ABSTRACT
Cardiovascular disease is an important cause of death in patients with chronic kidney disease (CKD). Protein-bound uremic toxins, such as p-cresyl and indoxyl sulfate (IS), are poorly removed during hemodialysis, leading to vascular endothelial dysfunction and leukocyte extravasation. These processes can be related to dynamic adhesion structures called podosomes. Several studies have indicated the role of integrin-linked kinase (ILK) in the accumulation of integrin-associated proteins in podosomes. Here, we investigated the involvement of ILK and podosome formation in the adhesion and extravasation of monocytes under p-cresol (pc) and IS exposure. Incubation of THP-1 human monocyte cells with these toxins upregulated ILK kinase activity. Together, both toxins increased cell adhesion, podosome formation, extracellular matrix degradation, and migration of THP-1 cells, whereas ILK depletion with specific small interfering RNAs suppressed these processes. Interestingly, F-actin colocalized with cortactin in podosome cores, while ILK was colocalized in podosome rings under toxin stimulation. Podosome Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) and AKT protein depletion demonstrated that monocyte adhesion depends on podosome formation and that the ILK/AKT signaling pathway is involved in these processes. Ex vivo experiments showed that both toxins induced adhesion and podosome formation in leukocytes from wild-type mice, whereas these effects were not observed in leukocytes of conditional ILK-knockdown animals. In summary, under pc and IS stimulation, monocytes increase podosome formation and transmigratory capacity through an ILK/AKT signaling pathway-dependent mechanism, which could lead to vascular injury. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.
Subject(s)
Podosomes , Protein Serine-Threonine Kinases , Animals , Cell Adhesion , Cresols , Cytoskeletal Proteins/metabolism , Humans , Indican/metabolism , Indican/pharmacology , Mice , Monocytes , Podosomes/metabolism , Protein Serine-Threonine Kinases/metabolism , THP-1 CellsABSTRACT
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-α 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.
Subject(s)
Diet, High-Fat/adverse effects , Down-Regulation , Metabolic Diseases/etiology , Protein Serine-Threonine Kinases/genetics , Animals , Female , Gluconeogenesis , Inflammation/etiology , Inflammation/genetics , Insulin Resistance , Lipolysis , Male , Metabolic Diseases/genetics , Mice , Mice, Inbred BALB CABSTRACT
In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.
Subject(s)
Antioxidants/metabolism , Cell-Derived Microparticles/metabolism , Endothelial Cells/metabolism , NADP/metabolism , HumansABSTRACT
ANTECEDENTES: En la hemodiafiltración posdilucional se han usado solo membranas sintéticas. Ahora contamos con un triacetato de celulosa asimétrico (ATA(R)) cuyas características lo hacen apto para esta técnica. OBJETIVOS: Describir las prestaciones y el comportamiento in vivo de esta membrana estudiando la eficacia depurativa y el uso clínico, además de su biocompatibilidad aguda tras un mes de tratamiento. MÉTODOS: Estudio prospectivo observacional en el que se incluyeron 23 pacientes que se dializaron durante 4 semanas con ATA(R) manteniendo su pauta previa. RESULTADOS: Se realizaron 287 sesiones y se recogieron 264 sesiones completas. Con un tiempo efectivo de 243,7 (17,6) min y un flujo medio de sangre de 371,7 (23) ml/min, se obtuvo un Kt medio de 56,3 (5,3) l, un volumen convectivo de 27,1 (4,2) l, con una fracción de filtración del 29,9 (3,7) %, un porcentaje de reducción (RR) de urea de 81 (5,2) %, un RR de creatinina de 74,7 (4,6) %, un RR de Beta2-microglobulina de 76,5 (4,8) % y un RR de proteína transportadora de retinol de 18,6 (7,6) %. No se produjeron problemas técnicos ni alarmas. No fue preciso cambiar la dosificación de heparina. A los 30 min de la sesión no se produjo ningún aumento de C3a, C5a ni leucopenia. Tampoco se modificaron de forma significativa las poblaciones monocitarias ni la IL-Beta1 ni IL-6 tras un mes de tratamiento. CONCLUSIONES: ATA(R) logra un Kt y un volumen convectivo adecuados, sin problemas técnicos y con buen perfil de biocompatibilidad e inflamatorio, lo que lo convierte en una posibilidad más de tratamiento para hemodiafiltración posdilucional, máxime en pacientes alérgicos a membranas sintéticas
BACKGROUND: In post-dilution haemodiafiltration only synthetic membranes have been used to date. Asymmetric cellulose triacetate (ATA(TM)) is now available, whose characteristics are suitable for this technique. OBJECTIVES: To describe the in vivo performance and behaviour of this membrane, to identify its depurative effectiveness, use in clinical practice and its biocompatibility, both acute and after one month of treatment. METHODS: Observational prospective study of 23 patients who were dialysed for 4 weeks using an ATA(TM) membrane and who maintained their prior regimen. RESULTS: A total of 287 sessions were performed and 264 complete sessions were collected. With an effective time of 243.7 (17.6) min and a mean blood flow of 371.7 (23) ml/min, an average Kt of 56.3 (5.3) l was observed, as well as a convection volume of 27.1 (4.2) l, a filtration fraction of 29.9 (3.7) %, a urea reduction ratio (RR) of 81 (5.2) %, a creatinine RR of 74.7 (4.6) %, a Beta2-microglobulin RR of 76.5 (4.8) % and a retinol binding protein RR of 18.6 (7.6) %. There were no technical problems or alarms. Changing the heparin dosage was not necessary. No increases in C3a or C5a concentrations or leukopenia were observed in the first 30min of the session. Neither the monocyte subpopulations nor IL-Beta1 or IL-6 were significantly altered after one month of treatment. CONCLUSIONS: The new ATA(TM) membrane achieves adequate Kt and convection volume, without technical problems and with good biocompatibility and inflammatory profiles. It is therefore a valid option for post-dilution haemodiafiltration, particularly in patients allergic to synthetic membranes
