Search

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. FGFR1 has been associated with tumorigenesis in a variety of tumor types, including lung cancer. As a therapeutic approach, their inhibition has been attempted and was initially focused on FGFR1-amplified tumors, though with limited success. Preliminary data of our group suggests that N-Cadherin play a key role for the oncogenicity of FGFR1 and predict FGFR-targeted therapy efficacy in Non-small cell lung cancer (NSCLC). However, it is possible that other biomarkers, together with N-Cadherin, can determine the response to anti-FGFR therapy. Therefore, it is essential the identification of new biomarkers that could help to predict more accurately those patients that could benefit from anti-FGFR therapy. Materials and Methods: We have treated 15 NSCLC PDX models with high FGFR1 expression levels and variable N-Cadherin expression levels, with the FGFR inhibitor (FGFRi) AZD4547. Based on their sensitivity to treatment, the PDX models were classified into two groups: Responders and Non-responders. All PDX models were characterized at the proteomic, genomic, and transcriptomic levels by western blot, whole exome sequencing, and RNA-Seq, respectively, and we used this information for defining the responder versus the non-responder group. Subsequently, the GDSC repository was used to validated our data in a cohort of lung adenocarcinoma (LUAD) cell lines. Results: Responder and non-responder groups revealed a distinct pattern of genomic alterations and gene expression profile. As expected, GSEA identified an enrichment in Responders group of pathways related to FGFR1 signaling and ECM proteins, among them N-Cadherin stands out. Moreover, we observed an enrichment of KRAS mutations and genes related to the KRAS signature and to the intrinsic resistance to FGFRi in the non-responder group. To validate our findings and using the GDSC repository, we observed an enriched resistance to inhibitors of FGFR1 signaling in a cohort of KRAS mutant LUAD cell lines. Furthermore, we confirm a tendency for increased sensitivity in high N-Cadherin expressing cell lines, but only in KRAS wildtype context. Conclusions: In conclusion, previous research had underlined N-Cadherin as a potential biomarker of response to FGFRi, while not successful in all contexts. Transcriptome study of PDXs classified as responder or non-responder to FGFRi revealed a differential gene expression pattern, with an upregulation of N-Cadherin pathway observed in the Responder group, while an enrichment of KRAS signaling and KRAS mutations in non-Responders. Furthermore, we validated our finding using a cell line repository that confirm that N-Cadherin could predict FGFR-targeted therapy efficacy but only in the KRAS wildtype context. Citation Format: Santiago G. Borrego, Cristina Cirauqui, David Gómez-Sánchez, Haiyun Wang, Alicia Luengo, Chiara Ambrogio, Luis Paz-Ares, Irene Ferrer. N-Cadherin acts as a predictive biomarker for anti-FGFR therapy in KRAS wild-type NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4489.

