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Integrin-linked kinase (ILK) has emerged as a controversial pseudokinase protein that plays a crucial role in the signaling process initiated by integrin-mediated signaling. However, ILK also exhibits a scaffolding protein function inside cells, controlling cytoskeletal dynamics, and has been related to non-neoplastic diseases such as chronic kidney disease (CKD). Although this protein always acts as a heterotrimeric complex bound to PINCH and parvin adaptor proteins, the role of parvin proteins is currently not well understood. Using in silico approaches for the design, we have generated and prepared a set of new tripeptides mimicking an alpha-parvin segment. These derivatives exhibit activity in phenotypic assays in an ILK-dependent manner without altering kinase activity, thus allowing the generation of new chemical probes and drug candidates with interesting ILK-modulating activities.

13 p.-9 fig.-3 tab., Protein tyrosine phosphatase 1B (PTP1B) is a very promising target for the treatment of metabolic disorders such as type II diabetes mellitus. Although it was validated as a promising target for this disease more than 30 years ago, as yet there is no drug in advanced clinical trials, and its biochemical mechanism and functions are still being studied. In the present study, based on our experience generating PTP1B inhibitors, we have developed and implemented a scaffold-hopping approach to vary the pyrrole ring of the pyrrolo[1,2-a]quinoxaline core, supported by extensive computational techniques aimed to explain the molecular interaction with PTP1B. Using a combination of docking, molecular dynamics and end-point free-energy calculations, we have rationally designed a hypothesis for new PTP1B inhibitors, supporting their recognition mechanism at a molecular level. After the design phase, we were able to easily synthesize proposed candidates and their evaluation against PTP1B was found to be in good concordance with our predictions. Moreover, the best candidates exhibited glucose uptake increments in cellulo model, thus confirming their utility for PTP1B inhibition and validating this approach for inhibitors design and molecules thus obtained., We are grateful for funding from the Spanish Ministerio de Economía y Competitividad (CTQ2017-85263-R MINECO/FEDER UE to J.J.V.); Instituto de Salud Carlos III and FEDER funds (RD16/0009/0015, RD16/0009/18, PI17/01513 and PI17/00625 to J.J.V., D.R.-P. and M.R.-P.). J. G−M was the recipient of an FPU fellowship (FPU16/01647) Spanish Ministerio de Educación y Cultura.

PTP1B dephosphorylates insulin receptor and substrates to modulate glucose metabolism. This enzyme is a validated therapeutic target for type 2 diabetes, but no current drug candidates have completed clinical trials. Pyrrolo[1,2-a]quinoxalines substituted at positions C1-C4 and/or C7-C8 were found to be nontoxic to cells and good inhibitors in the low- to sub-micromolar range, with the 4-benzyl derivative being the most potent inhibitor (0.24 μm). Some analogues bearing chlorine atoms at C7 and/or C8 kept potency and showed good selectivity compared to TCPTP (selectivity index >40). The most potent inhibitors behaved as insulin mimetics by increasing glucose uptake. The 4-benzyl derivative inhibited insulin receptor substrate 1 and AKT phosphorylation. Molecular docking and molecular dynamics simulations supported a putative binding mode for these compounds to the allosteric α3/α6/α7 pocket, but inconsistent results in enzyme inhibition kinetics were obtained due to the high tendency of these inhibitors to form stable aggregates. Computational calculations supported the druggability of inhibitors.

Background and Aims Hyperphosphatemia has been associated with aging and chronic kidney disease (CKD). Sarcopenia, which is a related condition of these pathologies, is defined by loss of force and muscular mass. During aging, chronic systemic inflammation appears, termed inflammaging, due to changes in immune system function. Inflammaging has been associated with many age-related diseases including Sarcopenia and CKD. The work aimed to evaluate the effect of hyperphosphatemia on proinflammatory profile of cultured myoblast cells and to analyze, in old mice, the effect of a dietary restriction in the phosphate intake on the aging-related sarcopenia. Methods Culture murine myoblast C2C12 cells were used for in vitro experiments. Cells were treated with 10 mM beta-glycerophosphate (BGP) as phosphate donor for 24, 48 or 72h. Inflammation was assessed through IL6, TNFα and MCP-1 expression by RT-qPCR. Twenty-four months old, C57BL6 mice were used for in vivo studies. Mice were fed with a normal diet containing 0.6% of phosphate until 21 months, after that, one group of mice continued with a normal diet and the other group was fed with a hypophosphatemic diet, containing a 0.2% of phosphate, for the following 3 months. Old mice were compared with 5 months old mice. Muscle force was measured by a grip strength test. Serum phosphate concentration was evaluated with a commercial kit and inflammation was assessed through IL-1β expression levels by RT-qPCR. Results Results showed that BGP treatment augmented pro-inflammatory cytokines levels, in myoblast, at 72h. On the other hand, old mice had a 40% increase in serum phosphate concentration regarding young mice, and, in parallel, they showed a reduction in forelimb strength. Old animals feeding with a hypophosphatemic diet showed a decreased level of phosphate serum linked to a better muscle function. Pro-inflammatory cytokines expression was higher in old mice compared to young mice; those values were reduced in 24-month-mice fed with a low phosphate diet. Furthermore, there was a positive correlation between IL-1β expression levels and serum phosphate levels, suggesting that high levels of serum phosphate were increasing inflammation in vivo and a negative correlation between IL-1β and grip strength test, which shows that high levels of inflammation decrease muscular function Conclusion In this work, we propose that high levels of phosphate are related to inflammation in vitro and in vivo. This increase of proinflammatory cytokines decreases muscle function whereas dietary restriction of phosphate decreases inflammation and improves muscle function. These results could point to a direct link between elevated serum phosphate levels and inflammaging presented in sarcopenic people such as CKD patients and aged people.

