MicroRNAs as Biomarkers and Therapeutic Targets for Acute Kidney Injury

Acute kidney injury (AKI) is a clinical syndrome where a rapid decrease in kidney function and/or urine output is observed, which may result in the imbalance of water, electrolytes and acid base. It is associated with poor prognosis and prolonged hospitalization. Therefore, an early diagnosis and treatment to avoid the severe AKI stage are important. While several biomarkers, such as urinary L-FABP and NGAL, can be clinically useful, there is still no gold standard for the early detection of AKI and there are limited therapeutic options against AKI. miRNAs are non-coding and single-stranded RNAs that silence their target genes in the post-transcriptional process and are involved in a wide range of biological processes. Recent accumulated evidence has revealed that miRNAs may be potential biomarkers and therapeutic targets for AKI. In this review article, we summarize the current knowledge about miRNAs as promising biomarkers and potential therapeutic targets for AKI, as well as the challenges in their clinical use.


Introduction
Acute kidney injury (AKI) is a clinical syndrome where a rapid decrease in kidney function and/or urine output is observed, which may result in the imbalance of water, electrolytes, and acid base, due to a variety of causes, including sepsis, major surgery, hypovolemia, drug toxicity, urinary tract obstruction, and rhabdomyolysis [1].It is reported that the prevalence of AKI is 23.2 % for inpatients [2] and 57.3% in intensive care unit (ICU) patients [3].The mortality of AKI is 4.9% in all AKI, 3.4 % in stage 1, 7.5% in stage 2, 13.2% in stage 3, and 24.1% in dialysis-dependent patients [2].AKI, which in outpatients is about 70% pre-renal and in inpatients is 55-60% intra-renal, is mostly induced either by acute tubular necrosis (ATN) caused by ischemia due to sepsis, or by NSAIDs, antibiotics, cisplatin or contrast agents [4].In addition, anti-cancer agents in current development increase drug-induced AKI.For example, an immune checkpoint inhibitor may cause tubulointerstitial nephritis [5], and a vascular endothelial growth factor (VEGF) inhibitor may cause thrombotic microangiopathy (TMA) [6].In addition, patients with AKI tend to require extended hospitalization, leading to a significant financial burden.Therefore, an early diagnosis and treatment to avoid the severe AKI stage would be important.For the purpose of an early diagnosis and the establishment of common diagnostic criteria, the concept of AKI was distinguished from acute renal failure (ARF) [7].Subsequently, several criteria for classifying AKI were developed: risk, injury, failure, loss of kidney function, and end-stage kidney disease (RIFLE); the acute kidney injury network (AKIN); and the kidney disease improving global outcomes (KDIGO) [1].In these guidelines, increases in serum creatinine (s-Cr) and/or decreases in urine output were the important criteria.An early diagnosis may not always be easy according to the guidelines, partly because s-Cr does not reflect direct tubular injury; thus, biomarkers over s-Cr and urine output would be required as biomarkers for AKI.Several biomarkers are reported for AKI, including neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), L-type fatty acid-binding protein (L-FABP), cystatin C, Clusterin, interleukin-18 (IL-18), Proenkephalin A 119-159 (Penkid), the product of insulin-like growth factor binding protein 7 (IGFBP-7), and the tissue inhibitor of metalloproteinase 2 (TIMP-2).However, there is still no gold standard for the early detection of AKI [8,9].
The long-term outcome is also problematic since patients who develop AKI do not necessarily experience a complete recovery of renal function; it may instead lead either to the development of chronic kidney disease (CKD) or to an exacerbation of the rate of progression of preexisting CKD or irreversible ESRD [10].Kidney cells have the potential to regenerate after AKI [11].The replacements of detached tubular cells have been analyzed for decades.There are three candidate cell types for regeneration: mesenchymal stem cells (MSCs), kidney stem cells, and remaining tubular cells.It is reported that bone-marrowderived MSCs transfer to the injured kidneys, differentiate into tubular cells, and become replacements [12].However, recent gene-tracking systems reveal that replacements by MSCs are less than 1%.Instead, MSCs have an important role in regeneration due to their paracrine effects [13,14].Adult kidney stem cells have also been explored, and several research groups have identified the role of kidney stem cells in regeneration [15][16][17].On the other hand, gene-tracking systems reveal that a majority of regenerative cells after AKI were tubular cells that had survived via de-differentiation and gained the stem cell phenotype [18,19].Further exploration of regenerative mechanisms may lead to a novel therapy for AKI, and microRNAs (miRNAs) are among the important candidates for that.
miRNAs are non-coding and single-stranded RNAs of 19-23 nucleotides [20].miRNAs silence their target genes in the post-transcriptional process through 3 -UTR binding.It is also known that miRNAs are involved in a wide range of biological processes, including development, differentiation, cell proliferation, apoptosis, cancer metastasis, inflammation, and fibrosis [20].miRNAs were discovered in 1993 [21] and confirmed to exist in mammals in 2000 [22].Currently, more than 2000 types of human miRNAs are registered in miRBase, a database of pre-miRNAs and miRNAs.It is reported that miRNAs work even if they are not necessarily complementary except for 2-8 on the 5 side [23,24].It is estimated that more than 60% of human translated genes have at least one conserved miRNA-binding site [25].It is also suggested that one miRNA has more than one hundred target mRNAs and controls various regulations [26].Recently accumulated evidence reveals that miRNAs may be the potential biomarkers as well as therapeutic targets for AKI and AKI-CKD transition.In this review article, we summarize the current knowledge about miRNAs as promising biomarkers and potential therapeutic targets for AKI.

