Angiotensin II activates the Smad pathway during epithelial mesenchymal transdifferentiation.

Epithelial to mesenchymal transdifferentiation is a novel mechanism that promotes renal fibrosis and here we investigated whether known causes of renal fibrosis (angiotensin II and transforming growth factor beta, TGFbeta) act through this pathway. We infused angiotensin II into rats for 1 day and found that it activated the Smad pathway which persisted for up to 2 weeks in chronically infused rats. Renal TGF-beta mRNA expression was increased at 3 days and its protein at 2 weeks suggesting Smad pathway activation occurred earlier than TGF-beta upregulation. In cultured human tubuloepithelial cells, angiotensin II caused a rapid activation of Smad signaling independent of TGF-beta however, Smad-dependent transcription after 1 day was TGF-beta mediated. Two weeks of angiotensin II infusion activated genes associated with epithelial mesenchymal transdifferentiation. Stimulation with angiotensin II for 3 days caused transdifferentiation of the cultured epithelial cells by TGF-beta-mediated processes; however, early changes were independent of endogenous TGF-beta. Smad7 overexpression, which blocks Smad2/3 activation, diminished angiotensin II-induced epithelial mesenchymal transdifferentiation. Our results show that angiotensin II activates the Smad signaling system by TGF-beta-independent processes, in vivo and in vitro, causing renal fibrosis.

Interactions between aldosterone and connective tissue growth factor in vascular and renal damage in spontaneously hypertensive rats.

OBJECTIVE: The aim of the present study was to investigate possible inter-relationships between connective tissue growth factor (CTGF) and aldosterone in vascular and renal damage associated with hypertension. METHOD: Spontaneously hypertensive rats (SHR) were treated with two doses (100 and 30 mg/kg per day) of the mineralocorticoid receptor antagonist eplerenone, or with antihypertensive therapy (HHR) (20 mg/kg per day hydralazine + 7 mg/kg per day hydrochlorothiazide + 0.15 mg/kg per day reserpine). RESULTS: CTGF mRNA expression and protein levels in the aorta of SHR were upregulated (P < 0.05) compared with Wistar-Kyoto rats. Both doses of eplerenone similarly and significantly diminished CTGF upregulation, correlated with amelioration of aortic remodelling and endothelium-dependent relaxations. Only high-dose eplerenone and HHR significantly reduced arterial blood pressure. HHR treatment also diminished CTGF overexpression, suggesting a blood-pressure-mediated effect in CTGF regulation. This reduction, however, was lower (P < 0.05) than that produced by eplerenone (100 mg/kg per day). The direct effect of aldosterone on vascular smooth muscle cells was also studied. Incubation of cultured vascular smooth muscle cells with aldosterone increased CTGF production in a dose-related manner, but was reduced (P < 0.05) by the mineralocorticoid receptor antagonist spironolactone. Renal CTGF mRNA and protein levels were higher in SHR than in Wistar-Kyoto rats (P < 0.05), and were similarly diminished by all treatments (P < 0.05). CONCLUSIONS: These data show that aldosterone and haemodynamic stress from elevated blood pressure levels regulate vascular and renal CTGF in SHR. The results suggest that aldosterone, through CTGF stimulation, could participate in vascular and renal structural alterations associated with hypertension, describing a novel mechanism of aldosterone in hypertensive target organ damage.

Endothelin-1, via ETA receptor and independently of transforming growth factor-beta, increases the connective tissue growth factor in vascular smooth muscle cells.

