Circular RNA-based biomarkers in blood of patients with Fabry disease and related phenotypes.

BACKGROUND: Fabry disease is a rare X-linked lysosomal storage disease caused by mutations in the galactosidase α gene. Deficient activity of α-galactosidase A leads to glycosphingolipid accumulations in multiple organs. Circular RNAs represent strong regulators of gene expression. Their circular structure ensures high stability in blood. We hypothesised that blood-based circular RNA profiles improve phenotypic assignment and therapeutic monitoring of Fabry disease. METHODS: A genome-wide circular RNA expression analysis was performed in blood of genetically diagnosed patients with Fabry disease (n=58), age-matched and sex-matched healthy volunteers (n=14) and disease control patients with acute kidney injury (n=109). Most highly dysregulated circular RNAs were validated by quantitative real-time PCR. Circular RNA biomarker sensitivity, specificity, predictive values and area under the curve (AUC) were determined. Linear regression analyses were conducted for validated circular RNA biomarkers and clinical patient characteristics. RESULTS: A distinct circular RNA transcriptome signature identified patients with Fabry disease. Level of circular RNAs hsa_circ_0006853 (AUC=0.73), hsa_circ_0083766 (AUC=0.8) and hsa_circ_0002397 (AUC=0.8) distinguished patients with Fabry disease from both healthy controls and patients with acute kidney injury. Hsa_circ_0002397 was, furthermore, female-specifically expressed. Circular RNA level were significantly related to galactosidase α gene mutations, early symptoms, phenotypes, disease severities, specific therapies and long-term complications of Fabry disease. CONCLUSION: The discovery of circular RNA-based and Fabry disease-specific biomarkers may advance future diagnosis of Fabry disease and help to distinguish related phenotypes.

Circular RNAs in kidney disease and cancer.

Circular RNAs (circRNAs) are a class of endogenously expressed regulatory RNAs with a single-stranded circular structure. They are generated by back splicing and their expression can be tightly regulated by RNA binding proteins. Cytoplasmic circRNAs can function as molecular sponges that inhibit microRNA-target interactions and protein function or as templates for the efficient generation of peptides via rolling circle amplification. They can also act as molecular scaffolds that enhance the reaction kinetics of enzyme-substrate interactions. In the nucleus, circRNAs might facilitate chromatin modifications and promote gene expression. CircRNAs are resistant to degradation and can be packaged in extracellular vesicles and transported in the circulation. Initial studies suggest that circRNAs have roles in kidney disease and associated cardiovascular complications. They have been implicated in hypertensive nephropathy, diabetic kidney disease, glomerular disease, acute kidney injury and kidney allograft rejection, as well as in microvascular and macrovascular complications of chronic kidney disease, including atherosclerotic vascular disease. In addition, several circRNAs have been reported to have oncogenic or tumour suppressor roles or to regulate drug resistance in kidney cancer. The available data suggest that circRNAs could be promising diagnostic and/or prognostic biomarkers and potential therapeutic targets for kidney disease, cardiovascular disease and kidney cancer.

Safety of Kidney Biopsy when Performed as an Outpatient Procedure.

INTRODUCTION: Kidney biopsy remains the gold standard for the diagnosis of most renal diseases. A major obstacle to performing a biopsy is safety concerns. However, many safety measures are not evidence based and therefore vary widely between centers. We sought to determine the rate and timing of kidney biopsy complications in our center, to compare the complication rate between native and transplant kidney biopsies, to evaluate the feasibility of performing kidney biopsies as an outpatient procedure and the value of a postbiopsy ultrasound before discharge, and to identify risk factors for complications. METHODS: We performed a single-center, retrospective, observational study at the Division of Nephrology of the University Hospital Zurich including all patients who underwent renal biopsy between January 2005 and December 2017. Major bleeding (primary outcome) and any other bleeding or nonbleeding complications (secondary outcomes) were compared between native and transplant kidney biopsies and between inpatient and outpatient procedures and correlated with clinical factors possibly affecting bleeding risk. RESULTS: Overall, 2,239 biopsies were performed in 1,468 patients, 732 as inpatient and 1,507 as outpatient procedures. Major bleeding was observed in 28 (3.8%) inpatient and in 15 (1.0%) outpatient procedures, totaling to 43 (1.9%) of all biopsies. Major bleeding requiring intervention amounted to 1.0% (0.5% of outpatient procedures). Rate of major bleeding was similar between native and transplant kidneys. 13/15 (87%) bleeding episodes in planned outpatient procedures were detected during the 4-h surveillance period. Risk factors for bleeding were aspirin use, low eGFR, anemia, cirrhosis, and amyloidosis. Routine postbiopsy ultrasound did not change management. CONCLUSIONS: Kidney biopsy is an overall safe procedure and can be performed as an outpatient procedure in most patients with an observation period as short as 4 h. The value of routine postbiopsy ultrasound is questionable.

