1777
J Physiol 593.8 (2015) pp 1777–1784
SYMPOSIUM REVIEW
Vascular and circulating microRNAs in renal ischaemia–reperfusion injury Johan M. Lorenzen1,2 1
The Journal of Physiology
2
Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover, Hannover Medical School, Germany Department of Medicine, Division of Nephrology, Hannover Medical School, Hannover, Germany
Abstract Ischaemia–reperfusion (I/R) injury of the kidney is a major cause of acute kidney injury. It may result in worsening or even loss of organ function. Transient occlusion of the renal vessel is followed by a reperfusion period, which induces further tissue damage by release of reactive oxygen and nitrogen species. Ischaemia–reperfusion injury of the kidney may be associated with surgical interventions in native kidneys and is also a common and unavoidable phenomenon in kidney transplantation. MicroRNAs are fascinating modulators of gene expression. They are capable of post-transcriptional silencing of genetic information by targeting the 3 -untranslated region of mRNAs, culminating in a suppression of protein synthesis or an increase in mRNA degradation. They might therefore be useful diagnostic and therapeutic entities during renal I/R injury; for instance, miR-21 has been shown to be enriched in kidney tissue in mice and humans with acute kidney injury. Interestingly, most recent literature suggests that modulation of vascular microRNAs might result in the amelioration of kidney function during renal I/R injury. To that end, miR-126 and miR-24, which have been demonstrated to be highly enriched in endothelial cells, were therapeutically modulated and shown to ameliorate renal I/R injury in mice. MicroRNAs in plasma, urine or enriched in microvesicles have been shown to serve as non-invasive tools for disease monitoring and to have potential impact on downstream mechanisms in recipient cells. This review highlights the latest developments regarding the role of microRNAs in renal I/R injury. (Received 19 November 2014; accepted after revision 13 February 2015; first published online 18 February 2015) Corresponding author J. M. Lorenzen: Department of Medicine, Division of Nephrology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Email: [email protected] Abbreviations I/R, ischaemia–reperfusion; miRNA, microRNA.
Pathophysiology of ischaemia–reperfusion injury of the kidney
Ischaemia–reperfusion (I/R) injury of the kidney is a major cause of acute kidney injury (Kelly, 2006). It is a common
phenomenon associated with a multitude of different insults in native kidneys (e.g. during cardiac surgery). Moreover, it is associated with the transplantation procedure and is therefore an unavoidable consequence in transplanted kidneys (Bon et al. 2012). During ischaemic
Johan Lorenzen is currently an assistant professor of medicine, a research group leader at the Institute of Molecular and Translational Therapeutic Studies (IMTTS) and a clinical specialist registrar at the Department of Nephrology and Hypertension at Hanover Medical School, Germany. Before becoming a research group leader at IMTTS he trained in the laboratory of Professor Katalin Susztak at Albert Einstein College of Medicine, New York City, U.S.A. and under the auspices of Professor Hermann Haller at the Department of Nephrology and Hypertension, Hanover Medical School, Germany. Scientifically he is interested in the regulation and therapeutic modulation of non-coding RNAs in various mouse models of renal injury. Various non-coding RNAs are either pharmacologically or genetically manipulated in mice. In addition, the research group is interested in circulating and urinary non-coding RNAs as biomarkers in patients with kidney disease.
This review was presented at the symposium Epigenetic regulation of cardiovascular development and disease, which took place at Physiology 2014, the annual meeting of The Physiological Society, London, UK on 1 July 2014. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
DOI: 10.1113/JP270318
1778
J. M. Lorenzen
acute kidney injury, a temporary impairment of blood flow to the kidney is followed by a reperfusion period. Reperfusion itself, though vital to restoration of kidney function, is associated with significant additional cellular injury (Weight et al. 1996). We have recently summarized the deleterious events resulting from I/R injury (Lorenzen et al. 2013). The damage inflicted by tissue ischaemia is subsequently aggravated by a dramatic surge in reactive oxygen and nitrogen species during reperfusion. These induce protein modifications, lipid oxidations and DNA double-strand breaks, finally culminating in endothelial dysfunction, neutrophil adherence to the endothelium and transendothelial migration, the release of inflammatory mediators and eventual cell death (Lorenzen et al. 2013). In the kidney, blood flow to the outer medulla is disproportionately reduced with respect to the reduction in total blood flow (Bonventre & Yang, 2011). Thus, epithelial cell injury is mainly detected in the S3 segment of the proximal tubule, located in the outer medulla (Bonventre & Yang, 2011). Interplay of several events contributes to the cellular injury observed in the kidney. The damaged endothelium interacts with and activates inflammatory cells through enhanced expression of adhesion molecules (e.g. intercellular adhesion molecule 1, selectins; Kelly et al. 1996; Bonventre & Yang, 2011). This interaction in turn contributes to obstruction of capillaries and postcapillary venules, further activation and transmigration of leucocytes, production of cytokines and inflammation in tubular epithelial cells (Bonventre & Yang, 2011). Capillary rarefaction in the inner stripe of the outer medulla ensues; due to the development of chronic hypoxia, this is an important contributor to tubulointerstitial fibrosis and progression to chronic kidney disease after acute kidney injury (Bonventre & Yang, 2011). Cell polarity and cytoskeletal arrangement are severely impaired in proximal tubular epithelial cells during ischaemia (Sutton & Molitoris, 1998; Bonventre & Yang, 2011). Important phenotypical changes are loss of the proximal tubule brush border as well as loss of polarity and derangement of adhesion molecules and other membrane proteins and disruption of cell–cell interactions at adherens and tight junctions (Bonventre & Yang, 2011). MicroRNA biogenesis and function
MicroRNAs (miRNAs) are currently under intense investigation as powerful regulators of various diseases with potential critical impact on disease initiation and/or progression, including kidney disease (Lorenzen et al. 2011a). MicroRNAs represent small non-coding RNA transcripts with a length of 22 nucleotides, which through post-transcriptional binding of the 3 -untranslated region of mRNA targets lead to the repression of
J Physiol 593.8
gene/protein expression and/or translational inhibition of protein synthesis (Lorenzen et al. 2011a; Lorenzen & Thum, 2012). The first miRNA, lin-4, was discovered while investigating genetic loci responsible for temporal patterning in Caenorrhabdidits elegans (Ambros, 1989; Lee et al. 1993) Intriguingly, a single microRNA may alter the expression of a large number of target genes, thus influencing a specific pathology by regulating whole disease-specific pathways and signalling cascades rather than a single gene. This unique function underlines the immense importance of these small molecules. The biogenesis of microRNAs follows a tightly regulated pattern (Lorenzen et al. 2011a). Transcribed as primary miRNA transcripts (pri-miRNAs), they are further processed in the nucleus by the ribonuclease Drosha to precursor miRNAs (pre-miRNAs) with a length of 70 nucleotides. The pre-miRNA is then shuttled out of the nucleus into the cytosol by a Ran-guanosine-5 -triphosphate-dependent transporter, exportin 5. A second ribonuclease, Dicer, gives rise to a double-stranded RNA duplex (miRNA:miRNA∗ ). It is composed of a miRNA guide strand and its complementary strand (miRNA∗ ), each consisting of 22 nucleotides. While the guide strand is incorporated into an RNA-induced silencing complex, where it interacts and cleaves its target mRNA, the complementary strand is believed to be degraded. However, recent evidence suggests that the complementary strand also carries functional importance (Yang et al. 2011). There are also several non-canonical pathways of miRNA biogenesis, which have been elegantly reviewed elsewhere (Ha & Kim, 2014). Some examples are listed here. i. The 7-methylguanosine-capped pre-miR-320 bypasses Drosha processing and is generated directly through transcription and exported to the cytoplasm by exportin 1. ii. Mirtrons are located in the introns of mRNA encoding host genes. Here, pre-miRNAs can be generated directly by splicing and debranching. iii. Some small nucleolar RNAs may also be cleaved to produce pre-miRNAs. iv. In terminal uridylyl transferase-dependent group II pri-miRNAs, pre-miRNAs are generated with shorter 3 overhangs, which have first to be monouridylated for efficient Dicer processing. v. There are also Dicer-independent pathways, e.g. pre-miR-451 is produced by Drosha and exported to the cytoplasm. Here, it interacts with Argonaute 2 without Dicer processing. Argonaute 2 further cleaves pre-miR-451. Subsequently, pre-miR-451 is modified by the 3 ,5 -exonuclease poly(A)-specific ribonuclease. Cellular miRNA function can be silenced by miRNA antagonists termed ‘antagomirs’ (or ‘antimiRs’), which are C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
J Physiol 593.8
Vascular and circulating microRNAs in acute kidney injury
chemically engineered oligonucleotides targeting specific miRNAs (Kr¨utzfeldt et al. 2005). For in vivo delivery, these antimiRs have to be modified in order to increase stability. These include cholesterol modification, modifications or such as 2 -O-methoxyethylphosphorothioate 2 -fluoro/2 -methoxyethyl substitutions (2 (F)-MOE), as well as oligonucleotides stabilized by locked nucleic acid phosphorothioate chemistries (Lorenzen et al. 2011a). If overexpression of a specific microRNA is the desired approach, miRNA expression can be enhanced by double-stranded miRNA mimics (Lorenzen et al. 2011a). An elucidation of the mechanisms of microRNA-mediated kidney injury is thus of utmost importance.