Background and Aims Insulin resistance (IR)-related metabolic disorders, such as diabetes type 2 and obesity, are linked to renal and cardiovascular risk. Unfortunately, they lack a better prognosis before their full establishment. An important mechanism associated to the IR development is the dysregulation of lipolysis in white adipose tissue (WAT), which is driven by the excessive lipid accumulation, together with inflammation and the excessive deposition of ECM components such as collagens and fibronectin, known as fibrosis. The intracellular lipid adjustment is affected by changes in the expression and activity of the hormone-sensitive lipase (HSL) and the intracellular and circulating balance of the lipolysis products glycerol and free fatty acids. We published that transgenic depletion of the scaffold intracellular protein Integrin-linked kinase (ILK), one of the adhesome translators of the ECM signals, modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017]. We also published that ILK downregulation in WAT is an early event in a short-term high fat diet (HFD) mice model that correlates with a faster insulin sensitivity loss, accompanied with an early increase of body weight and the erratic expression of WAT adipokines and metabolites transporters. [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Here we used in vivo and in vitro approaches to decode the role of ILK downregulation during WAT expansion and altered lipolysis. Method Adult mice with global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. We determined the weight changes on body and WAT depots, epidydimal (eWAT) and subcutaneous (scWAT), and the circulating lypolisis product glycerol. The expression of ECM component fibronectin (FN), fibrosis marker TGF-beta and the glycerol transporter AQP3 were determined in eWAT by RT-qPCR or Western blot. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). We determined the expression of ILK, total and active HSL isoforms (HSL, p-HSL s660) by RT-qPCR or Western blot and the intracellular fat droplet content (by adipo-red dye) Results HFD increased progressively CT eWAT, scWAT and whole body weight gains. HFD-fed cKD-ILK have an earlier increase of body and eWAT weights. However, the scWAT weight ratio against eWAT was inverted in the same cKD-ILK animals. This early depot-specific response to HFD coincide with increase in IR [Wajchenberg BL. Endocr Rev 2000]. HFD-fed cKD-ILK WAT early expressed altered levels of fibrotic markers (TGF-beta and Fibronectin). HFD-fed cKD-ILK shown a temporary increase of circulating glycerol and its WAT transporter AQP3. In cultured adipocytes with downregulated expression of ILK, the intracellular fat droplet was increased and the HSL contain and its lipolysis activity (measured as levels of p-HSL isoform) were reduced. Conclusion We suggest ILK as a modulator during the obesity establishment. Considering the observed relation between ILK expression with the altered lipolysis and fibrosis patterns observed in visceral WAT, ILK downregulation may be a predictive value and probably its pharmacological upregulation may be a novel therapeutic strategy against obesity and IR.

Background and Aims Important mechanism associated to the development of Insulin resistance (IR) are dysregulation of lipid adjustment and excessive deposition of extracellular matrix (ECM) in the visceral white adipose tissue (WAT). The transgenic depletion of the ECM-to-cell scaffold intracellular protein Integrin-linked kinase (ILK) modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017] and body weight gain and adipose malfunction correlates with early downregulation of ILK expression in WAT during a short-term high fat diet (HFD) mice model [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Thus, we demonstrated that transcriptional regulation of ILK might govern the expansion of adipocytes. Between several transcriptional factors implicated in lipid metabolism, the activation of the transcription factor peroxisome proliferator activated receptor (PPAR) family member PPARβ/δ ameliorates IR. Taking in consideration that it modulates the expression of ILK in other contexts aside metabolism [Di-Poï N et al. J Steroid Biochem Mol Biol. 2003, Zhu B et al. Oncogene. 2014], here we activated PPARβ/δ in vivo and in vitro to upregulate adipose ILK expression and prevent WAT expansion. Method Adult mice were fed with either standard (STD) or HFD diets during 4 weeks and divided into groups treated with PPARβ/δ activator L-165041 (i.p. 2 mg/kg b.w per day) or vehicle for 2 additional weeks with maintained diet challenges [Lim HJ et al. Eur J Pharmacol. 2009]. Body and epidydimal WAT depot (eWAT) weights changes were compared and ILK expression was determined by RT-qPCR. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). After cells were treated with L-165041 (1 microM, 24h), we determined the intracellular triglyceride content (by adipo-red dye), the expression of ILK and its downstream substrate AKT (total and active p-AKT in ser473) by RT-qPCR or Western blot. Results HFD increased progressively eWAT and whole body weight gains, which coincide with increase in IR. PPARβ/δ-activated mice under STD or HFD have preventive reduction of weight gains and increased expression of ILK in eWAT. Downregulated ILK expression in culture adipocytes coincide with increased triglyceride content, whereas the activation of PPARβ/δ reduced it at the same time that increased ILK expression and activity. Conclusion We suggest ILK modulation as a novel therapeutic strategy during the obesity establishment. The pharmacological activation of PPARβ/δ increases ILK expression and activity, which correlates with a weight loss in IR-based models in vivo. Although the increased presence of ILK induces the phosphorylation of AKT, in turn one of the main mediators during insulin signalling, we cannot conclude the role of ILK-mediated AKT modulation during the reduction of the adipocyte expansion observed