Spectrum and dosing of urate-lowering drugs in a large cohort of chronic kidney disease patients and their effect on serum urate levels: a cross-sectional analysis from the German Chronic Kidney Disease study.

BACKGROUND: Despite a plethora of studies on the effect of urate-lowering therapy (ULT) in patients with chronic kidney disease (CKD), current guidelines on the treatment of hyperuricaemia and gout vary, especially concerning the need for dose adjustment of allopurinol, whose main metabolite is accumulating with declining renal function. Data on allopurinol dosing and its relationship to renal function, co-medication and sex and the resulting urate level in large cohorts are missing. METHODS: We studied a subgroup of 2378 patients of the German Chronic Kidney Disease (GCKD) study to determine prescription patterns of ULT among CKD patients under nephrological care and the relationship of ULT dose to urate levels. Prescription and dosing of ULT were manually abstracted from the patient's paper charts at the baseline visit, in which all currently used medications and their dosing were recorded. RESULTS: In this cohort, 39.6% were women, the mean estimated glomerular filtration rate (eGFR) was 51.3 ± 19.3 mL/min/1.73 m2 and the mean age was 59.0 ± 12.4 years. Of the 2378 examined patients, 666 (28.0%) received ULT. The dose of ULT was available for 572 patients. The main ULT agent was allopurinol (94.4%), followed by febuxostat (2.9%) and benzbromarone (2.6%). Of the 540 patients who used allopurinol with a reported daily dose, 480 had an eGFR <60 mL/min/1.73 m2 and 320 had an eGFR <45 mL/min/1.73 m2, 31.5% of the latter (n = 101) received a dose >150 mg/day, the recommended maximal dose for this level of eGFR. The prescribed dose was not related to eGFR: the median eGFR for patients taking 100, 150 and 300 mg/day was 40 [interquartile range (IQR) 32-49], 43 (34-52) and 42 (35-54) mL/min/1.73 m2, respectively. Patients with lower doses of allopurinol had higher serum urate levels than patients with higher (than recommended) allopurinol doses. Sex, alcohol intake, eGFR, use of diuretics and treatment with allopurinol were independent determinants of serum urate levels in multivariate regression analysis. CONCLUSIONS: The most frequently used drug to lower serum urate levels in this CKD cohort was allopurinol. Even in patients regularly seen by nephrologists, the dose of allopurinol is often not adjusted to the current eGFR. Patients with higher ULT doses achieved better control of their serum urate levels. Lowering of serum urate in CKD patients requires balancing potential adverse effects of allopurinol with suboptimal control of serum urate levels.

Renal AAV2-Mediated Overexpression of Long Non-Coding RNA H19 Attenuates Ischemic Acute Kidney Injury Through Sponging of microRNA-30a-5p.

BACKGROUND: Renal ischemia-reperfusion (I/R) injury is a major cause of AKI. Noncoding RNAs are intricately involved in the pathophysiology of this form of AKI. Transcription of hypoxia-induced, long noncoding RNA H19, which shows high embryonic expression and is silenced in adults, is upregulated in renal I/R injury. METHODS: Lentivirus-mediated overexpression, as well as antisense oligonucleotide-based silencing, modulated H19 in vitro. In vivo analyses used constitutive H19 knockout mice. In addition, renal vein injection of adeno-associated virus 2 (AAV2) carrying H19 caused overexpression in the kidney. Expression of H19 in kidney transplant patients with I/R injury was investigated. RESULTS: H19 is upregulated in kidney biopsies of patients with AKI, in murine ischemic kidney tissue, and in cultured and ex vivo sorted hypoxic endothelial cells (ECs) and tubular epithelial cells (TECs). Transcription factors hypoxia-inducible factor 1-α, LHX8, and SPI1 activate H19 in ECs and TECs. H19 overexpression promotes angiogenesis in vitro and in vivo. In vivo, transient AAV2-mediated H19 overexpression significantly improved kidney function, reduced apoptosis, and reduced inflammation, as well as preserving capillary density and tubular epithelial integrity. Sponging of miR-30a-5p mediated the effects, which, in turn, led to target regulation of Dll4, ATG5, and Snai1. CONCLUSIONS: H19 overexpression confers protection against renal injury by stimulating proangiogenic signaling. H19 overexpression may be a promising future therapeutic option in the treatment of patients with ischemic AKI.

