Circular RNAs in urine of kidney transplant patients with acute T Cell-mediated allograft rejection

BACKGROUND: Circular RNAs (circRNAs) have recently been described as novel noncoding regulators of gene expression. They are detectable in the blood of patients with acute kidney injury. We tested whether circRNAs were present in urine and could serve as new predictors of outcome in renal transplant patients with acute rejection. METHODS: A global circRNA expression analysis using RNA from urine of patients with acute T cell-mediated renal allograft rejection and control transplant patients was performed. Dysregulated circRNAs were confirmed in a cohort of 62 patients with acute rejection, 10 patients after successful antirejection therapy, 18 control transplant patients without rejection, and 13 stable transplant patients with urinary tract infection. RESULTS: A global screen revealed several circRNAs to be altered in urine of patients with acute rejection. Concentrations of 2 circRNAs including hsa_circ_0001334 and hsa_circ_0071475 were significantly increased. These were validated in the whole cohort of patients. hsa_circ_0001334 was upregulated in patients with acute rejection compared with controls. Concentrations of hsa_circ_0001334 normalized in patients with acute rejection following successful antirejection therapy. hsa_circ_0001334 was associated with higher decline in glomerular filtration rate 1 year after transplantation. CONCLUSIONS: CircRNA concentrations are significantly dysregulated in patients with acute rejection at subclinical time points. Urinary hsa_circ_0001334 is a novel biomarker of acute kidney rejection, identifying patients with acute rejection and predicting loss of kidney function

The function of IQD-KLCR modules in macromolecular complex assembly

The putative scaffold proteins KLCR1 and IQD2 form the basic module of a regulatory multi-protein complex linking the actin- and microtubule cytoskeleton to the plasma membrane. The exact composition and dynamics of this complex, however, remain elusive to date. This thesis aims to characterise the interactions between KLCR1 and IQD2 as well as possible mechanisms of Ca2+-mediated regulation through the archetypal Ca2+-sensor Calmodulin (CaM). It shows for the first time dimerisation of KLCR1 with itself and other KLCRs, and uncovers the modes of interaction between KLCRs and CaMs. Further, KLCR- and CaM-interaction domains in IQD2 are mapped and ternary interactions between KLCR1, IQD2, and CaMs are shown. KLCRs interact with CaMs via the C-terminal helices of their central TPR-domain and likely recruit IQDs through its internal groove. IQD2 binds CaM and KLCRs through its central region. IQD2 putatively recruits KLCRs to CaM for Ca2+-signal transfer at the cytoskeleton-plasma membrane interface.Die mutmaßlichen Gerüstproteine KLCR1 und IQD2 stellen das Kernmodul eines regulatorischen Multiproteinkomplex, der das Aktin- und Miktotubulizytoskelett mit der Plasmamembran verbindet. Die Zusammensetzung und Dynamik dieses Komplexes ist allerdings bislang unbekannt. Diese Arbeit widmet sich der Untersuchung der Interaktionen von KLCR1 und IQD2 sowie möglichen Mechanismen der Ca2+-vermittelten Regulation durch den Ca2+-Sensor Calmodulin (CaM). Die Dimerisierung von KLCR1 mit sich selbst und anderen KLCRs und die Interaktionsmodi von KLCRs mit CaM werden gezeigt. Auch werden Interaktionsdomänen für KLCRs und CaMs in IQD2 kartiert sowie trinäre Interaktionen zwischen KLCR1, IQD2 und CaMs untersucht. KLCRs interagieren mit CaMs durch die C-terminalen Helices ihrer zentralen TPR-Domäne und rekrutieren IQDs vermutlich durch deren innere Krümmung. IQD2 interagiert mit CaM und KLCRs durch seinen zentralen Bereich. Vermutlich rekrutiert IQD2 KLCRs an CaM für Ca2+-Signaltransfer am Übergang zwischen Zytoskelett und Zellmembran

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

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. Keywords: gene expression profiling; genetics; molecular biology; molecular diagnostic techniques; phenotype

Therapeutic miR-21 Silencing Ameliorates Diabetic Kidney Disease in Mice

Diabetic nephropathy is the main cause of end-stage renal disease. MicroRNAs are powerful regulators of the genome, and global expression profiling revealed miR-21 to be among the most highly regulated microRNAs in kidneys of mice with diabetic nephropathy. In kidney biopsies of diabetic patients, miR-21 correlated with tubulointerstitial injury. In situ PCR analysis showed a specific enrichment of miR-21 in glomerular cells. We identified cell division cycle 25a (Cdc25a) and cyclin-dependent kinase 6 (Cdk6) as novel miR-21 targets in mesangial cells. miR-21-mediated repression of Cdc25a and Cdk6 resulted in impaired cell cycle progression and subsequent mesangial cell hypertrophy. miR-21 increased podocyte motility by regulating phosphatase and tensin homolog (Pten). miR-21 antagonism in vitro and in vivo in streptozotocin-induced diabetic mice decreased mesangial expansion, interstitial fibrosis, macrophage infiltration, podocyte loss, albuminuria, and fibrotic- and inflammatory gene expression. In conclusion, miR-21 antagonism rescued various functional and structural parameters in mice with diabetic nephropathy and, thus, might be a viable option in the treatment of patients with diabetic kidney disease

The Circular RNA 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. (or ) was most highly altered compared to healthy controls and disease controls (fold change of 52.1). 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 as an independent predictor of 28-day survival ( < 0.01). Conclusion Circulating concentrations of circRNAs in patients with AKI are detectable. may potentially sponge miR-126-5p and acts as a predictor of mortality in this patient cohort

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

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

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. Keywords: Chronic kidney disease; Complement; Glycobiology; Immunology; Nephrology