Background and Aims Patients with chronic kidney disease (CKD) present a high rate of cardiovascular mortality mainly associated with endothelial dysfunction, which causes more cardiovascular events in presence of atherosclerosis. Atherosclerosis is characterized by a significant increase of low density lipoproteins (LDL), reactive oxygen species (ROS) and inflammation. ROS can oxidize LDL generating oxidized-LDL (oxLDL) that promotes the development of cardiovascular pathologies. The aim of this study was to evaluate whether oxLDL induce endothelial dysfunction analysing the involvement of vascular fibrosis. Method The model used for in vivo studies was the Knockout apolipoprotein E (KO-apoE) mice, which resemble human atherosclerosis and shown high levels of cholesterol (LDL) that can be oxidized to oxLDL. In mice, blood pressure was registered before sacrificed them. After that, we measured different parameters as serum cholesterol levels, vascular function by vascular reactivity in mesenteric arteries and vascular fibrosis in aorta by Sirius Red staining and by the protein expression of fibronectin and collagen-I by immunohistochemistry. In order to investigate the mechanism of action of oxLDL, in vitro studios were performed on human smooth muscle cells (SMC) incubated with oxLDL at different times. Fibrosis was evaluated by the expression of TGF- β and extracellular matrix proteins such as fibronectin and collagen-I by Western blot and by immunofluorescence. ROS production was also measured by fluorescence confocal microscopy, using the CellROX Deep Red probe. Results KO-apoE mice shown higher levels of serum cholesterol and blood pressure than WT animals. Moreover, KO-apoE mice showed endothelial dysfunction since their arteries were less relaxed and more contracted. In addition, these mice presented thickening of vascular wall (SMC layer), more fibrosis showing intense Sirius Red staining and less expression of elastin, all compatible with their vascular dysfunction compared to WT mice. Furthermore, aortas from KO-apoE mice showed a slight increase in fibronectin and collagen-I expression assessed by immunohistochemistry. In vivo studies were confirmed in vitro after treating SMC with oxLDL. oxLDL induced fibrosis in SMC by increasing TGF-β, fibronectin and collagen-I protein expressions evaluated by Western blot and immunofluorescence assays. Treatment with oxLDL also increased ROS production, which seem to be responsible of oxLDL-induced fibrosis in human SMC, as it was blocked in the presence of the antioxidant N-Acetyl-cysteine. Conclusion In summary, these results point to endothelial dysfunction associated to atherosclerosis (oxLDL) could be mediated by an increase in the development of vascular fibrosis where ROS could play an important role. Therefore, the endothelial dysfunction typical of CKD patients could impair with more vascular fibrosis when atherosclerosis is also present.

Background and Aims The loss of muscle mass and function has been related to chronic kidney disease (CKD). About 37% of dialysis patients show symptoms of sarcopenia and this has been related to an increased risk of mortality. Changes in sarcopenic muscle include the loss of its regenerative capacity due to a reduction in the number and function of satellite cells, the muscle stem cells. The concentration of serum uremic toxins (UT) increases in parallel to a decline in the glomerular filtration rate in patients with CKD and this uremia may be involved in the development of sarcopenia. Previous studies showed as serum concentration of UT found in the early stages of CKD inhibits myogenic differentiation of cultured myoblasts. Nevertheless, the effect of those concentrations found in the advanced stages of CKD has not been described. The study aimed to analyse whether UT affect the muscular regeneration process by modifying the proliferation capacity of myoblasts (activated satellite cells). Method Cultured mouse myoblasts C2C12 cells were used for all experiments. Cells were grown with 0% or 10% FBS culture media in the presence or absence of indoxyl sulphate and para-cresol at doses of 100µg/ml each one, which are similar to ones found in the advanced stages of CKD. Proliferation was evaluated by scratch wound healing and cell cycle by flow cytometry with propidium iodide and the fluorescent probe CFSE, an intracellular protein binding dye that is divided equally between daughter cells, allowing the discrimination of successive rounds of cell division. Chromosome condensation was assessed by immunofluorescence staining by confocal microscopy. Apoptosis was analysed by annexin V staining. Results C2C12 cells treated with UT shown a significant decrease in the proliferation rate. A significant delay in wound closure was observed in cells treated with UT compared to control cells. Myoblasts treated with UT suffered a significant decrease in the proliferation rate since the probe remained higher than in the vehicle-treated cells. Proliferating cells treated with UT suffered a dramatic cell cycle arrest between the phases S and G2/M. Chromosome condensation was also analysed, finding that in the presence of colcemid, vehicle-treated cells condensed their chromosomes, as expected, whereas UT-treated cells did not, suggesting that UT stop the cell cycle at any point before the entry of cells in the mitosis phase. Besides, there was strong phosphorylation of cdc2 in the presence of UT indicating that cdc2 and the complex cdc2-cyclin B were inactive. This result explains why cells did not enter in the mitosis phase under UT exposition. Finally, UT induced the death of proliferating C2C12 cells by apoptosis. Conclusion In the advanced stages of CKD, uremic toxins concentration increases, thereby inducing a dramatic arrest in the cell cycle of myoblasts, inactivating the cdc2-cyclin B complex, interrupting their proliferation and leading them towards cell apoptosis. These results point to a role of uremic toxins impairing the skeletal muscle regeneration process, which could be involved in CKD-related sarcopenia and frailty.

