32 research outputs found
Gremlin, un nuevo mediador en la progresión de la enfermedad renal
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 18/07/2014Gremlin es un miembro de la superfamilia de TGF‐ que actúa como antagonista de las
proteínas morfogenéticas de hueso (BMPs), participando en desarrollo embrionario y fibrosis.
Recientemente se han demostrado efectos celulares directos de Gremlin independientes de BMPs,
algunos de ellos mediados por el receptor VEGFR2 (Vascular Endotelial growth factor receptor‐2).
Gremlin se re‐expresa en adultos en condiciones patológicas, y como ha descrito nuestro grupo,
en enfermedades renales crónicas asociado a fibrosis y pérdida de funcionalidad. El objetivo de
esta tesis ha sido determinar los efectos de Gremlin en riñón en condiciones fisiológicas y
patológicas, investigando el receptor implicado y los mecanismos intracelulares activados, con
especial atención a la vía Notch. Los resultados demuestran que Gremlin se une y activa el
receptor VEGFR2 y modula la respuesta inflamatoria renal por activación temprana de la vía
canónica de NF‐κB. Esta activación induce la expresión de factores pro‐inflamatorios e infiltración
renal de monocitos/macrófagos y linfocitos. In vitro hemos observado que Gremlin, vía VEGFR2
regula factores pro‐fibróticos, componentes de matriz extracelular y eventos asociados a
transición epitelio‐mesenquimal. Además, Gremlin es un mediador de las acciones pro‐fibróticas
de TGF‐β. En modelos experimentales de daño renal y en patologías renales crónicas humanas se
induce Gremlin y se activa la vía del VEGFR2 en riñón, sugiriendo que el eje Gremlin/VEGFR2
podría contribuir a la progresión del daño renal. Así, el bloqueo de VEGFR2 retardó la progresión
del daño renal experimental, inhibiendo Gremlin y disminuyendo inflamación y fibrosis, mientras
que el ligando canónico de VEGFR2 no fue modificado. La regulación de estos procesos es
compleja e involucra la activación y la integración de otros sistemas de señalización intracelular.
Gremlin activa la vía de señalización Notch asociado a la regulación de factores pro‐fibróticos y
marcadores de transición epitelio mesenquimal. El bloqueo de la ruta Notch mejoró el daño renal
experimental inhibiendo la fibrosis. En pacientes con nefropatías progresivas se observó activación
de la vía Notch, pero no en nefropatía hipertensiva o en modelos experimentales de hipertensión.
En estudios in vivo e in vitro, Angiotensina II no activó la ruta Notch, mostrando una clara
diferencia con otros factores pro‐fibróticos, como TGF‐β y Gremlin. En conjunto, estos datos
sugieren que Gremlin podría ser considerado un nuevo mediador de daño renal a través de la
activación de VEGFR2, y sugieren el eje Gremlin/VEGFR2 como una nueva diana terapéutica para
enfermedades renales crónicas.Gremlin is a member of the TGF‐β superfamily and a bone morphogenetic proteins (BMPs)
antagonist involved in embryonic development and fibrosis. BMPs independent Gremlin cellular
effects have recently been described, some of them mediated by binding to VEGFR2 receptor
(vascular endothelial growth factor receptor‐2). Gremlin is re‐expressed in pathological conditions
in adults, as previously described by our group in chronic kidney diseases associated with fibrosis
and renal failure. The aim of this thesis was to determine the Gremlin actions in the kidney,
investigating the receptor involved and the intracellular mechanisms activated, with special
attention to the Notch pathway. The results demonstrate that Gremlin binds to and activates the
VEGFR2 receptor signaling linked to renal inflammation. Gremlin caused an early activation of the
canonical NF‐kB pathway and upregulation of pro‐inflammatory factors, leading to infiltration of
monocytes/macrophages and lymphocytes into the kidney. In vitro Gremlin, via VEGFR2, regulated
pro‐fibrotic factors, extracellular matrix components and epithelial‐mesenchymal transition
related‐events. Moreover, Gremlin is a downstream mediator of TGF‐β pro‐fibrotic actions. In
experimental models of renal injury and in human chronic kidney pathologies, Gremlin induction
and VEGFR2 pathway activation in the kidney was found, suggesting that Gremlin/VEGFR2 axis