Resumen: Antecedentes: Los pacientes con enfermedad renal crónica presentan una acumulación de toxinas urémicas, las cuales han sido identificadas como agentes patogénicos asociados con la mortalidad cardiovascular, muy elevada en este grupo de enfermos. Un fenómeno común a la disfunción renal progresiva y al daño vascular asociado es la acumulación anormal de proteínas de la matriz extracelular (MEC) en las estructuras renales o vasculares. Objetivo: Estudiar la contribución de la uremia o las toxinas urémicas a la producción de citocinas y MEC en aortas de animales urémicos o células de músculo liso de aorta humana (HAOSMC). Materiales y métodos: Se utilizaron ratones con uremia inducida por una dieta rica en adenina (0,2%) durante 2, 4 o 6 semanas. Se evaluó la función renal mediante la diuresis, los niveles plasmáticos de creatinina y nitrógeno ureico plasmático, y la excreción fraccional de sodio y el daño vascular mediante histología y expresión proteica por RT-qPCR. In vitro, las HAOSMC se incubaron con toxinas urémicas: p-cresol 10-100 (μg/ml) e indoxil-sulfato 25-100 (μg/ml), solas o simultáneamente. La expresión proteica se evaluó por Western blot y microscopia confocal. Resultados: La administración de adenina produjo un progresivo daño renal en los ratones, un engrosamiento de la pared aórtica y un incremento de la expresión de TGF-β1 y proteínas de MEC. Las toxinas a dosis altas y combinadas también indujeron expresión de TGF-β1 y proteínas de MEC por las células HAOSMC. Conclusiones: La uremia producida por una dieta rica en adenina o las dosis altas de toxinas urémicas indujeron el depósito anormal de proteínas de MEC en las paredes vasculares o su producción por HAOSMC. La comprensión de los mecanismos que subyacen a este proceso fisiopatológico puede resultar de utilidad en la prevención del daño cardiovascular asociado a la progresión de la enfermedad renal crónica, una dolencia, de momento, irreversible y, en ocasiones, silenciosa hasta su diagnóstico en etapas avanzadas. Abstract: Background: Patients with chronic kidney disease present with an accumulation of uraemic toxins, which have been identified as pathogenic agents associated with cardiovascular mortality, which is very high is this patient group. A phenomenon common to the progressive renal dysfunction and associated vascular damage, is the abnormal accumulation of extracellular matrix (ECM) proteins in the renal or vascular structures. Objective: To determine the contribution of uraemia or the uraemic toxins to the production of cytokinins and ECM in aortas of uraemic animals or human aortic smooth muscle cells (HASMCs). Materials and methods: Mice were used with uraemia induced by a diet rich in adenine (0.2%) for 2, 4 or 6 weeks. Kidney function was evaluated by means of urine volume, plasma levels of creatinine, urea, fractional excretion of sodium, and vascular damage using histology, as well as protein expression using RT-qPCR. The HASMCs were incubated in vitro with uraemic toxins: p-cresol 10-100 (μg/ml) and indoxyl-sulphate25-100 (μg/ml) alone or simultaneously. The protein expression was evaluated using Western blot and confocal microscopy. Results: The administration of adenine produced progressive kidney damage in the mice, thickening of the aortic wall, and increasing the expression of TGF-β1 and ECM proteins. The toxins at high doses and combined also induced the expression of TGF-β1 and ECM proteins by the HASMCs. Conclusions: The uraemia produced by an adenine rich diet or high doses of uraemic toxins induced the abnormal deposit of ECM proteins in the vascular wall or its production by HASMCs. The understanding of the mechanisms that underlie this pathophysiological process may be useful in the prevention of cardiovascular damage associated with the progress of chronic kidney disease, a disease, at the moment that is irreversible and occasional silent until its diagnosis in advanced stages. Palabras clave: Enfermedad renal crónica, Daño vascular, Toxinas urémicas, Adenina, Fibrosis, Transforming Growth factor beta-1, Keywords: Chronic kidney disease, Vascular damage, Uraemic toxins, Adenine, Fibrosis, Transforming Growth factor beta-1

Two processes are associated with progressive loss of renal function: 1) decreased aquaporin-2 (AQP2) expression and urinary concentrating capacity (Nephrogenic Diabetes Insipidus, NDI); and 2) changes in extracellular matrix (ECM) composition, e.g. increased collagen I (Col I) deposition, characteristic of tubule-interstitial fibrosis. AQP2 expression is regulated by both the ECM-to-intracellular scaffold protein integrin-linked kinase (ILK) by NFATc/AP1 and other transcription factors. In the present work, we used in vivo and in vitro approaches to examine ILK participation in NFATc3/AP-1-mediated increases in AQP2 gene expression. Both NFATc3 knock-out mice and ILK conditional-knockdown mice (cKD-ILK) display symptoms of NDI (polyuria and reduced AQP2 expression). NFATc3 is upregulated in the renal medulla tubular cells of cKD-ILK mice but with reduced nuclear localization. Inner medullary collecting duct mIMCD3 cells were subjected to ILK depletion and transfected with reporter plasmids. Pharmacological activators or inhibitors determined the effect of ILK activity on NFATc/AP-1-dependent increases in transcription of AQP2. Finally, mIMCD3 cultured on Col I showed reduced activity of the ILK/GSK3β/NFATc/AQP2 axis, suggesting this pathway is a potential target for therapeutic treatment of NDI.