Background and Aims Calpains are intracellular cysteine proteases that play a critical role in cell remodeling, being involved in multiple biological processes linked to tissue damage and repair mechanisms. In addition, they are released into the circulation, being able to carry out systemic actions with pathological consequences. The aim of this study was to investigate the role of calpains in the progression of chronic kidney disease (CKD) in an experimental model of chronic renal damage induced by adenine. Method We induced an experimental model of CKD, in mice fed for 2 weeks with an adenine-supplemented diet (0.2% adenine) (A). Animals receiving this diet develop a tubulointerstitial damage resembling that is observed in human CKD. Mice with standard diet were used as controls (C). Renal function was assessed by measuring serum blood urea nitrogen (BUN) and creatinine (mg/dl). Fibrosis markers (collagen type I and fibronectin) were determined by RT-qPCR. Changes in the renal content of calpains 1 and 2 were analyzed by western blot (protein content), and RT-qPCR (mRNA expression). Results Our results show functional and structural changes at renal level in the adenine-fed mice, with increased BUN (A: 72 mg/dl, C: 28 mg/dl, p < 0.05), creatinine (A: 0.58 mg/dl, C: 0.25 mg/dl, p < 0.05), collagen type I mRNA expression (A: 12.9 units, C: 1.2 units, p < 0.05) and fibronectin mRNA expression (A: 3.46 units, C: 1.3 units, p < 0.05). Furthermore, protein content of calpains 1 (A: 1.27 units, C: 0.78 units, p < 0.05) and 2 (A: 1.30 units, C: 0.66 units, p < 0.05) was significantly higher in adenine-fed mice when compared to control. At the same time, we observed a significant increase in gene expression of both calpain 1 (A: 4.21 units, C: 0.51 units, p < 0.05) and 2 (A: 4.93 units, C: 0.56 units, p < 0.05) in the adenine model regarding to mice with standard diet. Our results demonstrate that calpain 1 and 2 expression in renal tissue increases as CKD progresses. Interestingly, we found statistically significant correlations between renal calpains 1 and 2 protein and mRNA content and plasma BUN and creatinine (p < 0.05, r between 0.79 and 0.92), as well as protein expression of calpain 2 and mRNA expression of collagen type I (p < 0.05, r = 0.76). These data suggest a potential direct relationship between renal calpain 1 and 2 content and loss of renal function, in part due probably to the modulation of the fibrotic changes, in adenine fed mice. Conclusion We suggest an implication of calpains 1 and 2 in the development of CKD. Thus, effective calpain blockade or downregulation could be useful as a therapeutic strategy to prevent CKD. Further experiments will be necessary to establish the relationship between these factors.

Background/Aims: Diabetes type 2, metabolic syndrome or non-alcoholic fatty liver disease are insulin resistance-related metabolic disorders, which lack a better prognosis before their full establishment. We studied the importance of the intracellular scaffold protein integrin linked kinaes (ILK) as a key modulator in the initial pathogenesis and the early progression of those insulin resistance- related disorders. Methods: Adult mice with a global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. Weights, blood glucose and other systemic biochemical parameters were determined in animals under fasting conditions and after glucose or pyruvate intraperitoneal injections to test their tolerance. In RNA or proteins extracted from insulin-sensitive tissues, we determined by reverse transcription?quantitative PCR and western blot the expression of ILK, metabolites transporters and other metabolism and inflammatory markers. Glucose uptake capacity was studied in freshly isolated tissues. Results: HFD feeding was able to early and progressively increase glycaemia, insulinemia, circulating glycerol, body weight gain, liver-mediated gluconeogenesis along this time lapse, but cKD-ILK have all these systemic misbalances exacerbated compared to CT in the same HFD time lapse. Interestingly, the tisular expression of ILK in HFD-fed CT was dramatically downregulated in white adipose tissue (WAT), skeletal muscle and liver at the same extent of the original ILK downregulation of cKD-ILK. We previously published that basal STD-fed cKD-ILK compared to basal STD-CT have different expression of glucose transporters GLUT4 in WAT and skeletal muscle. In the same STD-fed cKD-ILK, we observed here the increased expressions of hepatic GLUT2 and WAT pro-inflammatory cytokines TNF-? and MCP-1. The administration of HFD exacerbated the expression changes in cKD-ILK of these and other markers related to the imbalanced metabolism observed, such as WAT lipolysis (HSL), hepatic gluconeogenesis (PCK-1) and glycerol transport (AQP9). Conclusion: ILK expression may be taken as a predictive determinant of metabolic disorders establishment, because its downregulation seems to correlate with the early imbalance of glucose and glycerol transport and the subsequent loss of systemic homeostasis of these metabolites., Instituto de Salud Carlos III-ISCIII, Comunidad de Madrid, Fondo Europeo de Desarrollo Regional-FEDER, Instituto Ramon y Cajal de Investigación Sanitária-IRYCIS, Fundación Renal Iñigo Álvarez de Toledo-FRIAT

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

A series of new azolopyrimidine-peptide hybrids and indolomethylideneimidazolones were obtained and evaluated as calpain inhibitors. The hybrid compounds were inactive, whereas some members of the initial azolomethylideneimidazolone series showed interesting calpain inhibitory activity. By using 4b as a hit compound, a new series of analogs were synthesized by an efficient synthetic procedure based on a multicomponent reaction followed by an unprecedented reaction at the methylene position of the molecule. The best inhibitor found for calpain I (IC50 = 20 nM) was about 20 times more potent than the hit compound. Studies on 4b showed that its inhibition is consistent with an uncompetitive inhibition mode. This compound did not exhibit cellular toxicity at any of the doses tested (0.1–10 μM) and further studies indicated that it was capable of blockading chemical ischemia induction of apoptosis by preventing sodium azide-dependent calpain activation in intact human kidney tubular epithelial cells. The results of molecular modeling studies rationalized the inhibitory activity found for this series and account, from a structural point of view, for the most active compound identified (4j).

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.

