MicroRNAs in acute kidney injury

Acute kidney injury (AKI) is an important clinical issue that is associated with significant morbidity and mortality. Despite research advances over the past decades, the complex pathophysiology of AKI is not fully understood. The regulatory mechanisms underlying post-AKI repair and fibrosis have not been clarified either. Furthermore, there is no definitively effective treatment for AKI. MicroRNAs (miRNAs) are endogenous single-stranded noncoding RNAs of 19~23 nucleotides that have been shown to be crucial to the post-transcriptional regulation of various cellular biological functions, including proliferation, differentiation, metabolism, and apoptosis. In addition to being fundamental to normal development and physiology, miRNAs also play important roles in various human diseases. In AKI, some miRNAs appear to act pathogenically by promoting inflammation, apoptosis, and fibrosis, while others may act protectively by exerting anti-inflammatory, anti-apoptotic, anti-fibrotic, and pro-angiogenic effects. Thus, miRNAs have not only emerged as novel biomarkers for AKI; they also hold promise to be potential therapeutic targets.


Acute kidney injury
Acute kidney injury (AKI) is a complex syndrome that occurs in a variety of settings with clinical manifestations ranging from a minimal elevation in serum creatinine to anuric renal failure. AKI conveys significant morbidity and mortality, is a major risk factor of chronic kidney disease, and is thus associated with huge health and socioeconomic burdens [1,2]. Despite research advances in the past decades, however, the complex pathophysiology of AKI is not fully understood. The regulatory mechanisms underlying post-AKI repair and fibrosis remain to be clarified. Furthermore, there is no definitively effective treatment for AKI.

MicroRNA biogenesis and function
MicroRNAs (miRNAs) are endogenous single-stranded noncoding mRNAs of 19~23 nucleotides. They were first discovered in Caenorhabditis elegans by Ambros's group in 1993 [3] and show surprisingly high conservation across species. The evidence accumulated over the past two decades shows that miRNAs play a critical role in the post-transcriptional regulation of almost all biological cell functions, including proliferation, differentiation, metabolism, and apoptosis [4]. miRNAs, which are expressed in a tissue-specific manner, are fundamental to normal development and physiology [4] and are involved in the pathologic pathways of many disease models.
To date, more than 2000 miRNAs have been discovered in the human genome. The miRNA-encoded genes are found as either independent genes having their own promoters, or as sequences in the introns of proteincoding genes [5]. RNA polymerase II transcribes an miRNA gene into a primary transcript (called a pri-miRNA) of several kilobases that can encode either an individual miRNA or a polycistronic cluster of two or more miRNAs. The RNase III enzyme, DROSHA, and its cofactor DGCR8 (Di-George syndrome critical region gene 8 or Pasha), cleave a pri-miRNA at its stem-loop structure, generating an approximately 70-nucleotide intermediate called the pre-miRNA. Exportin-5 exports the pre-miRNA from the nucleus to the cytoplasm, and the RNase III enzyme, DICER, further cleaves it to yield a single-stranded mature miRNA. To perform its function, an miRNA is incorporated along with the argonaute (AGO) protein to form an effector complex called the RNA-induced silencing complex (RISC). RISC binds to the 3′-untranslated region (UTR) of a target messenger RNA (mRNA), leading to the repression of either protein translation or mRNA degradation. Unlike small interfering RNAs in plants, miRNAs do not require complete complementarity to bind their targets. Instead, the evidence suggests that the "seed sequence" (nucleotides 2 through 8 of the miRNA) is the most important region for the ability of an miRNA to bind and regulate its target gene(s). Once bound, miRNAs induce repression by blocking the initiation or elongation of translation or de-adenylating the mRNA transcripts. Because miRNAs do not require complete complementarity to repress gene expression, a given miRNA can regulate multiple mRNA transcripts and a given mRNA transcript can be repressed by multiple miRNAs. It is estimated that miRNAs regulate more than half of the protein-coding genes in human [6]. Moreover, miRNAs have been implicated in various human diseases [7,8], including kidney diseases, such as polycystic kidney disease [9], renal cell carcinoma [10], diabetic nephropathy [11], lupus nephritis, [12] and renal allograft rejection [13]. In the past few years, researchers have begun to address the relevance of miRNAs to AKI.