1779
identified miR-127 to contribute to rat renal I/R injury (Aguado-Fraile et al. 2012). MiR-127 was postulated to be regulated by hypoxia-inducible factor 1α and to target kinesin family member 3B. Modulation of miR-127 resulted in changes in cell adhesion and cytoskeletal structure. Regulation of its target, kinesin family member 3B, resulted in modulation of endocytosis and microtubular transport in rat renal proximal tubular cells. MiR-494 was shown to exacerbate renal I/R injury in mice by targeting activating transcription factor 3, thereby promoting apoptosis and further impairing renal function (Lan et al. 2012).
MicroRNAs and renal ischaemia–reperfusion injury
Novel vascular microRNAs and renal ischaemia–reperfusion injury
The contribution of specific miRNAs to the development of renal I/R injury has been investigated in several studies so far. It has recently been shown that targeted deletion of Dicer from the proximal tubular epithelium protects against I/R-induced renal injury through changes in expression of various microRNAs (Wei et al. 2010). Interestingly, Dicer knockdown was associated with reduced expression of miR-27a. This microRNA belongs to a microRNA cluster, which is simultaneously transcribed. The miR-23/27/24 gene cluster is composed of two separate gene clusters located at different genomic loci (Bang et al. 2012). The intergenic miR-24-2 cluster is encoded on human chromosome 19p13.13, expressing miR-23a, miR-27a and miR-24-2 (Chan et al. 2010; Bang et al. 2012). This cluster has been demonstrated to be involved in angiogenesis and endothelial apoptosis in cardiac ischaemia and retinal vascular development (Bang et al. 2012). Moreover, miR-24 has been demonstrated to regulate apoptosis of cancer and T cells (Brunner et al. 2012; Xie et al. 2012). In the kidney, overexpression of the miR-23a, miR-27a and miR-24-2 cluster induces apoptosis of human embryonic kidney cells (Chhabra et al. 2009). Changes in expression of other distinct microRNAs during I/R injury have been shown (e.g. miR-132, miR-362 and miR-379; Wei et al. 2010). Godwin et al. (2010) elegantly identified miR-21 as one of the most highly induced miRNAs during renal I/R injury. Moreover, miR-21 expression was shown to be increased in proliferating tubular epithelial cells, while knockdown of miR-21 in these cells resulted in enhanced apoptosis (Godwin et al. 2010). Recently, Duffield and co-workers also identified miR-21 as one of the most highly upregulated microRNAs in humans with acute kidney injury and in mice subjected to renal I/R injury (Chau et al. 2012). MiR-21 was found to target peroxisome proliferator-activated receptor α, thereby regulating metabolic pathways (Chau et al. 2012). Another group
Another interesting microRNA in I/R injury of the kidney is miR-126. As mentioned above (see section on pathophysiology of ischemia-reperfusion “injury of the kidney”), integrity of the renal peritubular capillary network is an important limiting factor in the recovery from renal I/R injury. MiR-126 has been shown to improve vascular regeneration by mobilizing haematopoietic stem/progenitor cells (van Solingen et al. 2011; Salvucci et al. 2012). Viral overexpression of miR-126 in the haematopoietic system was shown to improve neovascularization in mice (Bijkerk et al. 2014). Moreover, following renal I/R injury, mice overexpressing miR-126 displayed a significant improvement of kidney function, fibrosis and kidney injury. This was associated with a reduction of capillary rarefaction in the corticomedullary junction and an increase in the number of bone marrow-derived endothelial cells. In addition, the number of circulating Lin− /Sca-1+ /cKit+ haematopoietic stem and progenitor cells was increased, partly by attenuated expression of the chemokine receptor CXCR4 on haematopoietic stem and progenitor cells in the bone marrow. Renal expression of its ligand, stromal cell-derived factor 1, was increased, thus favouring mobilization of Lin− /Sca-1+ /cKit+ cells towards the kidney. Very recently, we found miR-24 to be enriched in the kidney following I/R injury in mice (Lorenzen et al. 2014, see Fig. 1). Kidney transplant patients whose transplanted kidneys were exposed to long versus short cold ischaemia times also presented with increased intragraft miR-24 levels. Cell sorting experiments using specific cell surface markers or lectins revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after induction of I/R. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Enrichment of miR-24 induced apoptosis, whereas its silencing ameliorated apoptotic responses. The effects of miR-24
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
1780
J. M. Lorenzen
were mediated through regulation of H2A histone family, member X, the sphingosine-1-phopshate receptor 1 and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. In vivo, silencing of miR-24 in mice following I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histological tubular epithelial injury and less infiltration of inflammatory cells. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites along with miR-24 precursors rescued various functional parameters in endothelial and tubular epithelial cells. Heme oxygenase 1 and H2A histone family, member X were also found to be regulated by miR-24 in vivo. Intriguingly, miR-24 was also enriched in kidney biopsies of renal transplant patients with prolonged cold ischaemia time, underlining its potential significance in humans. Inhibition of miR-24 is therefore a promising future therapeutic option in the treatment of patients with ischaemic acute kidney injury. It is conceivable that Tubular epithelial cell
J Physiol 593.8
miR-24 antagonism in transplant kidneys might result in an amelioration of kidney injury and thus prolonged long-term organ survival. Here, the first study to be published evaluating a microRNA-based therapy in renal I/R injury is presented. Previously, my colleagues and I were able to demonstrate that miR-24 was also specifically enriched in cardiac endothelial cells in the infarct border zone after myocardial infarction (Fiedler et al. 2011). Transient overexpression of miR-24 in human umbilical vein endothelial cells induced apoptosis, loss of capillary network formation capacity and migratory capacity in a modified Boyden chamber assay, and impaired endothelial spheroid formation, sprouting capacity and proliferation. These effects of miR-24 in endothelial cells were mediated by modulation of the transcription factor GATA2 and the p21-activated kinase PAK4, which were identified bioinformatically and subsequently validated by luciferase gene reporter assays. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blood vessel
miR-126 overexpression in haematopoietic compartment
Hypoxia/anoxia-induced miR-24
Renal expression of stromal cell-derived factor 1
Regulation of HO-1, S1PR1, H2A.X
Tubular epithelial / Endothelial cell apoptosis
miR-24 silencing
mobilization of Lin2/Sca-1+/cKit+ cells
- improvement of kidney function - Reduction of fibrosis - reduction of capillary rarefaction
Figure 1. Novel vascular microRNAs in renal ischaemia–reperfusion injury MiR-24 is induced in hypoxic/anoxic tubular epithelial and endothelial cells, targeting and silencing haem oxygenase 1 (HO-1), the sphingosine-1-phopshate receptor 1 (S1PR1) and H2A histone family, member X (H2A.X), thereby leading to increased apoptosis of these cells. Overexpression of miR-126 in the haematopoietic compartment in mice results in enhanced renal expression of stromal cell-derived factor 1, culminating in the mobilization of Lin2/Sca-1+ /cKit+ haematopoietic stem and progenitor cells to the kidney. Silencing of miR-24 and overexpression of miR-126 in mice subjected to renal ischaemia–reperfusion injury are finally characterized by an improvement of kidney function, a reduction of fibrosis and a reduction of capillary rarefaction. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