Aim To analyse the ability of TWEAK to modify the endothelin system, particularly endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1), studying the intracellular mechanisms implied. TNF-like weak inducer of apoptosis (TWEAK) is a member of TNF superfamily; it has different biological functions such as inflammation, angiogenesis, proliferation, and apoptosis. TWEAK and fibroblast growth-factor-inducible 14 are expressed in different cell types, including endothelial and smooth muscle cells. Despite their presence in endothelial cells, the effect of TWEAK on endothelial function is incompletely defined. Methods and results In cells, TWEAK induced protein (Western blot) and mRNA (quantitative polymerase chain reaction) expression of ECE-1. Results were related to transcriptional changes, as ECE-1 promoter activity (transfection assays) was also increased. Transfections with serial deletions of ECE-1 promoter suggest a potential role for AP-1 and NFkB, which were confirmed by electrophoretic mobility shift assays. When AP-1 or NFkB activations were inhibited by specific inhibitors of AP-1, PD-98059 (Erk1/2 inhibitor), or SP-600125 (JNK inhibitor), and also with an inhibitor of NFKB and PDTC, TWEAK effect was partially blocked in both cases, suggesting that both transcription factors are implied in ECE-1 regulation. Moreover, the endothelial changes induced by TWEAK were also tested in vivo , using 3-month-old male CD-1 mice treated with TWEAK 10 µg/kg body weight for 24 h, finding similar effects, a rise in ET-1 production (enzyme-linked immunosorbent assay), and ECE-1 expression in aorta and lung tissues. Mice showed slight hypertension after 4 h of treatment, which disappeared at 24 h. Conclusions In pathological situations such as chronic inflammation, TWEAK could be more harmful through this effect at endothelial level. Pharmacological blockade of this cytokine could prevent the haemodynamic and structural changes related to an increased ET-1 synthesis.

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.

Despite the presence of vitamin D receptor (VDR) in endothelial cells, the effect of vitamin D on endothelial function is unknown. An unbalanced production of vasoactive endothelial factors such as nitric oxide (NO) or endothelin-1 (ET-1) results in endothelial dysfunction, which can alter the normal cardiovascular function. Present experiments were devoted to assess the effect of active vitamin D (calcitriol) on the synthesis of endothelial vasoactive factors. The results were that, in cells, calcitriol increased ET-1 and NO productions, which were measured by ELISA and fluorimetric assay, respectively. Calcitriol also increased endothelin-converting enzyme-1 (ECE-1) and endothelial-nitric oxide synthase (eNOS) activities, their mRNA (qPCR), their protein expressions (Western-blot), and their promoter activities (transfection assays). Calcitriol did not change prepro-ET-1 mRNA. The effect was specific to VDR activation because when VDR was silenced by siRNA, the observed effects disappeared. Mechanisms involved in each upregulation differed. ECE-1 upregulation depended on AP-1 activation, whereas eNOS upregulation depended directly on VDR activation. To evaluate the in vivo consequences of acute calcitriol treatment, normal Wistar rats were treated with a single ip injection of 400 ng/kg calcitriol and euthanized 24 h later. Results confirmed those observed in cells, that production and expression of both factors were increased by calcitriol. Besides, calcitriol-treated rats showed a slight rise in mean blood pressure, which decreased when pretreated with FR-901533, an ECE-1 antagonist. We conclude that calcitriol increases the synthesis of both ET-1 and NO in endothelial cells. However, the ET-1 upregulation seems to be biologically more relevant, as animals acutely treated with calcitriol show slight increases in blood pressure.

Hyperglycemia is involved in the diabetic complication of different organs and can elevate serum osmolarity. Here, we tested whether hyperosmolarity promoted by high glucose levels induces cellular senescence in renal cells. We treated Wistar rats with streptozotocin to induce diabetes or with consecutive daily injections of mannitol to increase serum osmolarity and analyzed p53 and p16 genes in renal cortex by immunohistochemistry. Both diabetic and mannitol treated rats showed a significant increase in serum osmolarity, without significant signs of renal dysfunction, but associated with increased staining for p53 and p16 in the renal cortex. An increase in p53 and p16 expression was also found in renal cortex slices and glomeruli isolated from healthy rats, which were later treated with 30 mM glucose or mannitol. Intracellular mechanisms involved were analyzed in cultured human glomerular mesangial cells treated with 30 mM glucose or mannitol. After treatments, cells showed increased p53, p21 and p16 expression and elevated senescence-associated β-galactosidase activity. Senescence was prevented when myo-inositol was added before treatment. High glucose or mannitol induced constitutive activation of Ras and ERK pathways which, in turn, were activated by oxidative stress. In summary, hyperosmolarity induced renal senescence, particularly in glomerular mesangial cells, increasing oxidative stress, which constitutively activated Ras-ERK 1/2 pathway. Cellular senescence could contribute to the organ dysfunction associated with diabetes.

Under a Creative Commons license.-- et al., Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-α trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs., This work has been supported by grants from the Burton Myeloma Programme (YC), Ministerio Español de Ciencia e Innovación (BFU2010-21374/BMC (IMA)), the Medical Research Council (G1100041, GEJ, IMA) Fundación Científica de la Asociación Española Contra el Cáncer (AECC) (EM-V and IB-R) and the Wellcome Trust (080373) (YC, MPB, AJT and GEJ).

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular-to-intracellular scaffold protein integrin-linked kinase (ILK) regulates expression of tubular water channel aquaporin-2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional-knockdown (cKD-ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine-vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD-ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD-ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI.