miRNA Production and Dynamics
In the maturation of miRNAs, most primary miRNA transcripts (pri-miRNAs) are transcribed by RNA polymerase II, followed by cleavage by the microprocessor complex Drosha-DiGeorge syndrome critical region 8 (DGCr8) to precursor miRNAs (pre-miRNAs) [27].After moving to the cytoplasm from the nucleus, the pre-miRNAs are cleaved by Dicer and form a miRNA duplex.The miRNA duplex interacts with Argonaute (AGO) proteins and forms a RNA-induced silencing complex (RISC), finally forming a mature single miRNA while the other strand is degraded.The mature miRNAs target mRNAs in the cytosol, leading to the inhibition of protein translation or mRNA degradation [28].miRNAs can also be sorted into extracellular vesicles (EVs), such as exosomes, macrovesicles, and apoptotic bodies.These EVs may be secreted, circulating in the blood or urine, transferring into the recipient cells, and acting on them [28].miRNAs also secrete RNA-binding proteins, such as high-density lipoprotein (HDL) and AGO2.These miR-NAs in the blood are called circulating miRNAs [28].Especially in cancer research, these circulating miRNAs have been reported to be important biomarkers in liquid biopsy [29].
In kidney diseases, miRNAs in serum, plasma, and urine, as well as in kidney tissue, have been explored as potential biomarkers, for example, for nephrotic syndrome, IgA nephropathy, hypertensive nephropathy, diabetic nephropathy, CKD, and AKI [30].In addition, miRNAs are also explored as therapeutic targets for kidney diseases, including AKI.