Altered glycosylation of IgG4 promotes lectin complement pathway activation in anti-PLA2R1-associated membranous nephropathy.

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

Biogenesis and Function of Circular RNAs in Health and in Disease.

Circular RNAs (circRNAs) are a class of non-coding RNA that were previously thought to be insignificant byproducts of splicing errors. However, recent advances in RNA sequencing confirmed the presence of circRNAs in multiple cell lines and across different species suggesting a functional role of this RNA species. CircRNAs arise from back-splicing events resulting in a circular RNA that is stable, specific and conserved. They can be generated from exons, exon-introns, or introns. CircRNAs have multifaceted functions. They are likely part of the competing endogenous RNA class. They can regulate gene expression by sponging microRNAs, binding proteins or they can be translated into a protein themselves. CircRNAs have been associated with health and disease, some with disease protective effects, some with disease promoting functions. The widespread expression and disease regulatory mechanisms endow circRNAs to be used as functional biomarkers and therapeutic targets for a variety of different disorders. In this concise article we provide an overview of the association of circRNAs with various diseases including cancer, cardiovascular and kidney disease as well as cellular senescence. We conclude with an assessment of the current status and future outlook of this new field of research that carries immense potential with respect to diagnostic and therapeutic approaches of a variety of diseases.

The hypoxic kidney: pathogenesis and noncoding RNA-based therapeutic strategies.

Acute kidney injury (AKI) is a disease entity of major importance, affecting approximately 6% of all patients on the intensive care unit. The mortality rate exceeds 60%. AKI is related to several underlying conditions, including sepsis, nephrotoxicity or major surgery. Ischaemia reperfusion injury or hypoxic conditions may lead to severe injury of the kidney and is associated with a steep decline in survival rates of patients. At present, AKI is diagnosed on the basis of creatinine levels and urine output. Novel markers and knowledge of their pathophysiological role is of major importance for targeted therapeutic interventions. Noncoding RNAs (ncRNAs) have recently been introduced and are the subject of intensive research initiatives. They are arbitrarily separated into small ncRNAs (≤200 nucleotides) and long ncRNAs (lncRNAs, ≥200 nucleotides). Whereas small ncRNAs such as microRNAs have been extensively studied over the past several years, investigations into the role of linear lncRNAs and circular RNAs (circRNAs) are largely lacking. The present review article therefore aims to elucidate in detail the role of microRNAs, lncRNAs and circRNAs in animal models as well as patients with ischaemic AKI and to describe their use as biomarkers as well as their potential use as therapeutics.

The Circular RNA ciRs-126 Predicts Survival in Critically Ill Patients With Acute Kidney Injury.

INTRODUCTION: Circular RNAs (circRNAs) have recently been described as novel noncoding regulators of gene expression. They might have an impact on microRNA expression by their sponging activity. The detectability in blood of these RNA transcripts has been demonstrated in patients with cancer and cardiovascular disease. We tested the hypothesis that circulating circRNAs in blood of critically ill patients with acute kidney injury (AKI) at inception of renal replacement therapy may also be dysregulated and associated with patient survival. METHODS: We performed a global circRNA expression analysis using RNA isolated from blood of patients with AKI as well as controls. This global screen revealed several dysregulated circRNAs in patients with AKI. Most highly increased circRNA-array-based transcripts as well as expression of the circRNA target miR-126-5p were confirmed in blood of 109 patients with AKI, 30 age-matched healthy controls, 25 critically ill non-AKI patients, and 20 patients on maintenance hemodialysis by quantitative real-time polymerase chain reaction. RESULTS: Circulating concentrations of 3 novel circRNAs were amplified in blood of patients with AKI and in controls. Circular RNA sponge of miR-126 (or ciRs-126) was most highly altered compared to healthy controls and disease controls (fold change of 52.1). ciRs-126 was shown to bioinformatically sponge miR-126-5p, which was found to be highly suppressed in AKI patients and hypoxic endothelial cells. Cox regression and Kaplan-Meier curve analysis revealed ciRs-126 as an independent predictor of 28-day survival (P < 0.01). CONCLUSION: Circulating concentrations of circRNAs in patients with AKI are detectable. ciRs-126 may potentially sponge miR-126-5p and acts as a predictor of mortality in this patient cohort.

