La interleuquina-17A es un mediador y una potencial diana terapéutica en la hipertensión y en la nefropatía diabética

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología y Terapéutica. Fecha de lectura: 22-11-2019Esta tesis tiene embargado el acceso al texto completo hasta el 22-05-2021La hipertensión y la diabetes son las causas más comunes de enfermedad renal crónica (ERC). Los actuales tratamientos en estas patologías solo consiguen retardar la progresión de la enfermedad siendo necesarias mejores opciones terapéuticas. Estudios recientes sugieren que el sistema inmune juega un papel clave en la patogenia de las enfermedades renales, y remarcan la implicación de la activación de la respuesta inmune Th17, y de su citoquina efectora interleuquina-17A (IL-17A), en enfermedades inflamatorias crónicas. En pacientes hipertensos y diabéticos se han observado niveles elevados de citoquinas proinflamatorias circulantes. Los pacientes hipertensos presentan un número mas elevado de células circulantes T CD4+ productoras de IL-17A comparado con individuos normotensos. En pacientes con nefropatía diabética (ND) se han evaluado los niveles de IL-17A circulante o urinaria, pero no hay datos sobre los niveles renales. Los estudios preclínicos realizados hasta la fecha no han investigado los efectos de la modulación de IL-17A en el riñón en condiciones fisiológicas y los resultados obtenidos en nefropatía diabética experimental son contradictorios. En esta tesis se evalúa la hipótesis de que la activación de la respuesta inmune Th17, y por tanto, el aumento de la producción de IL-17A puede contribuir a la progresión del daño renal y vascular asociado a hipertensión y diabetes. El primer objetivo ha sido estudiar la implicación de IL17A en la patogenia de la hipertensión y en la lesión de órganos diana, como son las arterias mesentéricas de resistencia (AMRs), aorta y el riñón, así como el papel potencial del bloqueo de IL-17A en situaciones de daño renal asociado a hipertensión. Los estudios preclínicos realizados en el modelo murino de administración sistémica de IL17A, empleando una dosis que se asemeja a los niveles circulantes elevados descritos en pacientes prehipertensos, mostraron un aumento de la presión arterial sistólica después de 2 semanas de tratamiento. A nivel vascular se observó a un remodelado hipertrófico interno de las AMRs, presentando las células de músculo liso vascular (CMLVs) cambios en el fenotipo e hipertrofia y aumento de la rigidez arterial, en ausencia de variaciones en los principales componentes de la matriz extracelular, como son la elastina y el colágeno. A nivel renal, se observó un aumento del infiltrado inflamatorio, incluyendo linfocitos CD3+ y CD4+ y neutrófilos, asociado a inducción de genes proinflamatorios, como MCP-1. Sin embargo, no se encontró ni daño ni fibrosis renal. Además, en estudios in vitro se demostró que la IL-17A tiene efectos directos sobre la regulación del crecimiento celular, y la regulación de mediadores proinflamatorios, sin inducir cambios en la síntesis de proteínas de matriz extracelular. Posteriormente, en un modelo de infusión sistémica de Angiotensina II (Ang II) se observó que el bloqueo de la IL-17A disminuye las alteraciones en la estructura de las arterias meséntericas de resistencia, la hipertrofia vascular y el infiltrado inflamatorio renal, sin modificar los niveles de colágeno a nivel vascular o renal. El último objetivo de esta tesis fue evaluar si IL-17A podría ser una diana terapéutica en la nefropatía diabética (ND). Para ello se empleó un modelo de nefropatía diabética experimental en ratones BTBR ob/ob, caracterizado por lesiones que imitan características clave de la ND humana avanzada. Los estudios preclínicos en el modelo BTBR ob/ob mostraron un aumento de IL17A y una activación en los factores que controlan la diferenciación Th17 en los riñones diabéticos, que se asoció a infiltración de macrófagos y daño renal incipiente. El tratamiento terapéutico con un anticuerpo neutralizante de IL-17A, empezando cuando el daño renal por la diabetes ya ha comenzado, mejoró la función renal y retardó la progresión de la enfermedad. Estos efectos beneficiosos se correlacionan con la disminución de la inflamación renal, proceso este último regulado por la inhibición de la vía NF-κB y de factores proinflamatorios. Estos datos demuestran que IL-17A está implicada en el daño renal experimental mediado por la diabetes y podría ser una diana terapéutica prometedora para mejorar la nefropatía diabética. Como objetivo traslacional se ha evaluado si pacientes con daño renal asociado a hipertensión y diabetes presentan niveles elevados de IL-17A en el riñón, lo que podría confirmar el papel de esta citoquina en la patogenia de ambas enfermedades. En biopsias renales de pacientes con nefroesclerosis hipertensiva se han detectado células que expresan IL-17A, principalmente linfocitos CD4+ y γδ. En biopsias renales de pacientes ND con también se observaron células IL-17A+. En conjunto, estos datos experimentales apoyan el papel patogénico de la IL-17A en el daño tisular en hipertensión y diabetes, y sugieren que el bloqueo de IL-17A podría ser una opción terapéutica potencial para estas enfermedade

