The protective arm of the renin -angiotensin system may counteract the intense inflammatory process in fetuses with posterior urethral valves / O braço protetor do sistema renina-angiotensina pode contrapor-se ao intenso processo inflamatório em fetos com válvula de uretra posterior

Abstract Objective: Posterior urethral valve is the most common lower urinary tract obstruction in male children. A high percentage of patients with posterior urethral valve evolve to end‐stage renal disease. Previous studies showed that cytokines, chemokines, and components of the renin-angiotensin system contribute to the renal damage in obstructive uropathies. The authors recently found that urine samples from fetuses with posterior urethral valve have increased levels of inflammatory molecules. The aim of this study was to measure renin-angiotensin system molecules and to investigate their correlation with previously detected inflammatory markers in the same urine samples of fetuses with posterior urethral valve. Methods: Urine samples from 24 fetuses with posterior urethral valve were collected and compared to those from 22 healthy male newborns at the same gestational age (controls). Renin-angiotensin system components levels were measured by enzyme‐linked immunosorbent assay. Results: Fetuses with posterior urethral valve presented increased urinary levels of angiotensin (Ang) I, Ang‐(1‐7) and angiotensin‐converting enzyme 2 in comparison with controls. ACE levels were significantly reduced and Ang II levels were similar in fetuses with posterior urethral valve in comparison with controls. Conclusions: Increased urinary levels of angiotensin‐converting enzyme 2 and of Ang‐(1‐7) in fetuses with posterior urethral valve could represent a regulatory response to the intense inflammatory process triggered by posterior urethral valve.

Resumo Objetivo: A válvula de uretra posterior é a obstrução do trato urinário inferior mais comum em crianças do sexo masculino. Uma alta porcentagem de pacientes com válvula de uretra posterior evolui para doença renal em estágio final. Estudos anteriores mostraram que citocinas, quimiocinas e componentes do sistema renina-angiotensina contribuem para o dano renal em uropatias obstrutivas. Recentemente, descobrimos que amostras de urina de fetos com válvula de uretra posterior tinham níveis aumentados de moléculas inflamatórias. O objetivo deste estudo foi medir as moléculas de renina-angiotensina e investigar sua correlação com marcadores inflamatórios previamente detectados nas mesmas amostras de urina de fetos com válvula de uretra posterior. Métodos: Amostras de urina de 24 fetos com válvula de uretra posterior foram coletadas e comparadas com amostras de urina de 22 recém-nascidos saudáveis de mesma idade gestacional (controles). Os níveis dos componentes de SRA foram medidos por ensaio de imunoabsorção enzimática. Resultados: Os fetos com válvula de uretra posterior apresentaram níveis urinários aumentados de angiotensina (Ang) I, Ang-(1-7) e enzima conversora de angiotensina 2 em comparação com os controles. Os níveis de enzima conversora de angiotensina eram significativamente menores e os níveis de Ang II eram semelhantes nos fetos com válvula de uretra posterior em comparação com os controles. Conclusões: O aumento dos níveis urinários de enzima conversora de angiotensina 2 e de Ang-(1-7) em fetos com válvula de uretra posterior poderia representar uma resposta regulatória ao intenso processo inflamatório desencadeado pela válvula de uretra posterior.

The protective arm of the renin-angiotensin system may counteract the intense inflammatory process in fetuses with posterior urethral valves.

