miR-127 protects proximal tubule cells against ischemia/reperfusion : identification of Kinesin family member 3B as miR-127 target

Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management

Hypoxia Inducible Factor 1-Alpha (HIF-1 Alpha) Is Induced during Reperfusion after Renal Ischemia and Is Critical for Proximal Tubule Cell Survival

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant

Estudio de microRNAs implicados en la respuesta renal a isquemia-reperfusión : identificación como nuevos biomarcadores de daño renal agudo

La insuficiencia renal aguda (IRA) es un síndrome clínico complejo que presenta una alta tasa de morbilidad y mortalidad en los países desarrollados. A pesar de la intensa investigación de las últimas décadas, los métodos de diagnóstico del daño renal y su terapéutica han experimentado pocos avances, probablemente debido a que las herramientas clínicas actuales ofrecen una información de diagnóstico tardía. La identificación de nuevos mecanismos moleculares implicados en la patofisiología de la IRA es esencial para el desarrollo de nuevas herramientas de diagnóstico y el descubrimiento de nuevas dianas terapéuticas. Los microRNAs son reguladores post-transcripcionales implicados prácticamente en todos los procesos celulares. Son reguladores finos de la información genética y crecientes evidencias ha demostrado que están implicados en los mecanismos fisiopatológicos subyacentes a numerosas de enfermedades, incluyendo nefropatías. En este trabajo hemos identificado y caracterizado varios miRNAs como mediadores clave de la respuesta del túbulo proximal al daño por Isquemia/Reperfusión (I/R). Así mismo, hemos desvelado el papel de estos miRNAs como biomarcadores diagnósticos, pronósticos y de predisposición de IRA. Para ello hemos utilizado un modelo in vivo de I/R renal en rata, así como un modelo in vitro en células proximales tubulares que reproduce todos los estímulos y eventos observados in vivo. Adicionalmente, hemos utilizado muestras de suero de pacientes con IRA procedentes de dos cohortes: Pacientes de unidades de cuidados intensivos y pacientes sometidos a cirugía cardiaca. Utilizando estos modelos experimentales de I/R y por medio de microarrays y análisis mediante qRT-PCR, hemos demostrado que miR-127 se modula durante isquemia y también durante la reperfusión tanto in vivo como in vitro. Experimentos de interferencia in vitro demostraron que la inducción isquémica de miR-127 es mediada por la estabilización del Factor Inducible por Hipoxia-1α (HIF-1α). Así mismo, miR-127 está implicado en el mantenimiento de la adhesión célula-célula y célula-matriz, ya que la sobreexpresión de miR-127 mantiene el ensamblaje de los complejos de adhesión focal y la integridad de las uniones estrechas. miR-127 regula también el tráfico intracelular. De hecho, hemos identificado por primera vez en este trabajo el miembro 3B de la familia de la Kinesina (KIF3B), molécula clave para el tráfico intracelular, como una diana real de miR-127 en células proximales tubulares de rata. Así mismo, como los miRNAs pueden ser detectados en fluidos corporales extracelulares, y basándonos en nuestros resultados obtenidos en modelos experimentales, hemos identificado y validado un panel de 10 miRNAs en suero, incluyendo miR-127, como biomarcadores de IRA en pacientes. Un experimento de cribado inicial llevó a la selección de un panel de miRNAs en suero que fueron posteriormente validados en una cohorte de pacientes de UCI con daño renal, así como en una cohorte de pacientes de cirugía cardiaca con circulación extracorpórea. Los experimentos de validación demostraron que nuestro panel de miRNAs en suero son biomarcadores diagnósticos de IRA con una especificidad y sensibilidad cercana al 100%. Además, los miRNAs séricos pueden detectar el desarrollo de IRA antes que el incremento de creatinina, demostrando que son herramientas de diagnóstico precoz. Los miRNAs séricos proporcionan información clínica adicional valiosa, ya que los niveles de miRNAs en suero correlacionan con la severidad del daño renal y pueden discriminar entre daño renal de origen renal o pre-renal, así como entre sus etiologías. Así mismo, los niveles de miRNAs en suero, estimados antes de la cirugía, pueden predecir el desarrollo posterior de daño renal, lo que demuestra su valor como biomarcadores de predisposición de IRA. [ABSTRACT]Acute Kidney Injury (AKI) is a complex clinical syndrome which presents very high morbidity and mortality rates in developed countries. Despite the intense research of the last decades, AKI diagnosis and therapeutic approaches have undergone few advances, probably due to the fact that current clinical tools offer late diagnosis information. Identification of new molecular mechanisms involved in AKI pathophisiology is essential for development of new diagnostic and prognostic tools as well as discovery of new therapeutic targets. microRNAs (miRNAs) are post-transcriptional regulators of almost every cellular process. They have been unveiled as fine-tuners of genetic information and accumulating evidence has demonstrated that they are at the bases of the pathophysiological mechanism of a wide range of disorders, including nephropaties. In this work, we have identified and characterized several miRNAs as key mediators of the proximal tubule response to I/R injury. Moreover we have point out these miRNAs as AKI diagnostic, prognostic and predisposition biomarkers. For this purpose, we have used an in vivo model of renal ischemia/reperfusion in rat as well as an in vitro model in proximal tubule cells which closely mimics the stimuli and features observed in vivo. Additionally, we have used serum samples of AKI patients from two cohorts: Intensive Care Unit patients and cardiac surgery patients. Using these experimental models of I/R and by means of microarrays and qRT-PCR analysis, we demonstrated that miR-127 is modulated during ischemia and also during reperfusion, in vivo and in vitro. In vitro interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and tight junctions’ integrity. miR-127 also regulates intracellular trafficking. In fact, we have identified for the first time in this work the Kinesin Family Member 3B (KIF3B), key molecule in cell trafficking, as a target of miR-127 in rat proximal tubule cells. Moreover, since miRNAs can be detected in extracellular body fluids, and based in our findings using experimental models, we have identified and validated a panel of 10 serum miRNAs, including miR-127, as biomarkers of AKI in patients. An initial screening experiment led to a panel of serum miRNAs which were validated in bigger cohorts of ICU patients with AKI and patients who underwent cardiac surgery with cardiopulmonary bypass. Validation experiments demonstrated that our panel of miRNAs are powerful diagnostic biomarkers of AKI with sensitivity and specificity close to 100%. Moreover, serum miRNAs detect AKI development before serum creatinine increases, becoming early diagnostic tools. Serum miRNAs provide additional valuable clinical information since miRNA levels in serum correlate with AKI grade and can distinguish between pre-renal and intrinsic AKI origin as well as among AKI etiologies. Moreover, serum miRNAs levels estimated before surgery can predict AKI development later on, becoming biomarkers of AKI predisposition