Subject(s)
Humans , Hemodiafiltration/instrumentation , Hemodiafiltration/methods , Cellulose , Equipment Design , Prospective Studies , Observational Study , Renal Insufficiency, Chronic/therapyABSTRACT
BACKGROUND: In post-dilution haemodiafiltration only synthetic membranes have been used to date. Asymmetric cellulose triacetate (ATA™) is now available, whose characteristics are suitable for this technique. OBJECTIVES: To describe the in vivo performance and behaviour of this membrane, to identify its depurative effectiveness, use in clinical practice and its biocompatibility, both acute and after one month of treatment. METHODS: Observational prospective study of 23 patients who were dialysed for 4 weeks using an ATA™ membrane and who maintained their prior regimen. RESULTS: A total of 287 sessions were performed and 264 complete sessions were collected. With an effective time of 243.7 (17.6) min and a mean blood flow of 371.7 (23) ml/min, an average Kt of 56.3 (5.3) l was observed, as well as a convection volume of 27.1 (4.2) l, a filtration fraction of 29.9 (3.7) %, a urea reduction ratio (RR) of 81 (5.2) %, a creatinine RR of 74.7 (4.6) %, a ß2-microglobulin RR of 76.5 (4.8) % and a retinol binding protein RR of 18.6 (7.6) %. There were no technical problems or alarms. Changing the heparin dosage was not necessary. No increases in C3a or C5a concentrations or leukopenia were observed in the first 30min of the session. Neither the monocyte subpopulations nor IL-ß1 or IL-6 were significantly altered after one month of treatment. CONCLUSIONS: The new ATA™ membrane achieves adequate Kt and convection volume, without technical problems and with good biocompatibility and inflammatory profiles. It is therefore a valid option for post-dilution haemodiafiltration, particularly in patients allergic to synthetic membranes.
Subject(s)
Cellulose/analogs & derivatives , Hemodiafiltration/methods , Membranes, Artificial , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Prospective StudiesABSTRACT
Residual renal function (RRF) has an important effect on uremic toxin clearance, on volume control, on quality of life, and on mortality. In patients with chronic kidney disease (CKD), microinflammation with an increased percentage of CD14+ /CD16++ inflammatory monocytes has been reported, even with no clinical evidence of inflammation. No correlation has been established between these and RRF in hemodialysis (HD) patients. Our objective was to assess the relationship between RRF and the inflammatory parameters in HD patients. Cross-sectional observational study was carried out on 69 adult patients on chronic HD for at least 6 months, from which demographic, analytic and HD-technique data were collected and the following were measured: (i) RRF with average urea and creatinine clearance ((CCr + CU)/2) in 24-h urine (if >1 mL/min and diuresis >100 mL/day, RRF was considered); (ii) Inflammation through biochemical parameters (C-reactive protein, ß2 microglobulin, albumin) and monocyte subpopulations in peripheral blood. The average age was 70.9 [40-88] years old; 38 (55.1%) were male; and 25 (36.2%) were diabetic. 43.5% (30/69) presented RRF, with an average of ((CCr + CU)/2): 1.8 (2.6) mL/min and diuresis: 454.5 (569) mL /24 h. Patients with RRF presented lower concentrations of C-reactive protein (6.2 vs 21.4 mg/L) (P = 0.038) and a lower percentage of non-classical CD14+ /CD16++ monocytes (14.6 vs. 28.3%, P = 0.02). In our study, patients with RRF present lower concentrations of inflammatory parameters, which is another reason why its preservation is an essential objective in HD.
Subject(s)
Inflammation/physiopathology , Quality of Life , Renal Dialysis/methods , Renal Insufficiency, Chronic/therapy , Adult , Aged , Aged, 80 and over , C-Reactive Protein/metabolism , Creatinine/urine , Cross-Sectional Studies , Female , Humans , Inflammation/epidemiology , Kidney Function Tests , Lipopolysaccharide Receptors/metabolism , Male , Middle Aged , Monocytes/metabolism , Receptors, IgG/metabolism , Urea/metabolismABSTRACT
We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs) and their microvesicles (MVs). Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E) activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.
Subject(s)
Antioxidants/metabolism , Cell-Derived Microparticles/metabolism , Extracellular Vesicles/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Proteomics/methods , Humans , Reactive Oxygen SpeciesABSTRACT
Vascular calcification is commonly seen in elderly people, though it can also appear in middle-aged subjects affected by premature vascular aging. The aim of this work is to test the involvement of microvesicles (MVs) produced by senescent endothelial cells (EC) and from plasma of elderly people in vascular calcification. The present work shows that MVs produced by senescent cultured ECs, plus those found in the plasma of elderly subjects, promote calcification in vascular smooth muscle cells. Only MVs from senescent ECs, and from elderly subjects' plasma, induced calcification. This ability correlated with these types of MVs' carriage of: a) increased quantities of annexins (which might act as nucleation sites for calcification), b) increased quantities of bone-morphogenic protein, and c) larger Ca contents. The MVs of senescent, cultured ECs, and those present in the plasma of elderly subjects, promote vascular calcification. The present results provide mechanistic insights into the observed increase in vascular calcification-related diseases in the elderly, and in younger patients with premature vascular aging, paving the way towards novel therapeutic strategies.