The renin–angiotensin system in kidney development

All components of the renin–angiotensin system (RAS) are highly expressed in the developing kidney in a pattern suggesting a role for angiotensin II in renal development. In support of this notion, pharmacological interruption of angiotensin II type‐1 (AT₁) receptor signalling in animals with an ongoing nephrogenesis produces specific renal abnormalities characterized by papillary atrophy, abnormal wall thickening of intrarenal arterioles, tubular atrophy associated with expansion of the interstitium, and a marked impairment in urinary concentrating ability. Similar changes in renal morphology and function develop also in mice with targeted inactivation of genes encoding renin, angiotensinogen, angiotensin‐converting enzyme, or both AT₁ receptor isoforms simultaneously. Taken together, these results clearly indicate that an intact signalling through AT₁ receptors is a prerequisite for normal renal development. The present report mainly reviews the renal abnormalities induced by blocking the RAS pharmacologically in experimental animal models. In addition, pathogenetic mechanisms are discussed.     

Angiotensin II type 2 receptor regulates the development of pancreatic endocrine cells in mouse embryos

Background : We previously identified a local renin‐angiotensin system (RAS) regulating the differentiation of an isolated population of human pancreatic progenitor cells. Major RAS components that regulate organogenesis have been also described in embryos; however, it is not known whether a local RAS is present in the fetal pancreas. We now hypothesize that angiotensin II type 1 (AT₁) and type 2 (AT₂) receptors are expressed in mouse embryonic pancreas and involved in regulating endocrine cell development. Results: Differential expression of AT₁ and AT₂ receptors was observed in the mouse pancreata in late embryogenesis. Systemic AT₂, but not AT₁, receptor blockade during the second transition in pancreatic development (from embryonic day 12.0 onward) reduced the β‐cell to α‐cell ratio of the neonate islets, impaired their insulin secretory function and the glucose tolerance of the pups. Studies with pancreas explants ex vivo revealed regulation by AT₂ receptors of the differentiation of pancreatic progenitors into insulin‐producing cells and of the proliferation of the differentiated cell, actions that did not result from reduced angiogenesis as a secondary effect of AT₂ receptor antagonism. Conclusions: These data revealed an AT₂ receptor‐mediated mechanism regulating pancreatic endocrine cell development in vivo. Developmental Dynamics 243:415–427, 2014. © 2013 Wiley Periodicals, Inc.     

Repression of c-fos and c-jun gene expression is not part of AT2 receptor coupled signal transduction

 The signal transduction mechanism coupled to angiotensin AT₂ receptors is still a matter of debate. Based on the findings that AT₂ receptor stimulation causes inhibition of proliferation, and that other antiproliferative agents such as transforming growth factor-β, retinoic acid, and MyoD act via repression of immediate early gene (IEG) expression, this study was aimed at elucidating whether downregulation of IEG expression is also part of the AT₂ receptor coupled signaling mechanism. Stimulation of angiotensin AT₂ receptors in the rat pheochromocytoma cell line PC12 W following pretreatment with growth factors was able to counteract growth factor induced proliferation but not to repress growth factor induced c-fos and c-jun expression; neither did AT₂ receptor stimulation cause an induction of c-fos expression. We conclude that, in contrast to other growth-inhibiting agents, the antiproliferative effect of angiotensin II via the AT₂ receptor is not mediated by repression of the immediate early genes c-fos and c-jun. Received: 11 March 1996 / Accepted: 13 October 1997     

Nongenomic action of aldosterone on colocalization of angiotensin II type 1 and type 2 receptors in rat kidney

Previous in vitro studies have demonstrated that angiotensin II type 1 and type 2 receptors (AT₁R and AT₂R) are co-localized and can form AT₁R/AT₂R dimerization in rat proximal tubular cells. Aldosterone non-genomically enhances angiotensin II receptor dimerization. We found no other in vivo studies in the literature regarding the effect of aldosterone on colocalization of AT₁R and AT₂R in whole kidney. Male Wistar rats were intraperitoneally injected with either normal saline solution (sham group) or aldosterone (experimental group). Colocalization of renal AT₁R and AT₂R proteins was examined by double immunohistochemical staining. The colocalization of AT₁R and AT₂R proteins was more prominent in the glomerulus, distal convoluted tubules, and cortical collecting ducts while colocalization was weak and diffused in the proximal convoluted tubules and peritubular capillaries in both groups. Our in vivo study showed aldosterone did not alter a constitutive colocalization of AT₁R and AT₂R proteins in the renal cortex and medulla. However, these proteins were colocalized more prominently in the renal cortex.     