could contribute to the progression of renal damage. Thus, VEGFR2 blockade delayed renal
damage progression, associated to Gremlin downregulation, while the canonical VEGFR2 ligand
was not modified. The regulation of this process is complex and involves the activation and
integration of other intracellular signaling systems. Gremlin activated the Notch signaling pathway
in the kidney associated to the up‐regulation of pro‐fibrotic factors and changes in epithelialmesenchymal
transition markers. Notch pathway blockade improved experimental renal damage,
diminishing fibrosis. In patients with several progressive nephropathies, except in hypertensive
nephropathy, renal Notch pathway activation was found. In response to Angiotensin II in vivo and
in vitro Notch pathway was not activated, showing a clear difference with others pro‐fibrotic
factors, such as TGF‐β and Gremlin. In sumary, these data suggest that Gremlin through VEGFR2
activation could be considered a novel mediator of kidney damage, and suggests that the
Gremlin/VEGFR2 axis could be as a novel therapeutic target for chronic kidney diseases
Special issue “Diabetic nephropathy: Diagnosis, prevention and treatment”
Diabetic nephropathy (DN) is the main cause of end-stage renal disease. DN is a complex
disease mediated by genetic and environmental factors, and many cellular and molecular mechanisms
are involved in renal damage in diabetes. There are no biomarkers that reflect the severity of the
underlying renal histopathological changes and can e ectively predict the progression of renal
damage and stratify the risk of DN among individuals with diabetes mellitus. Current therapeutic
strategies are based on the strict control of glucose and blood pressure levels and, although there
are new anti-diabetic drugs, these treatments only retard renal damage progression, being necessary
novel therapies. In this Special Issue, there are several comprehensive reviews and interesting original
papers covering all these topics, which would be of interest to the growing number of readers of the Journal of Clinical MedicineEditors are funding by Grants from the Instituto de Salud Carlos III(ISCIII) and Fondos FEDER European
Union (PI17/00119 and Red de Investigación Renal (REDINREN): RD16/0009, to M.R-O), Comunidad de Madrid
(“NOVELREN” B2017/BMD-3751 to M.R-O); the José Castillejo grant (CAS19/00133 to R.R.R-D); the “Juan de
la Cierva Formacion” training program of the Ministerio de Economia, Industria y Competitividad (MINECO)
supported the salary of SR-M (FJCI-2016-29050); Sociedad Española de Nefrologia (S.E.N. to M.R-O). Grants PAI
82140017 to C.L. of Chile; IMPROVE-PD project (“Identification and Management of Patients atRisk–Outcome
and Vascular Events in Peritoneal Dialysis”) funding from the European Union’s Horizon 2020 research and
innovation program under the Marie Skłodowska-Curie Grant Agreement No. 812699 to M.R.O
VEGFR2 blockade improves renal damage in an experimental model of type 2 diabetic nephropathy
The absence of optimal treatments for Diabetic Nephropathy (DN) highlights the importance
of the search for novel therapeutic targets. The vascular endothelial growth factor receptor 2
(VEGFR2) pathway is activated in experimental and human DN, but the e ects of its blockade
in experimental models of DN is still controversial. Here, we test the e ects of a therapeutic
anti-VEGFR2 treatment, using a VEGFR2 kinase inhibitor, on the progression of renal damage in the
BTBR ob/ob (leptin deficiency mutation) mice. This experimental diabetic model develops histological
characteristics mimicking the key features of advanced human DN. A VEGFR2 pathway-activation
blockade using the VEGFR2 kinase inhibitor SU5416, starting after kidney disease development,
improves renal function, glomerular damage (mesangial matrix expansion and basement membrane
thickening), tubulointerstitial inflammation and tubular atrophy, compared to untreated diabetic
mice. The downstream mechanisms involved in these beneficial e ects of VEGFR2 blockade include
gene expression restoration of podocyte markers and downregulation of renal injury biomarkers