OBJECTIVE: Posterior urethral valve is the most common lower urinary tract obstruction in male children. A high percentage of patients with posterior urethral valve evolve to end-stage renal disease. Previous studies showed that cytokines, chemokines, and components of the renin-angiotensin system contribute to the renal damage in obstructive uropathies. The authors recently found that urine samples from fetuses with posterior urethral valve have increased levels of inflammatory molecules. The aim of this study was to measure renin-angiotensin system molecules and to investigate their correlation with previously detected inflammatory markers in the same urine samples of fetuses with posterior urethral valve. METHODS: Urine samples from 24 fetuses with posterior urethral valve were collected and compared to those from 22 healthy male newborns at the same gestational age (controls). Renin-angiotensin system components levels were measured by enzyme-linked immunosorbent assay. RESULTS: Fetuses with posterior urethral valve presented increased urinary levels of angiotensin (Ang) I, Ang-(1-7) and angiotensin-converting enzyme 2 in comparison with controls. ACE levels were significantly reduced and Ang II levels were similar in fetuses with posterior urethral valve in comparison with controls. CONCLUSIONS: Increased urinary levels of angiotensin-converting enzyme 2 and of Ang-(1-7) in fetuses with posterior urethral valve could represent a regulatory response to the intense inflammatory process triggered by posterior urethral valve.

Bj-PRO-5a and Bj-PRO 10c found at C-type natriuretic peptide precursor of bothrops jararaca change renal function of hypertensive rats renal function of hypertensive rats

Proline-rich oligopeptides from Bothrops jararaca (Bj-PROs) produce potent and long-lasting antihypertensive effect through mechanisms that go beyond ACE inhibition. In this study we evaluated the renal function parameters of spontaneously hypertensive rats (SHR) injected with Bj-PRO-5a and -10c (0.47, 71 or 710 nmol/kg) found in the CNP-precursor of the snake. At 71 and 710 nmol/kg, Bj-PROs increased urinary flow rate (18.1-43.5%). At 71 nmol/kg, Bj-PRO 5a and 10c elevated sodium excretion (68.1 and 40.9%, respectively) and Bj-PRO-5a also increased urinary sodium/creatinine ratio (56.5%). At 0.47 nmol/kg, Bj-PROs did not change renal function. All doses of Bj-PROs reduced blood pressure (Delta = -13 to -24mmHg). We conclude that Bj-PROs reduce blood pressure and improve renal function of SHRs through diuretic and natriuretic mechanisms.

Immune markers in the RASopathy neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a genetic disorder with an early mortality determined mostly by malignancy. Little is known about the immunosurveillance factors in NF1 patients. In this study we evaluated inflammatory markers and their cellular sources in NF1 patients to try understanding the relation of immune factors and the tumorigenesis that characterizes the disease. Using flow cytometry and ELISA, we assayed cytokines, co-stimulatory molecules, the functional state of circulating blood cells and cytokine plasma levels in a case-control transversal study. The frequency of CD4+ T cells seems reduced. In addition, a shift towards an anti-inflammatory profile was observed in cells expressing cytokines, except for a small subpopulation of CD8+ T cells that displayed an increased frequency of cells expressing the pro-inflammatory cytokine Tumor necrosis factor (TNF-α), while plasma soluble levels of Transforming growth factor-beta (TGF-ß) and interleukin-6 (IL-6) were increased in NF1 patients. Knowledge of the regulation of NF1 and the role of TGF-beta signaling pathway in malignant peripheral nerve sheath tumor pathogenesis might shed light on molecular carcinogenesis mechanisms and lead to putative interventions both in prevention and treatment of malignant tumors.

Lithothamnion muelleri treatment ameliorates inflammatory and hypernociceptive responses in antigen-induced arthritis in mice.

Rheumatoid Arthritis (RA) is a chronic disease characterized by persistent inflammation and pain. Alternative therapies to reduce these symptoms are needed. Marine algae are valuable sources of diverse bioactive compounds. Lithothamnion muelleri (Hapalidiaceae) is a marine algae with anti-inflammatory, antitumor, and immunomodulatory properties. Here, we investigated the potential anti-inflammatory and analgesic activities of L. muelleri in a murine model of antigen-induced arthritis (AIA) in mice. Our results demonstrate that treatment with L. muelleri prevented inflammation and hypernociception in arthritic mice. Mechanistically, the crude extract and the polysaccharide-rich fractions of L. muelleri may act impairing the production of the chemokines CXCL1 and CXCL2, and consequently inhibit neutrophil influx to the knee joint by dampening the adhesion step of leukocyte recruitment in the knee microvessels. Altogether our results suggest that treatment with L.muelleri has a potential therapeutic application in arthritis treatment.