Endothelin (ET)-1 is a potent vasoconstrictor that participates in cardiovascular diseases. Connective tissue growth factor (CTGF) is a novel fibrotic mediator that is overexpressed in human atherosclerotic lesions, myocardial infarction, and experimental models of hypertension. In vascular smooth muscle cells (VSMCs), CTGF regulates cell proliferation/apoptosis, migration, and extracellular matrix (ECM) accumulation. Our aim was to investigate whether ET-1 could regulate CTGF and to investigate the potential role of ET-1 in vascular fibrosis. In growth-arrested rat VSMCs, ET-1 upregulated CTGF mRNA expression, promoter activity, and protein production. The blockade of CTGF by a CTGF antisense oligonucleotide decreased FN and type I collagen expression in ET-1-treated cells, showing that CTGF participates in ET-1-induced ECM accumulation. The ETA, but not ETB, antagonist diminished ET-1-induced CTGF expression gene and production. Several intracellular signals elicited by ET-1, via ETA receptors, are involved in CTGF synthesis, including activation of RhoA/Rho-kinase and mitogen-activated protein kinase and production of reactive oxygen species. CTGF is a mediator of TGF-beta- and angiotensin (Ang) II-induced fibrosis. In VSMCs, ET-1 did not upregulate TGF-beta gene or protein. The presence of neutralizing transforming growth factor (TGF)-beta antibody did not modify ET-1-induced CTGF production, showing a TGF-beta-independent regulation. We have also found an interrelationship between Ang II and ET-1 because the ETA antagonist diminished CTGF upregulation caused by Ang II. Collectively, our results show that, in cultured VSMCs, ET-1, independently of TGF-beta and through the activation of several intracellular signals via ETA receptors, regulates CTGF. This novel finding suggests that CTGF could be a mediator of the profibrotic effects of ET-1 in vascular diseases.

Angiotensin IV activates the nuclear transcription factor-kappaB and related proinflammatory genes in vascular smooth muscle cells.

Inflammation is a key event in the development of atherosclerosis. Nuclear factor-kappaB (NF-kappaB) is important in the inflammatory response regulation. The effector peptide of the renin angiotensin system Angiotensin II (Ang II) activates NF-kappaB and upregulates some related proinflammatory genes. Our aim was to investigate whether other angiotensin-related peptides, as the N-terminal degradation peptide Ang IV, could regulate proinflammatory factors (activation of NF-kappaB and related genes) in cultured vascular smooth muscle cells (VSMCs). In these cells, Ang IV increased NF-kappaB DNA binding activity, caused nuclear translocation of p50/p65 subunits, cytosolic IkappaB degradation and induced NF-kappaB-dependent gene transcription. Ang II activates NF-kappaB via AT1 and AT2 receptors, but AT1 or AT2 antagonists did not inhibit NF-kappaB activation caused by Ang IV. In VSMC from AT1a receptor knockout mice, Ang IV also activated NF-kappaB pathway. In those cells, the AT4 antagonist divalinal diminished dose-dependently Ang IV-induced NF-kappaB activation and prevented IkappaB degradation, but had no effect on the Ang II response, indicating that Ang IV activates the NF-kappaB pathway via AT4 receptors. Ang IV also increased the expression of proinflammatory factors under NF-kappaB control, such as MCP-1, IL-6, TNF-alpha, ICAM-1, and PAI-1, which were blocked by the AT4 antagonist. Our results reveal that Ang IV, via AT4 receptors, activates NF-kappaB pathway and increases proinflammatory genes. These data indicate that Ang IV possesses proinflammatory properties, suggesting that this Ang degradation peptide could participate in the pathogenesis of cardiovascular diseases.

Angiotensin II activates the Smad pathway in vascular smooth muscle cells by a transforming growth factor-beta-independent mechanism.

BACKGROUND: Angiotensin II (Ang II) participates in vascular fibrosis. Transforming growth factor-beta (TGF-beta) is considered the most important fibrotic factor, and Smad proteins are essential components of the TGF-beta signaling system. Our aim was to investigate whether Ang II activates the Smad pathway in vascular cells and its potential role in fibrosis, evaluating connective tissue growth factor (CTGF) and extracellular matrix (ECM) proteins. METHODS AND RESULTS: Systemic infusion of Ang II into Wistar rats increased aortic Smad2, phosphorylated-Smad2, and Smad4 expression, associated with CTGF upregulation. In growth-arrested vascular smooth muscle cells, Ang II treatment for 20 minutes caused Smad2 phosphorylation, nuclear translocation of phosphorylated-Smad2 and Smad4, and increased Smad DNA-binding activity. Ang II also caused Smad overexpression and Smad-dependent gene transcription. The AT1 antagonist losartan diminished Ang II-induced Smad activation. The blockade of endogenous TGF-beta did not modify the activation of Smad caused by Ang II. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 diminished Ang II-induced Smad2 phosphorylation. These data show that Ang II activates the Smad pathway via AT1 receptors and MAPK activation independently of TGF-beta. Transient transfection with Smad7, which interferes with receptor-mediated activation of Smad2, diminished Ang II-induced CTGF promoter activation, gene and protein expression, and fibronectin and type-1 procollagen overexpression, showing that Smad activation is involved in Ang II-induced fibrosis. CONCLUSIONS: Our results show that Ang II activates the Smad signaling system in vascular cells in vivo and in vitro. Smad proteins are involved in Ang II-induced CTGF and ECM overexpression independently of TGF-beta. This novel finding suggests that Smad activation could be involved in the profibrogenic effects of Ang II in vascular diseases.