Activation of the insulin growth factor receptor-1 signaling pathways has been largely related to the aging process. Amadori products are produced in pathological conditions such as diabetes and aging, and are potentially involved in diabetic nephropathy or age-associated decline of renal function. We hypothesize that Amadori products induce senescence in primary human mesangial cells through the activation of IGF-1 receptor and investigate, in the present work, the intracellular mechanism involved after this activation. We treated cultured human mesangial cells with glycated albumin, one of the most abundant Amadori product, and senescence was assessed by determining the senescence associated β-galactosidase activity and the expression of the cell cycle regulators p53 and p21. We demonstrated that prolonged exposition (more than 24h) to glycated albumin induced senescence and, in parallel, incremented the release of IGF-1 and the activation of the IGF-1 receptor. Inhibition of the IGF-1 activation prevented the GA induced senescence. Activation of IGF-1R, after GA addition, promoted a reduction in the catalase content through the constitutive activation of Ras and erk1/2 proteins which were, in turn, responsible of the observed GA-induced senescence. In conclusion, we propose that the Amadori product, glycated albumin, promotes premature cell senescence in mesangial cells through the activation of the IGF-1 receptor and the subsequent reduction in the antioxidant enzyme catalase.

Aims Integrin-linked kinase (ILK) regulates proliferation, differentiation, cell adhesion, and motility in many cell types and has been related to cancer progression, fibrosis, and vascular diseases. We designed the present study to directly explore the effect of ILK deletion on the regulation of vascular tone through the soluble guanylate cyclase (sGC) /protein kinase G (PKG) pathway in healthy adult mice. Methods and results Experiments were carried out using a tamoxifen-inducible CRE-LOX system to conditionally delete the ILK gene in adult mice. Mice lacking ILK expression (cKO) presented increased vascular content and increased activity of sGC and PKG, resulting in a more intense vasodilatory response to a single dose of a nitric oxide (NO) donor [sodium nitroprusside (SNP)] or PKG agonist [8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt (8-Br)]. Five minutes after SNP or 8-Br administration the reduction in the systolic arterial pressure was enhanced in cKO mice (SNP WT: −7.4 ± 1.2 mmHG; SNP cKO: −14.0 ± 2.5; 8-Br WT: −2.9 ± 1.5 mmHG; 8-Br cKO: −10.0 ± 3.4 mmHG). ILK deletion restored the vascular response to SNP after chronic oral nitrite administration. In addition, ILK deletion also increased hypotensive SNP effect in angiotensin II-treated animals, suggesting a role for ILK in basal and pathological states. Conclusion Deletion of ILK in adult animals increased the vascular response to NO. These findings show, for the first time, a requirement for ILK in regulating sGC–PKG expression in vivo .

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

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and produces cGMP, which activates cGMP-dependent protein kinases (PKG) and is hydrolyzed by specific phosphodiesterases (PDE). The vasodilatory and cytoprotective capacity of cGMP-axis activation results in a therapeutic strategy for several pathologies. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix and intracellular signaling pathways, may modulate the expression and functionality of the cGMP-axis–related proteins. We introduce ILK as a novel modulator in renal homeostasis as well as a potential target for cisplatin (CIS)-induced acute kidney injury (AKI) improvement. We used an adult mice model of depletion of ILK (cKD-ILK), which showed basal increase of sGC and PKG expressions and activities in renal cortex when compared with wildtype (WT) littermates. Twenty-four h activation of sGC activation with NO enhanced the filtration rate in cKD-ILK. During AKI, cKD-ILK maintained the cGMP-axis upregulation with consequent filtration rates enhancement and ameliorated CIS-dependent tubular epithelial-to-mesenchymal transition and inflammation and markers. To emphasize the role of cGMP-axis upregulation due to ILK depletion, we modulated the cGMP axis under AKI in vivo and in renal cultured cells. A suboptimal dose of the PDE inhibitor ZAP enhanced the beneficial effects of the ILK depletion in AKI mice. On the other hand, CIS increased contractility-related events in cultured glomerular mesangial cells and necrosis rates in cultured tubular cells; ILK depletion protected the cells while sGC blockade with ODQ fully recovered the damage.

Rationale: Atherosclerotic lesions develop in regions of disturbed flow, whereas laminar flow protects from atherogenesis; however, the mechanisms involved are not completely elucidated. Integrins are mechanosensors of shear stress in endothelial cells, and integrin-linked kinase (ILK) is important for blood vessel integrity and cardiovascular development. Objectives: To explore the role of ILK in vascular function by studying conditionally ILK-deficient (cKO) mice and human atherosclerotic arteries. Results: ILK expression was detected in the endothelial cell layer of nonatherosclerotic vessels but was absent from the endothelium of atherosclerotic arteries. Live ultrasound imaging revealed that acetylcholine-mediated vasodilatation was impaired in cKO mice. These mice exhibited lowered agonist-induced nitric oxide synthase (NOS) activity and decreased cyclic guanosine monophosphate and nitrite production. ILK deletion caused endothelial NOS (eNOS) uncoupling, reflected in reduced tetrahydrobiopterin (BH4) levels, increased BH2 levels, decreased dihydrofolate reductase expression, and increased eNOS-dependent generation of superoxide accompanied by extensive vascular protein nitration. ILK reexpression prevented eNOS uncoupling in cKO cells, whereas superoxide formation was unaffected by ILK depletion in eNOS-KO cells, indicating eNOS as a primary source of superoxide anion. eNOS and ILK coimmunoprecipitated in aortic lysates from control animals, and eNOS-ILK–shock protein 90 interaction was detected in human normal mammary arteries but was absent from human atherosclerotic carotid arteries. eNOS-ILK interaction in endothelial cells was prevented by geldanamycin, suggesting heat shock protein 90 as a binding partner. Conclusions: Our results identify ILK as a regulatory partner of eNOS in vivo that prevents eNOS uncoupling, and suggest ILK as a therapeutic target for prevention of endothelial dysfunction related to shear stress–induced vascular diseases.

