Identificación de nuevos mecanismos patogénicos de la inflamación renal por inhibidores de calcineurina

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología. Fecha de lectura: 26-06-2015La inflamación es un importante factor patogénico de la enfermedad renal y como tal constituye un potencial objetivo terapéutico. En este trabajo de tesis nos propusimos caracterizar los mecanismos moleculares de la nefrotoxicidad de los inhibidores de calcineurina. Los inhibidores de calcineurina son unos fármacos inmunosupresores ampliamente utilizados en diversos contextos clínicos cuya toxicidad limitante de dosis es renal. En concreto hipotetizamos que la inducción de una respuesta inflamatoria puede contribuir a inducir o amplificar el daño renal. Nos propusimos identificar mediadores moleculares y mecanismos de la inflamación renal producida por los inhibidores de calcineurina para desarrollas nuevas aproximaciones terapéuticas. En células cultivadas hemos observado que tanto ciclosporina como tacrolimus inducen la expresión de genes que codifican citoquinas proinflamatorias y moléculas de adhesión en células tubulares renales y células endoteliales. El efecto proinflamatorio de los inhibidores de calcineurina está mediado por la receptor de inmunidad innata TLR4 y el reclutamiento de las vías de señalización intracelular IκBα/NF-κB, JNK y JAK/STAT. El adaptador MyD88 es necesario para la señalización de TLR4. La represión de la expresión de calcineurina mediante silenciamiento génico limitó la señalización por la ruta de JNK y por la rama adaptada por TRIF en la señalización por TLR4. Los efectos proinflamatorios tempranos de los inhibidores de calcineurina son reproducibles in vivo, en ratones a los que se les administró ciclosporina. El tratamiento de estos animales con un inhibidor farmacológico de TLR4 redujo la respuesta inflamatoria y el daño tubular. En conclusión, los inhibidores de calcineurina desencadenan una respuesta inflamatoria en células renales y activación endotelial a través del reclutamiento de diversas rutas de señalización dependientes del receptor de inmunidad innata TLR4. TLR4 es una posible diana terapéutica para prevenir los efectos nefrotóxicos de estos fármacosInflammation is an important pathogenic factor in kidney disease and constitutes a potential therapeutic target. In this thesis work, we focused on the characterization of the molecular mechanisms of nephrotoxicity induced by calcineurin inhibitors. Calcineurin inhibitors are immunesuppressant drugs whose dose limiting factor is nephrotoxicity. We hypothesized that induction of an inflammatory response may contribute to promote or amplify nephrotoxic kidney injury. We aimed to identify molecular mediators and characterize the molecular mechanisms of kidney inflammation in response to calcineurin inhibitors in order to develop novel therapeutic strategies. In cultured cells we have observed that both cyclosporine and tacrolimus induce the expression of genes encoding proinflammatory cytokines and adhesion molecules in kidney tubular and endothelial cells. The proinflammatory effect of calcineurin inhibitors requires the activation of the innate immunity receptor TLR4 and TLR4-dependent signallign pathways including IκBα/NF-κB, JNK and JAK/STAT. Silencing of the MyD88 adaptor protein via specific siRNA confirms the requirement for MyD88. Silencing calcineurin partially inhibited the inflammatory response and signaling through JNK and the TRIF branch of the TLR4 pathway. We confirmed the early proinflammatory effects of calcineurin inhibitors in vivo in kidneys of mice treated with cyclosporine. A pharmacological inhibitor of the TLR4 reduced the magnitude of the inflammatory response and tubular damage. In conclusion, calcineurin inhibitors induce inflammation and endothelial activation through the engagement of several innate immunity receptor TLR4-dependent signaling pathways. TLR4 is a possible therapeutic target to prevent anticalcineurin nephrotoxicity

Calcineurin inhibitors cyclosporine A and tacrolimus induce vascular inflammation and endothelial activation through TLR4 signaling