Role of connective tissue growth factor in vascular and renal damage associated with hypertension in rats. Interactions with angiotensin II.

We have evaluated the role of connective tissue growth factor (CTGF) in vascular and renal damage associated with hypertension and possible interactions with angiotensin II (Ang II). Spontaneously hypertensive rats (SHR) were treated with either the Ang II receptor antagonist candesartan (C;2 mg/Kg(-1)/day(-1)) or antihypertensive triple therapy (TT; in mg/Kg(-1)/day(-1);20 hydralazine +7 hydrochlorothiazide +0.15 reserpine) for 10 weeks. Wistar Kyoto rats were used as a normotensive control group. Hypertension was associated with an increase in aortic media area, media-to-lumen ratio and collagen density. Kidneys from SHR showed minimum renal alterations. Aorta and renal gene expression and immunostaining of CTGF were higher in SHR. Candesartan decreased arterial pressure, aortic media area, media-to-lumen ratio and collagen density. However, although arterial pressure decrease was comparable for both treatments, TT partially reduced these parameters. Candesartan-treated rats showed lower levels of vascular CTGF expression, aortic media area, media-to-lumen ratio and collagen density than TT-treated animals. Treatments improve renal damage and reduce renal gene expression and CTGF immunostaining in SHR in a similar manner. The results show that vascular and renal damage is associated with stimulation of CTGF gene and protein content. These results also might suggest that CTGF could be one downstream mediator of Ang II in hypertension-associated organ damage in SHR.

Connective tissue growth factor is a mediator of angiotensin II-induced fibrosis.

BACKGROUND: Angiotensin II (Ang II) participates in the development of fibrosis during vascular damage. Connective tissue growth factor (CTGF) is a novel fibrotic mediator. However, the potential link between CTGF and Ang II has not been investigated. METHODS AND RESULTS: In vivo Ang II effects were studied by systemic infusion into normal rats to evaluate CTGF and extracellular matrix protein (ECM) expression by immunohistochemistry. In aorta of Ang II-infused rats, CTGF staining was markedly increased and ECM overexpression was observed. An AT1 antagonist diminished CTGF and ECM. In growth-arrested vascular smooth muscle cells, Ang II induced CTGF mRNA expression after 1 hour, remained elevated up to 24 hours, and increased CTGF protein production, which was increased up to 72 hours. The AT1 antagonist blocked Ang II-induced CTGF gene and protein expression. Early CTGF upregulation is independent of new protein synthesis. Several intracellular signals elicited by Ang II are involved in CTGF synthesis, including protein kinase C activation, reactive oxygen species, and transforming growth factor-beta endogenous production. Incubation with a CTGF antisense oligonucleotide decreased CTGF and fibronectin upregulation caused by Ang II. CONCLUSIONS: Our results show that Ang II, via AT1, increases CTGF in vascular cells both in vivo and in vitro. This novel finding suggests that CTGF may be a mediator of the profibrogenic effects of Ang II in vascular diseases.

The Rho-kinase pathway regulates angiotensin II-induced renal damage.