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.

The aim of the present experiments was to evaluate the differences in arterial pressure between H-Ras lacking mice and control mice and to analyze the mechanisms involved in the genesis of the differences. H-Ras lacking mice and mouse embryonic fibroblasts from these animals were used. Blood pressure was measured using 3 different methods: direct intraarterial measurement in anesthetized animals, tail-cuff sphygmomanometer, and radiotelemetry. H-Ras lacking mice showed lower blood pressure than control animals. Moreover, the aorta protein content of endothelial nitric oxide synthase, soluble guanylyl cyclase, and cyclic guanosine monophosphate–dependent protein kinase was higher in H-Ras knockout mice than in control animals. The activity of these enzymes was increased, because urinary nitrite excretion, sodium nitroprusside–stimulated vascular cyclic guanosine monophosphate synthesis, and phosphorylated vasoactive-stimulated phosphoprotein in aortic tissue increased in these animals. Furthermore, mouse embryonic fibroblasts from H-Ras lacking mice showed higher cyclic guanosine monophosphate–dependent protein kinase promoter activity than control cells. These results strongly support the upregulation of the nitric oxide-cyclic guanosine monophosphate pathway in H-Ras–deficient mice. Moreover, they suggest that H-Ras pathway could be considered as a therapeutic target for hypertension treatment.

The aim of our study was to analyze the relationships between atherosclerosis and endothelin-converting enzyme-1 (ECE-1). Four-week-old C57BL/6J [wild-type (WT)] and apolipoprotein E-deficient (apoE) mice were fed with a standard or Western-type fat diet for 8 wks. ApoE showed atherosclerotic lesions in the aorta, higher blood pressure and vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein content than WT. ApoE showed a significant increase in ECE-1 protein content and mRNA expression in aorta, lung, and kidney, without changes in heart. When an ECE-1 inhibitor, FR-901533, was administered to them, blood pressure decreased in apoE on fat diet versus apoE on normal diet and WT. ECE-1 and LOX-1 protein content were elevated in peripheral blood mononuclear cells (PBMC) from hypercholesterolemic patients. In order to study the mechanism involved in this ECE-1 up-regulation, bovine aortic endothelial cells (BAEC) were treated with oxidized-low density lipoproteins (oxLDL). OxLDL, but not LDL, increased ECE-1 protein content, mRNA expression and promoter activity. Our results demonstrate that ECE-1 increases in different atherosclerosis situations. Up-regulation of ECE-1 could contribute, at least partially, to the development of hypertension seen in apoE mice, because FR-901533 avoided it. Probably, atherosclerotic situations course with an increase of oxLDL, which is able to induce ECE-1 expression with the subsequent potential pathological effects.

Telomere shortening and redox imbalance have been related to the aging process. We used cultured mouse embryonic fibroblasts (MEF) isolated from mice lacking telomerase activity (Terc(-/-)) to analyze the redox balance and the functional consequences promoted by telomerase deficiency. Comparison with wild-type (WT) MEF showed that Terc(-/-) MEF had greater oxidant damage, showing higher superoxide anion and hydrogen peroxide production and lower catalase activity. Restoration of telomerase activity in Terc(-/-) MEF increased catalase expression and activity. TGF-beta1 and collagen type IV levels were higher in Terc(-/-) than in WT MEF. TGF-beta1 promoter activity decreased when Terc(-/-) MEF were incubated with exogenous catalase, suggesting that catalase deficiency is the cause of the TGF-beta1 increase. Similar results were obtained in vivo. Homogenized renal cortex from 6-month-old Terc(-/-) showed higher oxidant capacity, lower catalase activity, greater oxidative damage, and higher TGF-beta1 and fibronectin levels than that from WT mice. In summary, telomerase deficiency reduces catalase activity, determining a redox imbalance that promotes overexpression of TGF-beta1 and extracellular matrix proteins.

While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.

Chronic activation of the acute phase response (APR) is associated with atherosclerosis. Elevated levels of interleukin-6, the major inducer of the APR, are associated with an increased risk of cardiovascular events. One of the clinical hallmarks of atherogenesis is endothelial dysfunction, characterized by a decrease in endothelial production of nitric oxide (NO). We hypothesized that interleukin-6 (IL-6) decreases endothelial NO synthase (eNOS) expression. We now show that IL-6 treatment of human aortic endothelial cells (HAEC) decreases steady-state levels of human eNOS mRNA and protein. This decrease in eNOS expression is caused in part by IL-6 inhibition of transactivation of the human eNOS promoter. To explore the mechanism by which IL-6 affects eNOS expression, we examined activation of signal transducer and transactivator-3 (Stat3). The IL-6 receptor (IL-6R) is expressed in HAEC, and Stat3 is phosphorylated in response to IL-6 stimulation of the IL-6R. We identified four consensus sequences for Stat3 binding (SIE) in the eNOS promoter at positions -1520, -1024, -840, and -540. Transfection of eNOS promoter mutants revealed that the SIE at -1024 mediates Stat3 inhibition of eNOS promoter activity. Gel-shift analysis of nuclear extracts from HAEC treated with IL-6 confirms that Stat3 binds to a complex containing the SIE at -1024. RNA silencing of STAT3 blocks the inhibitory effect of IL-6 on eNOS expression. Our data show that IL-6 has direct effects upon endothelial cells, inhibiting eNOS expression in part through Stat3. Decreased levels of eNOS may be an important component of the pro-atherogenic effect of the APR.

