angiotensinogen and Fibrosis

Activation of the renin-angiotensin system has a pivotal role in the pathogenesis of diabetic complications. However, recent evidence suggests that it may also contribute to the development of diabetes itself. In the endocrine pancreas, all the components of an active renin-angiotensin system are present, which modulate a range of activities including local blood flow, hormone release and prostaglandin synthesis. In both types 1 and 2 diabetes, there is an up-regulation of its expression and activity in the endocrine pancreas. Whether these changes have a direct pathogenetic role or reflect a response to local stress or tissue injury remains to be established. Angiotensin-mediated increases in oxidative stress, inflammation and free fatty acids levels potentially contribute to beta-cell dysfunction in diabetes. In addition, activation of the renin-angiotensin system appears to potentiate the action of other pathogenic pathways including glucotoxicity, lipotoxicity and advanced glycation. In experimental models of type 2 diabetes, blockade of the renin-angiotensin system with angiotensin converting enzyme inhibitors or angiotensin receptor antagonists results in the improvement of islet structure and function. Moreover, the incidence of de novo diabetes appears to be significantly reduced by blockade of the renin-angiotensin system in clinical studies. At least two large controlled trials are currently underway to study the role of renin-angiotensin system in the development of diabetes. It is hoped that these studies will demonstrate the true potential of the blockade of the renin-angiotensin system for the prevention of diabetes.

Topics: Angiotensin I; Angiotensin II; Angiotensinogen; Animals; Diabetes Mellitus; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Fibrosis; Humans; Hyperglycemia; Hypoglycemia; Islets of Langerhans; Pancreas; Peptidyl-Dipeptidase A; PPAR gamma; Receptors, Angiotensin; Renin; Renin-Angiotensin System

Recent progress in adipocyte biology delineates that adipocytes not only store excess energy, but also respond to metabolic signals by secreting proteins that exert local, central, and peripheral effects. Among these adipokines are free fatty acids, plasminogen activator inhibitor-1, angiotensinogen, TNFa, leptin and adiponectin. Dysregulation of production of these adipokines and/or imbalance of their actions lead to a wide array of liver and systemic pathophysiology related to NASH such as 1) development of systemic and hepatic insulin resistance, 2) progression from benign fatty liver to steatohepatitis and 3) activation of hepatic fibrogenesis. This review deals with the emerging concept of the adipokine interrelationship with the liver.

Topics: Adipocytes; Adiponectin; Angiotensinogen; Animals; Fatty Liver; Fibrosis; Humans; Insulin Resistance; Leptin; Liver; Plasminogen Activator Inhibitor 1; Tumor Necrosis Factor-alpha

Tissue repair is a property of all vascularized tissues. A complex yet co-ordinated series of molecular and cellular events regulates repair, including its fibrogenic component that eventuates in fibrous tissue formation. This report suggests that phenotypically transformed fibroblast-like cells, termed myofibroblasts (myoFb) because they express alpha-smooth muscle actin, are responsible for collagen turnover at sites of repair. They impart extracellular matrix with metabolic and contractile activity. De novo generation of angiotensin II by myoFb at sites of repair has important autocrine and paracrine functions. Regressive, persistent and progressive forms of fibrosis are related to the fate of myoFb and the signals they generate.

Topics: Angiotensinogen; Animals; Apoptosis; Collagen; Extracellular Matrix; Fibroblasts; Fibrosis; Humans; Peptidyl-Dipeptidase A; RNA, Messenger; Wound Healing

The purpose of current study was to elucidate polyphenol tannic acid effect on renal function and activity of the renin-angiotensin system after unilateral ureteral obstruction (UUO). Male Wistar rats were divided into three groups of six randomly: 1) Sham, 2) UUO, and 3) UUO + Tannic acid. Rats in the UUO and UUO + Tannic acid groups experienced unilateral ureteral obstruction. In the Sham group, the abdominal cavity was exposed without UUO induction. In the UUO + Tannic acid group, animals received tannic acid (20 mg/kg) intraperitoneally, 6 and 12 h after clamping the left ureter and 6 and 12 h after the right nephrectomy. Blood samples were taken to measure blood urea nitrogen (BUN) and creatinine levels. Kidney tissue samples were obtained for assessment of oxidative stress, inflammatory indices and the levels of renin-angiotensin system components. Tannic acid administration significantly improved UUO-induced kidney dysfunction (serum BUN: 66.42 ± 14.414 mg/dl, p < 0.05; serum creatinine: 1.67 ± 0.258 mg/dl, p < 0.05), oxidative stress (MDA level: 95.29 ± 37.35 µmol/g tissue, p < 0.05; SOD activity: 59.82 ± 13.41 U/g protein, p < 0.01) and inflammation (renal TNF-α: 57.05 ± 15.653 pg/g tissue, p < 0.05; renal IL-6: 117.015 ± 24.076 pg/g tissue, p < 0.001). The treatment caused a reduction in the amount of renal angiotensinogen, renin and ACE genes expression compared to the UUO group (Angiotensinogen: 8.9 ± onefold, p < 0.05, Renin: 6.5 ± 1.14 fold, p < 0.05, ACE: 4.9 ± 0.64 fold, p < 0.05). Angiotensin II type 1 receptor protein levels decreased in the tannic acid-treated rats in comparison with the UUO group (0.61 ± 0.136, p < 0.05). According to the result of the current study, tannic acid considerably attenuated the complications of unilateral ureteral obstruction through renin-angiotensin system modulation. Trial registration: IR.TUMS.MEDICINE.REC.1400.802.

Topics: Angiotensinogen; Animals; Fibrosis; Kidney; Male; Rats; Rats, Wistar; Renin; Signal Transduction; Ureteral Obstruction

Components of the renin-angiotensin system, including angiotensinogen (AGT), are critical contributors to chronic kidney disease (CKD) development and progression. However, the specific role of tissue-derived AGTs in CKD has not been fully understood. To define the contribution of liver versus kidney AGT in the CKD development, we performed 5/6 nephrectomy (Nx), an established CKD model, in wild-type (WT), proximal tubule (PT)- or liver-specific AGT knockout (KO) mice. Nx significantly elevated intrarenal AGT expression and elevated blood pressure (BP) in WT mice. The increase of intrarenal AGT protein was completely blocked in liver-specific AGT KO mice with BP reduction, suggesting a crucial role for liver AGT in BP regulation during CKD. Nx-induced glomerular and kidney injury and dysfunction, as well as fibrosis, were all attenuated to a greater extent in liver-specific AGT KO mice compared with PT-specific AGT KO and WT mice. However, the suppression of interstitial fibrosis in PT- and liver-specific AGT KO mouse kidneys was comparable. Our findings demonstrate that liver AGT acts as a critical contributor in driving glomerular and tubular injury, renal dysfunction, and fibrosis progression, whereas the role of PT AGT was limited to interstitial fibrosis progression in chronic renal insufficiency. Our results provide new insights for the development of tissue-targeted renin-angiotensin system intervention in the treatment of CKD.

Topics: Angiotensinogen; Animals; Fibrosis; Kidney; Liver; Mice; Renal Insufficiency; Renal Insufficiency, Chronic; Renin-Angiotensin System

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes with prominent morbidity and mortality. At present, there are hardly any effective drugs to treat DN. Epiberberine (EPI), an isoquinoline alkaloid, has attracted considerable attention due to its anti-hyperglycemic, anti-hyperlipidemic, and anti-inflammatory functions. However, whether there is a protective effect of EPI on DN has not been reported.. The research was aimed to investigate the activities of EPI alleviating kidney damage in db/db mice and to explore its possible mechanisms.. The db/db mice and high-glucose (HG) induced glomerular mesangial cells (GMCs) were used to explore the protective effect of EPI on DN in vivo and in vitro.. The changes in fasting blood glucose, metabolic index, renal function, and histopathological morphology in db/db mice were detected to evaluate the therapeutic effect of EPI. Then, renal transcriptome and molecular docking were used to screen the key targets. Subsequently, HG-induced GMCs through mimicing the pathological changes in DN were utilized to study the renal protective effects of EPI and its potential mechanism.. The results in vivo showed that EPI administration for 8 weeks significantly alleviated diabetes-related metabolic disorders, improved renal functions, and relieved the histopathological abnormalities of renal tissue, especially renal fibrosis in db/db mice. The results in vitro showed that EPI inhibited the proliferation and induced the G2/M phase arrest of HG-induced GMCs. Moreover, a key gene Angiotensinogen (Agt) was screen out by the RNA-seq of kidney and molecular docking, and EPI reduced Agt, TGFβ1, and Smad2 expression in vitro and in vivo. Noteworthy, Agt knockdown by siRNA significantly attenuated these beneficial efficacies exerted by EPI, indicating that Agt played a crucial role in the process of EPI improving DN.. These findings suggested that EPI might be a potential drug for the treatment of DN dependent on the Agt-TGFβ/Smad2 pathway.

