Differential regulation of Nitric Oxide Synthase isoforms : in vitro and in vivo studies

Le développement de la dyalise péritonéale (DP) comme traitement substitutif de l'insuffisance rénale a été possible grâce à la structure unique de la membrane péritonéeale (MP). Malheureusement, l'utilisation de la MP à long terme est limitée par une altération de sa structure et de ses propriétés de transport lorsqu'elle est exposé à des concentrations élevées en glucose. L'urémie et les épisodes de péritonite contribuent aussi à la détérioration de la MP. Nous avons utilisé un modèle expérimental de péritonite chez le rat pour étudier comment les différentes isoformes de NOS, induites par l'infection, sont impliquées dans les modifications structurelles (y compris la nitrosylation des protéines) et fonctionnelles de la MP. La péritonite est caractérisée par une perte de l'ultrafiltration (UF), l'augmentation des activités de NOS liées à une surexpression de iNOS et eNOS, de même qu'un marquage plus intense pour la S-nitrosocystéine. L'ajout de L-NAME, un inhibiteur de NOS, dans le dialysat est capable de rétablir l'UF. Dans l'urémie, une prolifération vasculaire et une augmentation de la perméabilité de la membrane péritonéale ont été également retrouvées, cette fois-ci en association avec une surexpression de eNOS et nNOS. Dans les deux modèles, l'augmentation de la nitrosocysteine associée à une augmentation de l'activité NOS illustre le rôle important du NOS dans le MP, le production de radicaux peroxynitrites en présence des anions superoxyde ayant pour conséquence des modifications post-translationnelles des proteines. Dans le modèle de diabète induit par la streptozocine, le diabète est associé à une augmentation progressive de la perméabilité du péritoine pour les petites molécules, une augmentation significative de l'expréssion et de l'activité NOS ainsi qu'une densité capillaire accrue. Le traitement par insulin normalise les tests de la fonction péritonale et prévient la prolifération vasculaire et la surexpression de eNOS. Des études in vitro (cellules endothéliales aortiques bovines) et in vivo (rats soumis à un apport chronique en urée) nous ont permis d'excure un effet de l'hyperosmolarité per se à l'origine des modifications observées. Les hormones corticostéroïdes ont un effet modulateur connu sur la transcription du gène iNOS, mais leurs effets sur l'expression de eNOS ne sont pas clairement définis. Dans la MP, eNOS et l'aquaporine-1, qui forme le canal à eau (AQP1) sont tous deux exprimés au niveau de la membrane des cellules endothéliales qui tapissent les capillaires. Dans le même modèle, nous montrons que l'administration de dexaméthasone peut augmenter la perméabilité de l'eau à travers la membrane péritonéale par une augmentation de l'expression d'AQP1. Contrairement à AQP1, l'expression et l'activité de NOS n'est pas augmentée par l'administration de la dexaméthasone aux doses utilisées. Même si les hormones corticotéroïdes ont des nombreux effets secondaires, cette étude ouvre des perspectives pour modifier la perméabilité à l'eau et augmenter l'efficacité de la DP. La régulation différentielle de NOS a également été étudié dans d'autres systèmes (cellules thyroïdiennes en culture, vascularisation rénale, artères rénales dans la polykystose autosomique dominante).Thèse de doctorat en sciences biomédicales (physiologie, physiopathologie) -- UCL, 200

Regulation of NO synthase isoforms in the peritoneum: implications for ultrafiltration failure in peritoneal dialysis.