Effects of golimumab and ustekinumab on circulating dendritic cell migratory capacity in inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic condition which includes ulcerative colitis (UC) and Crohn’s disease (CD), the origins of which are not yet fully understood. Both conditions involve an exacerbated immune response in the intestinal tract, leading to tissue inflammation. Dendritic cells (DCs) are antigen-presenting cells crucial for maintaining tolerance in the gastrointestinal mucosa. Previous research has indicated that DC recruitment to the intestinal mucosa is more pronounced in individuals with IBD, but the specific mechanisms governing this migration remain unclear. This study aimed to assess the expression of various homing markers and the migratory abilities of circulating DC subsets in response to intestinal chemotactic signals. Additionally, this study examined how golimumab and ustekinumab impact these characteristics in individuals with IBD compared to healthy controls. The findings revealed that a particular subset of DCs known as type 2 conventional DCs (cDC2) displayed a more pronounced migratory profile compared to other DC subsets. Furthermore, the study observed that golimumab and ustekinumab had varying effects on the migratory profile of cDC1 in individuals with CD and UC. While CCL2 did not exert a chemoattractant effect on DC subsets in this patient cohort, treatment with golimumab and ustekinumab enhanced their migratory capacity towards CCL2 and CCL25 while reducing their migration towards MadCam1. In conclusion, this study highlights that cDC2 exhibits a heightened migratory profile towards the gastrointestinal mucosa compared to other DC subsets. This finding could be explored further for the development of new diagnostic biomarkers or the identification of potential immunomodulatory targets in the context of IB

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)

Nephroprotective effects of synthetic flavonoid hidrosmin in experimental diabetic nephropathy

Diabetes mellitus (DM) is a high‐impact disease commonly characterized by hyperglycemia, inflammation, and oxidative stress. Diabetic nephropathy (DN) is a common diabetic microvascular complication and the leading cause of chronic kidney disease worldwide. This study investigates the protective effects of the synthetic flavonoid hidrosmin (5‐O‐(beta-hydroxyethyl) diosmin) in experimental DN induced by streptozotocin injection in apolipoprotein E deficient mice. Oral administration of hidrosmin (300 mg/kg/day, n = 11) to diabetic mice for 7 weeks markedly reduced albuminuria (albumin‐to‐creatinine ratio: 47 ± 11% vs. control) and ameliorated renal pathological damage and expression of kidney injury markers. Kidneys of hidrosmin‐treated mice exhibited lower content of macrophages and T cells, reduced expression of cytokines and chemokines, and attenuated inflammatory signaling pathways. Hidrosmin treatment improved the redox balance by reducing prooxidant enzymes and enhancing antioxidant genes, and also decreased senescence markers in diabetic kidneys. In vitro, hidrosmin dose‐dependently reduced the expression of inflammatory and oxidative genes in tubuloepithelial cells exposed to either high‐glucose or cytokines, with no evidence of cytotoxicity at effective concentrations. In conclusion, the synthetic flavonoid hidrosmin exerts a beneficial effect against DN by reducing inflammation, oxidative stress, and senescence pathways. Hidrosmin could have a potential role as a coadjutant therapy for the chronic complications of DM.This work was supported by grants from the Spanish Ministry of Science and Innovation- FEDER funds (Retos Colaboración RTC2017-6089-1 and Retos Investigación RTI2018-098788-B-I00) and Instituto de Salud Carlos III (PI20/00487 and DTS 19/00093