BACKGROUND: Angiotensin II (AngII) is a key factor in the pathogenesis of renal damage. AngII via AngII type 1 receptors activates several intracellular signaling systems, including the small guanosine triphosphatase Rho and its downstream effector Rho-dependent serine-threonine kinase (Rho-kinase). The Rho/Rho-kinase pathway contributes to inflammatory and proliferative changes observed in cardiovascular diseases. However, the data on renal diseases are scarce. The aim of this study was to investigate the effect of Rho-kinase inhibition in AngII-induced renal damage. METHODS: We used the model of systemic AngII infusion into normal rats (100 ng/kg per minute; subcutaneous osmotic minipumps), and some animals were treated with the Rho-kinase inhibitor Y-27632 (30 mg/kg per day). In the kidneys of these animals, we evaluated renal lesions, transcription factor activity (by electrophoretic mobility shift assay), and messenger RNA (by polymerase chain reaction) and protein expression levels (by Western blot and/or immunohistochemistry) of proinflammatory and profibrotic factors. RESULTS: Rats infused with AngII for three days present renal inflammatory cell infiltration and slight tubular damage, which were diminished by treatment with the Rho-kinase inhibitor Y-27632. AngII activates nuclear factor-kappaB and causes overexpression of proinflammatory factors, including cytokines (tumor necrosis factor alpha) and chemokines (monocyte chemotactic protein-1), and of profibrotic factors (connective tissue growth factor). Treatment of AngII-infused rats with Y-27632 decreases the upregulation of these proinflammatory and profibrotic mediators. CONCLUSION: These data demonstrate that the Rho-kinase pathway is involved in renal damage caused by AngII through the regulation of proinflammatory and profibrotic mediators. These results suggest that inhibition of the Rho-kinase pathway represents a novel therapy for renal diseases associated with local AngII generation.

Inflammation and angiotensin II.

Angiotensin II (AngII), the major effector peptide of renin-angiotensin system (RAS), is now recognized as a growth factor that regulates cell growth and fibrosis, besides being a physiological mediator restoring circulatory integrity. In the last few years, a large number of experimental studies has further demonstrated that AngII is involved in key events of the inflammatory process. Here, we summarize the wide variety of AngII functions and discuss them in relation with the inflammatory cascade. AngII increases vascular permeability (via the release of prostaglandins and vascular endothelial cell growth factor or rearrangement of cytoskeletal proteins) that initiates the inflammatory process. AngII could contribute to the recruitment of inflammatory cells into the tissue through the regulation of adhesion molecules and chemokines by resident cells. Moreover, AngII could directly activate infiltrating immunocompetent cells, including chemotaxis, differentiation and proliferation. Recent data also suggest that RAS activation could play a certain role even in immunologically-induced inflammation. Transcriptional regulation, predominantly via nuclear factor-kappaB (NF-kappaB) and AP-1 activation, and second mediator systems, such as endothelin-1, the small G protein (Rho) and redox-pathways are shown to be involved in the molecular mechanism by which AngII exerts those functions. Finally, AngII participates in tissue repair and remodeling, through the regulation of cell growth and matrix synthesis. In summary, recent data support the hypothesis that RAS is key mediator of inflammation. Further understanding of the role of the RAS in this process may provide important opportunities for clinical research and treatment of inflammatory diseases.

Angiotensin II regulates the synthesis of proinflammatory cytokines and chemokines in the kidney.