miRNAs in acute kidney injury
The miRNAs that have been implicated in AKI are summarized in Tables 1 and 2, and those with potential pathological or protective roles are summarized in Table 3. The first evidence of miRNAs having pathological roles in AKI was reported by Wei et al. who developed a Dicer-knockout mouse model, in which Dicer was specifically deleted from proximal tubular cells. These mice exhibit a global down-regulation of microRNAs in the renal cortex. They have normal renal function and histology under control conditions but show resistance to the AKI that follows bilateral renal ischemia-reperfusion (IRI). Under the latter conditions, Dicer-null mice show significantly better renal function, less tissue damage, less tubular apoptosis, and better survival than their wild-type counterparts [14]. miR-10a is renal tubule-specific miRNA that is released from kidney tissues upon injury. In rodent models of renal IRI and streptozocin (STZ)-induced diabetic nephropathy, the levels of miR-10a are increased decreased in urine and kidney tissue, respectively [15,16]. miR-10a is thought to exert protective actions during injury by targeting IL-12/IL-23p40 and the pro-apoptotic protein BIM [17]. In humans, decreased plasma levels of miR-10a have been shown to predict AKI in critical patients of intensive care units (ICUs) [18].
The members of the miR-17 family have been shown to be induced by pro-inflammatory cytokines, and their tissue expressions are increased in rodent models of renal IRI [19,20].
miR-101-3p is highly expressed in the kidney, and decreased serum levels of this miRNA have been shown to predict AKI in the ICU [18]. miR-122 is down-regulated in the mice kidneys of mice subjected to cisplatin-induced AKI [35]. It exerts anti-apoptotic effects by down-regulating forkhead box O3 (Foxo3). miR-127a, which is induced by HIF-1α, participates in protecting the cytoskeleton protection (by preventing actin depolmerization), maintaining cell-matrix and cell-cell adhesion maintenance (by preventing focal adhesion complexes disassembly and tight junctions disorganization), and promoting intracellular trafficking (by targeting kinesin family member 3B) [36]. Decreased blood levels of miR-127a were shown to predict AKI in the ICU. Decreased blood levels of miR-127a prior to cardiac surgery were found be predict AKI later on [18]. miR-146a is down-and up-regulated in the blood and kidney, respectively, during AKI. Decreased blood levels have been shown to predict AKI in the ICU and correlate with the severity of AKI [18]. It is induced by NF-kB and exerts anti-inflammatory effect by down-regulating TNF receptor-associated factor 6 (TRAF-6) and interleukin-1 receptor-associated kinase 1 (IRAK-1) [37]. miR-192 is enriched in kidneys and the small intestine. It is induced by TGF-β during the stress response. It promotes fibrosis by down-regulating SIP1. It also downregulates E3 ubiquitin ligase and murine double-minute 2 (MDM2) and results in de-repression of p53 and G2/M arrest [38]. miR-194 is also enriched in kidneys and small intestine. It is induced during the stress response, and its levels in tissue, blood, and urine levels are increased during AKI [15,38,39].
miR-126 and miR-296 have been identified in microvesicles from endothelial progenitor cells and are thought to exert renoprotective effects via their abilities to decrease apoptosis and leukocyte infiltration, while promotes angiogenesis and tubular cell proliferation [43]. Hematopoietic overexpression of miR-126 enhances stromal cell-derived factor 1/chemokine receptor type 4 (CXCR4) -dependent vasculogenic progenitor cell mobilization and promotes  [44]. Decreased serum levels of miR-126 have been shown to predict AKI in ICU patients, and correlate with the severity of AKI [18]. Members of the miR-200 family are highly expressed in tubular structures such as renal tubules, lungs, the small intestine, and various exocrine glands. miR-200b and miR-200c have been proposed to be anti-fibrotic. They down-regulate TGFβR1 and zinc finger E-boxbinding homeobox (ZEB1/ZEB2), which are transcriptional repressors of E-cadherin, and thereby prevent the epithelial-to-mesenchymal transition (EMT) induced by TGF-β [45].
miR-210 is induced by HIF1-α and released by renal endothelial cell. It regulates angiogenesis by downregulating ephrin-A3 and up-regulating vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2). It also regulates mitochondria ROS production. Increased blood levels of miR-210 was shown to predict post-AKI mortality in critically ill patients [46]. In another study, decreased blood levels of miR-210 were shown to predict AKI in the ICU and correlate with the severity of AKI [18]. miR-214 is induced by TGF-β and promotes fibrosis; it has been shown to down-regulate PTEN, up-regulate the AKT pathway and inhibit apoptosis of monocytes and macrophages. miR-214 is up-regulated in various models of AKI and renal fibrosis [24,45,47] as well as in monocytes of animal with chronic kidney disease. Experimental antagonism of miR-214 has been shown to ameliorate renal fibrosis [24]. miR-494 is up-regulated early in AKI, with increased urine levels detected in rodent models of renal IRI and patients with AKI. It has been reported to promote apoptosis and inflammation by down-regulating activating transcription factor 3 (ATF3) and increasing IL-6, monocyte chemoattractant protein-1 (MCP-1), p-selectin [48]. Pathway analysis has suggested that it also targets adiponectin receptor 2 (ADIPOR2), BCL-2 facilitator, and insulin-like growth factor 1 receptor (IGF1R), which would increase inflammation and lead to more damage. However, miR-494 also targets pro-apoptotic proteins in the AKT pathway, and to exert protective effects. The mechanism responsible for regulating the balance between these antiand pro-apoptotic effects requires further study.
Finally, miR-687 is induced by HIF-1, and enhances apoptosis by down-regulating PTEN. Animal studies have shown that miR-687 blockade preserves PTEN expression and attenuates cell cycle activation and decreases apoptosis, resulting in protection against kidney injury [49].