The introduction of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus greatly reduced the rate of allograft rejection, although their chronic use is marred by a range of side effects, among them vascular toxicity. In transplant patients, it is proved that innate immunity promotes vascular injury triggered by ischemia-reperfusion damage, atherosclerosis and hypertension. We hypothesized that activation of the innate immunity and inflammation may contribute to CNI toxicity, therefore we investigated whether TLR4 mediates toxic responses of CNIs in the vasculature. Cyclosporine and tacrolimus increased the production of proinflammatory cytokines and endothelial activation markers in cultured murine endothelial and vascular smooth muscle cells as well as in ex vivo cultures of murine aortas. CNI-induced proinflammatory events were prevented by pharmacological inhibition of TLR4. Moreover, CNIs were unable to induce inflammation and endothelial activation in aortas from TLR4−/− mice. CNI-induced cytokine and adhesion molecules synthesis in endothelial cells occurred even in the absence of calcineurin, although its expression was required for maximal effect through upregulation of TLR4 signaling. CNI-induced TLR4 activity increased O2 −/ROS production and NF-κB-regulated synthesis of proinflammatory factors in cultured as well as aortic endothelial and VSMCs. These data provide new insight into the mechanisms associated with CNI vascular inflammationThis work was supported by grants from the Instituto de Salud Carlos III (Ministerio de Economía Competitividad, Gobierno de España): FEDER funds ISCIII RETIC REDINREN RD12/0021, PI11/02242, PI13/00047, PI14/0041, PI14/00386, PI15/01460; Comunidad de Madrid (CIFRA S2010/BMD-2378); Sociedad Española de Nefrología. Salary support: RR-D: CIFRA; CO-S: Fundación Conchita Rábago de Jiménez Díaz; CG-G and RRR-D: REDINREN; AO: Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM); JE and MRO: Universidad Autónoma de Madrid; AMR: Contrato Miguel Serve (ISCIII

A primary healthcare information intervention for communicating cardiovascular risk to patients with poorly controlled hypertension: The Education and Coronary Risk Evaluation (Educore) study-A pragmatic, cluster-randomized trial

PURPOSE: Uncertainty exists regarding the best way to communicate cardiovascular risk (CVR) to patients, and it is unclear whether the comprehension and perception of CVR varies according to the format used. The aim of the present work was to determine whether a strategy designed for communicating CVR information to patients with poorly controlled high blood pressure (HBP), but with no background of cardiovascular disease, was more effective than usual care in the control of blood pressure (BP) over the course of a year. METHODS: A pragmatic, two-arm, cluster-randomized controlled trial was performed. Consecutive patients aged 40-65 years, all diagnosed with HBP in the last 12 months, and all of whom showed poor control of their condition (systolic BP ≥140 mmHg and/or diastolic BP ≥90 mmHg), were recruited at 22 primary healthcare centres. Eleven centres were randomly assigned to the usual care arm, and 11 to the informative intervention arm (Educore arm). At the start of the study, the Educore arm subjects were shown the "low risk SCORE table", along with impacting images and information pamphlets encouraging the maintenance of good cardiovascular health. The main outcome variable measured was the control of HBP; the secondary outcome variables were SCORE table score, total plasma cholesterol concentration, use of tobacco, adherence to prescribed treatment, and quality of life. RESULTS: The study participants were 411 patients (185 in the Educore arm and 226 in the usual care arm). Multilevel logistic regression showed that, at 12 months, the Educore intervention achieved better control of HBP (OR = 1.57; 1.02 to 2.41). No statistically significant differences were seen between the two arms at 12 months with respect to the secondary outcomes. CONCLUSIONS: Compared to usual care, the Educore intervention was associated with better control of HBP after adjusting for age, baseline SBP and plasma cholesterol, at 12 months.This study was funded by the Spanish Ministry of Science and Innovation via the Instituto de Salud Carlos III, Subprograma de Proyectos de Investigación en Evaluación de Tecnologías Sanitarias y Servicios de Salud (PI 09/90354), and the Fundación de Investigación e Innovación Biomédica en Atención Primaria (FIIBAP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptS

A polymeric nanomedicine diminishes inflammatory events in renal tubular cells

The polyglutamic acid/peptoid 1 (QM56) nanoconjugate inhibits apoptosis by interfering with Apaf-1 binding to procaspase-9. We now describe anti-inflammatory properties of QM56 in mouse kidney and renal cell models. In cultured murine tubular cells, QM56 inhibited the inflammatory response to Tweak, a non-apoptotic stimulus. Tweak induced MCP-1 and Rantes synthesis through JAK2 kinase and NF-kB activation. Similar to JAK2 kinase inhibitors, QM56 inhibited Tweak-induced NF-kB transcriptional activity and chemokine expression, despite failing to inhibit NF-kB-p65 nuclear translocation and NF-kB DNA binding. QM56 prevented JAK2 activation and NF-kB-p65(Ser536) phosphorylation. The anti-inflammatory effect and JAK2 inhibition by QM56 were observed in Apaf-12/2 cells. In murine acute kidney injury, QM56 decreased tubular cell apoptosis and kidney inflammation as measured by downmodulations of MCP-1 and Rantes mRNA expression, immune cell infiltration and activation of the JAK2-dependent inflammatory pathway. In conclusion, QM56 has an anti-inflammatory activity which is independent from its role as inhibitor of Apaf-1 and apoptosis and may have potential therapeutic relevance.This work was supported by grants from the Instituto de Salud Carlos III (www.isciii.es), FIS: PI07/0020, CP08/1083, PS09/00447 and ISCIII-RETICS REDINREN RD 06/0016; Sociedad Española de Nefrología (www.senefro.org). Álvaro Ucero, Sergio Berzal and Carlos Ocaña supported by Fundacion Conchita Rabago (www.fundacionconchitarabago.net), Alberto Ortiz by the Programa de Intensificación de la Actividad Investigadora in the Sistema Nacional de Salud of the Instituto de Salud Carlos III and the Agencia ‘‘Pedro Lain Entralgo’’ of the Comunidad de Madrid and CIFRA S-BIO 0283/2006 www.madrid.org/lainentralgo) and Adrián Ramos, by FIS (Programa Miguel Servet)

QM56 and JAK2 inhibition negatively regulate Tweak-induced p65 phosphorylation.

