Papel del ligando no canónico de NOTCH parecido a Delta 1 (DLK1) en el daño renal experimental

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina. Fecha de lectura: 09-07-2021La vía de señalización de NOTCH se activa durante el desarrollo embrionario, aunque se encuentra silenciada en la etapa adulta. Durante diversos procesos patológicos, algunos de sus componentes se reexpresan y dan lugar a la activación de esta vía de señalización. En el daño renal se ha descrito la activación de la ruta de NOTCH1 y NOTCH3, asociados a aumento de expresión del ligando canónico Jagged-1, y varios componentes de esta ruta se han postulado como potenciales biomarcadores de daño renal. Sin embargo, hasta hoy, los ligandos no canónicos de esta vía han sido poco estudiados, siendo el ligando no canónico de NOTCH parecido a Delta 1 (DLK1) el más explorado debido a su papel clave para el desarrollo embrionario de multitud de órganos. A nivel molecular, se ha descrito que los ligandos no canónicos son capaces de unirse al receptor de NOTCH e inhibirlo, actuando como antagonistas competitivos en la señalización de la vía. Sin embargo, se desconoce si DLK1 puede actuar también como un antagonista de la vía de NOTCH en situaciones de daño renal. El objetivo principal de esta tesis fue investigar la contribución del ligando no canónico DLK1 en el proceso patológico renal y los mecanismos moleculares subsecuentes. Para ello, se usaron ratones de la cepa 129/SvJ con una deleción sistémica del gen del Dlk1 los cuales fueron sometidos a una obstrucción unilateral del uréter (UUO) y los resultados fueron estudiados tras 2, 5, 10 y 14 días de obstrucción. En un primer lugar, el estudio en los ratones de la misma cepa no modificados genéticamente, reveló un aumento en la expresión génica de los ligandos no canónicos Dlk1 y Dlk2 a partir del quinto día del daño renal en los riñones obstruidos. La exploración de las lesiones renales, realizada por técnicas clásicas de histología, mostró un mayor aumento de infiltrado inflamatorio a partir de los 5 días tras la obstrucción en los ratones deficientes en Dlk1, destacando además la presencia de una infiltración perivascular hallada a los 14 días de obstrucción. Sin embargo, no se observó un mayor daño tubular o cambios en la acumulación de matriz extracelular (niveles de colágenos y presencia de células -SMA positivas). La caracterización del infiltrado renal demostró una mayor infiltración de macrófagos F4/80+, neutrófilos, y linfocitos T CD3+ y CD4+ en los riñones dañados en ausencia de Dkl1. Por otra parte, en ausencia de Dlk1 la vía de señalización de NOTCH1 se encontró sobreactivada también a los 14 días de obstrucción. Estos datos confirman que DLK1 es un antagonista de NOTCH en situaciones de daño renal. Además, otros componentes de la vía se encontraron modificados en los ratones deficientes en Dlk1, donde se observó una mayor expresión génica del ligando no canónico Dlk2 y del efector Hey-1 así como una disminución de la expresión proteica de NOTCH2. Sin embargo, no se encontraron diferencias significativas en los niveles de expresión del ligando canónico Jagged-1, del efector Hes-1, ni de los receptores NOTCH3 y NOTCH4. Además, el estudio de la vía de señalización de NF-B no mostró cambios en su activación entre los genotipos estudiados, aunque sí se observó un aumento en la expresión de la quimioquina regulada por esta vía, el MCP-1 a los 10 y 14 días de obstrucción en los ratones deficientes en Dlk1. Por otra parte, estudios recientes han demostrado que la respuesta inmune Th17 juega un papel fundamental en diferentes enfermedades renales crónicas, y que la quimioquina MCP-1 se encuentra asociada al aumento de la citoquina efectora de esta respuesta, la IL-17A. Por ello, se decidió estudiar los factores de transcripción implicados en la diferenciación de linfocitos Th17, RORt y STAT3, así como la IL-17A. Todos ellos se encontraron sobreexpresados en los riñones obstruidos en ausencia de Dlk1 a los 14 días de obstrucción. Por último, se evaluó el efecto del bloqueo de la ruta de NOTCH en el daño renal experimental por UUO en ratones de la cepa C57BL/6 mediante en el tratamiento preventivo con un inhibidor de la -secretasa, el DAPT, la cual es esencial en la activación de la ruta de NOTCH, con el objetivo de estudiar si este bloqueo disminuía la respuesta inmunológica Th17 renal. Los resultados mostraron que la inhibición de la vía de señalización de NOTCH por DAPT disminuyó la respuesta inmune Th17 al disminuir la expresión de RORt y de IL-17A en los riñones dañados. Teniendo en cuenta todos estos resultados se puede concluir que el DLK1 actúa como un inhibidor natural de esta vía y está asociado a la regulación de la respuesta inmune Th17 en la patología rena