Topics: Angiotensinogen; Animals; Berberine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibrosis; Gene Expression Regulation; Kidney; Male; Mesangial Cells; Mice, Obese; Molecular Docking Simulation; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

To explore the relationship between the degree of renal fibrosis in patients with immunoglobulin A nephropathy (IgAN) and their levels of Apelin-12, Average Optical Density of angiotensin II type 1 receptor (AODAT1R), and angiotensinogen (AGT).. A total of 156 patients with IgAN diagnosed by renal biopsy in our hospital were selected and divided into a T0 group (54 cases), T1 group (49 cases) and T2 group (53 cases). The levels of Apelin-12, AT1R, and AGT were compared among the three groups, and the relationship between the above three indicators and degree of renal fibrosis was analyzed among patients with IgAN.. The AODAT1R and AGT level in the T2 group and T1 groups were significantly higher than those of the T0 group, and the Apelin-12 level of patients in the T2 group and T1 groups were significantly lower than that in T0 group. Significances of the same trend were observed among all the above indicators between the T2 group and T1 group. ROC curves showed that when the cutoff value of Apelin-12 was 2.36 µg/L, the area under curve (AUC), sensitivity, and specificity of T0-T1T2 were 0.889, 92.00%, and 88.00%, respectively. When the cut-off value of AODAT1R was 0.065, the AUC, sensitivity, and specificity were 0.706, 76.00%, and 76.00%, respectively, and when the cut-off value of AGT was 47.26 ng/mL, the AUC, sensitivity, and specificity were 0.899, 84.00%, and 88.00%, respectively. When the cutoff value of Apelin-12 was 0.92 µg/L, the AUC, sensitivity, and specificity of T0T1-T2 were 0.819, 84.62%, and 87.50%, respectively, and when the cutoff value of AODAT1R was 0.079, the AUC, sensitivity, and specificity were 0.699, 76.92%, and 79.17%, respectively. When the cut-off value of AGT was 92.96 ng/mL, the AUC, sensitivity, and specificity were 0.893, 84.62%, and 91.67%, respectively.. Apelin-12 decreased with disease progression, while AT1R and AGT increased. The changes of levels of Apelin-12, AT1R, and AGT have certain significance in judging the degree of renal fibrosis in patients with IgA nephropathy, and the change of level of AGT has the highest correlation with the degree of renal fibrosis.

Topics: Angiotensinogen; Fibrosis; Glomerulonephritis, IGA; Humans; Intercellular Signaling Peptides and Proteins; Receptor, Angiotensin, Type 1

This study aimed to investigate the effect of combination of high-salt intake and hypertension on renal functional and histological damage, associated with renal (pro)renin receptor [(P)RR] and AT1 receptor in rats.. Wistar Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) received regular rat chow (normal-salt diet 0.9%) or high-salt rat chow (high-salt diet 8.9%) for 6 weeks from 6 to 12 weeks of age. Systolic blood pressure, serum creatinine and blood urea nitrogen (BUN) were measured. Histological analysis of the kidney was performed. Western blot analysis was performed on the expressions of (P)RR, angiotensinogen and AT1 receptor in the kidney.. High-salt intake significantly increased systolic blood pressure in WKYs and especially in SHRs. High-salt intake significantly increased serum creatinine and BUN, and accelerated renal tubulointerstitial fibrosis and glomerular sclerosis in SHRs. High-salt intake significantly enhanced the renal tissue expressions of (P)RR, angiotensinogen and AT1 receptor in SHRs.. High-salt intake accelerates functional and histological renal damage associated with renal tissue overexpression of (P)RR and AT1 receptors in SHRs.

Topics: Angiotensinogen; Animals; Blood Pressure; Blood Urea Nitrogen; Body Weight; Creatinine; Fibrosis; Glomerulosclerosis, Focal Segmental; Hypertension; Kidney; Male; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prorenin Receptor; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Angiotensin, Type 1; Receptors, Cell Surface; Signal Transduction; Sodium Chloride, Dietary; Systole

Congestive cardiac failure has become one of the major health challenges of the 21st century and new therapies are needed to address this problem. The concentration of vasoactive intestinal peptide (VIP) in the heart has been shown to decrease as fibrosis (the pathology leading to heart failure) increases and to become undetectable in end stage cardiomyopathy. We sought to determine whether replenishment of myocardial VIP might treat myocardial fibrosis and therefore represent a new therapeutic target. Wistar Kyoto rats on a high (4.4%) salt diet were randomised to zero time control, 4 week infusion of VIP (5 pmol/kg/min) or vehicle control infusion. Myocardial VIP concentration was measured by radioimmunoassay, fibrosis was quantitated by computerised histomorphometry and changes in pro-fibrotic mediators were measured by quantitative rt-PCR. Myocardial VIP increased significantly in VIP treated rats compared with vehicle treated controls (P < 0.01) while fibrosis in the VIP treated rats was significantly lower than in both the zero time control (P < 0.05) and the vehicle infused control (P < 0.0005). Although all six profibrotic mediators which were measured increased over the 4 week experimental period VIP infusion only affected angiotensinogen (Agt) and angiotensin receptor type 1a (AT

Topics: Angiotensinogen; Animals; Biomarkers; Cardiomyopathies; Disease Models, Animal; Fibrosis; Humans; Infusions, Intravenous; Male; Myocardium; Rats; Rats, Inbred WKY; Receptor, Angiotensin, Type 1; Sodium, Dietary; Vasoactive Intestinal Peptide

Topics: Angiotensinogen; Animals; Blood Pressure; Cells, Cultured; Collagen Type IV; Disease Models, Animal; Fibrosis; Hepatocytes; Humans; Hypertension; Kidney; Lymphokines; Male; Mice; Mice, Knockout; Platelet-Derived Growth Factor; Primary Cell Culture; Up-Regulation; Ureter

Hypertension and renal injury are common complications of type 2 diabetes mellitus (T2DM). Hyperglycemia stimulates renal proximal tubular angiotensinogen (AGT) expression via elevated oxidative stress contributing to the development of high blood pressure and diabetic nephropathy. The sodium glucose cotransporter 2 (SGLT2) in proximal tubules is responsible for the majority of glucose reabsorption by renal tubules. We tested the hypothesis that SGLT2 inhibition with canagliflozin (CANA) prevents intrarenal AGT augmentation and ameliorates kidney injury and hypertension in T2DM.. We induced T2DM in New Zealand obese mice with a high fat diet (DM, 30% fat) with control mice receiving regular fat diet (ND, 4% fat). When DM mice exhibited > 350 mg/dL blood glucose levels, both DM- and ND-fed mice were treated with 10 mg/kg/day CANA or vehicle by oral gavage for 6 weeks. We evaluated intrarenal AGT, blood pressure, and the development of kidney injury.. Systolic blood pressure in DM mice (133.9 ± 2.0 mm Hg) was normalized by CANA (113.9 ± 4.0 mm Hg). CANA treatment ameliorated hyperglycemia-associated augmentation of renal AGT mRNA (148 ± 21 copies/ng RNA in DM, and 90 ± 16 copies/ng RNA in DM + CANA) and protein levels as well as elevation of urinary 8-isoprostane levels. Tubular fibrosis in DM mice (3.4 ± 0.9-fold, fibrotic score, ratio to ND) was suppressed by CANA (0.9 ± 0.3-fold). Furthermore, CANA attenuated DM associated increased macrophage infiltration and cell proliferation in kidneys of DM mice.. CANA prevents intrarenal AGT upregulation and oxidative stress and which may mitigate high blood pressure, renal tubular fibrosis, and renal inflammation in T2DM.