Expression of angiotensin II and its receptors in the normal and hypoxic amoeboid microglial cells and murine BV-2 cells

Involvement of the local angiotensin receptor system in the central nervous system is well documented, yet its cellular localization and role in the glial cells have remained elusive. This study reports expression of angiotensin II and its receptors namely, angiotensin II receptor type 1 (AT₁) and angiotensin II receptor type 2 (AT₂) in the amoeboid microglial cells in the neonatal rat brain. In rats subjected to hypoxia, the amount of angiotensin II released in the corpus callosal tissue was reduced as revealed by enzyme immunoassay. Expression of AT₁ mRNA and protein was down-regulated after hypoxic exposure, but AT₂ was up-regulated. In BV-2 cells exposed to hypoxia for 4 h, expression of AT₁ mRNA was reduced but AT₂ was increased. These changes were further intensified respectively in LPS-stimulated microglia. Edaravone enhanced AT₁ expression but suppressed AT₂ expression significantly in lipopolysaccharide-stimulated cells. Neutralization of AT₂ with its antiserum significantly increased mRNA expression of tumor necrosis factor-α and interleukin-1β but decreased that of transforming growth factor-beta1. In conclusion, the present results suggest that AT₁ may be linked to regulation of vasodilation for increase of blood flow in hypoxic conditions, while up-regulated expression of AT₂ may reduce inflammatory responses through suppression of proinflammatory cytokines and elimination of free radicals.     

Evaluation of the brain and kidney renin-angiotensin system and oxidative stress in neonatal handled rats

The aim of this study was to test the hypothesis that the renin‐angiotensin system (RAS) components, as well as the oxidative stress system, would respond to early environmental changes. Thus, we have evaluated the effects of neonatal handling on both brain and kidney RAS and oxidative stress. Pups were divided into two groups: nonhandled and handled. The procedure consisted of handling them for 1 min/day in the first 10 days of life. On days 1, 5, and 10, animals were killed by decapitation. Blood samples were collected and the brain and kidneys were removed. Renin, AT₁, and AT₂ mRNA expression were evaluated through RT‐PCR. Angiotensin II (ANG II) serum concentration was also measured. An increased ANG II concentration, brain and kidney AT₂ mRNA expression were demonstrated. The kidney mRNA AT₁ expression was decreased. There was also a kidney lipid peroxidation increase and a brain superoxide dismutase and catalase decrease. In conclusion, handling in the neonatal period induces the activation of the angiotensinergic system, as well as modulates its mRNA receptor expression. The oxidative stress balance system seems not to be involved. © 2011 Wiley Periodicals,Inc. Dev Psychobiol 54: 706–713, 2012.     

Enhanced protection from fibrosis and inflammation in the combined absence of IL‐13 and IFN‐γ