Vascular and circulating microRNAs in acute kidney injury
J Physiol 593.8
In summary, our group was able to describe miR-24 as an important vascular microRNA in cardiac and renal I/R injury. MiR-24 was found to be increased greatly in endothelial cells in both experimental models. In addition, we found that miR-24 was also enriched in damaged/injured tubular epithelial cells in renal I/R injury. Vascular miRNAs are summarised in Fig. 1. Novel circulating and urinary microRNAs in kidney injury
MicroRNAs are also released into the extracellular compartment (blood and urine) in patients (Lorenzen & Thum, 2012). Thus, circulating microRNAs may serve as a non-invasive tool to detect and monitor disease activity. The investigation and analysis of the release pattern of circulating microRNAs may thus enable the clinician to monitor certain patients adequately regarding disease progression and response to treatment. To that end, microRNA-enriched microvesicles, which can be detected in blood, secreted by endothelial progenitor cells, were shown to ameliorate I/R injury in the murine kidney (Cantaluppi et al. 2012). MiRNA array analysis revealed that the pro-angiogenic and anti-apoptotic
1781
miR-126 and miR-296 were greatly enriched in microvesicles. In a rat model of I/R injury, the addition of endothelial progenitor cell-derived microvesicles resulted in a proliferative response of tubular epithelial cells as well as a reduction in tubular epithelial cell apoptosis and infiltration of leucocytes. Our group was able to analyse the miRNA expression profile in plasma samples of critically ill patients with dialysis-dependent acute kidney injury before the inception of renal replacement therapy (Lorenzen et al. 2011b). Thirteen different miRNAs were found to be deregulated significantly. MiR-210 emerged as the most striking mediator of survival in this patient cohort in Cox proportional hazard and Kaplan–Meier curve analyses. In urine of patients with acute T-cell-mediated renal allograft rejection, we also found miR-210 to be deregulated (Lorenzen et al. 2011c). Specifically, miR-210 was found to be downregulated in urine of patients with rejection, as diagnosed by a kidney biopsy, in comparison to control patients with stable transplant function without signs of rejection. Successful antirejection therapy normalized miR-210 to the levels of control patients. Moreover, urinary miR-210 at the time of rejection predicted the decline in glomerular filtration rate at 1 year after transplantation. Interestingly,
B
A
MV
Altered trafficking in damaged tubular epithelial cells ? Plasma of patients with AKI on ICU - miR-210 - miR-320 - miR-16
Urine of transplant patients with kidney rejection - miR-210 - miR-10a - miR-10b
EPC
pro-angiogenic and anti-apoptotic miR-126 and miR-296 Addition of miRNA-enriched MV
Rat model of I/R -injury 1.) proliferative response of tubular epithelial cells 2.) reduction in tubular epithelial cell apoptosis 3.) less infiltration of leukocytes Prediction of outcome by miR-210 Figure 2. Circulating and urinary microRNAs in kidney disease A, in patients with dialysis-dependent acute kidney injury (AKI) on the intensive care unit (ICU), miR-210 is increased in plasma, while miR-16 and miR-320 are reduced. In patients with acute T-cell-mediated renal transplant rejection, miR-210 and miR-10b are downregulated and miR-10a is upregulated in urine. In both patient cohorts, miR-210 predicts outcome (for AKI, survival on the ICU; for transplant rejection, loss of glomerular filtration rate at 1 year after transplantation). B, miR-126 and miR-296 enriched in microvesicles (MV) derived from endothelial progenitor cells (EPC) ameliorate AKI in a rat model of ischaemia–reperfusion injury. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
1782
J. M. Lorenzen
J Physiol 593.8
Table 1. MicroRNAs, their targets and pathophysiological effects in renal ischaemia–reperfusion injury MicroRNA
Target
Organ/cell type
Pathophysiological effects
Organism
Reference
MiR-126
—
Kidney, bone marrow
Mouse
Bijkerk et al. (2014)
MiR-24
H2A histone family, member X, the sphingosine-1-phopshate receptor 1 and heme oxygenase 1
Kidney
Mouse
Lorenzen et al. (2014)
MiR-127
Kinesin family member 3B
Rat
Aguado-Fraile et al. (2012)
MiR-126 and miR-296
—
Mouse
Cantaluppi et al. (2012)
MiR-21
Peroxisome proliferator-activated receptor α Activating transcription factor 3 —
Kidney, proximal tubular cells Endothelial progenitor cell-derived microvesicles, proximal tubular cells Kidney
Inhibition of capillary rarefaction, mobilization of Lin− /Sca-1+ /cKit+ haematopoietic stem and progenitor cells Improvement of survival and kidney function, a reduction of apoptosis, improvement in histological tubular epithelial injury and less infiltration of inflammatory cells Changes in cell adhesion and cytoskeletal structure Inhibition of capillary rarefaction, glomerulosclerosis and tubulointerstitial fibrosis
Regulation of metabolic pathways
Mouse
Chau et al. (2012)
Exacerbation of renal injury Signature of kidney ischaemia–reperfusion injury Signature of kidney ischaemia–reperfusion injury
Mouse
Lan et al. (2012) Wei et al. (2010)
MiR-494 MiR-132, miR-362 and miR-379 MiR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194 MiR-210
Kidney
—
Kidney, proximal tubular cells Kidney
—
Kidney
in a mouse model of renal I/R injury, miRNA-210 was also shown to be upregulated in kidney tissue and to regulate renal angiogenesis by activation of the vascular
Biomarker of acute kidney kidney injury and acute T-cell-mediated rejection. Regulation of renal angiogenesis by activation of the vascular endothelial growth factor signalling pathway
Mouse
Mouse
Godwin et al. (2010)
Mouse, human plasma, human urine
Lorenzen et al. (2011b, c), Liu et al. (2012)
endothelial growth factor signalling pathway (Liu et al. 2012). Circulating and urinary miRNAs are summarised in Fig. 2.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
J Physiol 593.8
Vascular and circulating microRNAs in acute kidney injury
Conclusion
All discussed miRNAs, their known targets and biological roles are summarized in Table 1. An elucidation of the mechanisms of microRNA-mediated kidney injury is thus of utmost importance. Intriguingly, pathological microRNA expression might be modulated by RNA therapeutics, termed antagomirs, which enable specific targeting and cleavage of miRNAs and thus alteration of pathological signalling pathways in vivo. MiR-21 was one of the first miRNAs found to be altered during I/R injury of mice and humans; however, miR-21 seems to be indispensible for tubular regeneration during acute ischaemic injuries. Interestingly, miRNAs, which were first discovered to play a major role in the vasculature, e.g. miR-24 and miR-126, have been shown to be useful as therapeutics in mice with I/R injury. Modulation of vascular microRNAs might be a viable therapeutic option in the treatment of patients with the important clinical disorder of I/R injury of the kidney. Moreover, a few miRNAs have been characterized to be detectable in the circulation of patients and mice, including pro-angiogenic and anti-apoptotic miR-126 and miR-296 as well as miR-210. Detection and possible modulation of circulating miRNAs might thus be an interesting means of monitoring and treating disease activity.