BACKGROUND: Emerging evidence suggests that angiotensin II (Ang II) is not only a vasoactive peptide, but also a true cytokine that regulates cell growth, inflammation and fibrosis. Many studies have demonstrated that this peptide plays an active role in the progression of renal injury. Some of Ang II-induced effects are mediated by the production of a large array of growth factors. The aim of this study was to investigate whether Ang II could regulate the expression of cytokines and chemokines in the kidney and its correlation with the Ang II-induced renal damage. METHODS: The model of Ang II-induced renal damage was done by systemic Ang II infusion into normal rats (50 ng/kg/min; subcutaneous osmotic minipumps). In addition, the implication of Ang II was investigated in a model of immune complex nephritis in rats treated with the angiotensin converting enzyme (ACE) inhibitor quinapril. The mRNA expression was analyzed by RT-PCR and/or Northern blot, and protein levels by Western blot and/or immunohistochemistry. RESULTS: Rats infused with Ang II for 3 days caused elevated renal expression of tumor necrosis factor-alpha (TNF-alpha; gene and protein levels). TNF-alpha positive cells were observed in glomeruli (mainly in endothelial cells), tubules and vessels. In rats with immune complex nephritis, the renal overexpression of TNF-alpha was diminished by the ACE inhibitor quinapril. Systemic infusion of Ang II also increased renal synthesis of cytokines (interleukin-6, IL-6) and chemokines (monocyte chemoattractant protein-1; MCP-1) that were associated with elevated tissue levels of activated nuclear factor-kappaB (NF-kappaB) and the presence of inflammatory cell infiltration. CONCLUSIONS: Ang II in vivo increases TNF-alpha production in the kidney. Ang II also up-regulates other proinflammatory mediators, including IL-6, MCP-1 and NF-kappaB, coincidentally associated to the presence of glomerular and interstitial inflammatory cells in the kidney. All these data further strengthen the idea that Ang II plays an active role in the inflammatory response in renal diseases.

Renal expression of angiotensin type 2 (AT2) receptors during kidney damage.

BACKGROUND: Activation of the renin angiotensin system has been described in pathologic conditions, including kidney damage. Angiotensin II (Ang II) acts through two receptors, AT1 and AT2. Most of the known actions of Ang II, including vasoconstriction and fibrosis, are due to AT1 activation. Recent data suggest that AT2 participates in the regulation of cell growth and renal inflammatory infiltration. Therefore, we investigated the renal expression of AT2 receptors in several models of renal injury. METHODS: Investigations were done in the following experimental models of kidney damage: systemic infusion of Ang II (inflammation), folic acid nephropathy (tubular cell death), and protein overload proteinuria. AT2 expression was determined by immunohistochemistry (protein) and reverse transcription-polymerase chain reaction (RT-PCR) (gene). RESULTS: In control animals, low levels of renal expression of AT2 were found. Ang II infusion resulted in an up-regulation of AT2 in tubular cells and de novo AT2 expression in glomeruli and vessels, associated with the presence of inflammatory cells. Acute tubular injury induced by folic acid was characterized by AT2 overexpression and apoptosis in tubular cells. Protein overload caused heavy proteinuria and tubular AT2 up-regulation. CONCLUSION: AT2 is re-expressed in pathologic conditions of kidney damage, such as inflammation, apoptosis, and proteinuria, suggesting a potential role of this receptor during renal injury.

Effect of simultaneous blockade of AT1 and AT2 receptors on the NFkappaB pathway and renal inflammatory response.

BACKGROUND: Angiotensin II (Ang II) is a cytokine that participates in the inflammatory response. The nuclear factor kappa B (NFkappaB) is involved in the regulation of many immune and inflammatory factors. Different works have shown that both angiotensin II receptor type 1 (AT1) and type 2 (AT2) receptors are involved in the NFkappaB pathway; however, some aspects remain mysterious. AT1 antagonists increased plasma Ang II levels that could bind to AT2, so understanding the clinical importance of AT2 stimulation or inhibition is an interesting unresolved point. METHODS: Experiments were done in wild-type (WT) and AT1a receptor knockout mice that received subcutaneous Ang II infusions (1000 ng/kg/min) for 3 days. Specific blockers of AT1 (losartan 10 mg/kg/day) and AT2 (PD123319 30 mg/kg/day) receptors were administered 1 day before and during Ang II infusion. NFkappaB activity was examined by electrophoretic mobility assay and inflammatory (monocyte/macrophage) cell infiltration by immunohistochemistry RESULTS: In WT mice, Ang II infusion caused renal NFkappaB activation that was partially diminished by either AT1 or AT2 antagonists. In AT1 knockout mice, Ang II also activated renal NFkappaB, which was only blocked by the AT2 antagonist. Both Ang II-infused WT and AT1 knockout mice showed inflammatory infiltration in tubulointerstitial areas that were suppressed by the AT2, but not AT1, antagonist. Combined therapy of both AT1 and AT2 antagonists blocked renal NFkappaB activation and inflammatory cell infiltration, both in WT and in AT1 knockout mice. CONCLUSION: Ang II, via AT1 and AT2 stimulation, leads to NFkappaB activation that was only blocked by combined therapy with both antagonists. The participation of AT2 receptors in the recruitment of inflammatory cells underscores the need of future studies that evaluate the clinical usefulness of this strategy.