Mechanisms of the anti-inflammatory actions of the angiotensin type 1 receptor antagonist losartan in experimental models of arthritis.

Angiotensin (Ang) II and its AT1 receptors have been implicated in the pathogenesis of rheumatoid arthritis. Activation of the counter-regulatory Ang-(1-7)-Mas receptor axis may contribute to some of the effects of AT1 receptor blockers (ARBs). In this study, we have used losartan, an ARB, to investigate the role of and the mechanisms by which AT1 receptors participated in two experimental models of arthritis: antigen-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Treatment with losartan decreased neutrophil recruitment, hypernociception and the production of TNF-α, IL-1ß and chemokine (C-X-C motif) ligand 1 in mice subjected to AIA. Histopathological analysis showed significant reduction of tissue injury and inflammation and decreased proteoglycan loss. In addition to decreasing cytokine production, losartan directly reduced leukocyte rolling and adhesion. Anti-inflammatory effects of losartan were not associated to Mas receptor activation and/or Ang-(1-7) production. Anti-inflammatory effects were reproduced in rats subjected to AdIA. This study shows that ARBs have potent anti-inflammatory effects in animal models of arthritis. Mechanistically, reduction of leukocyte accumulation and of joint damage was associated with local inhibition of cytokine production and direct inhibition of leukocyte-endothelium interactions. The anti-inflammatory actions of losartan were accompanied by functional improvement of the joint, as seen by reduced joint hypernociception. These findings support the use of ARBs for the treatment of human arthritis and provide potential mechanisms for the anti-inflammatory actions of these compounds.

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection.

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.

Treatment with Selemax®, a selenium-enriched yeast, ameliorates experimental arthritis in rats and mice.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that mainly targets the synovial membrane, cartilage and bone. It affects 1 % of the population and is associated with significant morbidity and increased mortality. Se is an essential trace element with antioxidant properties and the ability to modulate the immune responses. Selemax® is an inactive yeast (Saccharomyces cerevisiae) enriched with organic Se. The aim of the present study was to investigate the effects of Selemax® administration in models of an antigen-induced arthritis (AIA) in C57BL/6 mice, and of an adjuvant-induced arthritis (AdIA) in Holtzman rats. As control, the animals were treated with the same inactivated yeast species that was not enriched for Se. In the AIA model, treatment with different doses of Selemax® (0·01, 0·1, 1 and 10 % added to food) significantly decreased the number of inflammatory cells recruited to the knee cavity, essentially by reducing the number of neutrophils. Levels of proinflammatory cytokines, including TNF-α, IL-1ß and chemokine (C-X-C motif) ligand 1/keratinocyte chemoattractant (CXCL1/KC), were also reduced in the peri-articular tissue of mice treated with Selemax® at the tested dose (1 %). In the AdIA model in rats, Selemax® treatment decreased paw oedema and hypernociception. This reduction was associated with inhibition of the influx of proinflammatory cells. Therefore, treatment with Selemax® is associated with amelioration of several inflammatory and functional parameters in models of arthritis, suggesting that this Se-enriched yeast should be evaluated further in patients with RA.

Plasma and urinary levels of cytokines in patients with idiopathic hypercalciuria.

BACKGROUND: Recent studies suggest that cytokines modulate bone turnover. Idiopathic hypercalciuria (IH) seems to be associated with bone mineral loss. Therefore, the aim of this study was to assess cytokines involved in bone turnover in patients with IH. METHODS: Plasma and spot-urine levels of interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor alpha (TNF-α), transforming growth factor ß1 (TGF-ß1), and monocyte chemoattractant protein (MCP-1) were measured in 70 children and adolescents with IH and in 37 healthy controls. Patients with IH were subdivided according to their calciuria at the time of sample collection: ≥4 mg/kg/day (persistent IH, n=27) and below 4 mg/kg/day (controlled IH, n=43). Cytokines were determined by enzyme-linked immunoassay. RESULTS: Plasma and urinary concentrations of IL-1ß, IL-6, IL-8, and TNF-α were undetectable in all groups. No differences were found between controlled and persistent hypercalciuria for plasma and urinary levels of MCP-1 and TGF-ß1. On the other hand, MCP-1 levels were significantly higher in both subgroups of IH in comparison to healthy controls. Furthermore, urinary MCP-1 levels of IH patients correlated positively with bone mineral content (p=0.013). CONCLUSION: Although cytokine measurements did not allow the differentiation between persistent and controlled IH, our findings suggest that MCP-1 might play a role in patients with IH.