<p><b>A</b>) Tweak phosphorylates p65. MCT cells were treated with 100 ng/ml Tweak for the indicated times and phosphorylation of p65 in the Ser536 analyzed by Western blot. Representative figure of three individual experiments. <b>B</b>) QM56 and JAK2 inhibition prevent p65 phosphorylation. MCT cells were treated with 100 ng/ml Tweak for 3 h in the absence or in the presence of QM56 or AG490 (AG) added 1 h before the cytokine. Phosphorylated p65 was analyzed by Western blot. The figure shows a representative experiment and quantitation as Mean±SD of five independent experiments (*p<0.01 vs Control, **p<0.05 vs Tweak). <b>C</b>) QM56 and AG490 (AG) inhibit p65(Ser536) phosphorylation and nuclear translocation. MCT cells were treated as in B. Phospho-p65(Ser536) was studied by confocal microscopy. Arrows indicate representative cells with nuclear location of phospho-p65(Ser536) which are shown in detail in a merged green-propidium iodide image inset.</p

QM56 prevents Tweak-induced NF-κB-dependent transcription, but not NF-κB p65/relA nuclear translocation or NF-κB DNA-binding.

<p><b>A</b>) Tweak-induced NF-κB transcriptional activity in MCT cells is inhibited by QM56. NF-κB transcriptional activity was expressed as a percentage of Luciferase activity normalized to Renilla activity in a reporter gene assay. *p<0.01 vs Control; **p<0.05 vs Tweak (n = 3). <b>B</b>) Tweak increases IκBα phosphorylation after 30 minutes of incubation and pretreatment with QM56 does not preclude this effect. Representative Western blots are shown (n = 3). Protein bands in the sequence were arranged from non-consecutive lanes on the same membrane. <b>C</b>) QM56 does not prevent NF-κB p65/relA nuclear translocation in response to Tweak. MCT cells were stimulated for 30 minutes with the stimuli alone or in the presence of QM56 added 1 h before stimulation. Confocal microscopy: p65 (green), propidium iodide (red). Parthenolide was used as a control inhibitor of p65 nuclear translocation. Arrows indicate individual cells shown nuclear localization of p65 NF-κB subunit. <b>D</b>) QM56 does not prevent NF-κB DNA-binding activity in response to Tweak. Representative EMSA of nuclear extracts from MCT cells treated with Tweak alone or pretreated with QM56.</p

QM56 reduces renal inflammation in acute kidney injury.

<p><b>AKI was induced by a single folic acid (FA) overdose and mice were killed at 24 h</b>. <b>A</b>) renal expression of MCP-1 and Rantes as assessed by qRT-PCR (*p<0.05 vs Control, **p<0.05 vs FA). <b>B</b>) The increased number of interstitial macrophage stained with anti CD68 (left panel) or CD3 (right panel) in AKI kidneys was reduced by QM56. Representative images of groups of mice control (a) treated with FA (c and detail in e) and treated with FA and QM56 (d and detail in f). Controls for the technique were stained in the absence of primary antibodies (b). Arrows on c, d, e and f indicate macrophage infiltration (left panel) and lymphocyte infiltration (right panel). Delimited area in c and d are correspondingly amplified in e and f, respectively. Original magnifications ×200 and details ×400. Quantification as Means±SD of 5 mice per group (*p<0.02 vs Control, **p<0.02 vs FA, ^#p<0.01 vs Control, ^##p<0.01 vs FA) <b>C</b>) Western blots of nuclear (N) and cytoplasmatic (C) mouse kidney extracts from representative untreated control and FA and FA+QM56-treated mice. The phosphorylated form of JAK2 is increased in the folic acid-induced AKI group (1.4±0.1 fold increase vs Control group, p<0.02) and reduced by treatment with QM56 (1.1.±0.1 fold increase vs Control group, p<0.01 vs FA group). JAK2 activation induces both STAT3 nuclear translocation and phosphorylation of p65 in nuclei and they were prevented by QM56. However, IκBα phosphorylation and p65 nuclear translocation were not affected by QM56.</p