References Aguado-Fraile E, Ramos E, S´aenz-Morales D, Conde E, Blanco-S´anchez I, Stamatakis K, del Peso L, Cuppen E, Br¨une B & Bermejo ML (2012). MiR-127 protects proximal tubule cells against ischemia/reperfusion: identification of kinesin family member 3B as miR-127 target. PloS One 7, e44305. Ambros V (1989). A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans. Cell 57, 49–57. Bang C, Fiedler J & Thum T (2012). Cardiovascular importance of the microRNA-23/27/24 family. Microcirculation 19, 208–214. Bijkerk R, vanSolingen C, deBoer HC, vander Pol P, Khairoun M, deBruin RG, vanOeveren-Rietdijk AM, Lievers E, Schlagwein N, vanGijlswijk DJ, Roeten MK, Neshati Z, deVries AA, Rodijk M, Pike-Overzet K, vanden Berg YW, vander Veer EP, Versteeg HH, Reinders ME, Staal FJ, vanKooten C, Rabelink TJ & vanZonneveld AJ (2014). Hematopoietic microRNA-126 protects against renal ischemia/reperfusion injury by promoting vascular integrity. J Am Soc Nephrol 25, 1710–1722. Bon D, Chatauret N, Giraud S, Thuillier R, Favreau F & Hauet T (2012). New strategies to optimize kidney recovery and preservation in transplantation. Nat Rev Nephrol 8, 339–347. Bonventre JV & Yang L (2011). Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 121, 4210–4221.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
1783
Brunner S, Herndler-Brandstetter D, Arnold CR, Wiegers GJ, Villunger A, Hackl M, Grillari J, Moreno-Villanueva M, B¨urkle A & Grubeck-Loebenstein B (2012). Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8+ T cells sensitizing them to apoptotic cell death. Aging Cell 11, 579–587. Cantaluppi V, Gatti S, Medica D, Figliolini F, Bruno S, Deregibus MC, Sordi A, Biancone L, Tetta C & Camussi G (2012). Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia–reperfusion injury by microRNA-dependent reprogramming of resident renal cells. Kidney Int 82, 412–427. Chan MC, Hilyard AC, Wu C, Davis BN, Hill NS, Lal A, Lieberman J, Lagna G & Hata A (2010). Molecular basis for antagonism between PDGF and the TGFβ family of signalling pathways by control of miR-24 expression. EMBO J 29, 559–573. Chau BN, Xin C, Hartner J, Ren S, Castano AP, Linn G, Li J, Tran PT, Kaimal V, Huang X, Chang AN, Li S, Kalra A, Grafals M, Portilla D, MacKenna DA, Orkin SH & Duffield JS (2012). MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways. Sci Transl Med 4, 121ra18. Chhabra R, Adlakha YK, Hariharan M, Scaria V & Saini N (2009). Upregulation of miR-23a27a24-2 cluster induces caspase-dependent and -independent apoptosis in human embryonic kidney cells. PLoS One 4, e5848. Fiedler J, Jazbutyte V, Kirchmaier BC, Gupta SK, Lorenzen J, Hartmann D, Galuppo P, Kneitz S, Pena JT, Sohn-Lee C, Loyer X, Soutschek J, Brand T, Tuschl T, Heineke J, Martin U, Schulte-Merker S, Ertl G, Engelhardt S, Bauersachs J & Thum T (2011). MicroRNA-24 regulates vascularity after myocardial infarction. Circulation 124, 720– 730. Godwin JG, Ge X, Stephan K, Jurisch A, Tullius SG & Iacomini J (2010). Identification of a microRNA signature of renal ischemia reperfusion injury. Proc Natl Acad Sci U S A 107, 14339–14344. Ha M & Kim VN (2014). Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15, 509–524. Kelly KJ, Williams WW Jr, Colvin RB, Meehan SM, Springer TA, Gutierrez-Ramos JC & Bonventre JV (1996). Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. J Clin Invest 97, 1056–1063. Kelly KJ (2006). Acute renal failure: much more than a kidney disease. Semin Nephrol 26, 105–113. Kr¨utzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M & Stoffel M (2005). Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438, 685–689. Lan YF, Chen HH, Lai PF, Cheng CF, Huang YT, Lee YC, Chen TW & Lin H (2012). MicroRNA-494 reduces ATF3 expression and promotes AKI. J Am Soc Nephrol 23, 2012–2023. Lee RC, Feinbaum RL, Ambros V (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.
1784
J. M. Lorenzen
Liu F, Lou YL, Wu J, Ruan QF, Xie A, Guo F, Cui SP, Deng ZF & Wang Y (2012). Upregulation of microRNA-210 regulates renal angiogenesis mediated by activation of VEGF signaling pathway under ischemia/perfusion injury in vivo and in vitro. Kidney Blood Press Res 35, 182–191. Lorenzen JM, Batkai S & Thum T (2013). Regulation of cardiac and renal ischemia–reperfusion injury by microRNAs. Free Radic Biol Med 64, 78–84. Lorenzen JM, Haller H & Thum T (2011a). MicroRNAs as mediators and therapeutic targets in chronic kidney disease. Nat Rev Nephrol 7, 286–294. Lorenzen JM, Kaucsar T, Schauerte C, Schmitt R, Rong S, H¨ubner A, Scherf K, Fiedler J, Martino F, Kumarswamy R, K¨olling M, S¨orensen I, Hinz H, Heineke J, vanRooij E, Haller H & Thum T (2014). MicroRNA-24 antagonism prevents renal ischemia reperfusion injury. J Am Soc Nephrol 25, 2717–2729. Lorenzen JM, Kielstein JT, Hafer C, Gupta SK, Kumpers P, Faulhaber-Walter R, Haller H, Fliser D & Thum T (2011b). Circulating miR-210 predicts survival in critically ill patients with acute kidney injury. Clin J Am Soc Nephrol 6, 1540–1546. Lorenzen JM & Thum T (2012). Circulating and urinary microRNAs in kidney disease. Clin J Am Soc Nephrol 7, 1528–1533. Lorenzen JM, Volkmann I, Fiedler J, Schmidt M, Scheffner I, Haller H, Gwinner W & Thum T (2011c). Urinary miR-210 as a mediator of acute T-cell mediated rejection in renal allograft recipients. Am J Transplant 11, 2221–2227. Salvucci O, Jiang K, Gasperini P, Maric D, Zhu J, Sakakibara S, Espigol-Frigole G, Wang S & Tosato G (2012). MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1. Haematologica 97, 818–826.
J Physiol 593.8
Sutton TA & Molitoris BA (1998). Mechanisms of cellular injury in ischemic acute renal failure. Semin Nephrol 18, 490–497. vanSolingen C, deBoer HC, Bijkerk R, Monge M, vanOeveren-Rietdijk AM, Seghers L, de Vries MR, van derVeer EP, Quax PH, Rabelink TJ & van Zonneveld AJ (2011). MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+ /Lin− progenitor cells in ischaemia. Cardiovasc Res 92, 449–455. Wei Q, Bhatt K, He HZ, Mi QS, Haase VH & Dong Z (2010). Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury. J Am Soc Nephrol 21, 756–761. Weight SC, Bell PR & Nicholson ML (1996). Renal ischaemia–reperfusion injury. Br J Surg 83, 162–170. Xie Y, Tobin LA, Camps J, Wangsa D, Yang J, Rao M, Witasp E, Awad KS, Yoo N, Ried T & Kwong KF (2012). MicroRNA-24 regulates XIAP to reduce the apoptosis threshold in cancer cells. Oncogene 32, 2442–2451. Yang JS, Phillips MD, Betel D, Mu P, Ventura A, Siepel AC, Chen KC & Lai EC (2011). Widespread regulatory activity of vertebrate microRNA∗ species. RNA 17, 312–326.
Additional information Competing interests None declared.
Funding I wish to acknowledge funding by the German Research Council (DFG LO 1736/1-1) and the Else Kr¨oner-Fresenius Foundation (2011 A173).