Topics: Angiotensinogen; Animals; Blood Glucose; Blood Pressure; Canagliflozin; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diet, High-Fat; Fibrosis; Humans; Hypertension; Kidney Tubules, Proximal; Mice; Oxidative Stress; Sodium-Glucose Transporter 2 Inhibitors; Up-Regulation

Arterial hypertrophy and interstitial fibrosis are important characteristics in kidneys of angiotensinogen-knockout (Atg. We performed renal denervation and administered the α2-adrenergic receptor (AR) antagonist, atipamezole, to Atg. Norepinephrine content in kidneys of Atg. Alpha2-AR signaling is one of the causes of persistent renal arterial hypertrophy in Atg

Topics: Adrenergic alpha-2 Receptor Antagonists; Amides; Angiotensinogen; Animals; Fibrosis; Fumarates; Hypertrophy; Japan; Kidney; Mice; Mice, Inbred ICR; Mice, Knockout; Renal Artery; Renin; Tokyo; Transforming Growth Factor beta1

Despite the progress in the treatment of HF, its prognosis remains disappointing primarily due to the fact that important subgroups of patients with HF are not sufficiently investigated. This also applies to patients with HF and background metabolic disorders, in particular, type 2 diabetes. It is known that the polymorphism of the rs699 marker of the M235T ATG gene is associated with a tendency to arterial hypertension, coronary heart disease and atrial fibrillation. A relationship was found between the polymorphism of M235T and the risk of HF development. One of the promising new biomarkers is the fibrosis marker ST2. The purpose of our study was to evaluate the role of the biomarker ST2 and the genetic polymorphism of the AT2 gene M235T in the progression of CHF and the development of adverse events in patients with concomitant type 2 diabetes. We found that patients with HFpEF and T2DM with ATG TT + MT genotype have a higher level of ST2 and a higher probability of unfavorable cardiovascular events during 24 months of observation compared with MM genotype carriers.

Topics: Aged; Angiotensinogen; Biomarkers; Blood Glucose; Cholesterol, HDL; Cholesterol, LDL; Diabetes Mellitus, Type 2; Disease Progression; Female; Fibrosis; Follow-Up Studies; Gene Expression; Heart Failure; Humans; Interleukin-1 Receptor-Like 1 Protein; Male; Middle Aged; Polymorphism, Genetic; Prognosis; Survival Analysis; Triglycerides; Walk Test

Emerging evidence suggests that chronic metabolic acidosis (CMA) may have significant implications in terms of worsening renal disease in CKD patients, but the effect of CMA on renal function and structure has not been fully elucidated.. We studied the acute and chronic consequences of an acid load (AL) on glomerular filtration rate (GFR) and renal histology in C57BL/6 mice. FITC-inulin clearance was performed at several time points; markers of renal fibrosis were studied at mRNA and protein levels; finally, kidney expression of candidate molecules triggering changes in renal function was studied.. Glomerular hyperfiltration occurred within 1-3 days from AL; after 1 week, the GFR returned to baseline and then declined progressively within 15-21 days. The GFR decline was accompanied by the onset of renal fibrosis, as shown by Masson trichrome staining. Markers of renal fibrosis, namely α-smooth muscle actin and collagen-1, increased after 1 day of acid loading in both mRNA and protein levels and remained higher than baseline for up to 21 days. Well-known mediators of renal fibrosis, including transforming growth factor (TGF)-β and the intrarenal renin-angiotensin system (RAS) axis, were increased even before the decline of the GFR.. Acid load caused hyperfiltration acutely and a progressive decline of the GFR chronically; the evidence of renal fibrosis indicates that structural and not only functional renal changes occurred. The concomitant upregulation of TGF-β and intrarenal RAS axis indicates that those factors may be potentially involved in the progression of kidney disease in this setting.

Topics: Acidosis; Actins; Ammonium Chloride; Angiotensinogen; Animals; Chronic Disease; Collagen Type I; Disease Progression; Fibrosis; Gene Expression; Glomerular Filtration Rate; Hydrochloric Acid; Kidney; Male; Mice, Inbred C57BL; Renal Insufficiency, Chronic; Renin; Renin-Angiotensin System; RNA, Messenger; Transforming Growth Factor beta

One of the major obstacles to prevent AKI-CKD transition is the lack of effective methods to follow and predict the ongoing kidney injury after an AKI episode. In the present study, we test the utility of urinary angiotensinogen (UAGT) for dynamically evaluating renal structural changes and predicting AKI-CKD progression by using both mild and severe bilateral renal ischemia/reperfusion injury mice. UAGT returns to pre-ischemic levels 14 days after mild AKI followed by kidney architecture restoration, whereas sustained increase in UAGT accompanies by ongoing renal fibrosis after severe AKI. UAGT at day 14-42 correlates with renal fibrosis 84 days after AKI. For predicting fibrosis at day 84, the area under receiver operating characteristics curve of UAGT at day 14 is 0.81. Persistent elevation in UAGT correlates with sustained activation of intrarenal renin-angiotensin system (RAS) during AKI-CKD transition. Abrogating RAS activation post AKI markedly reduced renal fibrosis, with early RAS intervention (from 14 days after IRI) more beneficial than late intervention (from 42 days after IRI) in alleviating fibrosis. Importantly, UAGT decreases after RAS intervention, and its level at day 14-28 correlates with the extent of renal fibrosis at day 42 post RAS blockade. A pilot study conducted in patients with acute tubular necrosis finds that compared with those recovered, patients with AKI-CKD progression exhibits elevated UAGT during the 3-month follow-up after biopsy. Our study suggests that UAGT enables the dynamical monitoring of renal structural recovery after an AKI episode and may serve as an early predictor for AKI-CKD progression and treatment response.

Topics: Acute Kidney Injury; Angiotensinogen; Animals; Biomarkers; Disease Progression; Fibrosis; Humans; Kidney; Male; Mice, Inbred C57BL; Predictive Value of Tests; Renal Insufficiency, Chronic; Renin-Angiotensin System; Reperfusion Injury; ROC Curve; Time Factors

Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice.. Control, hypertensive, hypercholesterolemic (Apoe(-/-)), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice.. Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.

Topics: Angiotensinogen; Animals; Aortic Valve; Aortic Valve Stenosis; Apolipoproteins E; Disease Models, Animal; Female; Fibrosis; Gene Expression Regulation; Hypercholesterolemia; Hypertension; Male; Mice, Inbred C57BL; Mice, Knockout; Plasminogen Activator Inhibitor 1; Renin; Severity of Illness Index

In angiotensin II (ANG II)-dependent hypertension, there is an angiotensin type 1 receptor-dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion in the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of two-kidney, one-clip Sprague-Dawley hypertensive rats subjected to left renal arterial clipping (0.25-mm gap). By 18-21 days, systolic arterial pressure increased to 180 ± 3 mmHg, and uAGT increased. Water intake, body weights, 24-h urine volumes, and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate were similar in clipped and nonclipped kidneys and not different from those in sham rats, indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The nonclipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in nonclipped kidneys compared with clipped and sham kidneys. AGT mRNA was 2.15-fold greater in the nonclipped kidneys compared with sham (1.0 ± 0.1) or clipped (0.98 ± 0.15) kidneys. AGT protein levels were also greater in the nonclipped kidneys. The nonclipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated ANG II levels, the greater tissue injury in the nonclipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal ANG II to stimulate AGT production and exert greater renal injury.

Topics: Angiotensinogen; Animals; Arterial Pressure; Body Weight; Drinking; Fibrosis; Hypertension, Renovascular; Immunity, Cellular; Kidney; Kidney Glomerulus; Kidney Medulla; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium

The renal renin-angiotensin system (RAS) has been implicated in the pathogenesis of diabetic nephropathy. The aim of this study was to investigate sex differences in renal renin-angiotensin system (RAS) and the roles of androgens in diabetes-associated renal injury.. Renal injury and fibrosis were studied in streptozotocin-induced diabetic rats by albuminuria and by gene expression of collagen I and fibronectin. RAS was investigated by analysing the plasma angiotensinogen (AOGEN) and renin activity (PRA) and their renal gene expression. Also, a group of diabetic rats was treated with the anti-androgen flutamide.. Albuminuria was significantly lower in diabetic females than in males (1.2 [0.8-1.5] versus 4.4 [2.2-6.1] mg/24 h, data are median [IQR] values, P < 0.05). Renal AOGEN mRNA levels were increased by diabetes in males (8.1 ± 0.8% in diabetes versus 0.8 ± 0.2% in control, P < 0.001) but not in females (1.0 ± 0.1% in diabetes versus 0.8 ± 0.1% in control, P > 0.05), as were collagen I and fibronectin mRNAs. Furthermore, AOGEN mRNA levels were strongly correlated with albuminuria (Spearman r = 0.64, 95% [CI] 0.36-0.81, P < 0.0001). Diabetes decreased PRA, renal renin mRNA and plasma AOGEN in both females and males. Anti-androgen treatment decreased albuminuria only in diabetic males without affecting the endocrine or renal RAS.. These data indicate that renal but not hepatic AOGEN or renin is positively associated with diabetic albuminuria and contribute to the sex-dependent differences in renal injury. Androgens may contribute to albuminuria in male independently of the RAS.