Background: Endothelium is supported, in normal conditions, by a basement membrane composed, among others, by collagen IV and laminin. Changes in the basement membrane composition could induce changes in endothelial cell modifying their interactions with leukocytes. Methods and Results: Isolated polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) were added to cultured human umbilical endothelial cells (HuVEC) previously seeded on collagen IV, collagen I or gelatin. Adhesion of leukocytes to HUVEC and specific cytotoxicity were analysed. PMN adhesion and cytotoxicity were lower whereas those from PBMC were higher when HuVEC were seeded on collagen I, as compared with cells seeded on collagen IV. To analyse the mechanisms involved in these phenomena, P-selectin, ICAM-1, VCAM-1 and MCP- 1 expression were evaluated in HuVEC seeded on the different ECM components. P-selectin and mRNA expression of VCAM-1 were lower in cells seeded on collagen I. By contrast, MCP-1 expression was higher in collagen I. Collagen I-dependent effects were partially prevented when collagen I was treated with pepsin. ILK activity was lower in cells seeded on collagen I, whereas ERK 1/2 activity was enhanced. ILK overexpression reduced ERK 1/2 phosphorylation and this could promote the reduction in P-selectin and the increase in MCP-1. Conclusion: Collagen I decreased ILK activity and this would induce an increase in ERK 1/2 activity in HuVEC. As a consequence, the P-selectin content is diminished and, by contrast, the MCP-1 content is increased. The final effect is a lower recruitment of PMN and a higher adhesion of PBMC.

Glomerular diseases are characterized by an abnormal synthesis of extracellular matrix proteins, such as collagen type I. Evidence that growth on collagen type I downregulates soluble guanylyl cyclase (sGC) expression and the responsiveness of human mesangial cells to nitric oxide (NO) by activating specific integrin signals involving integrin-linked kinase (ILK) is presented. Human mesangial cells were grown on collagen type I or IV for 24 to 72 h. Compared with collagen IV, growth on collagen I reduced the protein expression and NO-stimulated enzyme activity of sGC. This downregulation was effected at the level of transcription, because steady-state sGC mRNA expression was reduced on collagen I, but inhibition of transcription with Actinomycin D revealed no differences in transcript stability between the two culture conditions. Collagen I also reduced the capacity of cells to relax in response to NO after H2O2 challenge and inhibited NO-induced phosphorylation of vasodilator-activated phosphoprotein, a target of cyclic guanine monophosphate-dependent protein kinase. Examination of the surface expression of integrins, the receptors for extracellular matrix components, revealed that alpha1 and alpha2 integrin subunits were more abundant on cells that were grown on collagen IV and that surface expression of beta1 integrin did not vary with collagen type. However, growth on collagen I induced beta1 integrin to adopt its active conformation, and this activation of beta1 integrin was accompanied by increased activity of its downstream effector ILK. Dominant-negative suppression of ILK signaling relieved the suppression of sGC expression and NO-induced vasodilator-activated phosphoprotein phosphorylation induced by collagen I.

Progressive renal diseases are characterized by an increased synthesis of extracellular matrix (ECM) components. The mechanisms involved in the development of these alterations are not completely known, but a crucial role for TGF-beta 1 has been suggested. Moreover, the ability of the ECM to modulate the phenotypic expression of different cell types has been widely described. In experiments presented here, human mesangial cells (HMC) were grown on collagen type I (COL I) or IV (COL IV). ECM protein and TGF-beta 1 mRNA expression were evaluated by Northern blot analysis, and TGF-beta 1 secretion was evaluated by ELISA. The involvement of tyrosine kinase and serine-threonine kinase pathways was studied by Western blot analysis, immunofluorescence, and in vitro kinase assays. HMC cultured on COL I showed an increased mRNA expression of COL I and COL IV, fibronectin, and TGF-beta 1. Both tyrosine phosphorylation and integrin-linked kinase (ILK) activity increased when HMC were cultured on COL I, and blockade of these pathways inhibited the increased secretion of TGF-beta 1. In conclusion, the present results support a role for extracellular COL I in the regulation of TGF-beta 1 synthesis during progressive renal sclerosis and fibrosis and the subsequent increase in newly synthesized ECM proteins. In addition, ILK, along with the tyrosine kinases, participates in the genesis of this effect.

Extracellular matrix (ECM) components, through specific peptide motifs such as Arg-Gly-Asp (RGD), interact with integrins and can modify the behavior of cells. Transforming growth factor-beta1 (TGF-beta1) is the main cytokine involved in the synthesis of ECM proteins. We analyzed the effect of a RGD-containing peptide, as Arg-Gly-Asp-Ser (RGDS), on the regulation of TGF-beta1 secretion in cultured human mesangial cells. We found that RGDS increased mRNA expression and secretion of TGF-beta1 by stimulating the TGF-beta1 gene promoter. This effect was dependent on the interaction of RGDS with integrins. We evaluated the signaling pathways implicated in TGF-beta1 production by analyzing the effect of RGDS on kinase-related integrins. RGDS stimulated tyrosine phosphorylation as well as integrin-linked kinase (ILK) activity. However, tyrosine kinase inhibitors did not prevent the RGDS effect. In contrast, the inhibition of ILK by cell transfection with a kinase dead-ILK completely abolished the increased TGF-beta1 secretion and promoter activity in the presence of RGDS. Thus RGDS modulates the secretion of TGF-beta1, probably through increased synthesis by interacting with integrins and activating ILK. This supports a role for ECM components in the regulation of their own secretion.