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
Copyright of Journal of Physiology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
J Physiol 593.8 (2015) pp 1777–1784
SYMPOSIUM REVIEW
Vascular and circulating microRNAs in renal ischaemia–reperfusion injury Johan M. Lorenzen1,2 1
The Journal of Physiology
2
Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover, Hannover Medical School, Germany Department of Medicine, Division of Nephrology, Hannover Medical School, Hannover, Germany
Abstract Ischaemia–reperfusion (I/R) injury of the kidney is a major cause of acute kidney injury. It may result in worsening or even loss of organ function. Transient occlusion of the renal vessel is followed by a reperfusion period, which induces further tissue damage by release of reactive oxygen and nitrogen species. Ischaemia–reperfusion injury of the kidney may be associated with surgical interventions in native kidneys and is also a common and unavoidable phenomenon in kidney transplantation. MicroRNAs are fascinating modulators of gene expression. They are capable of post-transcriptional silencing of genetic information by targeting the 3 -untranslated region of mRNAs, culminating in a suppression of protein synthesis or an increase in mRNA degradation. They might therefore be useful diagnostic and therapeutic entities during renal I/R injury; for instance, miR-21 has been shown to be enriched in kidney tissue in mice and humans with acute kidney injury. Interestingly, most recent literature suggests that modulation of vascular microRNAs might result in the amelioration of kidney function during renal I/R injury. To that end, miR-126 and miR-24, which have been demonstrated to be highly enriched in endothelial cells, were therapeutically modulated and shown to ameliorate renal I/R injury in mice. MicroRNAs in plasma, urine or enriched in microvesicles have been shown to serve as non-invasive tools for disease monitoring and to have potential impact on downstream mechanisms in recipient cells. This review highlights the latest developments regarding the role of microRNAs in renal I/R injury. (Received 19 November 2014; accepted after revision 13 February 2015; first published online 18 February 2015) Corresponding author J. M. Lorenzen: Department of Medicine, Division of Nephrology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Email: [email protected] Abbreviations I/R, ischaemia–reperfusion; miRNA, microRNA.
Pathophysiology of ischaemia–reperfusion injury of the kidney
Ischaemia–reperfusion (I/R) injury of the kidney is a major cause of acute kidney injury (Kelly, 2006). It is a common
phenomenon associated with a multitude of different insults in native kidneys (e.g. during cardiac surgery). Moreover, it is associated with the transplantation procedure and is therefore an unavoidable consequence in transplanted kidneys (Bon et al. 2012). During ischaemic
Johan Lorenzen is currently an assistant professor of medicine, a research group leader at the Institute of Molecular and Translational Therapeutic Studies (IMTTS) and a clinical specialist registrar at the Department of Nephrology and Hypertension at Hanover Medical School, Germany. Before becoming a research group leader at IMTTS he trained in the laboratory of Professor Katalin Susztak at Albert Einstein College of Medicine, New York City, U.S.A. and under the auspices of Professor Hermann Haller at the Department of Nephrology and Hypertension, Hanover Medical School, Germany. Scientifically he is interested in the regulation and therapeutic modulation of non-coding RNAs in various mouse models of renal injury. Various non-coding RNAs are either pharmacologically or genetically manipulated in mice. In addition, the research group is interested in circulating and urinary non-coding RNAs as biomarkers in patients with kidney disease.
This review was presented at the symposium Epigenetic regulation of cardiovascular development and disease, which took place at Physiology 2014, the annual meeting of The Physiological Society, London, UK on 1 July 2014. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
DOI: 10.1113/JP270318
1778
J. M. Lorenzen
acute kidney injury, a temporary impairment of blood flow to the kidney is followed by a reperfusion period. Reperfusion itself, though vital to restoration of kidney function, is associated with significant additional cellular injury (Weight et al. 1996). We have recently summarized the deleterious events resulting from I/R injury (Lorenzen et al. 2013). The damage inflicted by tissue ischaemia is subsequently aggravated by a dramatic surge in reactive oxygen and nitrogen species during reperfusion. These induce protein modifications, lipid oxidations and DNA double-strand breaks, finally culminating in endothelial dysfunction, neutrophil adherence to the endothelium and transendothelial migration, the release of inflammatory mediators and eventual cell death (Lorenzen et al. 2013). In the kidney, blood flow to the outer medulla is disproportionately reduced with respect to the reduction in total blood flow (Bonventre & Yang, 2011). Thus, epithelial cell injury is mainly detected in the S3 segment of the proximal tubule, located in the outer medulla (Bonventre & Yang, 2011). Interplay of several events contributes to the cellular injury observed in the kidney. The damaged endothelium interacts with and activates inflammatory cells through enhanced expression of adhesion molecules (e.g. intercellular adhesion molecule 1, selectins; Kelly et al. 1996; Bonventre & Yang, 2011). This interaction in turn contributes to obstruction of capillaries and postcapillary venules, further activation and transmigration of leucocytes, production of cytokines and inflammation in tubular epithelial cells (Bonventre & Yang, 2011). Capillary rarefaction in the inner stripe of the outer medulla ensues; due to the development of chronic hypoxia, this is an important contributor to tubulointerstitial fibrosis and progression to chronic kidney disease after acute kidney injury (Bonventre & Yang, 2011). Cell polarity and cytoskeletal arrangement are severely impaired in proximal tubular epithelial cells during ischaemia (Sutton & Molitoris, 1998; Bonventre & Yang, 2011). Important phenotypical changes are loss of the proximal tubule brush border as well as loss of polarity and derangement of adhesion molecules and other membrane proteins and disruption of cell–cell interactions at adherens and tight junctions (Bonventre & Yang, 2011). MicroRNA biogenesis and function
MicroRNAs (miRNAs) are currently under intense investigation as powerful regulators of various diseases with potential critical impact on disease initiation and/or progression, including kidney disease (Lorenzen et al. 2011a). MicroRNAs represent small non-coding RNA transcripts with a length of 22 nucleotides, which through post-transcriptional binding of the 3 -untranslated region of mRNA targets lead to the repression of
J Physiol 593.8
gene/protein expression and/or translational inhibition of protein synthesis (Lorenzen et al. 2011a; Lorenzen & Thum, 2012). The first miRNA, lin-4, was discovered while investigating genetic loci responsible for temporal patterning in Caenorrhabdidits elegans (Ambros, 1989; Lee et al. 1993) Intriguingly, a single microRNA may alter the expression of a large number of target genes, thus influencing a specific pathology by regulating whole disease-specific pathways and signalling cascades rather than a single gene. This unique function underlines the immense importance of these small molecules. The biogenesis of microRNAs follows a tightly regulated pattern (Lorenzen et al. 2011a). Transcribed as primary miRNA transcripts (pri-miRNAs), they are further processed in the nucleus by the ribonuclease Drosha to precursor miRNAs (pre-miRNAs) with a length of 70 nucleotides. The pre-miRNA is then shuttled out of the nucleus into the cytosol by a Ran-guanosine-5 -triphosphate-dependent transporter, exportin 5. A second ribonuclease, Dicer, gives rise to a double-stranded RNA duplex (miRNA:miRNA∗ ). It is composed of a miRNA guide strand and its complementary strand (miRNA∗ ), each consisting of 22 nucleotides. While the guide strand is incorporated into an RNA-induced silencing complex, where it interacts and cleaves its target mRNA, the complementary strand is believed to be degraded. However, recent evidence suggests that the complementary strand also carries functional importance (Yang et al. 2011). There are also several non-canonical pathways of miRNA biogenesis, which have been elegantly reviewed elsewhere (Ha & Kim, 2014). Some examples are listed here. i. The 7-methylguanosine-capped pre-miR-320 bypasses Drosha processing and is generated directly through transcription and exported to the cytoplasm by exportin 1. ii. Mirtrons are located in the introns of mRNA encoding host genes. Here, pre-miRNAs can be generated directly by splicing and debranching. iii. Some small nucleolar RNAs may also be cleaved to produce pre-miRNAs. iv. In terminal uridylyl transferase-dependent group II pri-miRNAs, pre-miRNAs are generated with shorter 3 overhangs, which have first to be monouridylated for efficient Dicer processing. v. There are also Dicer-independent pathways, e.g. pre-miR-451 is produced by Drosha and exported to the cytoplasm. Here, it interacts with Argonaute 2 without Dicer processing. Argonaute 2 further cleaves pre-miR-451. Subsequently, pre-miR-451 is modified by the 3 ,5 -exonuclease poly(A)-specific ribonuclease. Cellular miRNA function can be silenced by miRNA antagonists termed ‘antagomirs’ (or ‘antimiRs’), which are C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
J Physiol 593.8
Vascular and circulating microRNAs in acute kidney injury
chemically engineered oligonucleotides targeting specific miRNAs (Kr¨utzfeldt et al. 2005). For in vivo delivery, these antimiRs have to be modified in order to increase stability. These include cholesterol modification, modifications or such as 2 -O-methoxyethylphosphorothioate 2 -fluoro/2 -methoxyethyl substitutions (2 (F)-MOE), as well as oligonucleotides stabilized by locked nucleic acid phosphorothioate chemistries (Lorenzen et al. 2011a). If overexpression of a specific microRNA is the desired approach, miRNA expression can be enhanced by double-stranded miRNA mimics (Lorenzen et al. 2011a). An elucidation of the mechanisms of microRNA-mediated kidney injury is thus of utmost importance.