Inhibitory effect of interleukin-1beta on angiotensin II-induced connective tissue growth factor and type IV collagen production in cultured mesangial cells.

Connective tissue growth factor (CTGF) is overexpressed in kidney diseases associated with extracellular matrix accumulation. Angiotensin II (ANG II) participates in renal fibrosis by the upregulation of growth factors, including CTGF, and extracellular matrix proteins, such as type IV collagen. During renal injury, ANG II and the macrophage-produced cytokine interleukin-1beta (IL-1beta) may be present simultaneously in the glomerular environment. However, there are no studies about the interaction between ANG II and IL-1beta in renal fibrosis. For this reason, in cultured mesangial cells (MC), we investigated whether IL-1beta could regulate ANG II-mediated collagen accumulation and the mechanisms underlying this process. In MC, CTGF is a downstream mediator of type IV collagen production induced by ANG II. IL-1beta did not increase the production of CTGF and type IV collagen but significantly inhibited ANG II-induced CTGF and type IV collagen overexpression. Moreover, IL-1beta also inhibited type IV collagen upregulation caused by exogenous recombinant CTGF. Matrix metalloproteinase-9 (MMP-9) is the main enzyme involved in type IV collagen degradation. In MC, coincubation of IL-1beta and ANG II caused a synergistic increase in MMP-9 gene expression and activity, associated with type IV collagen inhibition. The described IL-1beta effects were dependent on activation of ERK/MAPK but independent p38-MAPK, JNK, phosphatidylinositol 3-kinase/Akt, and Rho-associated kinase pathways. In summary, these data indicate that IL-1beta inhibited ANG II-mediated type IV collagen production, via CTGF downregulation, and increased type IV collagen degradation, through MMP-9 upregulation. Our in vitro data show that the proinflammatory cytokine IL-1beta abrogates ANG II-induced CTGF production, describing antagonistic activities of proinflammatory cytokines on ANG II actions.

HMG-CoA reductase inhibitors decrease angiotensin II-induced vascular fibrosis: role of RhoA/ROCK and MAPK pathways.

3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) present beneficial effects in cardiovascular diseases. Angiotensin II (Ang II) contributes to cardiovascular damage through the production of profibrotic factors, such as connective tissue growth factor (CTGF). Our aim was to investigate whether HMG-CoA reductase inhibitors could modulate Ang II responses, evaluating CTGF expression and the mechanisms underlying this process. In cultured vascular smooth muscle cells (VSMCs) atorvastatin and simvastatin inhibited Ang II-induced CTGF production. The inhibitory effect of statins on CTGF upregulation was reversed by mevalonate and geranylgeranylpyrophosphate, suggesting that RhoA inhibition could be involved in this process. In VSMCs, statins inhibited Ang II-induced Rho membrane localization and activation. In these cells Ang II regulated CTGF via RhoA/Rho kinase activation, as shown by inhibition of Rho with C3 exoenzyme, RhoA dominant-negative overexpression, and Rho kinase inhibition. Furthermore, activation of p38MAPK and JNK, and redox process were also involved in Ang II-mediated CTGF upregulation, and were downregulated by statins. In rats infused with Ang II (100 ng/kg per minute) for 2 weeks, treatment with atorvastatin (5 mg/kg per day) diminished aortic CTGF and Rho activation without blood pressure modification. Rho kinase inhibition decreased CTGF upregulation in rat aorta, mimicking statin effect. CTGF is a vascular fibrosis mediator. Statins diminished extracellular matrix (ECM) overexpression caused by Ang II in vivo and in vitro. In summary, HMG-CoA reductase inhibitors inhibit several intracellular signaling systems activated by Ang II (RhoA/Rho kinase and MAPK pathways and redox process) involved in the regulation of CTGF. Our results may explain, at least in part, some beneficial effects of statins in cardiovascular diseases.