Vitamin D, cellular senescence and chronic kidney diseases: what is missing in the equation?

As life expectancy increases in many countries, the prevalence of age-related diseases also rises. Among these conditions, chronic kidney disease is predicted to become the second cause of death in some countries before the end of the century. An important problem with kidney diseases is the lack of biomarkers to detect early damage or to predict the progression to renal failure. In addition, current treatments only retard kidney disease progression, and better tools are needed. Preclinical research has shown the involvement of the activation of cellular senescence-related mechanisms in natural aging and kidney injury. Intensive research is searching for novel treatments for kidney diseases as well as for anti-aging therapies. In this sense, many experimental shreds of evidence support that treatment with vitamin D or its analogs can exert pleiotropic protective effects in kidney injury. Moreover, vitamin D deficiency has been described in patients with kidney diseases. Here, we review recent evidence about the relationship between vitamin D and kidney diseases, explaining the underlying mechanisms of the effect of vitamin D actions, with particular attention to the modulation of cellular senescence mechanismsThis research was funded by grants from the Instituto de Salud Carlos III (ISCIII) and Fondos FEDER European Union (PI20/00140, PI19/00240, PI19/00815, and DTS20/00083). Red de Investigación Renal REDINREN: RD16/0009/0003 to M.R.-O. and RICORS2040; RD21/0005/0002 funded by European Union—NextGenerationEU, INNOREN cm (P2022/BMD-7221) of the Comunidad de Madrid, Sociedad Española de Nefrología. Innovation programme under the Marie Skłodowska-Curie grant of the European Union’s Horizon 2020 (IMProve-PD ID: 812699) to M.R.-

Novel aspects of the immune response involved in the peritoneal damage in chronic Kkdney disease patients under dialysis

Chronic kidney disease (CKD) incidence is growing worldwide, with a significant percentage of CKD patients reaching end-stage renal disease (ESRD) and requiring kidney replacement therapies (KRT). Peritoneal dialysis (PD) is a convenient KRT presenting benefices as home therapy. In PD patients, the peritoneum is chronically exposed to PD fluids containing supraphysiologic concentrations of glucose or other osmotic agents, leading to the activation of cellular and molecular processes of damage, including inflammation and fibrosis. Importantly, peritonitis episodes enhance peritoneum inflammation status and accelerate peritoneal injury. Here, we review the role of immune cells in the damage of the peritoneal membrane (PM) by repeated exposure to PD fluids during KRT as well as by bacterial or viral infections. We also discuss the anti-inflammatory properties of current clinical treatments of CKD patients in KRT and their potential effect on preserving PM integrity. Finally, given the current importance of coronavirus disease 2019 (COVID-19) disease, we also analyze here the implications of this disease in CKD and KRTThis research was funded by grants from the Instituto de Salud Carlos III (ISCIII) and Fondos FEDER European Union (PI20/00140, PI19/00815, and DTS20/00083). Red de Investi gación Renal REDINREN: RD16/0009/0003 to M.R-O and RICORS2040; RD21/0005/0002 funded by European Union—NextGenerationEU, Sociedad Española de Nefrología. Innovation programme under the Marie Skłodowska-Curie grant of the European Union’s Horizon 2020 (IMPROVE-PD ID: 812699) to M.R-O. E.K. was supported by the grant from the Narodowe Centrum Nauki (NCN, Polish National Science Centre; 2018/29/N/NZ3/02504). R.S. was supported by Ministry for Health of Italy (Ricerca Corrente). This work was also supported by a grant (PID 2019-110132RB I00/AEI/10.13039/501100011033) from the Spanish Ministry of Science and Innovation/Fondo Europeo de Desarrollo Regional (MICINN/FEDER) to M.L.-C