Topics: Albuminuria; Androgen Antagonists; Angiotensinogen; Animals; Biomarkers; Collagen Type I; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Female; Fibronectins; Fibrosis; Flutamide; Kidney; Male; Rats, Sprague-Dawley; Renin; Renin-Angiotensin System; Risk Factors; RNA, Messenger; Sex Factors

Renal cyst enlargement is associated with the activation of both the circulating and intrarenal renin-angiotensin systems. Angiotensinogen (AGT) is the substrate for renin. The aim of the present study was to determine the effect of AGT inhibition on renal cyst enlargement. An AGT antisense oligonucleotide (ASO) that selectively inhibits AGT mRNA was injected once weekly in PKD2WS25 mice [an orthologous model of human autosmal dominant polycystic kidney disease (PKD) involving mutation of the Pkd2 gene] from 4 to 16 wk of age. The AGT ASO resulted in a 40% decrease in AGT RNA in the kidney, a 60% decrease in AGT RNA in the liver, and a significant decrease in AGT protein in the kidney and serum. The AGT ASO resulted in a significant decrease in kidney size, cyst volume density, and blood urea nitrogen. The AGT ASO resulted in a significant decrease in transforming growth factor-β and interstitial fibrosis in the kidney. Mice treated with the AGT ASO had a significant decrease in proinflammatory cytokines [chemokine (C-X-C motif) ligand (CXCL)1 and IL-12] in the kidney. Cluster of differentiation (CD)36 is a scavenger receptor found on tubular cells that can activate the renin-angiotensin system. Administration of a CD36 ASO had no effect on PKD and kidney function, suggesting that the effect of the AGT ASO is independent of CD36. In summary, AGT inhibition resulted in significant decreases in kidney size and cyst volume and an improvement in kidney function in PKD mice. The AGT ASO resulted in a decrease in transforming growth factor-β, interstitial fibrosis, and the proinflammatory cytokines CXCL1 and IL-12 in the kidney.

Topics: Angiotensinogen; Animals; Blood Urea Nitrogen; CD36 Antigens; Cells, Cultured; Chemokine CXCL1; Disease Models, Animal; Down-Regulation; Female; Fibrosis; Genetic Therapy; Interleukin-12; Kidney; Male; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation; Oligonucleotides, Antisense; Polycystic Kidney, Autosomal Dominant; Recovery of Function; RNA, Messenger; Transforming Growth Factor beta; TRPP Cation Channels

The intermediate-conductance Ca(2+)-activated K(+) (KCa3.1) channels play a pivotal role in the proliferation and collagen secretion of cardiac fibroblasts. However, their contribution in cardiac fibrosis remains unknown. This study was designed to investigate whether KCa3.1 channels mediate the development of cardiac fibrosis. Pressure-overloaded rats were induced by abdominal aortic constriction and treated without or with KCa3.1 blocker (TRAM-34) or angiotensin type 1 receptor blocker (losartan) for 2 weeks. Besides the increase of blood pressure, angiotensin (Ang) II level in the plasma and myocardium, left ventricle mass and hydroxyproline concentration, myocardial hypertrophy, as well as significant collagen deposition in the perivascular regions and interstitium of the myocardium were observed in pressure-overloaded rats. The expression of leukocyte differentiation antigens (CD45 and CD3), macrophage surface marker (F4/80), tumor necrosis factor alpha, and monocyte chemotactic protein-1 (MCP-1) also significantly increased. All these alterations were prevented by losartan and TRAM-34. TRAM-34 also reduced the increase of renin and angiotensinogen in the plasma and myocardium of pressure-overloaded rats. Ang II promoted the migration of monocytes through endothelial cells and the secretion of MCP-1 from human umbilical vein endothelial cells in vitro, which was inhibited by TRAM-34. In conclusion, the present study demonstrates that TRAM-34 alleviates cardiac fibrosis induced by pressure overload, which is related to its inhibitory action on KCa3.1 channels and Ang II level. Our findings indicate that the inhibition of KCa3.1 channels may represent a novel approach of preventing the progression of cardiac fibrosis, and also add to the already developing literature of promising targets for TRAM-34.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensinogen; Animals; Aorta, Abdominal; Blood Pressure; Cardiomegaly; Cytokines; Fibrosis; Hydroxyproline; Losartan; Male; Myocardium; Potassium Channel Blockers; Pyrazoles; Rats; Rats, Sprague-Dawley; Renin; Shaw Potassium Channels

Diabetic cardiomyopathy is a cascade of complex events leading to eventual failure of the heart and cardiac fibrosis being considered as one of its major causes. miR-133a is one of the most abundantly expressed microRNAs in the heart. We investigated the role of miR-133a during severe hyperglycaemia. And, our aim was to find out what role miR-133a plays during diabetes-induced cardiac fibrosis. We saw a drastic decrease in miR-133a expression in the hearts of streptozotocin-induced diabetic animals, as measured by RT-qPCR. This decrease was accompanied by an increase in the transcriptional co-activator EP300 mRNA and major markers of fibrosis [transforming growth factor-β1, connective tissue growth factor, fibronectin (FN1) and COL4A1]; in addition, focal cardiac fibrosis assessed by Masson's trichome stain was increased. Interestingly, in diabetic mice with cardiac-specific miR-133aa overexpression, cardiac fibrosis was significantly decreased, as observed by RT-qPCR and immunoblotting of COL4A1, ELISA for FN1 and microscopic examination. Furthermore, Cardiac miR-133a overexpression prevented ERK1/2 and SMAD-2 phosphorylation. These findings show that miR-133a could be a potential therapeutic target for diabetes-induced cardiac fibrosis and related cardiac dysfunction.

Topics: Angiotensinogen; Animals; Biomarkers; Diabetes Mellitus, Experimental; E1A-Associated p300 Protein; Endothelin-1; Fibrosis; Gene Expression Regulation; Mice; MicroRNAs; Myocardium; RNA, Messenger

We investigated the relationship among oxidative stress, hypertension, renal injury, and angiotensin-converting enzyme-2 (ACE2) expression in type 1 diabetic Akita mice. Blood glucose, blood pressure, and albuminuria were monitored for up to 5 mo in adult male Akita and Akita catalase (Cat) transgenic (Tg) mice specifically overexpressing Cat, a key antioxidant enzyme in their renal proximal tubular cells (RPTCs). Same-age non-Akita littermates and Cat-Tg mice served as controls. In separate studies, adult male Akita mice (14 wk) were treated with ANG 1-7 (500 μg·kg⁻¹·day⁻¹ sc) ± A-779, an antagonist of the Mas receptor (10 mg·kg⁻¹·day⁻¹ sc), and euthanized at the age of 18 wk. The left kidneys were processed for histology and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess protein and gene expression. Urinary angiotensinogen (AGT), angiotensin II (ANG II), and ANG 1-7 were quantified by specific ELISAs. Overexpression of Cat attenuated renal oxidative stress; prevented hypertension; normalized RPTC ACE2 expression and urinary ANG 1-7 levels (both were low in Akita mice); ameliorated glomerular filtration rate, albuminuria, kidney hypertrophy, tubulointerstitial fibrosis, and tubular apoptosis; and suppressed profibrotic and proapoptotic gene expression in RPTCs of Akita Cat-Tg mice compared with Akita mice. Furthermore, daily administration of ANG 1-7 normalized systemic hypertension in Akita mice, which was reversed by A-779. These data demonstrate that Cat overexpression prevents hypertension and progression of nephropathy and highlight the importance of intrarenal oxidative stress and ACE2 expression contributing to hypertension and renal injury in diabetes.

Topics: Albuminuria; Angiotensin I; Angiotensin-Converting Enzyme 2; Angiotensinogen; Animals; Apoptosis; Catalase; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Fibrosis; Hypertension; Kidney; Kidney Tubules; Kidney Tubules, Proximal; Male; Mice; Mice, Transgenic; Oxidative Stress; Peptide Fragments; Peptidyl-Dipeptidase A

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-β1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-β-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

Topics: Angiotensin II; Angiotensinogen; Animals; Cardiomegaly; Extracellular Matrix; Fibroblasts; Fibrosis; Heterozygote; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myosin Heavy Chains; RNA-Binding Proteins; Signal Transduction; Smad3 Protein; STAT3 Transcription Factor; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta1; Ventricular Remodeling

Uremic toxins are considered to have a determinant pathological role in the progression of chronic kidney disease. The aim of this study was to define the putative pathological roles of the renal renin-angiotensin-aldosterone system (RAAS) and renal tubular epithelial-to-mesenchymal transition (EMT) in kidney fibrosis induced by (indoxyl sulfate) IS and (p-cresol sulfate) PCS.. Mouse proximal renal tubular cells (PKSV-PRs) treated with IS or PCS were used. Half-nephrectomized B-6 mice were treated with IS or PCS for 4 weeks. In the losartan treatment study, the study animal was administrated with IS+losartan or PCS+losartan for 4 weeks.. IS and PCS significantly activated the intrarenal RAAS by increasing renin, angiotensinogen, and angiotensin 1 (AT1) receptor expression, and decreasing AT2 receptor expression in vitro and in vivo. IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression. The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. IS and PCS induced the phenotype of EMT-like transition in renal tubules by increasing the expression of fibronectin and α-smooth muscle actin and decreasing the expression of E-cadherin. Losartan significantly attenuated the expression of TGF-β1 and Snail, and decreased kidney fibrosis induced by IS and PCS in vivo.. Activating the renal RAAS/TGF-β pathway has an important pathological role in chronic kidney injury caused by IS and PCS. IS and PCS may increase Snail expression and induce EMT-like transition.