The development of insulin resistance is characterized by the impairment of glucose uptake mediated by glucose transporter 4 (GLUT4). Extracellular matrix changes are induced when the metabolic dysregulation is sustained. The present work was devoted to analyze the possible link between the extracellular-to-intracellular mediator integrin-linked kinase (ILK) and the peripheral tissue modification that leads to glucose homeostasis impairment. Mice with general depletion of ILK in adulthood (cKD-ILK) maintained in a chow diet exhibited increased glycemia and insulinemia concurrently with a reduction of the expression and membrane presence of GLUT4 in the insulin-sensitive peripheral tissues compared with their wild-type littermates (WT). Tolerance tests and insulin sensitivity indexes confirmed the insulin resistance in cKD-ILK, suggesting a similar stage to prediabetes in humans. Under randomly fed conditions, no differences between cKD-ILK and WT were observed in the expression of insulin receptor (IR-B) and its substrate IRS-1 expressions. The IR-B isoform phosphorylated at tyrosines 1150/1151 was increased, but the AKT phosphorylation in serine 473 was reduced in cKD-ILK tissues. Similarly, ILK-blocked myotubes reduced their GLUT4 promoter activity and GLUT4 expression levels. On the other hand, the glucose uptake capacity in response to exogenous insulin was impaired when ILK was blockedin vivoandin vitro, although IR/IRS/AKT phosphorylation states were increased but not different between groups. We conclude that ILK depletion modifies the transcription of GLUT4, which results in reduced peripheral insulin sensitivity and glucose uptake, suggesting ILK as a molecular target and a prognostic biomarker of insulin resistance.

Kidney fibrosis is one of the main pathological findings of progressive chronic kidney disease (CKD) although the pathogenesis of renal scar formation remains incompletely explained. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix (ECM) and intracellular signaling pathways, is involved in several pathophysiological processes during renal damage. However, ILK contribution in the CKD progress remains to be fully elucidated. In the present work, we studied 1) the renal functional and structural consequences of CKD genesis and progression when ILK is depleted and 2) the potential of ILK depletion as a therapeutic approach to delay CKD progression. We induced an experimental CKD model, based on an adenine-supplemented diet on adult wild-type (WT) and ILK-depleted mice, with a tubulointerstitial damage profile resembling that is observed in human CKD. The adenine diet induced in WT mice a progressive increase in plasma creatinine and urea concentrations. In the renal cortex it was also observed tubular damage, interstitial fibrosis and progressive increased ECM components, pro-inflammatory and chemo-attractant cytokines, EMT markers and TGF-β1 expressions. These observations were highly correlated to a simultaneous increase of ILK expression and activity. In adenine-fed transgenic ILK-depleted mice, all these changes were prevented. Additionally, we evaluated the potential role of ILK depletion to be applied after the disease induction, as an effective approach to interventions in human CKD subjects. In this scenario, two weeks after the establishment of adenine-induced CKD, ILK was abrogated in WT mice and stabilized renal damage, avoiding CKD progression. We propose ILK to be a potential target to delay renal disease progression.

Key points Patients with chronic kidney disease have a higher risk of developing cardiovascular diseases than the general population. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which have poor clearance by conventional dialysis. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity and in this study we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. For the first time, we demonstrate the implication of ILK in the protection against endothelial cell damage (inhibition of proliferation, toxicity, oxidative stress and programed cell death) induced by uraemic serum from chronic kidney disease patients and uraemic toxins. This molecular mechanism may have clinical relevance because it highlights the importance of maintaining high levels of ILK activity to help preserve endothelial integrity, at least in early stages of chronic kidney disease. Abstract Patients with chronic kidney disease (CKD) have a higher risk of developing cardiovascular diseases. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which, mostly protein-bound compounds such as indoxyl sulfate (IS) and p-cresyl sulphate, having poor clearance by conventional dialysis, induce endothelial toxicity. However, the molecular mechanism by which uraemic toxins regulate early stages of endothelial dysfunction remains unclear. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity. In this study, we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. First, we show that incubation of EA.hy926 cells with human uraemic serum from CKD patients upregulates ILK activity. This ILK activation also occurs when the cells are exposed to IS (25–100 μg ml−1), p-cresol (10–100 μg ml−1) or both combined, compared to human serum control. Next, we observed that high doses of both toxins together induce a slight decrease in cell proliferation and increase apoptosis and reactive oxygen species production. Interestingly, these toxic effects displayed a strong increase when the ILK protein is knocked down by small interfering RNA, even at low doses of uraemic toxins. Abrogation of AKT has demonstrated the ILK/AKT signalling pathway involved in these processes. This study has demonstrated the implication of ILK in the protection against endothelial cell damage induced by uraemic toxins, a molecular mechanism that could play a protective role in the early stages of endothelial dysfunction observed in uraemic patients.