1779
identified miR-127 to contribute to rat renal I/R injury (Aguado-Fraile et al. 2012). MiR-127 was postulated to be regulated by hypoxia-inducible factor 1α and to target kinesin family member 3B. Modulation of miR-127 resulted in changes in cell adhesion and cytoskeletal structure. Regulation of its target, kinesin family member 3B, resulted in modulation of endocytosis and microtubular transport in rat renal proximal tubular cells. MiR-494 was shown to exacerbate renal I/R injury in mice by targeting activating transcription factor 3, thereby promoting apoptosis and further impairing renal function (Lan et al. 2012).
MicroRNAs and renal ischaemia–reperfusion injury
Novel vascular microRNAs and renal ischaemia–reperfusion injury
The contribution of specific miRNAs to the development of renal I/R injury has been investigated in several studies so far. It has recently been shown that targeted deletion of Dicer from the proximal tubular epithelium protects against I/R-induced renal injury through changes in expression of various microRNAs (Wei et al. 2010). Interestingly, Dicer knockdown was associated with reduced expression of miR-27a. This microRNA belongs to a microRNA cluster, which is simultaneously transcribed. The miR-23/27/24 gene cluster is composed of two separate gene clusters located at different genomic loci (Bang et al. 2012). The intergenic miR-24-2 cluster is encoded on human chromosome 19p13.13, expressing miR-23a, miR-27a and miR-24-2 (Chan et al. 2010; Bang et al. 2012). This cluster has been demonstrated to be involved in angiogenesis and endothelial apoptosis in cardiac ischaemia and retinal vascular development (Bang et al. 2012). Moreover, miR-24 has been demonstrated to regulate apoptosis of cancer and T cells (Brunner et al. 2012; Xie et al. 2012). In the kidney, overexpression of the miR-23a, miR-27a and miR-24-2 cluster induces apoptosis of human embryonic kidney cells (Chhabra et al. 2009). Changes in expression of other distinct microRNAs during I/R injury have been shown (e.g. miR-132, miR-362 and miR-379; Wei et al. 2010). Godwin et al. (2010) elegantly identified miR-21 as one of the most highly induced miRNAs during renal I/R injury. Moreover, miR-21 expression was shown to be increased in proliferating tubular epithelial cells, while knockdown of miR-21 in these cells resulted in enhanced apoptosis (Godwin et al. 2010). Recently, Duffield and co-workers also identified miR-21 as one of the most highly upregulated microRNAs in humans with acute kidney injury and in mice subjected to renal I/R injury (Chau et al. 2012). MiR-21 was found to target peroxisome proliferator-activated receptor α, thereby regulating metabolic pathways (Chau et al. 2012). Another group
Another interesting microRNA in I/R injury of the kidney is miR-126. As mentioned above (see section on pathophysiology of ischemia-reperfusion “injury of the kidney”), integrity of the renal peritubular capillary network is an important limiting factor in the recovery from renal I/R injury. MiR-126 has been shown to improve vascular regeneration by mobilizing haematopoietic stem/progenitor cells (van Solingen et al. 2011; Salvucci et al. 2012). Viral overexpression of miR-126 in the haematopoietic system was shown to improve neovascularization in mice (Bijkerk et al. 2014). Moreover, following renal I/R injury, mice overexpressing miR-126 displayed a significant improvement of kidney function, fibrosis and kidney injury. This was associated with a reduction of capillary rarefaction in the corticomedullary junction and an increase in the number of bone marrow-derived endothelial cells. In addition, the number of circulating Lin− /Sca-1+ /cKit+ haematopoietic stem and progenitor cells was increased, partly by attenuated expression of the chemokine receptor CXCR4 on haematopoietic stem and progenitor cells in the bone marrow. Renal expression of its ligand, stromal cell-derived factor 1, was increased, thus favouring mobilization of Lin− /Sca-1+ /cKit+ cells towards the kidney. Very recently, we found miR-24 to be enriched in the kidney following I/R injury in mice (Lorenzen et al. 2014, see Fig. 1). Kidney transplant patients whose transplanted kidneys were exposed to long versus short cold ischaemia times also presented with increased intragraft miR-24 levels. Cell sorting experiments using specific cell surface markers or lectins revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after induction of I/R. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Enrichment of miR-24 induced apoptosis, whereas its silencing ameliorated apoptotic responses. The effects of miR-24
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
1780
J. M. Lorenzen
were mediated through regulation of H2A histone family, member X, the sphingosine-1-phopshate receptor 1 and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. In vivo, silencing of miR-24 in mice following I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histological tubular epithelial injury and less infiltration of inflammatory cells. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites along with miR-24 precursors rescued various functional parameters in endothelial and tubular epithelial cells. Heme oxygenase 1 and H2A histone family, member X were also found to be regulated by miR-24 in vivo. Intriguingly, miR-24 was also enriched in kidney biopsies of renal transplant patients with prolonged cold ischaemia time, underlining its potential significance in humans. Inhibition of miR-24 is therefore a promising future therapeutic option in the treatment of patients with ischaemic acute kidney injury. It is conceivable that Tubular epithelial cell
J Physiol 593.8
miR-24 antagonism in transplant kidneys might result in an amelioration of kidney injury and thus prolonged long-term organ survival. Here, the first study to be published evaluating a microRNA-based therapy in renal I/R injury is presented. Previously, my colleagues and I were able to demonstrate that miR-24 was also specifically enriched in cardiac endothelial cells in the infarct border zone after myocardial infarction (Fiedler et al. 2011). Transient overexpression of miR-24 in human umbilical vein endothelial cells induced apoptosis, loss of capillary network formation capacity and migratory capacity in a modified Boyden chamber assay, and impaired endothelial spheroid formation, sprouting capacity and proliferation. These effects of miR-24 in endothelial cells were mediated by modulation of the transcription factor GATA2 and the p21-activated kinase PAK4, which were identified bioinformatically and subsequently validated by luciferase gene reporter assays. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blood vessel
miR-126 overexpression in haematopoietic compartment
Hypoxia/anoxia-induced miR-24
Renal expression of stromal cell-derived factor 1
Regulation of HO-1, S1PR1, H2A.X
Tubular epithelial / Endothelial cell apoptosis
miR-24 silencing
mobilization of Lin2/Sca-1+/cKit+ cells
- improvement of kidney function - Reduction of fibrosis - reduction of capillary rarefaction
Figure 1. Novel vascular microRNAs in renal ischaemia–reperfusion injury MiR-24 is induced in hypoxic/anoxic tubular epithelial and endothelial cells, targeting and silencing haem oxygenase 1 (HO-1), the sphingosine-1-phopshate receptor 1 (S1PR1) and H2A histone family, member X (H2A.X), thereby leading to increased apoptosis of these cells. Overexpression of miR-126 in the haematopoietic compartment in mice results in enhanced renal expression of stromal cell-derived factor 1, culminating in the mobilization of Lin2/Sca-1+ /cKit+ haematopoietic stem and progenitor cells to the kidney. Silencing of miR-24 and overexpression of miR-126 in mice subjected to renal ischaemia–reperfusion injury are finally characterized by an improvement of kidney function, a reduction of fibrosis and a reduction of capillary rarefaction. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