Gremlin regulates tubular epithelial to mesenchymal transition via VEGFR2: Potential role in renal fibrosis

Chronic kidney disease (CKD) is emerging as an important health problem due to the increase number of CKD patients and the absence of an effective curative treatment. Gremlin has been proposed as a novel therapeutic target for renal inflammatory diseases, acting via Vascular Endothelial Growth Factor Receptor-2 (VEGFR2). Although many evidences suggest that Gremlin could regulate renal fibrosis, the receptor involved has not been yet clarified. Gremlin, as other TGF-β superfamily members, regulates tubular epithelial to mesenchymal transition (EMT) and, therefore, could contribute to renal fibrosis. In cultured tubular epithelial cells Gremlin binding to VEGFR2 is linked to proinflammatory responses. Now, we have found out that in these cells VEGFR2 is also involved in the profibrotic actions of Gremlin. VEGFR2 blockade by a pharmacological kinase inhibitor or gene silencing diminished Gremlin-mediated gene upregulation of profibrotic factors and restored changes in EMT-related genes. Moreover, VEGFR2 inhibition blocked EMT phenotypic changes and dampened the rate of wound healing in response to Gremlin. The role of VEGFR2 in experimental fibrosis was evaluated in experimental unilateral ureteral obstruction. VEFGR2 inhibition diminished the upregulation of profibrotic genes and EMT changes, as well as the accumulation of extracellular matrix proteins, such as fibronectin and collagens in the obstructed kidneys. Notch pathway activation participates in renal damage progression by regulating cell growth/proliferation, regeneration and inflammation. In cultured tubular epithelial cells, Notch inhibition markedly downregulated Gremlin-induced EMT changes and wound healing speed. These results show that Gremlin regulates the EMT process via VEGFR2 and Notch pathway activation, suggesting that the Gremlin/VEGFR2 axis could be a potential therapeutic target for CKD.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) and Fondos FEDER European Union (PI16/02057, PI17/00119, PI17/01495, and Red de Investigación Renal REDINREN: RD16/0009), Sociedad Española de Nefrologia, “NOVELREN-CM: Enfermedad renal crónica: nuevas Estrategias para la prevención, Diagnóstico y tratamiento”; B2017/BMD-3751, B2017/BMD-3686 CIFRA2-CM, PAI 82140017, and FONDECYT 1160465 (Chile) and Bayer HealthCare AG (Grants4Targets initiative, Berlin, Germany)

Deletion of delta-like 1 homologue accelerates renal inflammation by modulating the Th17 immune response

