However, knowledge about the functions and mechanisms of circRNA-derived pseudogenes is limited, and more work should be done in the future. In addition, circRNA have been identified as novel therapeutic targets and biomarkers for renal disease. Future studies should confirm the exact mechanisms through which circRNAs interact with specific proteins and other non-coding RNAs to mediate their effects. Recently, the mechanism of circRNA degradation was revealed by treating it with a number of stressors. 25 Despite recent discoveries in circRNA biogenesis and function, many challenges remain to be overcome. For example, few technologies exist for the investigation of the physicochemical properties and mechanisms of circRNAs. Moreover, we lack an understanding of how to further analyse kidney-derived samples to verify circRNAs related to renal disease. Though many circRNAs have been confirmed in kidney tissue, their expression profiles and potential roles during disease development and its inhibition or stimulation remain largely unknown. Increasing exploration into the predictive roles and functions of circRNAs in renal diseases, especially testing the therapeutic potential of circRNAs in animal models, will contribute to the better understanding of the pathophysiological and physiological processes of the kidney, and this will hopefully become an intense area of research.

ACK N OWLED G EM ENTS
This work is supported by Natural Science Foundation of Colleges and

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.