Persistent or dysregulated IL‐13 responses are key drivers of fibrosis in multiple organ systems, and this identifies this cytokine as an important therapeutic target. Nevertheless, the mechanisms by which IL‐13 blockade leads to the amelioration of fibrosis remain unclear. Because IFN‐γ exhibits potent anti‐fibrotic activity, and IL‐4Rα signalling antagonizes IFN‐γ effector function, compensatory increases in IFN‐γ activity following IL‐13/IL‐4Rα blockade might contribute to the reduction in fibrosis. To investigate the role of IFN‐γ, we developed novel IL‐13^?/?/IFN‐γ^?/? double cytokine‐deficient mice and examined disease progression in models of type 2‐driven fibrosis. As predicted, we showed that fibrosis in the lung and liver are both highly dependent on IL‐13. We also observed increased IFN‐γ production and inflammatory activity in the tissues of IL‐13‐deficient mice. Surprisingly, however, an even greater reduction in fibrosis was observed in IL‐13/IFN‐γ double deficient mice, most notably in the livers of mice chronically infected with Schistosoma mansoni. The increased protection was associated with marked decreases in Tgfb1, Mmp12, and Timp1 mRNA expression in the tissues; reduced inflammation; and decreased expression of important pro‐inflammatory mediators such as TNF‐α. Experiments conducted with neutralizing monoclonal antibodies to IL‐13 and IFN‐γ validated the findings with the genetically deficient mice. Together, these studies demonstrate that the reduction in fibrosis observed when IL‐13 signalling is suppressed is not dependent on increased IFN‐γ activity. Instead, by reducing compensatory increases in type 1‐associated inflammation, therapeutic strategies that block IFN‐γ and IL‐13 activity simultaneously can confer greater protection from progressive fibrosis than IL‐13 blockade alone. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

IL‐33 Receptor (ST2) Signalling is Important for Regulation of Th2‐Mediated Airway Inflammation in a Murine Model of Acute Respiratory Syncytial Virus Infection

T1/ST2, an orphan receptor with homology with the interleukin (IL)‐1 receptor family, is the ligand‐binding component of the receptor for the cytokine IL‐33, a newly identified cytokine known to amplify the Th2 cell‐dominant immune responses. The function of IL‐33/ST2 signalling during respiratory syncytial virus (RSV) infection is not fully known. In this study, following intranasal infection with RSV, BALB/c mice showed a marked increase in the production of IL‐33, with an elevated expression of ST2 mRNA as well as a massive infiltration of CD45⁺ST2⁺ cells in the lungs, suggesting that during the early phase of RSV infection, IL‐33 target cells which express ST2 on cell surface, may play a critical role for the development of RSV‐induced airway inflammation. Indeed, blocking ST2 signalling using anti‐ST2 monoclonal antibody diminished not only RSV‐induced eosinophil recruitment, but also the amounts of Th2‐associated cytokines, particularly IL‐13, and Th17‐type cytokine IL‐17A in the lungs of infected mice. However, anti‐ST2 antibody treatment did not affect the production of Th1‐type cytokine IFN‐γ as well as pulmonary viral growth and clearance. These results indicate that IL‐33/ST2 signalling is involved in RSV‐induced, Th2‐associated airway inflammation but not protective immunity.     

Resolvins E1 and D1 inhibit interstitial fibrosis in the obstructed kidney via inhibition of local fibroblast proliferation

Resolvin E1 (RvE1) is a naturally occurring lipid‐derived mediator generated during the resolution of inflammation. The anti‐inflammatory effects of RvE1 have been demonstrated in a variety of disease settings; however, it is not known whether RvE1 may also exert direct anti‐fibrotic effects. We examined the potential anti‐fibrotic actions of RvE1 in the mouse obstructed kidney—a model in which tissue fibrosis is driven by unilateral ureteric obstruction (UUO), an irreversible, non‐immune insult. Administration of RvE1 (300 ng/day) to mice significantly reduced accumulation of α‐smooth muscle actin (SMA)⁺ myofibroblasts and the deposition of collagen IV on day 6 after UUO. This protective effect was associated with a marked reduction of myofibroblast proliferation on days 2, 4 and 6 after UUO. RvE1 treatment also inhibited production of the major fibroblast mitogen, platelet‐derived growth factor‐BB (PDGF‐BB), in the obstructed kidney. Acute resolvin treatment over days 2–4 after UUO also had a profound inhibitory effect upon myofibroblast proliferation without affecting the PDGF expression, suggesting a direct effect upon fibroblast proliferation. In vitro studies established that RvE1 can directly inhibit PDGF‐BB‐induced proliferation in primary mouse fibroblasts. RvE1 induced transient, but not sustained, activation of the pro‐proliferative ERK and AKT signalling pathways. Of note, RvE1 inhibited the sustained activation of ERK and AKT pathways seen in response to PDGF stimulation, thereby preventing up‐regulation of molecules required for progression through the cell cycle (c‐Myc, cyclin D) and down‐regulation of inhibitors of cell cycle progression (p21, cip1). Finally, siRNA‐based knock‐down studies showed that the RvE1 receptor, ChemR23, is required for the anti‐proliferative actions of RvE1 in cultured fibroblasts. In conclusion, this study demonstrates that RvE1 can inhibit fibroblast proliferation in vivo and in vitro, identifying RvE1 as a novel anti‐fibrotic therapy. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Central AT1 and AT2 receptors mediate chronic intracerebroventricular angiotensin II‐induced drinking in rats fed high sodium chloride diet from weaning