Topics: Angiotensinogen; Animals; Cresols; Epithelial-Mesenchymal Transition; Fibrosis; Gene Expression Regulation; Indican; Kidney; Kidney Failure, Chronic; Kidney Tubules; Losartan; Male; Mice; Models, Biological; Receptor, Angiotensin, Type 1; Renin; Renin-Angiotensin System; Snail Family Transcription Factors; Sulfuric Acid Esters; Transcription Factors; Transforming Growth Factor beta1; Uremia

We investigated the impact of heterogeneous nuclear ribonucleoprotein F (hnRNP F) overexpression on angiotensinogen (Agt) gene expression, hypertension, and renal proximal tubular cell (RPTC) injury in high-glucose milieu both in vivo and in vitro. Diabetic Akita transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs were created, and the effects on systemic hypertension, Agt gene expression, renal hypertrophy, and interstitial fibrosis were studied. We also examined immortalized rat RPTCs stably transfected with control plasmid or plasmid containing hnRNP F cDNA in vitro. The results showed that hnRNP F overexpression attenuated systemic hypertension, suppressed Agt and transforming growth factor-β1 (TGF-β1) gene expression, and reduced urinary Agt and angiotensin II levels, renal hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg mice. In vitro, hnRNP F overexpression prevented the high-glucose stimulation of Agt and TGF-β1 mRNA expression and cellular hypertrophy in RPTCs. These data suggest that hnRNP F plays a modulatory role and can ameliorate hypertension, renal hypertrophy, and interstitial fibrosis in diabetes. The underlying mechanism is mediated, at least in part, via the suppression of intrarenal Agt gene expression in vivo. hnRNP F may be a potential target in the treatment of hypertension and kidney injury in diabetes.

Topics: Angiotensinogen; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibrosis; Gene Expression; Heterogeneous-Nuclear Ribonucleoprotein Group F-H; Hypertension; Kidney; Kidney Diseases; Mice; Mice, Transgenic; Renin-Angiotensin System; Transforming Growth Factor beta1

Renal dysfunction due to acute rejection (AR), acute tubular necrosis, or calcineurin inhibitors toxicity is related to development of interstitial fibrosis/tubular atrophy (IF/TA) and graft survival. Determination of serum creatinine (sCr) displays poor sensitivity as a marker for early detection of graft dysfunction. Kidney biopsy is an accurate but invasive procedure for the diagnosis. The levels of urinary mRNA of genes that regulate epithelial-mesenchymal transition (EMT) can reflect early damage and detect the development of IF/TA. Repeated studies of these genes can provide noninvasive information about the evolution of the graft, facilitating early diagnosis and treatment.. To analyze the relationships between early and 1-year graft evolution in relation to gene expression of EMT biomarkers.. Seventy-one kidney transplant recipients were monitored during 1 year recording analytical, clinical, and histological (if available) data. We determined RNA gene expression of EMT, angiotensinogen, E-cadherin, N-cadherin, transforming growth factor (TGF) beta and bone morphogenetic patients 7 (BMP7).. At 3 months, angiotensinogen (mean [standard deviation]), (2.42 [.66] versus 8.58 [3.24]; P = .017) and N-cadherin (0.59 [0.26] versus 3.15 [1.35]; P = .016) discriminate a good evolution from AR episodes BMP-7 discriminated a good evolution versus AR (0.72 [0.29] versus 4.53 [2.23]; P = .006) and delayed graft function versus AR (1.14 [0.79] versus 4.53 [2.23]; P = .049). After 1 year, the ratio TGF-beta/BMP7 discriminated patients with an sCr > 1.5 mg/dL (6614.6 [1063.6] versus 3378.7 [1019]; P = .034). There was a positive correlation between urinary and tissue TGF-beta [r = 59; P = .003].. The expression of studied genes reverting EMT at 3 months postransplantation showed differences in initial graft evolution. At 1 year, the TGF-beta/BMP7 ratio suggested activation of EMT, possible early marker of renal dysfunction.

Topics: Angiotensinogen; Antigens, CD; Bone Morphogenetic Protein 7; Cadherins; Creatinine; Delayed Graft Function; Epithelial-Mesenchymal Transition; Fibrosis; Gene Expression Regulation; Genetic Markers; Humans; Kidney; Kidney Diseases; Kidney Transplantation; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Treatment Outcome

The renin-angiotensin system plays an important role in the etiology of cardiovascular diseases. Three transgenic mouse lines overexpressing rat angiotensinogen (rAOGEN) were generated. The aim of our study was to characterize the originally undescribed second transgenic line TGM(rAOGEN)102. The transgene tissue distribution and expression of brain natriuretic peptide and collagen type III were investigated by ribonuclease protection assay. Catheter measurements of blood pressure and cardiac function were performed in anesthetized mice. End-organ fibrosis was further assessed by van Gieson staining. In line TGM(rAOGEN)102, the rAOGEN transgene was mainly expressed in liver and brain but could also be detected in hearts, kidneys, and lungs. Transgenic mice developed excessive chronic hypertension compared with their wild-type littermates. The rise of blood pressure was paralleled by cardiac hypertrophy, impaired cardiac function, and increased expression of brain natriuretic peptide. Pronounced fibrosis was detected in the hearts, lungs, and kidneys of transgenic mice. Our data indicate that overexpression of rAOGEN in mice leads to excessive hypertension, cardiac hypertrophy, impaired heart function, and pronounced fibrosis. Thus, this line TGM(rAOGEN)102 provides a new model to study hypertension-mediated end-organ damage and to evaluate new antihypertensive or cardioprotective drugs.

Topics: Angiotensinogen; Animals; Antihypertensive Agents; Blood Pressure; Cardiomegaly; Fibrosis; Genes; Heart; Hypertension; Kidney; Male; Mice; Mice, Transgenic; Rats; Rats, Transgenic; Renin-Angiotensin System

To determine how angiotensin II (Ang II) contributes to renal interstitial fibrosis, the inflammatory response, and tubular cell apoptosis and proliferation in unilateral ureteral obstruction using mice genetically deficient in angiotensinogen (Agt(-/-)).. The left kidney of wild-type mice (WT; C57BL/6) and Agt(-/-) mice was obstructed for 2 weeks, and then both kidneys were harvested. The serum Ang II levels were determined by radioimmunoassay. The expression of transforming growth factor-beta in renal tissue was assessed using enzyme-linked immunosorbent assay. The renal tissue was stained with Masson's trichrome. Renal tubular proliferation and apoptosis was detected by immunostaining for proliferating cell nuclear antigen and single-stranded DNA, respectively. Interstitial leukocyte and macrophage infiltration was investigated by immunostaining for CD45 and F4/80, respectively.. The serum Ang II levels in the Agt(-/-) mice were significantly lower than those in the WT mice (P < .01), and tissue transforming growth factor-beta in the obstructed kidney of Agt(-/-) mice was significantly lower than that in WT mice (P < .05). Interstitial collagen deposition was significantly lower in the Agt(-/-) obstructed kidneys than in the WT obstructed kidneys (P < .01). Tubular proliferation was significantly greater and tubular apoptosis was significantly lower in the Agt(-/-) obstructed kidneys than in the WT obstructed kidneys (P < .01 and P < .01, respectively). Interstitial infiltration by leukocytes and macrophages was significantly lower in the Agt(-/-) obstructed kidneys than in the WT obstructed kidneys (P < .01 and P < .01, respectively).. The results of the present study support the targeting of Ang II as a reasonable approach by which to prevent renal tissue damage in unilateral ureteral obstruction.