BACKGROUND: Ultrafiltration (UF) failure often complicates peritoneal dialysis (PD). At least two molecules might be involved in UF failure: aquaporin-1 (AQP1), a water channel thought to be the ultra small pore of the peritoneal membrane (PM), and nitric oxide (NO), which might regulate effective peritoneal surface area and microvascular permeability. METHODS: The contributions of AQP1 and NO in UF failure were evaluated by combining different experimental approaches. Specific antibodies were used to investigate the expression (immunoblotting) and localization (immunostaining) of AQP1 and NO synthase (NOS) isoforms in the peritoneum, in correlation with: (i) morphometric analyses; (ii) the l-citrulline assay, which specifically measures NOS enzymatic activities; and (iii) permeability parameters across the PM. RESULTS: AQP1 is located in the endothelium lining peritoneal capillaries, and its expression is remarkably stable in samples ranging from normal to highly inflamed peritoneum and even when transcellular water permeability is absent (loss of sodium sieving). A significant NOS activity, mediated by specific NOS isoforms, can be assayed in the peritoneum. The NOS activity significantly increases in conditions such as peritonitis and long-term PD, and this increase is mirrored by up-regulation of NOS isoforms, as well as angiogenesis and increased endothelial area. CONCLUSIONS: These data suggest that the NO-mediated increase in effective peritoneal surface area, followed by a dissipation of the osmotic gradient, is a major mechanism accounting for the loss of UF in PD. Other biological consequences of increased NO levels in the peritoneum might include initiation of angiogenesis or modification of functionally important proteins such as AQP1

Experimental diabetes induces functional and structural changes in the peritoneum.

BACKGROUND: Peritoneal dialysis (PD) is an established renal replacement therapy in diabetic patients, but the influence of diabetes on the peritoneal membrane (PM) remains debated. We have used functional, biochemical and molecular studies in vivo and in vitro to substantiate the changes induced by diabetes and hyperglycemia in the PM. METHODS: Peritoneal equilibration tests were performed 2, 4, and 6 weeks after induction of diabetes with streptozotocin (STZ) in rats. Morphological analyses, determination of nitric oxide synthase (NOS) activities, and expression studies for NOS isoforms and advanced glycation end products (AGE) were performed in parallel. Additional studies were conducted in diabetic rats treated with insulin, non-diabetic rats fed with urea, and cultured bovine aortic endothelial cells (BAEC). RESULTS: In comparison with controls, diabetic rats were characterized by: increased permeability for small solutes and decreased sodium sieving; capillary proliferation; increased endothelial NOS (eNOS) and AGE immunoreactivity; up-regulation of eNOS and down-regulation of neuronal NOS; and increased NOS activity in the PM. The changes, which culminated at week 6, were prevented by chronic insulin treatment in diabetic rats. In contrast to hyperglycemia, hyperosmolality alone did not induce functional or structural changes in the PM. Studies in BAEC showed that high glucose incubation led to increased activity and expression of eNOS, a prerequisite for vascular proliferation. CONCLUSIONS: These data demonstrate that chronic hyperglycemia is associated with functional and structural changes in the peritoneum that parallel with selective regulation of NOS isoforms and AGE deposits. The alterations are prevented by insulin treatment, which suggests that adequate control of diabetes can preserve PM integrity in diabetic patients prior to PD

Attenuated anti-SARS-CoV-2 antibody response to vaccination in patients with rheumatic diseases.

No abstract availabl

Inhibition of nitric oxide synthase reverses changes in peritoneal permeability in a rat model of acute peritonitis.

BACKGROUND: Acute peritonitis is the most frequent complication of peritoneal dialysis (PD), and nitric oxide (NO) is thought to play a role in the structural and permeability changes observed in this condition. We have used a combination of expression, enzymatic and pharmacological studies to substantiate the potential role(s) played by NO during peritonitis. METHODS: The peritoneal equilibration test was performed in control rats and rats with acute peritonitis (originating from skin flora), using standard dialysate supplemented or not with the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In parallel, peritoneal NOS enzymatic activities were measured and expression studies for NOS isoforms and S-nitrosocysteine reactivity performed in the peritoneum. RESULTS: In comparison with controls, rats with acute peritonitis were characterized by inflammatory changes, increased S-nitrosocysteine immunoreactivity, and increased NOS activities in the peritoneum, due to the up-regulation of endothelial and inducible NOS. In parallel, rats with acute peritonitis showed increased permeability for small solutes; decreased sodium sieving; loss of ultrafiltration (UF); and increased protein loss in the dialysate. Addition of L-NAME to the dialysate did not induce permeability changes in control rats, but significantly improved UF and reversed permeability modifications in rats with peritonitis. The effect of L-NAME was reflected by a mild but consistent increase in blood pressure during PD exchange. CONCLUSIONS: Our results demonstrate that local generation of NO, secondary to up-regulation of NOS isoforms, plays an important role in the regulation of peritoneal permeability during acute peritonitis in rats. By itself, NOS inhibition improves UF and reverses permeability changes, which might offer new therapeutic perspectives in acute peritonitis

Diabetes-induced microvascular dysfunction in the hydronephrotic kidney: role of nitric oxide.