Overview of Biomarkers in AKI
Under AKI, the decrease in the glomerular filtration rate leads to an increase in s-Cr after a delay of 24 to 48 h [31].A renal tubular disorder marker that precedes the s-Cr is thought to be useful for the early diagnosis of AKI.Several tubular injury markers have been reported as AKI biomarkers, including KIM-1, L-FABP, IL-18, NAG, and NGAL, which could indicate kidney damage prior to the increase in s-Cr [32,33].In addition, cystatin C, clusterin, and penkid have also been reported as the potential biomarkers [34,35].Among these, NGAL was reported as one of the fastest markers for detecting tubular injury, particularly in the distal tubular segments [34].In humans, elevated NGAL levels can be observed within 3 h after tubular injury and peak around 6-12 h.It is reported that IL-18 levels rise around 6 h after kidney damage and peak between 12 and 18 h [34].It is also reported that urinary L-FABP may be elevated 2 h postoperatively in AKI patients, suggesting that the 2 h postoperative urinary L-FABP may predict AKI [34].It is also reported that G1 cell cycle arrest markers, TIMP-2, and IGFBP7 expressions are increased during the early phase of cellular stress or injury.Indeed, the combination of TIMP2 and IGFBP7 ([TIMP-2] × [IGFBP7]) was reported as an accurate indicator for identifying the early phase of AKI [36].In spite of these advances in early AKI diagnosis, they are not widely applied in clinical practice.Therefore, novel biomarkers would still be desirable for the early AKI detection and prediction of its severity as well as for the differentiation of AKI etiologies, such as ischemic, drug-induced, contrast-induced, and septic.From this point of view, miRNAs would be potent and novel biomarkers to solve these problems.

miRNAs as Biomarkers for Contrast-Induced AKI
As biomarkers for contrast-induced AKI, plasma levels of miR-30a, miR-30c, and miR-30e showed > two-fold increase in patients with contrast-induced nephropathy (CIN) [47].Another study also indicated increased plasma levels of miR-30a and miR-30b as well as miR-188 in patients with CIN [48], suggesting a specific increase in the plasma miR-30 family in CIN.

miRNAs as Biomarkers for Sepsis-Induced AKI
As biomarkers for sepsis-induced AKI, serum levels of miR-29a and miR-10a-5p were increased in patients with sepsis-induced AKI, and these miRNAs were also detected as the predicting marker for 28-day survival [49].Urine miR-26b was also reported to be increased in patients with sepsis-induced AKI and was associated with mortality [50].Increased plasma miR-494 was also reported to predict 28-day survival in patients with sepsis-induced AKI [51].Serum miR-21 was also up-regulated in patients with sepsis-induced AKI [52].It was also reported that serum and urine miR-22-3p was down-regulated in sepsis-induced AKI patients, and may serve as a biomarker to predict 28-day survival [53].Regarding the early detection of AKI, increases in serum and urine levels of miR-452 in patients with sepsis-induced AKI were reported, where the sensitivity of urine miR-452 was higher than urine [TIMP-2] × [IGFBP7] [54].

miRNAs as Biomarkers for AKI Caused by Cardiac Surgery
As biomarkers for AKI caused by cardiac surgery, urine miR-30c-5p as well as miR-192-5p were increased in patients with AKI after cardiac surgery as early as 2 h post operation, suggesting a possible biomarker for predicting AKI after cardiac surgery [55].Similarly, plasma miR-192 was also increased in patients with AKI at the time of ICU admission, remaining stable for 2 h and decreasing after 24 h, suggesting a time-dependent change in miR-192 [56].Levels of serum, plasma and urine miR-21 were increased in patients with AKI after cardiac surgery [57,58].In addition, urine and plasma miR-21 levels also predicted the need for post-operative renal replacement therapy, 30-day in-hospital mortality, and prolonged stay in hospital or ICU [58].Serum miR-494 was up-regulated in child patients with AKI after cardiopulmonary bypass for congenital heart disease.In the report, the combination of NGAL, Kim-1, and miR-494 showed the high area under the curve (AUC) to predict the death of children with postoperative AKI [59].On the other hand, decreased miR-21 levels in patients with AKI after cardiac surgery were also reported [60], where reduced post-operative serum and urine miR-21 levels could predict AKI development.In addition, it is also reported that baseline miR-21 before cardiac surgery could predict AKI development [61].These results may reflect the complex regulation of miR-21 under AKI.