miR-127 Protects Proximal Tubule Cells against Ischemia/Reperfusion: Identification of Kinesin Family Member 3B as miR-127 Target

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management. © 2012 Aguado-Fraile et al.This work was supported by grant FIS PS09/02183 funding by Instituto de Salud Carlos III and Ayuda Intramural Fundación para la Investigación en Biomedicina del Hospital Universitario Ramón y Cajal 122/2009.Peer Reviewe

A pilot study identifying a set of microRNAs as precise diagnostic biomarkers of acute kidney injury

© 2015 Aguado-Fraile et al.In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.This work was supported by grants FIS PS09/02183 and PS12/00094 by Instituto de Salud Carlos III (ISCIII) and Comunidad de MAdrid-Consorcio de Investigación en Fracaso Renal Agudo (CAM-CIFRA, S2010/BMD-2378). EAF was supported by CAM-CIFRA (S2010/BMD-2378) and is currently funded by Eusebio Martín Escudero Foundation Fellowship, ER was funded by Red de Investigación Renal (REDINREN, RD12/0021/0020), MR was recipient of contract from ISCIII (CA11/00491)

HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p

© The Author(s) 2017.Ischemia/reperfusion (I/R) leads to Acute Kidney Injury. HIF-1α is a key factor during organ response to I/R. We previously demonstrated that HIF-1α is induced during renal reperfusion, after ischemia. Here we investigate the role of HIF-1α and the HIF-1α dependent mechanisms in renal repair after ischemia. By interference of HIF-1α in a rat model of renal I/R, we observed loss of expression and mis-localization of e-cadherin and induction of α-SMA, MMP-13, TGFβ, and collagen I. Moreover, we demonstrate that HIF-1α inhibition promotes renal cell infiltrates by inducing IL-1β, TNF-α, MCP-1 and VCAM-1, through NFkB activity. In addition, HIF-1α inhibition induced proximal tubule cells proliferation but it did not induce compensatory apoptosis, both in vivo. In vitro, HIF-1α knockdown in HK2 cells subjected to hypoxia/reoxygenation (H/R) promote cell entry into S phase, correlating with in vivo data. HIF-1α interference leads to downregulation of miR-127-3p and induction of its target gene Bcl6 in vivo. Moreover, modulation of miR-127-3p in HK2 cells subjected to H/R results in EMT regulation: miR127-3p inhibition promote loss of e-cadherin and induction of α-SMA and collagen I. In conclusion, HIF-1α induction during reperfusion is a protector mechanism implicated in a normal renal tissue repair after I/R.This work was supported by grants FIS PS09/02183, PS12/00094 and PI15/01715 by Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013–2016) and co-financed by the European Development Regional Fund “A way to achieve Europe” (ERDF), CAM-CIFRA (S2010/BMD-2378) from Comunidad de Madrid and FMMA 2013/0063 from Fundación de Investigación Mutua Madrileña, all to MLGB. E.C. was funded by CAMColomics2 (S2010/BMD-2344). L.M.-G. was funded by FMMA 2013/0063. M.R. was recipient of contract from ISCIII (CA11/00491), by SOMANE grant 2014 and currently is funded by REDINREN (RD12/0021/0020). E.R. was funded by REDINREN (RD12/0021/0020) and currently MINECO contract (PTA2014-09496-1) E.A.F. was supported by CAM-CIFRA (S2010/BMD-2378). We are also grateful to Dr J Earl and Dr B Saiz for editing assistance

Differential resolution of inflammation and recovery after renal ischemia-reperfusion injury in Brown Norway compared with Sprague Dawley rats

To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia-reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-gamma, IL-1beta, and TNF-alpha were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFkappaB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia-reperfusion injury

Serum miRNAs estimated before cardiac surgery identify patients in risk of AKI development.

<p>(A) miRNAs were detected by RT-qPCR in serum samples obtained before surgery, expressed as ΔCp. Data are presented as median and interquartile range. Asterisks indicate statistical significance (*P<0.05; ** P<0.01). (B) Spike-In raw Cp values are shown as control.</p