Preclinical studies have demonstrated that activation of the NOTCH pathway plays a key role in the pathogenesis of kidney damage. There is currently no information on the role of the Delta-like homologue 1 (DLK1), a NOTCH inhibitor, in the regulation of renal damage. Here, we investigated the contribution of DLK1 to experimental renal damage and the underlying molecular mechanisms. Using a Dlk1-null mouse model in the experimental renal damage of unilateral ureteral obstruction, we found activation of NOTCH, as shown by increased nuclear translocation of the NOTCH1 intracellular domain, and upregulation of Dlk2/hey-1 expression compared to wild-type (WT) littermates. NOTCH1 over-activation in Dlk1-null injured kidneys was associated with a higher inflammatory response, characterized by infiltration of inflammatory cells, mainly CD4/IL17A + lymphocytes, and activation of the Th17 immune response. Furthermore, pharmacological NOTCH blockade inhibited the transcription factors controlling Th17 differentiation and gene expression of the Th17 effector cytokine IL-17A and other related-inflammatory factors, linked to a diminution of inflammation in the injured kidneys. We propose that the non-canonical NOTCH ligand DLK1 acts as a NOTCH antagonist in renal injury regulating the Th17-mediated inflammatory response.MINECO | Instituto de Salud Carlos III (ISCIII), Grant/Award Number: PI17/00119; Ministerio de Economia y Competitividad, Grant/Award Number: SAF2015-66107-R; Comunidad Autonoma de Madrid, Grant/Award Number: B2017/ BMD-3751; Fondo Nacional de Desarroll

CCN2 Binds to Tubular Epithelial Cells in the Kidney

Cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), is considered a fibrotic biomarker and has been suggested as a potential therapeutic target for kidney pathologies. CCN2 is a matricellular protein with four distinct structural modules that can exert a dual function as a matricellular protein and as a growth factor. Previous experiments using surface plasmon resonance and cultured renal cells have demonstrated that the C-terminal module of CCN2 (CCN2(IV)) interacts with the epidermal growth factor receptor (EGFR). Moreover, CCN2(IV) activates proinflammatory and profibrotic responses in the mouse kidney. The aim of this paper was to locate the in vivo cellular CCN2/EGFR binding sites in the kidney. To this aim, the C-terminal module CCN2(IV) was labeled with a fluorophore (Cy5), and two different administration routes were employed. Both intraperitoneal and direct intra-renal injection of Cy5-CCN2(IV) in mice demonstrated that CCN2(IV) preferentially binds to the tubular epithelial cells, while no signal was detected in glomeruli. Moreover, co-localization of Cy5-CCN2(IV) binding and activated EGFR was found in tubules. In cultured tubular epithelial cells, live-cell confocal microscopy experiments showed that EGFR gene silencing blocked Cy5-CCN2(IV) binding to tubuloepithelial cells. These data clearly show the existence of CCN2/EGFR binding sites in the kidney, mainly in tubular epithelial cells. In conclusion, these studies show that circulating CCN2(IV) can directly bind and activate tubular cells, supporting the role of CCN2 as a growth factor involved in kidney damage progression

Oxidative Stress and Cellular Senescence Are Involved in the Aging Kidney

Chronic kidney disease (CKD) can be considered as a clinical model for premature aging. However, non‐invasive biomarkers to detect early kidney damage and the onset of a senescent phenotype are lacking. Most of the preclinical senescence studies in aging have been done in very old mice. Furthermore, the precise characterization and over-time development of age-related senes-cence in the kidney remain unclear. To address these limitations, the age-related activation of cellular senescence-associated mechanisms and their correlation with early structural changes in the kidney were investigated in 3- to 18-month-old C57BL6 mice. Inflammatory cell infiltration was ob-served by 12 months, whereas tubular damage and collagen accumulation occurred later. Early activation of cellular-senescence-associated mechanisms was found in 12-month-old mice, character-ized by activation of the DNA-damage-response (DDR), mainly in tubular cells; activation of the antioxidant NRF2 pathway; and klotho downregulation. However, induction of tubular-cell-cycle-arrest (CCA) and overexpression of renal senescent-associated secretory phenotype (SASP) components was only found in 18-month-old mice. In aging mice, both inflammation and oxidative stress (marked by elevated lipid peroxidation and NRF2 inactivation) remained increased. These findings support the hypothesis that prolonged DDR and CCA, loss of nephroprotective factors (klotho), and dysfunctional redox regulatory mechanisms (NRF2/antioxidant defense) can be early drivers of age-related kidney-damage progressionThis research was funded by grants from the Instituto de Salud Carlos III (ISCIII); Fondos FEDER European Union (PI17/00119, PI20/00140; and DTS20/00083 to M.R.-O.; PI18/01133 to A.M.R.); Sara Borrell’ program from Instituto de Salud Carlos III (ISCIII) (grant number CD20/00042 to R.R.R.-D.); Red de Investigación Renal REDINREN: RD16/0009/0003 and RICORS program to RICORS2040 496 (RD21/0005), to M.R.-O., Sociedad Española de Nefrología; “NOVELREN-CM: Enfermedad renal crónica: nuevas Estrategias para la prevención, Diagnóstico y tratamiento” (B2017/BMD3751 to M.R.-O.); “Convocatoria Dinamización Europa Investigación 2019” MINECO (EIN2019-103294 to M.R.-O.); Juan de la Cierva incorporacion grant: IJC2018-035187-I to S.R.-M.; innovation program under the Marie Skłodowska-Curie grant of the European Union’s Horizon 2020 (IMProvePD ID: 812699) to M.R.-O.; and Fundacion Conchita Rabago to L.T.-