Topics: Angiotensin II; Angiotensinogen; Animals; Female; Fibrosis; Kidney Diseases; Mice; Mice, Inbred C57BL; Ureteral Obstruction

Hepatic stellate cells express all components of the renin-angiotensinogen (AGT) system and secrete active angiotensin II. Animal studies provided evidence that angiotensin II stimulates the accumulation of extracellular matrix by enhancing transforming growth factor beta1 production. A functional genetic alteration in the human AGT promoter (c.1-44G>A) has been linked to accelerated progression of fibrosis in hepatitis C virus infection.. We enrolled 2154 patients with chronic liver disease of various etiologies, including 1286 individuals with chronic hepatitis C virus infection as well as 207 healthy volunteers. We performed genotyping for two AGT variants, c.1-44G>A and c.803T>C (p.M268T), by melting curve analysis using fluorescence resonance energy transfer probes.. Allele frequencies and genotype distributions of both variants did not differ between patients and controls. Genotype frequencies of the c.1-44G>A variant were GG 31.0%, GA 45.6%, and AA 23.4% in patients and GG 30.0%, GA 47.8%, and AA 22.2% in controls. The genotype frequencies of p.M268T, which is in strong linkage disequilibrium with c.1-44G>A, were MM 30.8%, MT 45.5%, and TT 23.4% in patients and MM 29.0%, MT 48.8%, and TT 22.2% in controls. Both variants were associated with neither higher stages of fibrosis nor requirement for liver transplantation in any of the diagnosis subgroups. Particularly, these genetic alterations were not associated with progressive fibrosis in chronic HCV infection.. In contrast to previous reports, both AGT variants do not predispose to the progression of fibrosis in chronic liver disease.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Angiotensinogen; Case-Control Studies; Disease Progression; Female; Fibrosis; Gene Frequency; Genetic Variation; Genotype; Hepatic Stellate Cells; Hepatitis C, Chronic; Humans; Linkage Disequilibrium; Liver Diseases; Liver Diseases, Alcoholic; Male; Middle Aged; Risk Factors; Young Adult

The intrarenal renin-angiotensin system (RAS) plays a key role in the development of diabetic nephropathy. Recently, we showed that combination therapy with an AT(1) receptor blocker (ARB) and an activated vitamin D analog produced excellent synergistic effects against diabetic nephropathy, as a result of blockade of the ARB-induced compensatory renin increase. Given the diversity of vitamin D analogs, here we used a pro-drug vitamin D analog, doxercalciferol (1alpha-hydroxyvitamin D(2)), to further test the efficacy of the combination strategy in long-term treatment. Streptozotocin-induced diabetic DBA/2J mice were treated with vehicle, losartan, doxercalciferol (0.4 and 0.6 microg/kg), or losartan and doxercalciferol combinations for 20 wk. Vehicle-treated diabetic mice developed progressive albuminuria and glomerulosclerosis. Losartan alone moderately ameliorated kidney injury, with renin being drastically upregulated. A similar therapeutic effect was seen with doxercalciferol alone, which markedly suppressed renin and angiotensinogen expression. The losartan and doxercalciferol combination most effectively prevented albuminuria, restored glomerular filtration barrier structure, and dramatically reduced glomerulosclerosis in a dose-dependent manner. These effects were accompanied by blockade of intrarenal renin upregulation and ANG II accumulation. These data demonstrate an excellent therapeutic potential for doxercalciferol in diabetic renal disease and confirm the concept that blockade of the compensatory renin increase enhances the efficacy of RAS inhibition and produces synergistic therapeutic effects in combination therapy.

Topics: Albuminuria; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensinogen; Animals; Cytokines; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Ergocalciferols; Fibrosis; Glomerular Basement Membrane; Inflammation Mediators; Losartan; Macrophages; Mice; Mice, Inbred DBA; Podocytes; Renin; Renin-Angiotensin System; Time Factors; Vitamins

An understanding of the cellular physiology of cardiac myocytes (MCs) and non-myocytes (NMCs) may help to explain the mechanisms underlying cardiac hypertrophy. Despite numerous studies using MC/NMC co-culture systems, it is difficult to precisely evaluate the influence of each cell type because of the inherent cellular heterogeneity of such a system. Here we developed a co-culture system using Wistar rat neonatal MCs and NMCs isolated by discontinuous Percoll gradient and adhesion separation methods and cultured on either side of insert well membranes. Co-culture of MCs and NMCs resulted in significant increases in [3H]-leucine incorporation by MCs, in the amount of protein synthesized by MCs, and in the secretion of natriuretic peptides, while the addition of MCs to NMC cultures significantly reduced [3H]-thymidine incorporation by NMCs. Interestingly, the percentage of the brain natriuretic peptide (BNP) component of total natriuretic peptide secreted (atrial natriuretic peptide+BNP) increased as the number of NMCs placed in the MC/NMC co-culture system increased. However, MCs did not affect production of angiotensin II (Ang II) by NMCs or secretion of endothelin-1 and transforming growth factor-beta1 into the MC/NMC co-culture system. This finding was supported by the anti-hypertrophic and anti-fibrotic actions of RNH6270, an active form of olmesartan, on MCs in the MC/NMC co-culture system and on NMCs that may synthesize Ang II in the heart. The present data indicate that cardiac fibrosis may not only facilitate MC hypertrophy (possibly through the local angiotensin system) but may also change particular pathophysiological properties of MCs, such as the secretory pattern of natriuretic peptides.

Topics: Angiotensin II; Angiotensinogen; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cell Separation; Cells, Cultured; Coculture Techniques; Diuretics, Osmotic; Endothelin-1; Fibrosis; Leucine; Mannitol; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Peptidyl-Dipeptidase A; Rats; Rats, Wistar; Receptors, Angiotensin; Renin; Thymidine; Transforming Growth Factor beta1; Tritium

Patients with glycogen storage disease type Ia (GSD-Ia) develop renal disease of unknown etiology despite intensive dietary therapies. This renal disease shares many clinical and pathological similarities to diabetic nephropathy. We studied the expression of angiotensinogen, angiotensin type 1 receptor, transforming growth factor-beta1, and connective tissue growth factor in mice with GSD-Ia and found them to be elevated compared to controls. While increased renal expression of angiotensinogen was evident in 2-week-old mice with GSD-Ia, the renal expression of transforming growth factor-beta and connective tissue growth factor did not increase for another week; consistent with upregulation of these factors by angiotensin II. The expression of fibronectin and collagens I, III, and IV was also elevated in the kidneys of mice with GSD-Ia, compared to controls. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities including tubular basement membrane thickening, tubular atrophy, tubular dilation, and multifocal interstitial fibrosis. Our results suggest that activation of the angiotensin system has an important role in the pathophysiology of renal disease in patients with GSD-Ia.

Topics: Angiotensin II; Angiotensinogen; Angiotensins; Animals; Connective Tissue Growth Factor; Extracellular Matrix; Fibrosis; Glucose-6-Phosphatase; Glycogen Storage Disease Type I; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Mice; Mice, Mutant Strains; Receptor, Angiotensin, Type 1; Transforming Growth Factor beta1

Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline in renal function. Although several inflammatory mediators have been implicated in the pathophysiology of renal obstruction, the contribution of TNF-alpha to obstruction-induced fibrosis and renal dysfunction has not been thoroughly evaluated. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1) every 84 h. The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were analyzed for TNF-alpha expression (ELISA), macrophage infiltration (ED-1 staining), transforming growth factor-beta(1) expression (ELISA, RT-PCR), collagen I and IV activity (Western Blot, immunohistochemistry), alpha-smooth muscle actin accumulation (immunohistochemistry, Western blot analysis), and angiotensinogen expression (Western blot). In a separate arm, the glomerular filtration rate (inulin clearance) of rats subjected to unilateral ureteral obstruction in the presence of either vehicle or PEG-sTNFR1 was determined. Renal obstruction induced increased tissue TNF-alpha and transforming growth factor-beta(1) levels, collagen I and IV activity, interstitial volume, alpha-smooth muscle actin accumulation, angiotensinogen expression, and renal dysfunction, whereas treatment with PEG-sTNFR1 significantly reduced each of these markers of renal fibrosis. These results demonstrate that TNF-alpha mediates obstruction-induced renal fibrosis and identify TNF-alpha neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.