Intracerebroventricular (ICV) angiotensin (AIl) administration stimulates central AII receptors to induce water consumption in rats. The aim of this study was to determine the role of brain AT₁ and AT₂ receptors in mediating chronic ICV AII‐induced drinking in rats raised on normal or high sodium chloride diets from weaning. Rats were weaned at 21 days of age and placed on normal or high sodium chloride diet for 10–12 weeks. At adulthood, the animals were instrumented with brain lateral ventricular cannulas and femoral arterial catheters. Low dose chronic central AII infusion (20 ng min^?1) significantly (P < 0.05) increased water intake in both groups of rats when compared with their respective controls of 24 h artificial cerebrospinal fluid infusions. In a separate group of high sodium fed rats, coinfusion of AII with the AT₁ receptor antagonist, losartan (0.25 μg min^?1) or the AT₂ receptor blocker, PD 123319 (0.50 μg min^?1) blocked chronic ICV AII‐induced drinking. Upon reinfusion of AII water intake increased above control. Following the cessation of AII infusions, water intake returned to values not significantly different from control (P > 0.05). In contrast, in the normal sodium fed rats losartan, but not PD 123319, blocked the AII‐mediated water intake. The data demonstrate that in high sodium chloride fed rats AII stimulates both central AT₁ and AT₂ receptors to induce drinking, while in the normal sodium chloride fed rats the peptide activates the drinking response primarily by stimulation of central AT₁ receptors.     

The fish oil ingredient,docosahexaenoic acid,activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti‐inflammatory role in macrophages

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti‐inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A₂ (cPLA₂) and lipid mediators produced from cPLA₂ activation are involved in the anti‐inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA₂ and cyclooxygenase 2 (COX‐2), and cause prostaglandin E₂ (PGE₂) release in a murine macrophage cell line RAW264.7 and in human primary monocyte‐derived macrophages. DHA and GW9508 activate cPLA₂ via GPR120 receptor, G protein Gαq and scaffold protein β‐arrestin 2. Extracellular signal‐regulated kinase 1/2 activation is involved in DHA‐ and GW9508‐induced cPLA₂ activation, but not p38 mitogen‐activated protein kinase. The anti‐inflammatory role of DHA and GW9508 is in part via activation of cPLA₂, COX‐2 and production of PGE₂ as a cPLA₂ inhibitor or a COX‐2 inhibitor partially reverses the DHA‐ and GW9508‐induced inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. The cPLA₂ product arachidonic acid and PGE₂ also play an anti‐inflammatory role. This effect of PGE₂ is partially through inhibition of the nuclear factor‐κB signalling pathway and through the EP4 receptor of PGE₂ because an EP4 inhibitor or knock‐down of EP4 partially reverses DHA inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. Hence, DHA has an anti‐inflammatory effect partially through induction of PGE₂.     