Protein Kinase C δ Regulates the Depletion of Actin at the Immunological Synapse Required for Polarized Exosome Secretion by T Cells

Multivesicular bodies (MVB) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, ILVs are secreted as Fas ligand-bearing, pro-apoptotic exosomes following T cell receptor (TCR)-induced fusion of MVB with the plasma membrane at the immune synapse (IS). In this study we show that protein kinase C δ (PKCδ), a novel PKC isotype activated by diacylglycerol (DAG), regulates TCR-controlled MVB polarization toward the IS and exosome secretion. Concomitantly, we demonstrate that PKCδ-interfered T lymphocytes are defective in activation-induced cell death. Using a DAG sensor based on the C1 DAG-binding domain of PKCδ and a GFP-PKCδ chimera, we reveal that T lymphocyte activation enhances DAG levels at the MVB endomembranes which mediates the association of PKCδ to MVB. Spatiotemporal reorganization of F-actin at the IS is inhibited in PKCδ-interfered T lymphocytes. Therefore, we propose PKCδ as a DAG effector that regulates the actin reorganization necessary for MVB traffic and exosome secretion

Toward a Spanish SKA Regional Centre fully engaged with open science

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.The Square Kilometre Array Observatory (SKAO) will build the most sensitive radio telescopes on Earth. To address fundamental questions in astrophysics, fundamental physics, and astrobiology, it will require processing and handling complex and extremely massive data close to the exascale, hence constituting a technological challenge for the next decade. Approximately 600 Peta-bytes (PB) of calibrated data will be delivered to the network of SKA Regional Centers (SRCs) worldwide. As a world-leading scientific instrument, SKAO aims to pursue the best practices in scientific methodology. Remarkably, it includes the reproducibility of its data as a metric of success. We present the Spanish prototype of an SRC (SPSRC), which supports preparatory scientific activities for the future SKA projects. These include science with SKA precursors and pathfinders while promoting Open Science practices as a way to enable scientific reproducibility. We describe the key developments and components of the SPSRC that align with these objectives. In particular, we describe the performed work on hardware and cloud computing infrastructure, science archive, software and services, user support and training, and collaboration with other SRCs. The resulting SPSRC platform is flexible enough to host heterogeneous projects while being scalable toward the demanding SKA requirements. © The Authors. Published by SPIE.We acknowledge financial support from the State Agency for Research of the Spanish Ministry of Science, Innovation and Universities through the “Center of Excellence Severo Ochoa” awarded to the Instituto de Astrofísica de Andalucía (SEV-2017-0709) and from the Grant no. RTI2018-096228-B-C31 (Ministry of Science, Innovation and Universities/State Agency for Research/European Regional Development Funds, European Union). In addition, we acknowledge financial support from the Ministry of Science, Innovation and Universities and the European Regional Development Funds (EQC2019-005707-P) and the Regional Government of Andalusia (SOMM17-5208-IAA-2017). LVM, JG, SSE and SLV acknowledge The European Science Cluster of Astronomy and Particle Physics ESFRI Research Infrastructures project that has received funding from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 824064. We would like to explicitly acknowledge Dr. José Ruedas, head of the Computer Centre at IAA-CSIC, for his technical assistance. We acknowledge the contribution from Theresa Wiegert. LVM, SSE, and SLV acknowledge financial support from the Grant No. COOPB20448 (Spanish National Research Council Program of Scientific Cooperation for Development i-COOP+2019). LVM, JG, and JM acknowledge financial support from the Grant No. RED2018-102587-T (Spanish Ministry of Science, Innovation and Universities/State Agency for Research). LVM, JG, SSE, JM acknowledge financial support from the Grant No. IAA4SKA P18-RT-3082 (Regional Government of Andalusia). LVM acknowledges financial support from the Ministry of Science and Innovation, from the budgetary line 28.06.000x.430.09 of the General State Budgets of 2021, for the coordination of the participation in SKA-SPAIN. LD acknowledges financial support from the Grant No. PTA2018-015980-I (Ministry of Science, Innovation and Universities and the Spanish National Research Council). MP acknowledges financial support from the Grant No. 54A Scientific Research and Innovation Program (Regional Council of Economy, Knowledge, Business and Universities, Regional Government of Andalusia and the European Regional Development Funds 2014-2020, program D1113102E3).Peer reviewe