Podocytes regulate the glomerular basement membrane protein nephronectin by means of miR-378a-3p in glomerular diseases.

The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.

Antagonism of profibrotic microRNA-21 improves outcome of murine chronic renal allograft dysfunction.

Chronic renal allograft dysfunction (CAD) is a major limiting factor of long-term graft survival. It is characterized by interstitial fibrosis and tubular atrophy. The underlying pathomechanisms are incompletely understood. MicroRNAs are powerful regulators of gene expression and may have an impact on various diseases by direct mRNA decay or translational inhibition. A murine model of allogenic kidney transplantation was used resulting in CAD at 6 weeks after kidney transplantation. We identified fibrosis-associated miR-21a-5p by whole miRNAome expression analysis to be among the most highly upregulated miRNAs. In vitro in renal fibroblasts, miR-21a-5p was transcriptionally activated by interleukin 6-induced signal transducer and activator of transcription 3. Co-culture of LPS-activated macrophages with renal fibroblasts increased expression levels of miR-21a-5p and markers of fibrosis and inflammation. In addition, mature miR-21a-5p was secreted by macrophages in small vesicles, which were internalized by renal fibroblasts, thereby promoting profibrotic and proinflammatory effects. Notch2 receptor was identified as a potential target of miR-21a-5p and validated by luciferase gene reporter assays. Therapeutic silencing of miR-21a-5p in mice after allogenic kidney transplantation resulted in an amelioration of CAD, as indicated by a reduction in fibrosis development, inflammatory cell influx, tissue injury and BANFF lesion scoring. In a life-supporting model, miR-21a-5p antagonism had beneficial effects on kidney function. miR-21a-5p silencing may therefore be a viable therapeutic option in the treatment of patients following kidney transplantation to halt the development of CAD.

Long noncoding RNAs in kidney and cardiovascular diseases.

Transcription of a large part of the human genome results in RNA transcripts that have limited or no protein-coding potential. These include long noncoding RNAs (lncRNAs), which are defined as being ≥200 nucleotides long. Unlike microRNAs, which have been extensively studied, little is known about the functional role of lncRNAs. However, studies over the past 5 years have shown that lncRNAs interfere with tissue homeostasis and have a role in pathological processes, including in the kidney and heart. The developmental expression of the microRNA sponge H19, for example, is altered in the kidneys of embryos carried by hyperglycaemic mothers, and the lncRNA Malat1 regulates hyperglycaemia-induced inflammation in endothelial cells. Putative roles for other lncRNAs have been identified in conditions such as heart failure, cardiac autophagy, hypertension, acute kidney injury, glomerular diseases, acute allograft rejection and renal cell carcinoma. This Review outlines our current understanding of the role and function of lncRNAs in kidney and cardiovascular disease as novel important regulators and potential therapeutic entry points of disease progression.

Long Noncoding RNAs in Urine Are Detectable and May Enable Early Detection of Acute T Cell-Mediated Rejection of Renal Allografts.

BACKGROUND: Long noncoding RNAs (lncRNAs) are novel intracellular noncoding ribonucleotides regulating the genome and proteome. They are detectable in the blood of patients with acute kidney injury. We tested whether lncRNAs are present in urine and may serve as new predictors of outcome in renal transplant patients with acute rejection. METHODS: A global lncRNA expression analysis was performed with RNA from urine of patients with acute T cell-mediated renal allograft rejection and control transplant patients. Deregulated lncRNAs were confirmed in kidney biopsies and urine in a validation cohort of 62 patients with acute rejection, 10 of them after successful antirejection therapy, and 31 control transplant patients. RESULTS: A global screen revealed several lncRNAs to be deregulated in urine of patients with acute rejection. Three intergenic lncRNAs, LNC-MYH13-3:1, RP11-395P13.3-001, and RP11-354P17.15-001, were most strongly altered. These were validated in the whole cohort of patients. RP11-395P13.3-001 and RP11-354P17.15-001 were upregulated in patients with acute rejection compared with controls. Only levels of RP11-354P17.15-001 normalized in patients with acute rejection after successful antirejection therapy. RP11-354P17.15-001 was associated with higher decline in glomerular filtration rate 1 year after transplantation. In vitro, in tubular epithelial cells, all lncRNAs were enriched by interleukin-6 treatment, but only RP11-395P13.3-001 and RP11-354P17.15-001 increased in cell culture supernatant, indicating that these lncRNAs might be secreted under inflammatory conditions. CONCLUSIONS: lncRNAs are strongly altered in urine of patients with acute rejection. Urinary RP11-354P17.15-001 may serve as a novel biomarker of acute kidney rejection, identifying patients with acute rejection and predicting loss of kidney function.