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

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

Long non-coding RNA signatures in the Ileum and Colon of Crohn’s disease patients and effect of Anti-TNF-α treatment on their modulation

Biological therapies only benefit one-third of patients with Crohn’s disease (CD). For this reason, a deeper understanding of the mechanisms by which biologics elicit their effect on intestinal mucosa is needed. Increasing evidence points toward the involvement of long noncoding RNAs (lncRNAs) in the pathogenesis of CD, although their role remains poorly studied. We aimed to characterize lncRNA profiles in the ileum and colon from CD patients and evaluate the effect of anti-TNF-α treatment on their transcription. Terminal ileum and left colon samples from 30 patients (active CD = 10, quiescent CD = 10, and healthy controls (HCs) = 10) were collected for RNA-seq. The patients were classified according to endoscopic activity. Furthermore, biopsies were cultured with infliximab, and their transcriptome was determined by Illumina gene expression array. A total of 678 differentially expressed lncRNAs between the terminal ileum and left colon were identified in HCs, 438 in patients with quiescent CD, and 468 in patients with active CD. Additionally, we identified three new lncRNAs in the ileum associated with CD activity. No differences were observed when comparing the effect of infliximab according to intestinal location, presence of disease (CD vs. HC), and activity (active vs. quiescent). The expression profiles of lncRNAs are associated with the location of intestinal tissue, being very different in the ileum and colon. The presence of CD and disease activity are associated with the differential expression of lncRNAs. No modulatory effect of infliximab has been observed in the lncRNA transcriptom

TGF-Beta Blockade Increases Renal Inflammation Caused by the C-Terminal Module of the CCN2

The CCN family member 2 (CCN2, also known as connective tissue growth factor) may behave as a risk biomarker and a potential therapeutic target for renal disease. CCN2 participates in the regulation of inflammation and fibrosis. TGF-β is considered the main fibrogenic cytokine; however, in some pathological settings TGF-β also has anti-inflammatory properties. CCN2 has been proposed as a downstream profibrotic mediator of TGF-β, but data on TGF-β role in CCN2 actions are scarce. Our aim was to evaluate the effect of TGF-β blockade in CCN2-mediated experimental renal damage. Systemic administration of the C-terminal module of CCN2 to mice caused sustained renal inflammation. In these mice, TGF-β blockade, using an anti-TGF-β neutralizing antibody, significantly increased renal expression of the NGAL (a kidney injury biomarker), kidney infiltration by monocytes/macrophages, and upregulation of MCP-1 expression. The anti-inflammatory effect of TGF-β seems to be mediated by a dysregulation of the systemic Treg immune response, shown by decreased levels of circulating CD4+/Foxp3+Treg cells. Our experimental data support the idea that TGF-β exerts anti-inflammatory actions in the kidney and suggest that it is not an optimal therapeutic target

Meta-inflammation and de novo lipogenesis markers are involved in metabolic associated fatty liver disease progression in BTBR ob/ob mice

Metabolic associated fatty liver disease (MAFLD) is a hepatic manifestation of metabolic syndrome and usually associated with obesity and diabetes. Our aim is to characterize the pathophysiological mechanism involved in MAFLD development in Black Tan and brachyuric (BTBR) insulin-resistant mice in combination with leptin deficiency (ob/ob). We studied liver morphology and biochemistry on our diabetic and obese mice model (BTBR ob/ob) as well as a diabetic non-obese control (BTBR + streptozotocin) and non-diabetic control mice (BTBR wild type) from 4-22 weeks. Lipid composition was assessed, and lipid related pathways were studied at transcriptional and protein level. Microvesicular steatosis was evident in BTBR ob/ob from week 6, progressing to macrovesicular in the following weeks. At 12th week, inflammatory clusters, activation of STAT3 and Nrf2 signaling pathways, and hepatocellular ballooning. At 22 weeks, the histopathological features previously observed were maintained and no signs of fibrosis were detected. Lipidomic analysis showed profiles associated with de novo lipogenesis (DNL). BTBR ob/ob mice develop MAFLD profile that resemble pathological features observed in humans, with overactivation of inflammatory response, oxidative stress and DNL signaling pathways. Therefore, BTBR ob/ob mouse is an excellent model for the study of the steatosis to steatohepatitis transition