BACKGROUND: Renal hemodynamics in early diabetes are characterized by preglomerular and postglomerular vasodilation and increased glomerular capillary pressure, leading to hyperfiltration. Despite intensive research, the etiology of the renal vasodilation in diabetes remains a matter of debate. The present study investigated the controversial role of nitric oxide (NO) in the renal vasodilation in streptozotocin-induced diabetic rats. METHODS: In the renal microcirculation, basal tone and response to NO synthase blockade were studied using the in vivo hydronephrotic kidney technique. L-arginine analog N-nitro-L-arginine methyl ester (L-NAME) was administered locally to avoid confounding by systemic blood pressure effects. The expression of endothelial NO synthase (eNOS) was investigated in total kidney by immunocytochemistry and in isolated renal vascular trees by Western blotting. Urinary excretion of nitrites/nitrates was measured. RESULTS: Diabetic rats demonstrated a significant basal vasodilation of all preglomerular and postglomerular vessels versus control rats. Vasoconstriction to L-NAME was significantly increased in diabetic vessels. After high-dose L-NAME, there was no difference in diameter between diabetic and control vessels, suggesting that the basal vasodilation is mediated by NO. Immunocytochemically, the expression of eNOS was mainly localized in the endothelium of preglomerular and postglomerular vessels and glomerular capillaries, and was increased in the diabetic kidneys. Immunoblots on isolated renal vascular trees revealed an up-regulation of eNOS protein expression in diabetic animals. The urinary excretion of nitrites/nitrates was elevated in diabetic rats. CONCLUSION: The present study suggests that an up-regulation of eNOS in the renal microvasculature, resulting in an increased basal generation of NO, is responsible for the intrarenal vasodilation characteristic of early diabetes

Nitric oxide is involved in interleukin-1alpha-induced cytotoxicity in polarised human thyrocytes.

Nitric oxide (NO) is a well-known mediator of autoimmune processes. In the thyroid gland, it is produced in response to interleukin 1 (IL-1) and may mediate cytokine action at an early stage of autoimmune thyroiditis. In this study, we have investigated whether NO is involved in cytokine-induced cytotoxic effects and epithelial barrier alterations in thyrocytes. Human thyroid epithelial cells were cultured as tight polarised monolayers on a permeable support and exposed or not to IL-1alpha (100 U/ml), alone or in combination with interferon-gamma (IFN-gamma; 100 U/ml) added to the basal compartment. NO production was not detected in control thyrocytes, but was significantly induced by the combination of IL-1alpha with IFN-gamma, in a time-dependent fashion. Similarly, expression of the inducible isoform of nitric oxide synthase (NOSII), determined by immunoblot and immunofluorescence confocal microscopy, was not detected in control cells, but was markedly induced after 48-h exposure to both cytokines. This treatment significantly increased the release of cytosolic lactate dehydrogenase (LDH) in the apical and basolateral media and decreased transepithelial electrical resistance. Although IFN-gamma was not sufficient to induce NO production, it could by itself decrease transepithelial resistance and synergised the IL-1alpha effect on LDH release. The NOS inhibitor, L-nitro-arginine-methyl ester, suppressed the cytokine-induced NO production and decreased the LDH release, but failed to prevent the loss of transepithelial resistance. These results indicated that human thyrocytes express NOSII and produce NO in response to IL-1alpha+IFN-gamma and suggest that NO acts as a mediator of cytokine-induced cytotoxicity in the thyroid gland and may promote the exposure of autoantigens to the immune system. In contrast, NO does not appear to mediate the cytokine-induced disruption of the thyroid epithelial barrier

Chronic uremia induces permeability changes, increased nitric oxide synthase expression, and structural modifications in the peritoneum.