Renal and vascular hypertension-induced inflammation: role of angiotensin II.

PURPOSE OF REVIEW: We will focus on the recent findings concerning the inflammatory response in vascular and renal tissues caused by hypertension. RECENT FINDINGS: Angiotensin II is one of the main factors involved in hypertension-induced tissue damage. This peptide regulates the inflammatory process. Angiotensin II activates circulating cells, and participates in their adhesion to the activated endothelium and subsequent transmigration through the synthesis of adhesion molecules, chemokines and cytokines. Among the intracellular signals involved in angiotensin II-induced inflammation, the production of reactive oxygen species and the activation of nuclear factor-kappaB are the best known. SUMMARY: The pharmacological blockade of angiotensin II actions, by angiotensin-converting enzyme inhibitors or angiotensin receptor antagonists, results in beneficial organ protective effects, in addition to the effects of these agents on blood pressure control, that can be explained by the blockade of the angiotensin II-induced pro-inflammatory response. These data provide a rationale for the use of blockers of the renin-angiotensin system to prevent vascular and renal inflammation in patients with hypertension.

Angiotensin II: a key factor in the inflammatory and fibrotic response in kidney diseases.

Angiotensin II (AngII) participates in the pathogenesis of renal diseases, through the regulation of two key processes inflammation and fibrosis. AT1 and AT2 are the main receptors of AngII. AT1 mediates most of the actions of AngII. This receptor regulates the expression of profibrotic factors, such as connective tissue growth factor (CTGF). The Smad signalling pathway and the Rho/Rho kinase system are two novel mechanisms involved in AngII-induced matrix regulation recently described. The role of AT2 receptors in renal pathophysiological processes is not fully elucidated. Experimental data suggest that AT2 receptors through activation of nuclear factor-kappaB participate in renal inflammatory cell recruitment. Studies in animal models of kidney injury have shown that the combined blockade of both AT1 and AT2 receptors, as well as the inhibition of the NF-kappaB pathway are necessary to stop the inflammatory process fully. On the whole, these data highlight the complex signalling systems activated by AngII and suggest novel potential targets to block fibrosis and inflammation in renal diseases.

Suppressors of cytokine signaling regulate angiotensin II-activated Janus kinase-signal transducers and activators of transcription pathway in renal cells.

Suppressors of cytokine signaling (SOCS) family is constituted by cytokine-inducible proteins that modulate receptor signal transduction via tyrosine kinases, mainly the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Differential SOCS expression was noted in renal cells that were incubated with inflammatory stimuli, but the role of SOCS in the pathogenesis of renal diseases is not yet well defined. Because angiotensin II (Ang II) plays a key role in renal disease, SOCS proteins were studied as a novel mechanism involved in the negative regulation of Ang II-mediated processes. Systemic Ang II infusion for 3 d increased the renal mRNA expression of SOCS-3 and SOCS-1. SOCS protein synthesis was found in glomerular mesangial area and tubules. In cultured mesangial cells and tubular epithelial cells, Ang II induced a rapid and transient SOCS-3 and SOCS-1 expression in parallel with JAK2 and STAT1 activation. In both cell types, overexpression of SOCS proteins prevented the STAT activation in response to Ang II. SOCS expression observed in Ang II-infused rats and in Ang II-stimulated cells was significantly inhibited by treatment with AT(1) but not AT(2) receptor antagonist and was attenuated in mesangial cells from AT(1a)-deficient mice, demonstrating the implication of AT(1) in those responses. In SOCS-3 knockdown studies, antisense oligonucleotides inhibited the expression of SOCS-3 and increased the Ang II-induced STAT activation and c-Fos/c-Jun expression, then resulting in a more severe renal damage. These results suggest that SOCS proteins may act as negative regulators of Ang II signaling in renal cells and implicate SOCS as important modulators of renal damage.

Molecular mechanisms of angiotensin II-induced vascular injury.

