Kátia D. Silveira

ACE2, angiotensin-(1-7) and Mas receptor axis in inflammation and fibrosis.

Recent advances have improved our understanding of the renin‐angiotensin system (RAS). These have included the recognition that angiotensin (Ang)‐(1‐7) is a biologically active product of the RAS cascade. The identification of the ACE homologue ACE2, which forms Ang‐(1‐7) from Ang II, and the GPCR Mas as an Ang‐(1‐7) receptor have provided the necessary biochemical and molecular background and tools to study the biological significance of Ang‐(1‐7). Most available evidence supports a counter‐regulatory role for Ang‐(1‐7) by opposing many actions of Ang II on AT1 receptors, especially vasoconstriction and proliferation. Many studies have now shown that Ang‐(1‐7) by acting via Mas receptor exerts inhibitory effects on inflammation and on vascular and cellular growth mechanisms. Ang‐(1‐7) has also been shown to reduce key signalling pathways and molecules thought to be relevant for fibrogenesis. Here, we review recent findings related to the function of the ACE2/Ang‐(1‐7)/Mas axis and focus on the role of this axis in modifying processes associated with acute and chronic inflammation, including leukocyte influx, fibrogenesis and proliferation of certain cell types. More attention will be given to the involvement of the ACE2/Ang‐(1‐7)/Mas axis in the context of renal disease because of the known relevance of the RAS for the function of this organ and for the regulation of kidney inflammation and fibrosis. Taken together, this knowledge may help in paving the way for the development of novel treatments for chronic inflammatory and renal diseases.

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.

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.

Treatment with a novel chemokine-binding protein or eosinophil lineage-ablation protects mice from experimental colitis

Eosinophils are multifunctional leukocytes implicated in numerous inflammatory diseases. The present study was conducted to clarify the precise role of eosinophils in the development of colitis by using eosinophil-depleted mice and a novel chemokine-binding protein that neutralizes CCL11 action. Colitis was induced by administration of dextran sodium sulfate (DSS) to wild-type and eosinophil-deficient DeltadblGATA-1 mice. Accumulation of eosinophils in the gut of mice given DSS paralleled worsening of clinical score and weight loss. In response to DSS, DeltadblGATA-1 mice showed virtual absence of eosinophil recruitment, amelioration of clinical score, weight loss, and tissue destruction, and no lethality. There was a decrease in CXCL1 and CCL3 production and decreased neutrophil influx in the intestine of DeltadblGATA-1 mice. Transfer of bone marrow cells from wild-type mice reconstituted disease manifestation in DSS-treated DeltadblGATA-1 mice, and levels of CCL11 were increased after DSS treatment and localized to inflammatory cells. Treatment with the chemokine-binding protein evasin-4 at a dose that prevented the function of CCL11 greatly ameliorated clinical score, weight loss, overall tissue destruction, and death rates. In conclusion, the influx of eosinophils is critical for the induction of colitis by DSS. Treatment with a novel chemokine-binding protein decreased eosinophil influx and greatly ameliorated colitis, suggesting that strategies that interfere with the recruitment of eosinophils may be useful as therapy for colitis.

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.

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 β1 (TGF-β1), 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-β1 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-β1 are involved in the pathogenesis of INS: IL-8/CXCL8 associated with local changes in glomerular permeability and TGF-β1 could be related to worse response to corticosteroids.

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-1β 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.

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-1β 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.

Urinary levels of TGF β-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-β1 (TGF-β1) 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-β1, 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-β1, IL-6, and TNF-α levels in discriminating infants with nephrouropathies. No significant differences in urinary TGF- β1, IL-6, and TNF-α levels were found in the comparison between the groups. TGF-β1 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-β1 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-β1 pointed out to renal damage as indicated by reduced DMSA uptake.

Protective effects of the angiotensin type 1 receptor antagonist losartan in infection-induced and arthritis-associated alveolar bone loss

BACKGROUND AND OBJECTIVE The angiotensin type 1 (AT1) receptor has been implicated in the pathogenesis of inflammatory bone disorders. This study aimed to investigate the effect of an AT1 receptor antagonist in infection-induced and arthritis-associated alveolar bone loss in mice. MATERIAL AND METHODS Mice were subjected to Aggregatibacter actinomycetemcomitans oral infection or antigen-induced arthritis and treated daily with 10 mg/kg of the prototype AT1 antagonist, losartan. Treatment was conducted for 30 d in the infectious condition and for 17 d and 11 d in the preventive or therapeutic regimens in the arthritic model, respectively. The mice were then killed, and the maxillae, serum and knee joints were collected for histomorphometric and immunoenzymatic assays. In vitro osteoclast assays were performed using RAW 264.7 cells stimulated with A. actinomycetemcomitans lipopolysacharide (LPS). RESULTS Arthritis and A. actinomycetemcomitans infection triggered significant alveolar bone loss in mice and increased the levels of myeloperoxidase and of TRAP(+) osteoclasts in periodontal tissues. Losartan abolished such a phenotype, as well as the arthritis joint inflammation. Both arthritis and A. actinomycetemcomitans conditions were associated with the release of tumor necrosis factor alpha (TNF-α), interferon-gamma, interleukin-17 and chemokine (C-X-C motif) ligand 1 and an increased RANKL/osteoprotegerin ratio in periodontal tissues, but such expression decreased after losartan treatment, except for TNF-α. The therapeutic approach was as beneficial as the preventive one. In vitro, losartan prevented LPS-induced osteoclast differentiation and activity. CONCLUSION The blockade of AT1 receptor exerts anti-inflammatory and anti-osteoclastic effects, thus protecting periodontal tissues in distinct pathophysiological conditions of alveolar bone loss.