and pro-inflammatory mediators. Several ligands can activate VEGFR2, including the canonical
ligands VEGFs and GREMLIN. Activation of a GREMLIN/VEGFR2 pathway, but not other ligands, is
correlated with renal damage progression in BTBR ob/ob diabetic mice. RNA sequencing analysis of
GREMLIN-regulated genes confirm the modulation of proinflammatory genes and related-molecular
pathways. Overall, these data show that a GREMLIN/VEGFR2 pathway activation is involved in
diabetic kidney disease and could potentially be a novel therapeutic target in this clinical conditionThis research was funded by Fondecyt 1160465 to S.M., e Instituto de Salud Carlos III (ISCIII) and
Fondos FEDER European Union (Grants PI17/00119 and Red de Investigación Renal REDINREN: RD16/0009 to
M.R.-O; and PI17/01495 to J.E.). Comunidad de Madrid (Grant “NOVELREN” B2017/BMD-3751 to M.R.-O)
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)
Gremlin activates the smad pathway linked to epithelial mesenchymal transdifferentiation in cultured tubular epithelial cells
Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming
growth factor-(TGF-β). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial
cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate
whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2)
with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and
Smad-dependent gene transcription). The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify
Gremlin-induced early Smad activation.These data showthatGremlin directly, by a TGF-β independent process, activates the Smad
pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad
activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation,
diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlininduced
EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular
epithelial cells through activation of Smad pathway and induction of TGF-βThis work was supported by grants from the Instituto
de Salud Carlos III (PI11/01854 and REDINREN ISCIIIRETIC
RD12/0021/0002 and 0001), Sociedad Española de
Nefrología, PCI Iberoamerica (A/9571/07), CYTED IBERERC,
FONDECYT Chile 1080083 and 1120480, Comunidad
de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD-
2378), Programa Intensificación Actividad Investigadora
(ISCIII/Agencia Laín Entralgo/CM) to A.O. Fundación para
el fomento en Asturias de la investigaciónn cientíica aplicada
y la tecnología (FICYT)
Statins Inhibit Angiotensin II/Smad Pathway and Related Vascular Fibrosis, by a TGF-β-Independent Process
We have recently described that in an experimental model of atherosclerosis and in vascular smooth muscle cells (VSMCs) statins increased the activation of the Smad pathway by transforming growth factor-β (TGF-β), leading to an increase in TGF-β-dependent matrix accumulation and plaque stabilization. Angiotensin II (AngII) activates the Smad pathway and contributes to vascular fibrosis, although the in vivo contribution of TGF-β has not been completely elucidated. Our aim was to further investigate the mechanisms involved in AngII-induced Smad activation in the vasculature, and to clarify the beneficial effects of statins on AngII-induced vascular fibrosis. Infusion of AngII into rats for 3 days activates the Smad pathway and increases fibrotic-related factors, independently of TGF-β, in rat aorta. Treatment with atorvastatin or simvastatin inhibited AngII-induced Smad activation and related-fibrosis. In cultured rat VSMCs, direct AngII/Smad pathway activation was mediated by p38 MAPK and ROCK activation. Preincubation of VSMCs with statins inhibited AngII-induced Smad activation at all time points studied (from 20 minutes to 24 hours). All these data show that statins inhibited several AngII-activated intracellular signaling systems, including p38-MAPK and ROCK, which regulates the AngII/Smad pathway and related profibrotic factors and matrix proteins, independently of TGF-β responses. The inhibitory effect of statins on the AngII/Smad pathway could explain, at least in part, their beneficial effects on hypertension-induced vascular damage
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
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