The small heat‐shock protein αB‐crystallin is essential for the nuclear localization of Smad4: impact on pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the proliferation of myofibroblasts and the accumulation of extracellular matrix (ECM) in the lungs. TGF‐β1 is the major profibrotic cytokine involved in IPF and is responsible for myofibroblast proliferation and differentiation and ECM synthesis. αB‐crystallin is constitutively expressed in the lungs and is inducible by stress, acts as a chaperone and is known to play a role in cell cytoskeleton architecture homeostasis. The role of αB‐crystallin in fibrogenesis remains unknown. The principal signalling pathway involved in this process is the Smad‐dependent pathway. We demonstrate here that αB‐crystallin is strongly expressed in fibrotic lung tissue from IPF patients and in vivo rodent models of pulmonary fibrosis. We also show that αB‐crystallin‐deficient mice are protected from bleomycin‐induced fibrosis. Similar protection from fibrosis was observed in αB‐crystallin KO mice after transient adenoviral‐mediated over‐expression of IL‐1β or TGF‐β1. We show in vitro in primary epithelial cells and fibroblasts that αB‐crystallin increases the nuclear localization of Smad4, thereby enhancing the TGF‐β1–Smad pathway and the consequent activation of TGF‐β1 downstream genes. αB‐crystallin over‐expression disrupts Smad4 mono‐ubiquitination by interacting with its E3–ubiquitin ligase, TIF1γ, thus limiting its nuclear export. Conversely, in the absence of αB‐crystallin, TIF1γ can freely interact with Smad4. Consequently, Smad4 mono‐ubiquitination and nuclear export are favoured and thus TGF‐β1–Smad4 pro‐fibrotic activity is inhibited. This study demonstrates that αB‐crystallin may be a key target for the development of specific drugs in the treatment of IPF or other fibrotic diseases. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Fell‐Muir lecture: connective tissue growth factor (CCN2) – a pernicious and pleiotropic player in the development of kidney fibrosis

Connective tissue growth factor (CTGF, CCN2) is a member of the CCN family of matricellular proteins. It interacts with many other proteins, including plasma membrane proteins, modulating cell function. It is expressed at low levels in normal adult kidney cells but is increased in kidney diseases, playing important roles in inflammation and in the development of glomerular and interstitial fibrosis in chronic disease. This review reports the evidence for its expression in human and animal models of chronic kidney disease and summarizes data showing that anti‐CTGF therapy can successfully attenuate fibrotic changes in several such models, suggesting that therapies targeting CTGF and events downstream of it in renal cells may be useful for the treatment of human kidney fibrosis. Connective tissue growth factor stimulates the development of fibrosis in the kidney in many ways including activating cells to increase extracellular matrix synthesis, inducing cell cycle arrest and hypertrophy, and prolonging survival of activated cells. The relationship between CTGF and the pro‐fibrotic factor TGFβ is examined and mechanisms by which CTGF promotes signalling by the latter are discussed. No specific cellular receptors for CTGF have been discovered but it interacts with and activates several plasma membrane proteins including low‐density lipoprotein receptor‐related protein (LRP)‐1, LRP‐6, tropomyosin‐related kinase A, integrins and heparan sulphate proteoglycans. Intracellular signalling and downstream events triggered by such interactions are reviewed. Finally, the relationships between CTGF and several anti‐fibrotic factors, such as bone morphogenetic factor‐4 (BMP4), BMP7, hepatocyte growth factor, CCN3 and Oncostatin M, are discussed. These may determine whether injured tissue heals or progresses to fibrosis.     

Genetic deletion and overexpression of angiotensin II receptors

Angiotensin II receptors are essential components of the renin-angiotensin system transducing angiotensin II mediated signals across the plasma membrane of many cell types in the cardiovascular system. To date, three subtypes of angiotensin II receptors have been identified by molecular cloning, termed angiotensin II type 1 (AT_1A, AT_1B) and type 2 (AT₂) receptors. This review focuses on recent transgenic animal models which have been generated to study the in vivo significance of angiotensin receptor diversity. AT_1A receptors are the major blood pressure regulators and have a potent growth-stimulatory effect on cardiac myocytes in vivo. The AT_1B receptor subtype may participate in the control of vascular tone if AT_1A receptors are absent. AT₂ receptors are abundantly expressed during embryonic development and may also play a role in blood pressure regulation by influencing vascular development and differentiation. Received: 16 February 1998 / Accepted: 10 August 1998     

Lack of activity of 15‐epi‐lipoxin A4 on FPR2/ALX and CysLT1 receptors in interleukin‐8‐driven human neutrophil function