Topics: Actins; Angiotensinogen; Animals; Biomarkers; Blotting, Western; Collagen Type I; Collagen Type IV; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Fibrosis; Half-Life; Immunohistochemistry; Kidney Cortex; Kidney Diseases; Kinetics; Macrophages; Male; Molecular Weight; Polyethylene Glycols; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor, Type I; Recombinant Proteins; Solubility; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Ureter; Ureteral Obstruction

We previously reported that a high-sodium diet activates the local renin-angiotensin-aldosterone system (RAAS) in cardiovascular tissues of Dahl salt-sensitive hypertensive (DS) rats. Angiotensin-converting enzyme 2 (ACE2) is a novel regulator of blood pressure (BP) and cardiac function. The effect of blockade of aldosterone or angiotensin II (Ang II) on cardiac angiotensinogen and ACE2 in DS rats is unknown.. The BP, plasma renin activity (PRA), plasma aldosterone concentration (PAC), heart weight, endothelium-dependent relaxation (EDR), and messenger RNA (mRNA) levels of collagen III, angiotensinogen, ACE, and ACE2 in the heart were measured in DS rats and in Dahl salt-resistant (DR) rats fed high or low salt diets. The rats were treated orally with or without eplerenone (100 mg/kg/d), candesartan (10 mg/kg/d), or both drugs combined for 8 weeks.. A high salt diet increased BP (140%), heart/body weight (132%), and collagen III mRNA levels (146%) and decreased PRA and PAC concomitant with increased expression of cardiac angiotensinogen mRNA and decreased mRNA levels of ACE2 in DS rats. Eplerenone or candesartan significantly decreased the systolic BP from 240 +/- 5 mm Hg to 164 +/- 4 mm Hg or to 172 +/- 10 mm Hg, respectively (P < .05). Eplerenone or candesartan partially improved heart/body weight and cardiac fibrosis, improved EDR and decreased cardiac ACE and angiotensinogen mRNA levels in DS rats. Candesartan increased ACE2 mRNA levels in the heart. Combination therapy normalized BP and further improved cardiac hypertrophy, fibrosis, and EDR.. In DS rats, blockade of aldosterone or Ang II protects cardiac hypertrophy and fibrosis by inactivation of the local RAAS in the heart.

Topics: Aldosterone; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme 2; Angiotensinogen; Animals; Benzimidazoles; Biphenyl Compounds; Cardiomegaly; Endothelium, Vascular; Eplerenone; Fibrosis; Hypertension; Male; Mineralocorticoid Receptor Antagonists; Myocardium; Peptidyl-Dipeptidase A; Rats; Rats, Inbred Dahl; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; RNA, Messenger; Spironolactone; Tetrazoles

Magnesium deficiency promotes vasoconstriction and myocardial damage. Recent studies provide evidence that Ang II mobilizes intracellular Mg through AT1 receptor-mediated pathways. We tested the hypothesis of whether magnesium supplementation prevents Ang II-induced myocardial damage and induction of the profibrotic connective tissue growth factor (CTGF).. Four-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGR) were given dietary magnesium supplementation (0.6%) for 3 weeks. Control dTGR and normotensive Sprague-Dawley (SD) rats received normal diet (Mg 0.2%). Histopathological, immunohistochemical and mRNA analysis were used to detect the treatment-related effects of dietary magnesium in dTGR.. Magnesium (Mg) supplementation decreased blood pressure, ameliorated cardiac hypertrophy, protected against the development of Ang II-induced myocardial damage and increased serum ionized Mg2+ concentration (all variables P < 0.05). There was no difference in serum ionized Mg2+ concentration between dTGR and SD rats. Myocardial connective tissue growth factor (CTGF) mRNA and protein expressions were increased by 300% in dTGR (P < 0.05), especially in areas with myocardial infarctions and vascular inflammation. Magnesium supplementation prevented Ang II-induced myocardial CTGF overexpression (P < 0.05). Magnesium supplementation also improved the therapeutic effects of the calcineurin inhibitor tacrolimus, which produced marked hypomagnesemia when given as monotherapy.. Our findings suggest a salutary effect for magnesium supplementation in the treatment of Ang II-induced myocardial complications.

Topics: Angiotensin II; Angiotensinogen; Animals; Animals, Genetically Modified; Blood Pressure; Cardiomegaly; Connective Tissue Growth Factor; Dietary Supplements; Fibrosis; Humans; Immediate-Early Proteins; Immunosuppressive Agents; Intercellular Signaling Peptides and Proteins; Magnesium; Male; Myocardium; Rats; Rats, Sprague-Dawley; Renin; RNA, Messenger; Tacrolimus

Aldosterone and angiotensin (Ang) II both may cause organ damage. Circulating aldosterone is produced in the adrenals; however, local cardiac synthesis has been reported. Aldosterone concentrations depend on the activity of aldosterone synthase (CYP11B2). We tested the hypothesis that reducing aldosterone by inhibiting CYP11B2 or by adrenalectomy (ADX) may ameliorate organ damage. Furthermore, we investigated how much local cardiac aldosterone originates from the adrenal gland.. We investigated the effect of the CYP11B2 inhibitor FAD286, losartan, and the consequences of ADX in transgenic rats overexpressing both the human renin and angiotensinogen genes (dTGR). dTGR-ADX received dexamethasone and 1% salt. Dexamethasone-treated dTGR-salt served as a control group in the ADX protocol. Untreated dTGR developed hypertension and cardiac and renal damage and had a 40% mortality rate (5/13) at 7 weeks. FAD286 reduced mortality to 10% (1/10) and ameliorated cardiac hypertrophy, albuminuria, cell infiltration, and matrix deposition in the heart and kidney. FAD286 had no effect on blood pressure at weeks 5 and 6 but slightly reduced blood pressure at week 7 (177+/-6 mm Hg in dTGR+FAD286 and 200+/-5 mm Hg in dTGR). Losartan normalized blood pressure during the entire study. Circulating and cardiac aldosterone levels were reduced in FAD286 or losartan-treated dTGR. ADX combined with dexamethasone and salt treatment decreased circulating and cardiac aldosterone to barely detectable levels. At week 7, ADX-dTGR-dexamethasone-salt had a 22% mortality rate compared with 73% in dTGR-dexamethasone-salt. Both groups were similarly hypertensive (190+/-9 and 187+/-4 mm Hg). In contrast, cardiac hypertrophy index, albuminuria, cell infiltration, and matrix deposition were significantly reduced after ADX (P<0.05).. Aldosterone plays a key role in the pathogenesis of Ang II-induced organ damage. Both FAD286 and ADX reduced circulating and cardiac aldosterone levels. The present results show that aldosterone produced in the adrenals is the main source of cardiac aldosterone.

Topics: Adrenal Glands; Adrenalectomy; Aldosterone; Angiotensin II; Angiotensinogen; Animals; Animals, Genetically Modified; Cytochrome P-450 CYP11B2; Enzyme Inhibitors; Fibrosis; Heart Diseases; Humans; Inflammation; Kidney Diseases; Losartan; Mineralocorticoid Receptor Antagonists; Myocardium; Rats; Renin

The brain renin-angiotensin-aldosterone system (RAAS) plays a major role in cardiac remodeling after myocardial infarction (MI). To assess the contribution of the brain RAAS in the activation of the cardiac RAAS post-MI, transgenic (TG) rats deficient in brain angiotensinogen and Wistar rats with intracerebroventricular (ICV) infusion of spironolactone were studied. An MI was induced by acute coronary artery ligation. TG and control Sprague-Dawley (SD) rats were followed for 8 weeks and Wistar rats for 6 weeks. Infarct sizes, % of left ventricle (LV) area, were in the 30-33% range. In SD rats at 8 weeks post-MI, internal circumference, interstitial and perivascular fibrosis, cardiomyocyte diameter in the LV and right ventricle (RV), laminin and fibronectin in the LV, and lung weights were increased. Aldosterone was increased markedly in both the LV and RV at 8 weeks post-MI. In TG rats, the MI-induced increases of RV internal circumference and weight were prevented and increases of lung weight and LV internal circumference were significantly inhibited. In TG rats, the post-MI increases of interstitial fibrosis and cardiomyocyte diameter were prevented in septum and RV and significantly inhibited in the peri-infarct zone of the LV. The increases in perivascular fibrosis, laminin and fibronectin were prevented in the LV. In TG rats, cardiac aldosterone did not increase. In Wistar rats at 6 weeks post-MI, aldosterone was markedly increased in the LV, but not in the RV. This increase was prevented by ICV infusion of spironolactone. These findings support the pivotal role of locally produced angiotensin II in the brain in cardiac remodeling post-MI. The brain RAAS appears to activate a cascade of events, among others an increase in cardiac aldosterone, which play a major role in cardiac remodeling post-MI.