MiR‐9‐5p protects from kidney fibrosis by metabolic reprogramming

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally and control biological processes (BPs), including fibrogenesis. Kidney fibrosis remains a clinical challenge and miRNAs may represent a valid therapeutic avenue. We show that miR‐9‐5p protected from renal fibrosis in the mouse model of unilateral ureteral obstruction (UUO). This was reflected in reduced expression of pro‐fibrotic markers, decreased number of infiltrating monocytes/macrophages, and diminished tubular epithelial cell injury and transforming growth factor‐beta 1 (TGF‐β1)‐dependent de‐differentiation in human kidney proximal tubular (HKC‐8) cells. RNA‐sequencing (RNA‐Seq) studies in the UUO model revealed that treatment with miR‐9‐5p prevented the downregulation of genes related to key metabolic pathways, including mitochondrial function, oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and glycolysis. Studies in human tubular epithelial cells demonstrated that miR‐9‐5p impeded TGF‐β1‐induced bioenergetics derangement. The expression of the FAO‐related axis peroxisome proliferator‐activated receptor gamma coactivator 1 alpha (PGC‐1α)‐peroxisome proliferator‐activated receptor alpha (PPARα) was reduced by UUO, although preserved by the administration of miR‐9‐5p. We found that in mice null for the mitochondrial master regulator PGC‐1α, miR‐9‐5p was unable to promote a protective effect in the UUO model. We propose that miR‐9‐5p elicits a protective response to chronic kidney injury and renal fibrosis by inducing reprogramming of the metabolic derangement and mitochondrial dysfunction affecting tubular epithelial cells.This work was supported by Grants from the Ministerio de Economía y Competitividad (MINECO) SAF 2012‐31388 (SL) and SAF2015‐66107‐R (SL), both cofunded by the European Regional Development Fund, Instituto de Salud Carlos III REDinREN RD12/0021/0009 and RD16/0009/0016 (SL and MRO), PI17/00119 (MRO), SAF2015‐63904‐R (MM), cofunded by the European Regional Development Fund, European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement 721236‐TREATMENT (MM), Comunidad de Madrid “NOVELREN” B2017/BMD­3751 (SL and MRO), and Fundación Renal “Iñigo Alvarez de Toledo” (SL), all from Spain. The CBMSO receives institutional support from Fundación “Ramón Areces”. VM and CNT were supported by predoctoral fellowships of the FPI Program (BES‐2013‐065986 and BES‐2014‐068929) from MINECO.Peer reviewe