Urinary levels of TGF ß-1 and of cytokines in patients with prenatally detected nephrouropathies.

This study aimed to identify noninvasive biomarkers of clinically significant nephrouropathies in patients with antenatal renal and/or urinary tract alterations. Spot-urine levels of interleukin-6 (IL-6), transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) were measured in 100 patients with antenatal detected nephrouropathies. Patients were divided in idiopathic hydronephrosis (n = 47), urinary tract malformations (n = 35), and dysplastic kidneys (n = 18). Urinary concentrations of TGF-ß1, IL-6, and TNF-α were compared between groups according to clinical and image findings. Receiver-operating characteristic (ROC) curves were analyzed for the overall diagnostic accuracy of TGF-ß1, IL-6, and TNF-α levels in discriminating infants with nephrouropathies. No significant differences in urinary TGF- ß1, IL-6, and TNF-α levels were found in the comparison between the groups. TGF-ß1 levels tended to be higher in patients with renal hypodysplasia compared to idiopathic hydronephrosis (p = 0.07). Twenty-nine patients had reduced DMSA uptake. In these cases, absolute urinary concentration of TGF-ß1 and levels standardized for creatinine were significantly higher than in patients with normal DMSA uptake, while IL6 and TNF-α did not differ between groups. Urinary cytokine measurements were not useful as a screening test for clinically significant nephrouropathies. Conversely, increased concentrations of TGF-ß1 pointed out to renal damage as indicated by reduced DMSA uptake.

ACE2-angiotensin-(1-7)-Mas axis in renal ischaemia/reperfusion injury in rats.

AngII (angiotensin II), ACE (angiotensin I-converting enzyme) and the AT1 receptor (AngII type 1 receptor) are associated with the inflammatory process and microvascular dysfunction of AKI (acute kidney injury) induced by renal I/R (ischaemia/reperfusion). However, Ang-(1-7) [angiotensin-(1-7)], ACE2 (angiotensin I-converting enzyme 2) and the Mas receptor also play a role in renal disease models. Therefore, in the present study, we have examined the renal profile of Ang-(1-7), ACE2 and the Mas receptor in renal I/R and compared them with that of AngII, ACE and the AT1 receptor. Male Wistar rats were submitted to left nephrectomy and ischaemia (45 min) followed by reperfusion (2 or 4 h) in the right kidney. At 4 h of reperfusion, renal AngII was increased (P<0.01) and renal Ang-(1-7) was decreased substantially (P<0.05), although plasma levels of both angiotensins were unchanged. In addition, renal I/R decreased the renal mRNA expression of renin (P<0.05), AT1 receptors (P<0.001) and ACE2 (P<0.05). At 2 and 4 h of reperfusion, renal ACE activity was reduced (P<0.05). On the other hand, renal expression of the Mas receptor was greatly increased at 4 h of reperfusion (P<0.01), which was confirmed by immunohistochemical and Western blot analysis. In conclusion, increased renal expression of the Mas receptor associated with changes in the RAS (renin-angiotensin system)-related peptidases support an important role for the ACE2-Ang-(1-7)-Mas axis in AKI.

Genetic deletion of the angiotensin-(1-7) receptor Mas leads to glomerular hyperfiltration and microalbuminuria.