Blood-based microRNA signatures differentiate various forms of cardiac hypertrophy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by mutations in different structural genes and induces pathological hypertrophy with sudden cardiac death as a possible consequence. HCM can be separated into hypertrophic non-obstructive and obstructive cardiomyopathy (HNCM/HOCM) with different clinical treatment approaches. We here distinguished between HNCM, HOCM, cardiac amyloidosis and aortic stenosis by using microRNA profiling and investigated potential interactions between circulating miRNA levels and the most common mutations in MYH7and MYBPC3 genes. METHODS: Our study included 4 different groups: 23 patients with HNCM, 28 patients with HOCM, 47 patients with aortic stenosis and 22 healthy controls. Based on previous findings, 8 different cardiovascular known microRNAs (miR-1, miR-21, miR-29a, miR-29b, miR-29c, miR-133a, miR-155 and miR-499) were studied in serum of all patients and compared with clinically available patient data. RESULTS: We found miR-29a levels to be increased in patients with HOCM and correlating markers of cardiac hypertrophy. This was not the case in HNCM patients. In contrast, we identified miR-29c to be upregulated in aortic stenosis but not the other patient groups. ROC curve analysis of miR-29a/c distinguished between HOCM patients and aortic stenosis patients. MiR-29a and miR-155 levels discriminated HNCM patients from patients with senile cardiac amyloidosis. MiR-29a increased mainly in HOCM patients with a mutation in MYH7, whereas miR-155 was decreased in hypertrophic cardiomyopathy patients with a mutation in MYBPC3. CONCLUSION: We demonstrated that miR-29a and miR-29c show a specific signature to distinguish between aortic stenosis, hypertrophic non-obstructive and obstructive cardiomyopathies and thus could be developed into clinically useful biomarkers.

Osteopontin is indispensible for AP1-mediated angiotensin II-related miR-21 transcription during cardiac fibrosis.

AIMS: Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process. METHODS AND RESULTS: OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo. CONCLUSION: In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation.

Circulating long noncoding RNATapSaki is a predictor of mortality in critically ill patients with acute kidney injury.

BACKGROUND: Long noncoding RNAs (lncRNAs) are novel intracellular noncoding ribonucleotides regulating gene expression. Intriguingly, these RNA transcripts are detectable and stable in the blood of patients with cancer and cardiovascular disease. We tested whether circulating lncRNAs in plasma of critically ill patients with acute kidney injury (AKI) at inception of renal replacement therapy were deregulated and might predict survival. METHODS: We performed a global lncRNA expression analysis using RNA isolated from plasma of patients with AKI, healthy controls, and ischemic disease controls. This global screen revealed several deregulated lncRNAs in plasma samples of patients with AKI. lncRNA-array-based alterations were confirmed in kidney biopsies of patients as well as in plasma of 109 patients with AKI, 30 age-matched healthy controls, and 30 disease controls by quantitative real-time PCR. RESULTS: Circulating concentrations of the novel intronic antisense lncRNA TrAnscript Predicting Survival in AKI (TapSAKI) (P < 0.0001) were detectable in kidney biopsies and upregulated in plasma of patients with AKI. Cox regression and Kaplan-Meier curve analysis revealed TapSAKI as an independent predictor of 28-day survival (P < 0.01). TapSAKI was enriched in tubular epithelial cells subjected to ATP depletion (P = 0.03). CONCLUSIONS: The alteration of circulating concentrations of lncRNAs in patients with AKI supports TapSAKI as a predictor of mortality in this patient cohort.

Vascular importance of the miR-212/132 cluster.