Summary of miRNAs as Biomarkers for AKI
In summary, there are several promising miRNAs that could serve as possible biomarkers for early diagnosis, prediction of mortality, and the specific pathology in AKI.Nevertheless, there are several limitations and problems relating to clinical use.For example, several types of samples, such as serum, plasma, urine, and exosomal miRNAs, may be used for the analysis.Indeed, it is reported that serum and plasma may yield result differences.In addition, the collecting methods of miRNAs are different between reports, which may be one cause of the differences between the reports.Since miRNA volume is very limited, the methods of analysis and collection of EV may affect the results.Therefore, clinical procedures still need exploration.growth factors [14].In addition, MSCs secrete extracellular vesicles containing soluble proteins, mRNAs, and miRNAs [13].These factors transfer to the recipient cells, which mediate renoprotection (anti-apoptosis, anti-nectrosis, anti-inflammation, anti-oxidative stress, and anti-fibrosis) and regeneration (cell proliferation, cell migration, tubular de-differentiation, and angiogenesis) [67].It is likely that extracellular vesicles are the predominant paracrine effects in AKI [67].Which factors provide the dominant therapeutic effects?It was reported that EVs derived from MSCs with knockdown of Drosha, essential for miRNA production, failed to ameliorate I/R-induced AKI, while MSC-derived EVs without knockdown ameliorated AKI [68].These data suggest that miRNAs in EVs might be the most important factors for renal protection and regeneration in AKI.Renoprotective miRNAs from MSCs were previously reported [67].In the report, miR-21 and miR-30 mediated anti-apoptosis, miR-210 mediated angionegesis, miR-145 mediated autophagy, miR-15 and miR-16 ameliorated kidney fibrosis, and miR-15, miR-16, miR-21, and let-7 ameliorated inflammation through the regulation of macrophage.In addition to MSCs, secreted factors from other stem cells, such as kidney stem cells and iPS-derived nephron progenitor cells, have also been reported to be renoprotective [14,64,65].These trophic effects might be at least partly delivered via miRNAs.miRNAs from other cells, such as circulating inflammatory cells and tubular cells, might be involved in kidney damage and/or regeneration during AKI.
Researchers still need to explore miRNA dynamics.Nevertheless, miRNAs might be novel therapeutic targets for AKI.
In addition, a hypoxia-inducible factor (HIF)-prolyl hydroxylase (HIF-PHD) inhibitor has been developed as a therapy for renal anemia [69].The pharmacological activation of HIF regulates a variety of genes, including Epo, leading to hematopoiesis.Other than hematopoiesis, the HIF-PHD inhibitor has also been shown to ameliorate AKI in rodent experimental models [70], including I/R-induced and drug-induced models with cisplatin, gentamicin, and lipopolysaccharide (LPS).These protective mechanisms include antiapoptosis via miR-21, anti-inflammation by macrophage reduction, reduced VCAM1, up-regulation of angiogenesis via VEGF up-regulation, and anti-oxidative stress via upregulation of Heme Oxygenase 1 (HO-1) [70].As shown with increases in miR-21, these effects may be mediated at least partly via regulation of miRNAs.Indeed, it is reported that kidney ischemia activates HIF-1α, which in turn up-regulates miR-21, leading to antiapoptosis through the suppression of pro-apoptotic factor programmed cell death protein 4 (Pdcd4) and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) [71].HIF1 also increases miR-668 expression, which targets mitochondrial fission process protein 1 (MTP18), leading to the protection of kidney tubular cells via mitochondrial dynamics under ischemic AKI in humans and mice [72].Activation of HIF also increases miR-489, leading to anti-apoptosis in kidney tubular cells during ischemic AKI though targeting repair sensor poly(ADP-ribose) polymerase 1 (PARP1) [73].Taken together, these data suggest that the regulation of miRNA might be a novel and specific therapy against AKI.4.2.Therapeutic Targeting of miRNAs for AKI 4.2.1.Overview of Therapeutic miRNA Target for AKI Some of the most important evidence regarding miRNAs in AKI was reported in 2010, where knockouts of tubular miRNAs were analyzed in the rodent model [74].The loxp-cre system was used to produce mice lacking Dicer, a key enzyme for miRNA production, predominantly in kidney proximal tubular cells by crossing with phosphoenolpyruvate carboxykinase (PEPCK)-cre mice.The mice showed global down-regulation of miRNAs in the kidney cortex and had normal kidney function and histology under normal conditions.Importantly, the mice were resistant to renal ischemia-reperfusion (I/R) injury, demonstrating the involvement of miRNAs under AKI.Since then, there has been accumulating evidence supporting miRNAs as potential therapeutic targets in AKI.Several miRNAs have been reported to have protective and/or pathogenic roles in AKI, regulating tubular apoptosis, tubulointerstitial fibrosis, and inflammation in a variety of etiologies of AKI, including ischemia, drug, and sepsis.Some miRNAs have common gene targets.For example, miR-30 and miR-26a target Snai1, regulating epithelial-mesenchymal transition (EMT), while miR-21, miR-17, miR-188, and miR-378 target PTEN, which is implicated in cell apoptosis, proliferation, inflammation, and fibrosis [27].The potential therapeutic targets of miRNAs for AKI are summarized in Table 2.