Vascular and circulating microRNAs in acute kidney injury
J Physiol 593.8
In summary, our group was able to describe miR-24 as an important vascular microRNA in cardiac and renal I/R injury. MiR-24 was found to be increased greatly in endothelial cells in both experimental models. In addition, we found that miR-24 was also enriched in damaged/injured tubular epithelial cells in renal I/R injury. Vascular miRNAs are summarised in Fig. 1. Novel circulating and urinary microRNAs in kidney injury
MicroRNAs are also released into the extracellular compartment (blood and urine) in patients (Lorenzen & Thum, 2012). Thus, circulating microRNAs may serve as a non-invasive tool to detect and monitor disease activity. The investigation and analysis of the release pattern of circulating microRNAs may thus enable the clinician to monitor certain patients adequately regarding disease progression and response to treatment. To that end, microRNA-enriched microvesicles, which can be detected in blood, secreted by endothelial progenitor cells, were shown to ameliorate I/R injury in the murine kidney (Cantaluppi et al. 2012). MiRNA array analysis revealed that the pro-angiogenic and anti-apoptotic
1781
miR-126 and miR-296 were greatly enriched in microvesicles. In a rat model of I/R injury, the addition of endothelial progenitor cell-derived microvesicles resulted in a proliferative response of tubular epithelial cells as well as a reduction in tubular epithelial cell apoptosis and infiltration of leucocytes. Our group was able to analyse the miRNA expression profile in plasma samples of critically ill patients with dialysis-dependent acute kidney injury before the inception of renal replacement therapy (Lorenzen et al. 2011b). Thirteen different miRNAs were found to be deregulated significantly. MiR-210 emerged as the most striking mediator of survival in this patient cohort in Cox proportional hazard and Kaplan–Meier curve analyses. In urine of patients with acute T-cell-mediated renal allograft rejection, we also found miR-210 to be deregulated (Lorenzen et al. 2011c). Specifically, miR-210 was found to be downregulated in urine of patients with rejection, as diagnosed by a kidney biopsy, in comparison to control patients with stable transplant function without signs of rejection. Successful antirejection therapy normalized miR-210 to the levels of control patients. Moreover, urinary miR-210 at the time of rejection predicted the decline in glomerular filtration rate at 1 year after transplantation. Interestingly,
B
A
MV
Altered trafficking in damaged tubular epithelial cells ? Plasma of patients with AKI on ICU - miR-210 - miR-320 - miR-16
Urine of transplant patients with kidney rejection - miR-210 - miR-10a - miR-10b
EPC
pro-angiogenic and anti-apoptotic miR-126 and miR-296 Addition of miRNA-enriched MV
Rat model of I/R -injury 1.) proliferative response of tubular epithelial cells 2.) reduction in tubular epithelial cell apoptosis 3.) less infiltration of leukocytes Prediction of outcome by miR-210 Figure 2. Circulating and urinary microRNAs in kidney disease A, in patients with dialysis-dependent acute kidney injury (AKI) on the intensive care unit (ICU), miR-210 is increased in plasma, while miR-16 and miR-320 are reduced. In patients with acute T-cell-mediated renal transplant rejection, miR-210 and miR-10b are downregulated and miR-10a is upregulated in urine. In both patient cohorts, miR-210 predicts outcome (for AKI, survival on the ICU; for transplant rejection, loss of glomerular filtration rate at 1 year after transplantation). B, miR-126 and miR-296 enriched in microvesicles (MV) derived from endothelial progenitor cells (EPC) ameliorate AKI in a rat model of ischaemia–reperfusion injury. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
1782
J. M. Lorenzen
J Physiol 593.8
Table 1. MicroRNAs, their targets and pathophysiological effects in renal ischaemia–reperfusion injury MicroRNA
Target
Organ/cell type
Pathophysiological effects
Organism
Reference
MiR-126
—
Kidney, bone marrow
Mouse
Bijkerk et al. (2014)
MiR-24
H2A histone family, member X, the sphingosine-1-phopshate receptor 1 and heme oxygenase 1
Kidney
Mouse
Lorenzen et al. (2014)
MiR-127
Kinesin family member 3B
Rat
Aguado-Fraile et al. (2012)
MiR-126 and miR-296
—
Mouse
Cantaluppi et al. (2012)
MiR-21
Peroxisome proliferator-activated receptor α Activating transcription factor 3 —
Kidney, proximal tubular cells Endothelial progenitor cell-derived microvesicles, proximal tubular cells Kidney
Inhibition of capillary rarefaction, mobilization of Lin− /Sca-1+ /cKit+ haematopoietic stem and progenitor cells Improvement of survival and kidney function, a reduction of apoptosis, improvement in histological tubular epithelial injury and less infiltration of inflammatory cells Changes in cell adhesion and cytoskeletal structure Inhibition of capillary rarefaction, glomerulosclerosis and tubulointerstitial fibrosis
Regulation of metabolic pathways
Mouse
Chau et al. (2012)
Exacerbation of renal injury Signature of kidney ischaemia–reperfusion injury Signature of kidney ischaemia–reperfusion injury
Mouse
Lan et al. (2012) Wei et al. (2010)
MiR-494 MiR-132, miR-362 and miR-379 MiR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194 MiR-210
Kidney
—
Kidney, proximal tubular cells Kidney
—
Kidney
in a mouse model of renal I/R injury, miRNA-210 was also shown to be upregulated in kidney tissue and to regulate renal angiogenesis by activation of the vascular
Biomarker of acute kidney kidney injury and acute T-cell-mediated rejection. Regulation of renal angiogenesis by activation of the vascular endothelial growth factor signalling pathway
Mouse
Mouse
Godwin et al. (2010)
Mouse, human plasma, human urine
Lorenzen et al. (2011b, c), Liu et al. (2012)
endothelial growth factor signalling pathway (Liu et al. 2012). Circulating and urinary miRNAs are summarised in Fig. 2.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
J Physiol 593.8
Vascular and circulating microRNAs in acute kidney injury
Conclusion
All discussed miRNAs, their known targets and biological roles are summarized in Table 1. An elucidation of the mechanisms of microRNA-mediated kidney injury is thus of utmost importance. Intriguingly, pathological microRNA expression might be modulated by RNA therapeutics, termed antagomirs, which enable specific targeting and cleavage of miRNAs and thus alteration of pathological signalling pathways in vivo. MiR-21 was one of the first miRNAs found to be altered during I/R injury of mice and humans; however, miR-21 seems to be indispensible for tubular regeneration during acute ischaemic injuries. Interestingly, miRNAs, which were first discovered to play a major role in the vasculature, e.g. miR-24 and miR-126, have been shown to be useful as therapeutics in mice with I/R injury. Modulation of vascular microRNAs might be a viable therapeutic option in the treatment of patients with the important clinical disorder of I/R injury of the kidney. Moreover, a few miRNAs have been characterized to be detectable in the circulation of patients and mice, including pro-angiogenic and anti-apoptotic miR-126 and miR-296 as well as miR-210. Detection and possible modulation of circulating miRNAs might thus be an interesting means of monitoring and treating disease activity.