Conclusions
Many miRNAs have been implicated in the AKI. Some of them contribute to the pathogenesis by regulating apoptosis and inflammation, to amplifying or reduce acute injury responses, while others regulate fibrosis and angiogenesis, to participate in renal recovery or the progression to fibrosis. The biological and pathological functions of many miRNAs in AKI are still not fully understood in AKI. Some studies have yielded inconsistent data regarding the expression pattern of miRNAs across different samples, species, disease models, and time points. These discrepancies warrant investigations.
In addition to their tissue expressions, miRNAs may be detected in various extracellular human body fluids, such as serum, urine, saliva, and cerebral spinal fluid. miRNAs are contained in exosomes and may remained stable over prolonged periods. They may be specifically up-regulated or down-regulated in response to injury signals and/or released into body fluids from resident tissues. Certain miRNAs have been investigated for their potential to serve as novel biomarkers for the early detection or prognostication of AKI. Given the complex pathophysiology and the dynamic nature of AKI, an miRNA panel may be more feasible rather than a single miRNA. Further validation studies are needed to evaluate the clinical utility of such a panel.
Some miRNAs may be potential therapeutic targets for AKI. Recently, an miRNA inhibitor has been proven to successfully suppress the replication of hepatitis C virus in a clinical trial [50]. Systemic or local administration of specific miRNAs mimics or antagonists in vivo could offer a strategy for preventing or ameliorating AKI or barring its progression to chronic kidney disease.
In the post-genome era, miRNAs are promising rising stars in translational medicine as they offer the potential to guide the individualized diagnosis and treatment of human diseases including AKI.