RATIONALE: Many processes in endothelial cells including angiogenic responses are regulated by microRNAs. However, there is limited information available about their complex cross-talk in regulating certain endothelial functions. AIM: The objective of this study is to identify endothelial functions of the pro-hypertrophic miR-212/132 cluster and its cross-talk with other microRNAs during development and disease. METHODS AND RESULTS: We here show that anti-angiogenic stimulation by transforming growth factor-beta activates the microRNA-212/132 cluster by derepression of their transcriptional co-activator cAMP response element-binding protein (CREB)-binding protein (CBP) which is a novel target of a previously identified pro-angiogenic miRNA miR-30a-3p in endothelial cells. Surprisingly, despite having the same seed-sequence, miR-212 and miR-132 exerted differential effects on endothelial transcriptome regulation and cellular functions with stronger endothelial inhibitory effects caused by miR-212. These differences could be attributed to additional auxiliary binding of miR-212 to its targets. In vivo, deletion of the miR-212/132 cluster increased endothelial vasodilatory function, improved angiogenic responses during postnatal development and in adult mice. CONCLUSION: Our results identify (i) a novel miRNA-cross-talk involving miR-30a-3p and miR-212, which led to suppression of important endothelial genes such as GAB1 and SIRT1 finally culminating in impaired endothelial function; and (ii) microRNAs may have different biological roles despite having the same seed sequence.

MicroRNA-24 antagonism prevents renal ischemia reperfusion injury.

Ischemia-reperfusion (I/R) injury of the kidney is a major cause of AKI. MicroRNAs (miRs) are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R injury. miR-24 was upregulated in the kidney after I/R injury of mice and in patients after kidney transplantation. Cell-sorting experiments revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after I/R induction. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Transient overexpression of miR-24 alone induced apoptosis and altered functional parameters in these cells, whereas silencing of miR-24 ameliorated apoptotic responses and rescued functional parameters in hypoxic conditions. miR-24 effects were mediated through regulation of H2A histone family, member X, and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. 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. In vivo, silencing of miR-24 in mice before I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histologic tubular epithelial injury, and less infiltration of inflammatory cells. miR-24 also regulated heme oxygenase 1 and H2A histone family, member X, in vivo. Overall, these results indicate miR-24 promotes renal ischemic injury by stimulating apoptosis in endothelial and tubular epithelial cell. Therefore, miR-24 inhibition may be a promising future therapeutic option in the treatment of patients with ischemic AKI.

Regulation of cardiac and renal ischemia-reperfusion injury by microRNAs.

Tissue damage caused by ischemia-reperfusion (I/R) injury represents a serious event, which often leads to deterioration or even loss of organ function. I/R injury is associated with transient tissue oxygen deprivation due to vessel occlusion and a subsequent reperfusion period following restoration of blood flow. Initial tissue damage inflicted by ischemia is aggravated in the reperfusion period through mechanisms such as burst of reactive oxygen and nitrogen species and inflammatory reactions. I/R injury occurs during surgical interventions, organ transplantation, diseases such as myocardial infarction, circulatory shock, and toxic insults. Recently, microRNAs have come into focus as powerful regulators of gene expression and potential diagnostic tools during I/R injury. These small noncoding ribonucleotides (~22 nucleotides in length) posttranscriptionally target mRNAs, culminating in suppression of protein synthesis or increase in mRNA degradation, thus fundamentally influencing organ function. This review highlights the latest developments regarding the role of microRNAs in cardiac and renal I/R injury.

Pathologic endothelial response and impaired function of circulating angiogenic cells in patients with Fabry disease.

Fabry disease is an X-chromosomal recessive deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal). This results in an accumulation of globotriaosylceramide (GL-3) in a variety of cells often with subsequent functional impairment. Here, the impact of Fabry disease on the biology of circulating angiogenic cells (CACs) and the endothelial response to transient ischemia was investigated. Untreated patients with Fabry disease (n = 26), patients after initiation of alpha-Gal enzyme replacement therapy (ERT) (n = 16) and healthy controls (n = 26) were investigated. Endothelial function was assessed by the EndoPAT2000 device. CAC numbers were assessed by flow-cytometry, CAC function by a modified Boyden chamber assay. Fabry patients showed a pathologic endothelial response, which normalized after ERT. CACs were increased in number, but functionally impaired. Immunofluorescence and electron microscopy identified an accumulation of GL-3 in Fabry CACs. ERT attenuated CAC dysfunction and improved markers of oxidative stress response in Fabry patients via a reduction in GL-3 accumulation in vitro and in vivo. Silencing of alpha-Gal in healthy CACs impaired their migratory capacity underlining a key role of this enzyme for CAC function. CAC supernatant as well as CACs from Fabry patients impaired angiogenesis and migratory capacity of HUVECs providing a mechanistic link between CAC and endothelial dysfunction. CAC adhesion to TNF-α pre-stimulated HUVECs and tube formation was impaired by alpha-Gal knockdown. Fabry patients show a dysfunction of CAC and a pathologic endothelial response. ERT improves CAC and endothelial function and thus may attenuate development of cardiovascular disease in the long term in this patient population.