miRNAs with Both Protective and Pathogenic Effects for AKI
miR-21 is one of the most analyzed miRNAs, described as having double-edged-sword effects in kidneys [67] and both protective and pathogenic effects in kidney diseases.As a protective effect, miR-21 ameliorates I/R-induced AKI by inhibiting tubular cell apoptosis in I/R-and LPS-induced AKI mice [71,125], targeting PTEN/Akt/mammalian target of rapamycin (mTOR) signaling and Cyclin-dependent kinase 6 (CDK6).miR-21 is also shown to inhibit maturation of dendritic cells through the PDCD4/ NFκ-B pathway [71] and CCR7 [126], thereby mediating anti-inflammatory effects in I/R-induced AKI mice.In addition, miR-21 is also shown to target mitogen-activated protein kinase kinase 3 (MKK3), inhibiting the downstream factors IL-6 and TNF-α levels, mediating anti-inflammatory effects [127].On the other hand, as a pathogenic effect, miR-21 inhibits autophagy in I/R-AKI rats by targeting Rab11a [128].In addition, long-term elevation of miR-21 might promote kidney fibrosis, including via PPARα [129].Furthermore, miR-21-3p was shown to regulate energy metabolism via AKT/Cyclin-dependent kinase 2 (CDK2)-FOXO1 in a sepsis-induced rat AKI model, while it was unclear whether the regulation was protective or harmful for a long-term prognosis [130].Taken together, these data suggest that miR-21 may target several signaling pathways, involving cell apoptosis, inflammation, and autophagy, as well as energy metabolism under AKI.