Neutrophil recruitment and survival are important control points in the development and resolution of inflammatory processes. 15‐epi‐lipoxin (LX)A₄ interaction with formyl peptide receptor 2 (FPR2)/ALX receptor is suggested to enhance anti‐inflammatory neutrophil functions and mediate resolution of airway inflammation. However, it has been reported that 15‐epi‐LXA₄ analogues can also bind to cysteinyl leukotriene receptor 1 (CysLT1) and that the CysLT1 antagonist MK‐571 binds to FPR2/ALX, so cross‐reactivity between FPR2/ALX and CysLT1 ligands cannot be discarded. It is not well established whether the resolution properties reported for 15‐epi‐LXA₄ are mediated through FPR2/ALX, or if other receptors such as CysLT1 may also be involved. Evaluation of specific FPR2/ALX ligands and CysLT1 antagonists in functional biochemical and cellular assays were performed to establish a role for both receptors in 15‐epi‐LXA₄‐mediated signalling and function. In our study, a FPR2/ALX synthetic peptide (WKYMVm) and a small molecule FPR2/ALX agonist (compound 43) induced FPR2/ALX‐mediated signalling, enhancing guanosine triphosphate‐gamma (GTPγ) binding and decreasing cyclic adenosine monophosphate (cAMP) levels, whereas 15‐epi‐LXA₄ was inactive. Furthermore, 15‐epi‐LXA₄ showed neither binding affinity nor signalling towards CysLT1. In neutrophils, 15‐epi‐LXA₄ showed a moderate reduction of interleukin (IL)‐8‐mediated neutrophil chemotaxis but no effect on neutrophil survival was observed. In addition, CysLT1 antagonists were inactive in FPR2/ALX signalling or neutrophil assays. In conclusion, 15‐epi‐LXA₄ is not a functional agonist or an antagonist of FPR2/ALX or CysLT1, shows no effect on IL‐8‐induced neutrophil survival and produces only moderate inhibition in IL‐8‐mediated neutrophil migration. Our data do not support an anti‐inflammatory role of 15‐epi‐LXA₄‐ FPR2/ALX interaction in IL‐8‐induced neutrophil inflammation.     

Extracellular signal regulated kinase and SMAD signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR(mRen2)27

Angiotensin (Ang) II is a key player in left ventricular (LV) remodeling and cardiac fibrosis. Its effects are thought to be transferred at least in part by mitogen-activated protein kinases (MAPK), transforming growth factor (TGF) ₁, and the Smad pathway. In this study we sought to elucidate whether Ang II related effects on LV dysfunction and fibrosis in vivo are mediated via MAPK or rather via Smad stimulation. We treated homozygous REN2 rats (7–11 weeks) with placebo, Ang II type 1 (AT₁) receptor blocker or tyrphostin A46 (TYR), an inhibitor of epidermal growth factor receptor tyrosine kinase that blocks extracellular signal-regulated kinase (ERK) activity. REN2 rats had LV hypertrophy (LVH) and LV dysfunction that progressed to heart failure between 10 and 13 weeks. Blood pressure normalized over time. Renin, N-terminal atrial natriuretic peptide (N-ANP), and ERK were activated while p38 MAPK was not. Treatment with AT₁ receptor blockade prevented LVH and right ventricular hypertrophy, normalized systolic and diastolic dP/dt, N-ANP levels, and reduced collagen apposition. Similarly, TYR reduced LVH, N-ANP levels, and collagen apposition. Myocardial ERK activation did not depend on AT₁ receptor signaling as it was not affected by AT₁ receptor blockade. TYR abolished myocardial ERK activity. Smad2 activation was inhibited by AT₁ receptor blockade but was unaltered by TYR. Ang II induced LV remodeling and fibrosis are dependent on both ERK and Smad2 activation. This process is prevented by both AT₁ receptor blockade and TYR, and therefore inhibition of either pathway is equally efficacious in restoring LV function and architecture.R.A.D.B. and S.P. contributed equally to this work