The coordination compound of the antihypertensive ligand irbesartan (irb) with copper(II) (CuIrb) was synthesized and characterized by FTIR, FT-Raman, UV–visible, reflectance and EPR spectroscopies. Experimental evidence allowed the implementation of structural and vibrational studies by theoretical calculations made in the light of the density functional theory (DFT). This compound was designed to induce structural modifications on the ligand. No antioxidant effects were displayed by both compounds, though CuIrb behaved as a weak 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·) scavenger (IC₅₀ = 425 μM). The measurements of the contractile capacity on human mesangial cell lines showed that CuIrb improved the antihypertensive effects of the parent medication. In vitro cell growth inhibition against prostate cancer cell lines (LNCaP and DU 145) was measured for CuIrb, irbesartan and copper(II). These cell lines have been selected since the angiotensin II type 1 (AT1) receptor (that was blocked by the angiotensin receptor blockers, ARB) has been identified in them. The complex exerted anticancer behavior (at 100 μM) improving the activity of the ligand. Flow cytometry determinations were used to determine late apoptotic mechanisms of cell death., Facultad de Ciencias Exactas, Centro de Química Inorgánica

Biobanks are useful platforms to build bridges between basic, translational, and clinical research and clinical care. They are repositories of high-quality human biological samples ideal for evaluating their histological characteristics and also their genome, transcriptome, and proteome. The Spanish Renal Research Network Biobank contains more than 76,500 well-preserved frozen samples of a wide variety of kidney diseases, collected from 5450 patients seen by over 70 nephrology services throughout the Spanish territory.To determine and to report the results of the quality control of samples and processes conducted in our biobank, implemented in accordance with the requirements of the ISO 9001:2008 international standard.Two types of quality controls were performed: (1) systematic, that is, measurement of viable peripheral blood mononuclear cells (PBMCs) obtained and purity of nucleic acids and (2) ad-hoc, that is, viability of thawed PBMC, DNA extraction process reproducibility, and the integrity and functionality of nucleic acids, implemented on a routine basis.PBMC isolation by Ficoll yielded reproducible results and its cryopreserved viability was90%. Acceptable A260/A280 ratios were obtained for the vast majority of the DNA (n = 2328) and RNA (n = 78) samples analyzed. DNA integrity was demonstrated by agarose gels and by β-globulin gene polymerase chain reaction (PCR) amplification of 1327 and 989 bp fragments. DNA of acceptable quality had at least three bands of β-globulin amplified obtained (n = 26/30). RNA integrity number (RIN) determinations obtained RIN numbers ≥7 (n = 87/96). The amplifiability of nucleic acids was confirmed by qPCR and RT-qPCR of β-actin and GAPDH genes. Long storage or delayed processing time did not affect the quality of the samples analyzed. The processes of DNA extraction also yielded reproducible results.These results clearly indicate that our PBMC, DNA, and RNA stored samples meet the required quality standards to be used for biomedical research, ensuring their long-term preservation.

Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway., This study was supported by grant ISCIII-RETIC REDinREN/RD06/0001, by grants Fundación Mutua Madrileña (FMM2008 – 004) and Proyecto CAMUAH (CCG08-UAH/BIO-4237) to M. Alique, by grant Ministerio de Educación (SAF 2007 623471) to M. Rodríguez-Puyol, by grant Fondo de Investigaciones Sanitarias (FISS-PI070695) to D. Rodríguez-Puyol, and by grants Ministerio de Ciencia e Innovación (SAF2007-61716; BFU2007-62659/ BMC), ISCIII-RETIC RED RECAVA RD06/0014/1013, Comunidad Autónoma de Madrid, (S-SAL-0159/2006), CAM-UAM (CCG08-UAM/BIO-4299), and European Union (EICOSANOX LSH-CT-2004-005033) to M. Fresno and M. A. Iñiguez.

Biobank certification ISO 9001:2008 aims to improve the management of processes performed. This has two objectives: customer satisfaction and continuous improvement. This paper presents the impact of certification ISO 9001:2008 on the sample transfer process in a Spanish biobank specialising in kidney patient samples. The biobank experienced a large increase in the number of samples between 2009 (12,582 vials) and 2010 (37,042 vials).The biobank of the Spanish Renal Research Network (REDinREN), located at the University of Alcalaacute;, has implemented ISO standard 9001:2008 for the effective management of human material given to research centres. Using surveys, we analysed two periods in theldquo;sample transferrdquo; process. During the first period between 1-10-12 and 26-11-12 (8 weeks), minimal changes were made to correct isolated errors. In the second period, between 7-01-13 and 18-02-13 (6 weeks), we carried out general corrective actions.The identification of problems and implementation of corrective actions for certification allowed: a 70% reduction in the process execution time, a significant increase (200%) in the number of samples processed and a 25% improvement in the process. The increase in the number of samples processed was directly related to process improvement.The certification of ISO standard 9001:2008, obtained in July 2013, allowed an improvement of the REDinREN biobank processes to be achieved, which increased quality and customer satisfaction.

The geometry and electronic properties of several malonic acids receptors are studied with a view to optimizing their decarboxylative activity.

Renal fibrosis is the final outcome of many clinical conditions that lead to chronic renal failure, characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, in particular collagen type I. The aim of this study was to identify the mechanisms responsible for human mesangial cell survival, conditioned by changes in extracellular-matrix composition. Our results indicate that collagen I induces apoptosis in cells but only after inactivation of the pro-survival factor NFκB by either the super-repressor IκBα or the PDTC inhibitor. Collagen I activates a death pathway, through ILK/GSK-3β-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates β1 integrin and this is accompanied by increased activity of ILK which leads to AKT activation. Knockdown of ILK or AKT with small interfering RNA suppresses the increase in NFκB activity. NFκB mediates cell survival through the antiapoptotic protein Bcl-xL. Our data suggest that human mesangial cells exposed to abnormal collagen I are protected against apoptosis by a complex mechanism involving integrin β1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3β-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction. © Springer Science+Business Media New York 2012., This study was supported by Grant ISCIIIRETIC (REDinREN/RD06/0016/0002), by Grant Fundación Mutua Madrileña (FMM2011-001) to LC, by Grant Ministerio de Educación (SAF2010-16198) to MRP and by Grant Fondo de Investigaciones Sanitarias (FISSPI11/01630) to DRP.

The creation of the Biobank, a resource pertaining to the Spanish Renal Research Network (REDinREN) promotes advances in clinical research on kidney disease in Spain. The Biobank's aims are to generate an archive of clinical samples and associated data, furnish those samples to research teams, and coordinate with European biobanks.Applicable legislation had to be complied with in order to launch the Biobank project (Biomedical Research Law, Data Protection Law and Biological Sample Transport Regulations). A strict work protocol and a new database for the Network's clinical data were also implemented.Over time, the Biobank has acquired additional infrastructure and qualified personnel. In 2010, 2953 new patient samples were collected, giving a total of 37,043 stored vials containing different types of samples. Furthermore, the Biobank is currently participating in eleven research projects.Although the Biobank was originally designed for REDinREN use, we must take joint action to make this biological sample storage system and the many possibilities it offers available to the entire nephrological community with a view to promoting kidney disease research.

The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β1-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

The geometry and electronic properties of several malonic acids receptors are studied with a view to optimizing their decarboxylative activity.