Chronic kidney disease induced by an adenine rich diet upregulates integrin linked kinase (ILK) and its depletion prevents the disease progression.

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-ß1 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.

Contribución de las toxinas urémicas a la fibrosis vascular asociada a la enfermedad renal crónica / Contribution of uraemic toxins to the vascular fibrosis associated with chronic kidney disease

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-Beta1 y proteínas de MEC. Las toxinas a dosis altas y combinadas también indujeron expresión de TGF-Beta1 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

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 (Mig/ml) and indoxyl-sulphate 25-100 (Mig/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-Beta1 and ECM proteins. The toxins at high doses and combined also induced the expression of TGF-Beta1 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

Contribution of uraemic toxins to the vascular fibrosis associated with chronic kidney disease. / Contribución de las toxinas urémicas a la fibrosis vascular asociada a la enfermedad renal crónica.

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-ß1 and ECM proteins. The toxins at high doses and combined also induced the expression of TGF-ß1 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.

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iß pathway activation.

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras-/-) and control wild-type (H -ras+/+) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iß protein expression in H -ras-/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras-/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3ß-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iß overexpression in H -ras-/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iß overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iß pathway activation.

Integrin linked kinase regulates the transcription of AQP2 by NFATC3.

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/GSK3ß/NFATc/AQP2 axis, suggesting this pathway is a potential target for therapeutic treatment of NDI.

Peripheral insulin resistance in ILK-depleted mice by reduction of GLUT4 expression.

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 blocked in vivo and in 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.

Experimental and DFT characterization, antioxidant and anticancer activities of a Cu(II)-irbesartan complex: structure-antihypertensive activity relationships in Cu(II)-sartan complexes.

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. Experimental and DFT characterization of an irbesartan copper(II) complex has been performed. The complex exhibits low scavenging activity against DPPH(·) and significant growth inhibition of LNCaP and DU 145 prostate cancer cell lines. Flow cytometry determinations were used to determine late apoptotic mechanisms of cell death. This compound improved the antihypertensive effect of irbesartan. This effect was observed earlier for the mononuclear Cu-candesartan complex, but not in structurally modified sartans forming dinuclear or octanuclear Cu-sartan compounds.

Utilización de haloperidol en un paciente con síndrome de encefalomiopatía mitocondrial, acidosis láctica y episodios parecidos a ictus (síndrome MELAS) / Using haloperidol in a patient with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS síndrome)

El síndrome de MELAS (del inglés: mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) es una enfermedad de herencia materna caracterizada por una alteración en la cadena respiratoria mitocondrial. Además de la encefalopatía, la miopatía y los fenómenos que simulan ictus, entre sus síntomas también se encuentran manifestaciones psiquiátricas, sobre todo deterioro cognitivo, trastornos afectivos, psicosis y ansiedad. Debido a la escasa prevalencia de esta enfermedad, existen pocas referencias respecto al tratamiento de sus síntomas psiquiátricos. Muchos de los neurolépticos empleados en la práctica clínica habitual han demostrado toxicidad únicamente in vitro sobre la cadena respiratoria mitocondrial, por lo que su uso se desaconseja en estos pacientes. Presentamos un caso de un varón con diagnóstico de síndrome de MELAS mediante estudio genético que demostró la mutación A3243G de MELAS en el gen MT-TL1 del ADN mitocondrial. El paciente, además de las manifestaciones clásicas de la enfermedad, presentaba agitación psicomotriz, insomnio y alteraciones conductuales agudas con heteroagresividad, que, tras el ensayo de múltiples fármacos, únicamente lograron controlarse mediante la administración intravenosa de haloperidol, sin empeorar las manifestaciones neurológicas de la enfermedad. El presente caso evidencia que el empleo de haloperidol en el tratamiento agudo de las manifestaciones psiquiátricas de las enfermedades mitocondriales puede ser seguro y eficaz (AU)

Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome is a maternally inherited disease characterised by an anomaly in the mitochondrial respiratory chain complex. Apart from encephalopathy, myopathy and stroke-like episodes, these patients can also develop psychiatric symptoms such as dementia, affective disorders, psychosis, and anxiety phenomena. Because of the low prevalence of this syndrome, there are few references about the management of its psychiatric comorbidity. In mitochondrial diseases, neuroleptic agents are not recommended because they have demonstrated in vitro toxicity over the mitochondrial respiratory chain. The case is presented of a patient with a diagnosis of MELAS syndrome confirmed by the detection of a A3243G mutation in the MT-TL1 gene encoding part of the mitochondrial DNA. This patient did not only show the classic manifestations of the disease, but also presented with psychomotor agitation, insomnia and behavioural disturbances with aggressiveness. Several drugs were ineffective, but intravenous haloperidol induced remission without worsening of the neurological manifestations. This case suggests that haloperidol may be safe and effective for the acute control of psychiatric symptoms in mitochondrial syndromes (AU)

Effect of uraemia on endothelial cell damage is mediated by the integrin linked kinase pathway.

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.