Therapy Targeting miRNAs
Nucleic acid drugs can treat adults at the level of RNA.They are usually composed of oligonucleic acids linked by ten to several tens of bases, and act directly on the body without gene expression [151,152].Nucleic acid drugs include antisense, siRNA, miRNA, decoy, aptamer, and CpG oligodeoxynucleotides [153].Among these, two approaches can be used for targeting miRNAs: antisense for inhibition-specific miRNA and miRNA-mimic injection.Clinical trials targeting miRNAs as nucleic acid medicine are currently still limited.Anti-miR was first conducted with miR-122 with locked nucleic acid in the form of Miravirsen for treating type C hepatitis [154].Subsequently, antisense miR-155 (MRG-106) for T-cell lymphoma and mycosis fungoides was tested in a clinical trial [155].A miR-10b antisense was used in preclinical trials with dextran-coated iron oxide nanoparticles for multiple cancers [156][157][158][159]. miRNA mimic treatment was conducted with miR-34 in lipid nanoparticles in the form of MRX34 for targeting multiple cancers, including hepatic cancers [160][161][162].Subsequently, a clinical trial (Phase 1) with miR-16 mimics (TargomiR) was conducted for mesothelioma and lung cancer using the bacterial minicell EnGeneIC delivery system [163].
Currently, treatment targeting miRNAs in kidney diseases in the clinical stage is limited.A phase 2 placebo-controlled randomized controlled trial for Alport syndrome (NCT02855268) using oligonucleotides of miR-21 is currently ongoing [164], where the efficacy for the progression of kidney dysfunction, as well as the safety, pharmacodynamics, and pharmacokinetics, are being evaluated.A phase 1 clinical trial in polycystic kidney disease (PKD) patients using anti-miR-17 oligonucleotide RGLS4326 is also being conducted (NCT04536688) [165], where the primary objective is to assess the dose-response relationship between RGLS4326 and biomarkers of PKD.Referring to the miRNA treatment for AKI, miR-5100 was recently detected as a potential AKI biomarker as well as a therapy target [45].miR-5100 was down-regulated in a rodent AKI model, and a miR-5100 mimic ameliorated I/R-induced AKI.In addition, in human serum samples, miR-5100 was down-regulated; thus, it is possibly both a biomarker and a therapy target.
In spite of these potentials of miRNAs as the therapeutic targets under AKI, there are several limitations and problems relating to clinical use.One of the most important challenges for miRNA-targeting therapy is the use of a drug delivery system (DDS) to transfer these anti-miR or miR-mimics more efficiently.Antisense drugs composed of single-stranded oligonucleic acids must be translocated into cells because they function by complementary binding to intracellular RNA.However, the antisense is large in size and has a negative charge due to the phosphodiester bond, making it difficult for it to pass through biological membranes.Therefore, many antisense miRNAs are attached with a phosphorothioate modification (Sylation).In addition, chemical modifications are also introduced into the sugar moiety of nucleic acids, thereby exerting efficacy without using carriers, such as liposomes.In contrast, miRNA mimics are normally composed of a double strand, and are thus larger than antisense miRNAs, leading to further difficulty with cell membrane permeability.Therefore, miRNA mimic therapy in general requires a DDS, such as lipid nanoparticles, polyplexes, and polymeric micelles [166].Exosome may be the natural DDS to transfer anti-miR or miR-mimics.There are two approaches indicated, post-loading and pre-loading, both of which still have difficulties in the efficient incorporation of target nucleic acids [167].Receptors such as GalNACs can be used as asialoglycoporotein receptors [168].In addition, miRNA mimics need to be recognized by RISC, and thus the degree of nucleic acid modification possible is also limited.Treatment with nucleic acid drugs may also cause on-target toxicity (toxicity due to binding to target RNA) and off-target toxicity (toxicity due to binding to non-target RNA) [152].It is notable that treatment targeting miRNAs might cause unexpected side effects [169], considering that one type of miRNA may regulate more than one hundred genes, including genes of interest.

Conclusions
In the present review article, we summarize the current knowledge about miRNAs as promising biomarkers and potential therapeutic targets for AKI.Recent accumulated evidence has revealed the vital role of miRNAs in both protective and pathogenic mechanisms under AKI, and possible strategies for their application as biomarkers for the early diagnosis, prediction of mortality, and identification of the specific pathology in AKI.The identification of etiology-specific miRNAs may uncover the disease mechanisms, leading to novel therapy for these diseases.Regarding the possible strategies for therapeutic options, there are two approaches: antisense for inhibition of pathogenic miRNA, and protective miRNA-mimic injection.Although the investigation of miRNA-targeted therapy for kidney diseases has just started and needs to confront several challenges before clinical use, including DDS and off-target effects involving non-target genes and organs, this research may begin a new era in the management of AKI through the regulation of specific miRNAs in the future.

Table 1 .
miRNAs as potential biomarkers of AKI.

Table 2 .
Potential therapeutic targets of miRNAs for AKI.