Angiotensin-(1-7), an active fragment of both angiotensins I and II, generally opposes the vascular and proliferative actions of angiotensin II. Here we evaluated effects of the angiotensin-(1-7) receptor Mas on renal physiology and morphology using Mas-knockout mice. Compared to the wild-type animals, Mas knockout mice had significant reductions in urine volume and fractional sodium excretion without any significant change in free-water clearance. A significantly higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow suggest that glomerular hyperfiltration occurs in the knockout mice. Histological analysis found reduced glomerular tuft diameter and increased expression of collagen IV and fibronectin in the both the mesangium and interstitium, along with increased collagen III in the interstitium. These fibrogenic changes and the renal dysfunction of the knockout mice were associated with an upregulation of angiotensin II AT1 receptor and transforming growth factor-beta mRNA. Our study suggests that Mas acts as a critical regulator of renal fibrogenesis by controlling effects transduced through angiotensin II AT1 receptors in the kidney.

Immune mediators in idiopathic nephrotic syndrome: evidence for a relation between interleukin 8 and proteinuria.

The pathogenesis of idiopathic nephrotic syndrome (INS) remains unknown. Several findings suggest a role for the immune system. This study aimed to evaluate immune mediators in INS by measuring plasma and urinary levels of transforming growth factor beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1/CCL2), regulated on activation normal T-cell expressed and secreted (RANTES/CCL5) and IL-8 (IL-8/CXCL8) in pediatric patients with INS and in age-matched healthy controls. Patients were divided according to their response to corticosteroids: steroid-sensitive (SS, n = 8), or steroid-resistant (SR, n = 24). Immune mediators were also compared in regard with disease activity (relapse and remission). Immune mediators were measured by ELISA. Plasma TGF-beta1 levels in SR patients were approximately 2.8-fold higher than control values (p < 0.05). Urinary IL-8/CXCL8 was 2.9-fold higher in INS patients in relapse (proteinuria >100 mg/m2/24 h) when compared with patients in remission (p < 0.05), and levels had a positive correlation with individual proteinuria values (p < 0.05). Urinary IL-8/CXCL8 was significantly higher in relapsed SR than in SS patients in remission. No changes in MCP-1/CCL2 and RANTES/CCL5 levels were detected. Our findings suggest that IL-8/CXCL8 and TGF-beta1 are involved in the pathogenesis of INS: IL-8/CXCL8 associated with local changes in glomerular permeability and TGF-beta1 could be related to worse response to corticosteroids.

The ATP-sensitive potassium channel blocker glibenclamide prevents renal ischemia/reperfusion injury in rats.

BACKGROUND: Renal ischemia/reperfusion (I/R) is a complex neutrophil-mediated syndrome. Adenosine-triphosphate (ATP)-sensitive potassium (K(ATP)) channels are involved in neutrophil migration in vivo. In the present study, we have investigated the effects of glibenclamide, a K(ATP) channel blocker, in renal I/R injury in rats. METHODS: The left kidney of the rats was excised through a flank incision and ischemia was performed in the contralateral kidney by total interruption of renal artery flow for 45 minutes. Renal perfusion was reestablished, and the kidney and lungs were removed for analysis of vascular permeability, neutrophil accumulation, and content of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-10] 4 and 24 hours later. Renal function was assessed by measuring creatinine, Na(+), and K(+) levels in the plasma and by determination of creatinine clearance. Drugs were administered subcutaneously after the onset of ischemia. RESULTS: Reperfusion of the ischemic kidney induced local (kidney) and remote (lung) inflammatory injury and marked renal dysfunction. Glibenclamide (20 mg/kg) significantly inhibited the reperfusion-associated increase in vascular permeability, neutrophil accumulation, increase in TNF-alpha levels and nuclear factor-kappaB (NF-kappaB) translocation. These inhibitory effects were noticed in the kidney and lungs. Moreover, glibenclamide markedly ameliorated the renal dysfunction at 4 and 24 hours. CONCLUSION: Treatment with glibenclamide is associated with inhibition of neutrophil recruitment and amelioration of renal dysfunction following renal I/R. Glibenclamide may have a therapeutic role in the treatment of renal I/R injury, such as after renal transplantation.