Impacto de la implementación de la norma ISO 9001: 2008 en el proceso de cesión de muestras del biobanco Red de Investigación Renal española / Impact of implementing ISO 9001: 2008 standard on the Spanish Renal Research Network biobank sample transfer process

Antecedentes: La certificación de biobancos según la norma ISO 9001:2008 pretende mejorar la gestión de los procesos realizados en estos con dos objetivos: la satisfacción del cliente y la mejora continua. En este trabajo se presenta el impacto de la certificación ISO 9001:2008 sobre los procesos de cesión de muestras de un biobanco español especializado en muestras de pacientes renales y con un gran aumento del número de estas entre los años 2009 (12 582 viales) y 2010 (37 042 viales). Métodos: El biobanco de la Red de Investigación Renal española (REDinREN) situado en la Universidad de Alcalá ha puesto en marcha la norma ISO 9001:2008 para la gestión eficaz del material humano cedido a los centros de investigación. Se han analizado mediante encuestas dos períodos en el proceso «cesión de muestras». Durante el primer período, entre las fechas 1-10-12 y 26-11-12 (8 semanas), se han realizado cambios mínimos para corregir errores puntuales. En el segundo período, entre las fechas 7-01-13 y 18-02-13 (6 semanas), se han realizado acciones correctivas generales. Resultados: La identificación de inconvenientes y la puesta en marcha de acciones correctivas para la certificación permitieron: reducir el 70 % del tiempo de ejecución del proceso, aumentar significativamente (200 %) el número de muestras procesadas y mejorar un 25 % el proceso. El aumento del número de muestras procesadas estuvo directamente relacionado con la mejora del proceso. Conclusión: La certificación de la norma ISO 9001:2008, obtenida en julio de 2013, permitió la mejora de los procesos del biobanco REDinREN, aumentando la calidad y la satisfacción del cliente (AU)

Background: 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). Methods: The biobank of the Spanish Renal Research Network (REDinREN), located at the University of Alcalá, has implemented ISO standard 9001:2008 for the effective management of human material given to research centres. Using surveys, we analysed two periods in the "sample transfer" 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. Results: 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. Conclusion: 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 (AU)

Impact of implementing ISO 9001:2008 standard on the Spanish Renal Research Network biobank sample transfer process.

BACKGROUND: 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). METHODS: The biobank of the Spanish Renal Research Network (REDinREN), located at the University of Alcalá, has implemented ISO standard 9001:2008 for the effective management of human material given to research centres. Using surveys, we analysed two periods in the “sample transfer” 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. RESULTS: 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. CONCLUSION: 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.

Balance between apoptosis or survival induced by changes in extracellular-matrix composition in human mesangial cells: a key role for ILK-NFκB pathway.

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-3ß-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates ß1 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 ß1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3ß-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction.

Start-up of a clinical sample processing, storage and management platform: organisation and development of the REDinREN Biobank.

BACKGROUND: 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. METHOD: 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. RESULTS: 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. DISCUSSION: 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.

Puesta en marcha de una plataforma de proceso, almacenamiento y gestión de muestras clínicas: organización y desarrollo del Biobanco de REDinREN / Start-up of a clinical sample processing, storage and management platform: organisation and development of the Biobank of REDinREN

Antecedentes: La creación del Biobanco, como una plataforma dentro de la Red de Investigación Renal (REDinREN), impulsa el avance de la investigación clínica de la enfermedad renal en España. Los objetivos del Biobanco son la generación del archivo de muestras clínicas y de datos asociados, para su cesión a los grupos de investigación, y la coordinación con biobancos europeos. Métodos: Para su puesta en marcha, fue necesaria la implementación de la normativa vigente (Ley de Investigación Biomédica y de Protección de Datos y la Normativa del Transporte de Sustancias Biológicas), un estricto protocolo de trabajo y la creación de una base de datos clínicos de la Red. Resultados: En su evolución, el Biobanco ha adquirido infraestructura y personal cualificado, lo que permitió que en el año 2010 se obtuviera un total de 2.953 pacientes, lo que hace un total de 37.043 viales almacenados con muestras de diferentes naturalezas. Además, hasta la fecha, el Biobanco está incluido en 11 proyectos de investigación. Discusión: Aunque el Biobanco fue diseñado como una plataforma de soporte de la REDinREN, es necesario con una acción conjunta poner a disposición de toda la comunidad científica nefrológica las posibilidades que otorga este sistema de almacenamiento de muestras biológicas para potenciar la investigación de la enfermedad renal (AU)

Background: The Biobank creation in Network for the Kidney Research (REDinREN) promotes the advance of kidney disease clinic research 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. Methods: In the beginning were indispensable the implementation of in force normative (Bio-medic Investigation Law, Dates Protect Law and Biologic Samples Transport Normative), a work protocol and the creation of medical database of the Red. Results: The Biobank growth included infastructures and qualified personnel. In 2010 year, the patien samples increased in 2953 achieved 37,042 vials of differect nature stored. Mereover in this moment the Biobank participates in eleventh research projects. Discussion: Even if the Biobank was designed for the support to REDinREN is neccesary to opening his biologic samples to all scientific nephrology community for promote the kidney disease research (AU)

Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells.

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.

Fibronectin upregulates cGMP-dependent protein kinase type Iß through C/EBP transcription factor activation in contractile cells.

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 ß(1)-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.