References Aguado-Fraile E, Ramos E, S´aenz-Morales D, Conde E, Blanco-S´anchez I, Stamatakis K, del Peso L, Cuppen E, Br¨une B & Bermejo ML (2012). MiR-127 protects proximal tubule cells against ischemia/reperfusion: identification of kinesin family member 3B as miR-127 target. PloS One 7, e44305. Ambros V (1989). A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans. Cell 57, 49–57. Bang C, Fiedler J & Thum T (2012). Cardiovascular importance of the microRNA-23/27/24 family. Microcirculation 19, 208–214. Bijkerk R, vanSolingen C, deBoer HC, vander Pol P, Khairoun M, deBruin RG, vanOeveren-Rietdijk AM, Lievers E, Schlagwein N, vanGijlswijk DJ, Roeten MK, Neshati Z, deVries AA, Rodijk M, Pike-Overzet K, vanden Berg YW, vander Veer EP, Versteeg HH, Reinders ME, Staal FJ, vanKooten C, Rabelink TJ & vanZonneveld AJ (2014). Hematopoietic microRNA-126 protects against renal ischemia/reperfusion injury by promoting vascular integrity. J Am Soc Nephrol 25, 1710–1722. Bon D, Chatauret N, Giraud S, Thuillier R, Favreau F & Hauet T (2012). New strategies to optimize kidney recovery and preservation in transplantation. Nat Rev Nephrol 8, 339–347. Bonventre JV & Yang L (2011). Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 121, 4210–4221.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
1783
Brunner S, Herndler-Brandstetter D, Arnold CR, Wiegers GJ, Villunger A, Hackl M, Grillari J, Moreno-Villanueva M, B¨urkle A & Grubeck-Loebenstein B (2012). Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8+ T cells sensitizing them to apoptotic cell death. Aging Cell 11, 579–587. Cantaluppi V, Gatti S, Medica D, Figliolini F, Bruno S, Deregibus MC, Sordi A, Biancone L, Tetta C & Camussi G (2012). Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia–reperfusion injury by microRNA-dependent reprogramming of resident renal cells. Kidney Int 82, 412–427. Chan MC, Hilyard AC, Wu C, Davis BN, Hill NS, Lal A, Lieberman J, Lagna G & Hata A (2010). Molecular basis for antagonism between PDGF and the TGFβ family of signalling pathways by control of miR-24 expression. EMBO J 29, 559–573. Chau BN, Xin C, Hartner J, Ren S, Castano AP, Linn G, Li J, Tran PT, Kaimal V, Huang X, Chang AN, Li S, Kalra A, Grafals M, Portilla D, MacKenna DA, Orkin SH & Duffield JS (2012). MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways. Sci Transl Med 4, 121ra18. Chhabra R, Adlakha YK, Hariharan M, Scaria V & Saini N (2009). Upregulation of miR-23a27a24-2 cluster induces caspase-dependent and -independent apoptosis in human embryonic kidney cells. PLoS One 4, e5848. Fiedler J, Jazbutyte V, Kirchmaier BC, Gupta SK, Lorenzen J, Hartmann D, Galuppo P, Kneitz S, Pena JT, Sohn-Lee C, Loyer X, Soutschek J, Brand T, Tuschl T, Heineke J, Martin U, Schulte-Merker S, Ertl G, Engelhardt S, Bauersachs J & Thum T (2011). MicroRNA-24 regulates vascularity after myocardial infarction. Circulation 124, 720– 730. Godwin JG, Ge X, Stephan K, Jurisch A, Tullius SG & Iacomini J (2010). Identification of a microRNA signature of renal ischemia reperfusion injury. Proc Natl Acad Sci U S A 107, 14339–14344. Ha M & Kim VN (2014). Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15, 509–524. Kelly KJ, Williams WW Jr, Colvin RB, Meehan SM, Springer TA, Gutierrez-Ramos JC & Bonventre JV (1996). Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. J Clin Invest 97, 1056–1063. Kelly KJ (2006). Acute renal failure: much more than a kidney disease. Semin Nephrol 26, 105–113. Kr¨utzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M & Stoffel M (2005). Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438, 685–689. Lan YF, Chen HH, Lai PF, Cheng CF, Huang YT, Lee YC, Chen TW & Lin H (2012). MicroRNA-494 reduces ATF3 expression and promotes AKI. J Am Soc Nephrol 23, 2012–2023. Lee RC, Feinbaum RL, Ambros V (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.
1784
J. M. Lorenzen
Liu F, Lou YL, Wu J, Ruan QF, Xie A, Guo F, Cui SP, Deng ZF & Wang Y (2012). Upregulation of microRNA-210 regulates renal angiogenesis mediated by activation of VEGF signaling pathway under ischemia/perfusion injury in vivo and in vitro. Kidney Blood Press Res 35, 182–191. Lorenzen JM, Batkai S & Thum T (2013). Regulation of cardiac and renal ischemia–reperfusion injury by microRNAs. Free Radic Biol Med 64, 78–84. Lorenzen JM, Haller H & Thum T (2011a). MicroRNAs as mediators and therapeutic targets in chronic kidney disease. Nat Rev Nephrol 7, 286–294. Lorenzen JM, Kaucsar T, Schauerte C, Schmitt R, Rong S, H¨ubner A, Scherf K, Fiedler J, Martino F, Kumarswamy R, K¨olling M, S¨orensen I, Hinz H, Heineke J, vanRooij E, Haller H & Thum T (2014). MicroRNA-24 antagonism prevents renal ischemia reperfusion injury. J Am Soc Nephrol 25, 2717–2729. Lorenzen JM, Kielstein JT, Hafer C, Gupta SK, Kumpers P, Faulhaber-Walter R, Haller H, Fliser D & Thum T (2011b). Circulating miR-210 predicts survival in critically ill patients with acute kidney injury. Clin J Am Soc Nephrol 6, 1540–1546. Lorenzen JM & Thum T (2012). Circulating and urinary microRNAs in kidney disease. Clin J Am Soc Nephrol 7, 1528–1533. Lorenzen JM, Volkmann I, Fiedler J, Schmidt M, Scheffner I, Haller H, Gwinner W & Thum T (2011c). Urinary miR-210 as a mediator of acute T-cell mediated rejection in renal allograft recipients. Am J Transplant 11, 2221–2227. Salvucci O, Jiang K, Gasperini P, Maric D, Zhu J, Sakakibara S, Espigol-Frigole G, Wang S & Tosato G (2012). MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1. Haematologica 97, 818–826.
J Physiol 593.8
Sutton TA & Molitoris BA (1998). Mechanisms of cellular injury in ischemic acute renal failure. Semin Nephrol 18, 490–497. vanSolingen C, deBoer HC, Bijkerk R, Monge M, vanOeveren-Rietdijk AM, Seghers L, de Vries MR, van derVeer EP, Quax PH, Rabelink TJ & van Zonneveld AJ (2011). MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+ /Lin− progenitor cells in ischaemia. Cardiovasc Res 92, 449–455. Wei Q, Bhatt K, He HZ, Mi QS, Haase VH & Dong Z (2010). Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury. J Am Soc Nephrol 21, 756–761. Weight SC, Bell PR & Nicholson ML (1996). Renal ischaemia–reperfusion injury. Br J Surg 83, 162–170. Xie Y, Tobin LA, Camps J, Wangsa D, Yang J, Rao M, Witasp E, Awad KS, Yoo N, Ried T & Kwong KF (2012). MicroRNA-24 regulates XIAP to reduce the apoptosis threshold in cancer cells. Oncogene 32, 2442–2451. Yang JS, Phillips MD, Betel D, Mu P, Ventura A, Siepel AC, Chen KC & Lai EC (2011). Widespread regulatory activity of vertebrate microRNA∗ species. RNA 17, 312–326.
Additional information Competing interests None declared.
Funding I wish to acknowledge funding by the German Research Council (DFG LO 1736/1-1) and the Else Kr¨oner-Fresenius Foundation (2011 A173).
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
Copyright of Journal of Physiology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