Blockers of the renin-angiotensin system are used in the treatment of several cardiovascular and renal diseases, including hypertension, atherosclerosis, and cardiac failure. Angiotensin II plays an essential role in the pathogenesis of these diseases through the regulation of cell growth, inflammation, and fibrosis. There are two main angiotensin II receptors, AT(1) and AT(2). The AT(1) receptor is responsible for most of the pathophysiologic actions of angiotensin II, including cell proliferation, production of growth factors and cytokines, and fibrosis. AT(2) causes antiproliferation and counteracts the cell growth induced by AT(1) activation. We review the mechanisms whereby AT(1) and AT(2) receptors elicit their respective actions. We discuss the current understanding of the signaling mechanisms involved in angiotensin II-induced vascular damage, describing the mediators (growth factors and cytokines) and intracellular signals (activation of protein kinases, transcription factors, and redox pathways) implicated in these processes, with special emphasis on novel information and open questions.

Angiotensin II increases connective tissue growth factor in the kidney.

Connective tissue growth factor (CTGF) has been described as a novel fibrotic mediator. CTGF is overexpressed in several kidney diseases and is induced by different factors involved in renal injury. Angiotensin II (AngII) participates in the pathogenesis of kidney damage, contributing to fibrosis; however, whether AngII regulates CTGF in the kidney has not been explored. Systemic infusion of AngII into normal rats for 3 days increased renal CTGF mRNA and protein levels. At day 7, AngII-infused rats presented overexpression of CTGF in glomeruli, tubuli, and renal arteries, as well as tubular injury and elevated fibronectin deposition. Only treatment with an AT(1) receptor antagonist, but not an AT(2), diminished CTGF and fibronectin overexpression and ameliorated tubular damage. In rats with immune complex nephritis, renal overexpression of CTGF was diminished by the ACE inhibitor quinapril, correlated with a diminution in fibrosis. In cultured renal cells (mesangial and tubular epithelial cells) AngII, via AT(1), increased CTGF mRNA and protein production, and a CTGF antisense oligonucleotide decreased AngII-induced fibronectin synthesis. Our data show that AngII regulates CTGF in the kidney and cultured in mesangial and tubular cells. This novel finding suggests that CTGF could be a mediator of the profibrogenic effects of AngII in the kidney.

Angiotensin III activates nuclear transcription factor-kappaB in cultured mesangial cells mainly via AT(2) receptors: studies with AT(1) receptor-knockout mice.

Nuclear factor-kappaB (NF-kappaB) regulates many genes involved in renal pathophysiologic processes. It was previously demonstrated that angiotensin II (AngII) and its amino-terminal degradation product AngIII activate NF-kappaB in mesangial cells. However, which are the Ang receptor subtypes involved in the NF-kappaB pathway and whether these Ang peptides act through the same or different receptors in mesangial cells have not been evaluated. Under the culture conditions used, quiescent rat mesangial cells expressed both AT(1) and AT(2) receptors. To investigate the receptors involved in the NF-kappaB pathway, two different approaches were used, i.e., pharmacologic studies, using specific AT(1) and AT(2) receptor antagonists and agonists, and studies in AT(1) receptor-knockout mice. In cultured rat mesangial cells, both AT(1) and AT(2) receptor antagonists inhibited AngII-induced NF-kappaB DNA binding activity, whereas NF-kappaB activation elicited by AngIII was mainly blocked by the AT(2) receptor antagonist. Similar results were observed for cytosolic IkappaBalpha degradation. An AT(2) receptor agonist also activated NF-kappaB. In AT(1) receptor-knockout murine mesangial cells, AngIII and AngII increased NF-kappaB activity and degraded cytosolic IkappaBalpha; both processes were blocked by the AT(2) receptor antagonist. These data demonstrate that, in mesangial cells, NF-kappaB activation is mediated by AT(1) and AT(2) receptors, suggesting a novel intracellular signaling mechanism for AT(2) receptors in the kidney. Some differences in Ang peptide receptor-mediated responses were also observed. AngII activates NF-kappaB via AT(1) and AT(2) receptors, whereas AngIII acts mainly via AT(2) receptors. These results suggest the potential involvement of the AngIII/AT(2) receptor/NF-kappaB pathway in pathophysiologic processes in the kidney and provide a better understanding of the renin-angiotensin system.