Advanced glycation end products (AGE), growth factors, and nitric oxide contribute to alterations of the peritoneum during peritoneal dialysis (PD). These mediators are also involved in chronic uremia, a condition associated with increased permeability of serosal membranes. It is unknown whether chronic uremia per se modifies the peritoneum before PD initiation. A rat model of subtotal nephrectomy was used to measure peritoneal permeability after 3, 6, and 9 wk, in parallel with peritoneal nitric oxide synthase (NOS) isoform expression and activity and structural changes. Uremic rats were characterized by a higher peritoneal permeability for small solutes and an increased NOS activity due to the up-regulation of endothelial and neuronal NOS. The permeability changes and increased NOS activities correlated with the degree of renal failure. Focal areas of vascular proliferation and fibrosis were detected in uremic rats, in relation with a transient up-regulation of vascular endothelial growth factor and basic fibroblast growth factor, as well as vascular deposits of the AGE carboxymethyllysine and pentosidine. Correction of anemia with erythropoietin did not prevent the permeability or structural changes in uremic rats. Thus, in this rat model, uremia induces permeability and structural changes in the peritoneum, in parallel with AGE deposits and up-regulation of specific NOS isoforms and growth factors. These data suggest an independent contribution of uremia in the peritoneal changes during PD and offer a paradigm to better understand the modifications of serosal membranes in uremia

Modifier effect of ENOS in autosomal dominant polycystic kidney disease.

A significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is associated with altered endothelial-dependent vasodilation and decreased vascular production of nitric oxide (NO). Thus, ENOS, the gene coding for the endothelial nitric oxide synthase (eNOS), could have a modifier effect in ADPKD. In order to test this hypothesis, we genotyped 173 unrelated ADPKD patients from Belgium and the north of France for the Glu298Asp, intron 4 VNTR and T-786C polymorphisms of ENOS and looked for their influence on the age at end-stage renal disease (ESRD). In males (n = 93), the Glu298Asp polymorphism was associated with a lower age at ESRD (Glu/Asp + Asp/Asp: 49.0 +/- 1.2 years, n = 53; Glu/Glu: 53.5 +/- 1.5 years, n = 40; simple regression, P = 0.02; multiple regression, P = 0.006). This effect was confirmed in a subset of males linked to PKD1 and reaching ESRD before age 45, and by a cumulative renal survival analysis in PKD1-linked families. Further studies demonstrated that NO synthase (NOS) activity was decreased in renal artery samples from ADPKD males harbouring the Asp298 allele, in association with post-translational modifications and partial cleavage of eNOS. No significant effect of the other polymorphisms was found in males, and no polymorphism influenced the age at ESRD in females. In conclusion, the frequent Glu298Asp polymorphism of ENOS is associated with a 5 year lower mean age at ESRD in this subset of ADPKD males. This effect could be due to a decreased NOS activity and a partial cleavage of eNOS, leading to a further decrease in the vascular production of NO

The OMERACT Ultrasound Group: A Report from the OMERACT 2016 Meeting and Perspectives

Objective To provide an update from the Outcome Measures in Rheumatology (OMERACT) Ultrasound Working Group on the progress for defining ultrasound (US) minimal disease activity threshold at joint level in rheumatoid arthritis (RA) and for standardization of US application in juvenile idiopathic arthritis (JIA). Methods For minimal disease activity, healthy controls (HC) and patients with early arthritis (EA) who were naive to disease-modifying antirheumatic drugs were recruited from 2 centers. US was performed of the hands and feet, and scored semiquantitatively (0–3) for synovial hypertrophy (SH) and power Doppler (PD). Synovial effusion (SE) was scored a binary variable. For JIA, a Delphi approach and subsequent validation in static images and patient-based exercises were used to developed preliminary definitions for synovitis and a scoring system. Results For minimal disease activity, 7% HC had at least 1 joint abnormality versus 30% in the EA group. In HC, the findings of SH and PD were predominantly grade 1 whereas all grades were seen in the EA cohort, but SE was rare. In JIA, synovitis can be diagnosed based on B-mode findings alone because of the presence of physiological vascularization. A semiquantitative scoring system (0–3) for synovitis for both B-mode and Doppler were developed in which the cutoff between Doppler grade 2 and grade 3 was 30%. Conclusion The first step has been taken to define the threshold for minimal disease activity in RA by US and to define and develop a scoring system for synovitis in JIA. Further steps are planned for the continuous validation of US in these areas