Topics: Aldosterone; Angiotensinogen; Animals; Animals, Genetically Modified; Brain; Cell Size; Fibronectins; Fibrosis; Heart Ventricles; Immunohistochemistry; Laminin; Male; Myocardial Infarction; Myocytes, Cardiac; Organ Size; Radioimmunoassay; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Spironolactone; Time Factors; Ventricular Function, Left; Ventricular Remodeling

Tissue fibrosis in systemic sclerosis (SSc) is attributed to excessive deposition of extracellular matrix components produced by fibroblasts in skin lesions. Angiotensin II (Ang II), a vasoconstrictive peptide, is reported to have profibrotic activity as a result of induction of the extracellular matrix. The aim of the present study was to examine the expression of Ang II and its type 1 (AT(1)) and type 2 (AT(2)) receptors in affected skin and dermal fibroblasts from patients with SSc and to study the role of Ang II in collagen production by SSc dermal fibroblasts.. Levels of Ang II in sera from SSc patients and normal subjects were measured by a solid-phase immobilized-epitope immunoassay. Expression of angiotensinogen (Angt) in the skin was evaluated by immunohistochemistry. Expression of Angt, AT(1), and AT(2) in cultured dermal fibroblasts was analyzed by reverse transcription-polymerase chain reaction and immunohistochemistry. Levels of type I procollagen produced by cultured dermal fibroblasts were measured by enzyme-linked immunosorbent assay.. Serum Ang II levels in patients with diffuse cutaneous SSc were significantly higher than those in patients with limited cutaneous SSc and in healthy donors. Immunohistochemical and immunoblotting analyses showed that Angt was present in skin from SSc patients, but not in normal skin. Angt messenger RNA (mRNA) was expressed in fibroblasts from patients with diffuse cutaneous SSc who had high levels of serum Ang II, but not in normal fibroblasts. AT(1) mRNA expression was found in both SSc and normal fibroblasts, whereas AT(2) mRNA was found only in SSc fibroblasts. Exogenous Ang II augmented the production of type I procollagen and transforming growth factor beta1 by cultured fibroblasts via activation of AT(1).. Aberrant Ang II production may be involved in tissue fibrosis through excessive production of the extracellular matrix components in SSc dermal fibroblasts. This suggests that the use of AT(1) receptor antagonists may be a novel strategy for the treatment of tissue fibrosis in SSc patients.

Topics: Adult; Aged; Angiotensin II; Angiotensinogen; Autocrine Communication; Blotting, Western; Cathepsin D; Cells, Cultured; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; Immunohistochemistry; Male; Middle Aged; Procollagen; Receptor, Angiotensin, Type 1; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Adrenomedullin (AM) is a novel vasodilating peptide thought to have important effects on cardiovascular function. The aim of this study was to assess the activity of endogenous AM in the cardiovascular system using AM knockout mice.. Mice heterozygous for an AM-null mutation (AM+/-) and their wild-type littermates were subjected to aortic constriction or angiotensin II (Ang II) infusion. The resultant cardiovascular stress led to increases in heart weight/body weight ratios, left ventricular wall thickness, and perivascular fibrosis, as well as expression of genes encoding angiotensinogen, ACE, transforming growth factor-beta, collagen type I, brain natriuretic peptide, and c-fos. In addition, renal damage characterized by decreased creatinine clearance with glomerular sclerosis was noted. In all cases, the effects were significantly more pronounced in AM+/- mice. Hearts from adult mice subjected to aortic constriction showed enhanced extracellular signal-regulated kinase (ERK) activation, as did cardiac myocytes from neonates treated acutely with Ang II. Again the effect was more pronounced in AM+/- mice, which showed increases in cardiac myocyte size, protein synthesis, and fibroblast proliferation. ERK activation was suppressed by protein kinase C inhibition to a greater degree in AM+/- myocytes. In addition, treatment of cardiac myocytes with recombinant AM suppressed Ang II-induced ERK activation via a protein kinase A-dependent pathway.. Endogenous AM exerts a protective effect against stress-induced cardiac hypertrophy via protein kinase C- and protein kinase A-dependent regulation of ERK activation. AM may thus represent a useful new tool for the treatment of cardiovascular disease.

Topics: Adrenomedullin; Angiotensin II; Angiotensinogen; Animals; Aorta, Abdominal; Cardiomegaly; Collagen Type I; Constriction; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Fibrosis; Gene Expression Regulation; Genes, fos; Genes, Lethal; Glomerulosclerosis, Focal Segmental; Heterozygote; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Natriuretic Peptide, Brain; Peptides; Peptidyl-Dipeptidase A; Protein Kinase C; Proto-Oncogene Proteins c-fos; Transforming Growth Factor beta; Ventricular Remodeling

The renin-angiotensin system (RAS) seems to play a pivotal role in progression of immunoglobulin A (IgA) nephropathy (IgAN). Accordingly, in patients with IgAN a relationship between the RAS and the fibrogenic cascade triggered by transforming growth factor-beta1 (TGF-beta1) should be observed. This study was carried out to obtain deeper insight into the regulation of RAS and the interaction with TGF-beta1 in the diseased kidney.. Twenty renal biopsies from IgAN patients and five from renal cancer patients (controls) were analyzed in both microdissected glomerular and tubulointerstitial compartments by reverse transcription-polymerase chain reaction (RT-PCR). All patients had normal renal function. The expression of the following genes was determined: angiotensinogen (Agtg), renin, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 and type II (AT1 and AT2 receptors), TGF-beta1, collagen IV (Coll IV), alpha-smooth muscle actin (alpha-SMA). Quantitative data were confirmed for TGF-beta1 and ACE genes by real-time PCR. Results. RAS genes were overexpressed in IgAN patients vs. control subjects. There was no difference between glomerular and tubulointerstitial RAS gene expression levels. On the contrary, the overactivation of fibrogenic cascade genes (TGF-beta1, Coll IV, alpha-SMA) in the tubulointerstitium was observed (TGF-beta1, glomerular 0.14 +/- 0.10 SD; tubulointerstial 0.34 +/- 0.20; P = 0.000) (alpha-SMA, glomerular 0.08 +/- 0.07; tubulointerstitial 0.35 +/- 0.19; P = 0.000) (Coll IV, glomerular 0.12 +/- 0.11; tubulointerstitial 0.22 +/- 0.10; P = 0.03). This fibrogenic cascade seems to be triggered by RAS as indicated by statistically significant correlations between the expression of their respective genes. A direct relationship between the putative Ang II activity and the expression of AT receptor genes was found in the tubulointerstitium, whereas in the glomeruli this relationship was negative. In the interstitium, statistically significant positive relationships emerged between interstitial infiltrates and the gene expression of Agtg, AT1 receptor, Coll IV, and TGF-beta1.. This study demonstrates that a tight regulation of the intrarenal RAS exists in IgAN and that it follows the general rules disclosed in animal models. Moreover, the RAS seems to be activated early in the diseased kidney and it appears that such activation drives inflammation and a parallel stimulation of the TGF-beta fibrogenic loop, particularly at the tubulointerstitial level.

Topics: Adult; Angiotensin II; Angiotensinogen; Case-Control Studies; Collagen Type IV; Fibrosis; Gene Expression; Gene Expression Regulation; Glomerulonephritis, IGA; Humans; Kidney; Kidney Glomerulus; Kidney Tubules; Male; Middle Aged; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Renin-Angiotensin System; Transforming Growth Factor beta; Transforming Growth Factor beta1

Guanylyl cyclase (GC)-A, a natriuretic peptide receptor, lowers blood pressure and inhibits the growth of cardiac myocytes and fibroblasts. Angiotensin II (Ang II) type 1A (AT1A), an Ang II receptor, regulates cardiovascular homeostasis oppositely. Disruption of GC-A induces cardiac hypertrophy and fibrosis, suggesting that GC-A protects the heart from abnormal remodeling. We investigated whether GC-A interacts with AT1A signaling in the heart by target deletion and pharmacological blockade or stimulation of AT1A in mice.. We generated double-knockout (KO) mice for GC-A and AT1A by crossing GC-A-KO mice and AT1A-KO mice and blocked AT1 with a selective antagonist, CS-866. The cardiac hypertrophy and fibrosis of GC-A-KO mice were greatly improved by deletion or pharmacological blockade of AT1A. Overexpression of mRNAs encoding atrial natriuretic peptide, brain natriuretic peptide, collagens I and III, transforming growth factors beta1 and beta3, were also strongly inhibited. Furthermore, stimulation of AT1A by exogenous Ang II at a subpressor dose significantly exacerbated cardiac hypertrophy and dramatically augmented interstitial fibrosis in GC-A-KO mice but not in wild-type animals.. These results suggest that cardiac hypertrophy and fibrosis of GC-A-deficient mice are partially ascribed to an augmented cardiac AT1A signaling and that GC-A inhibits AT1A signaling-mediated excessive remodeling.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Angiotensinogen; Animals; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiomegaly; Collagen; Fibrosis; Gene Targeting; Guanylate Cyclase; Heart Rate; Heart Ventricles; Hypertension; Imidazoles; Mice; Mice, Knockout; Myocardium; Natriuretic Peptide, Brain; Olmesartan Medoxomil; Organ Size; Peptidyl-Dipeptidase A; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Tetrazoles; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Ventricular Remodeling

Hypertension-induced damage of kidney and heart is of major clinical relevance, but its pathophysiology is only partially understood. As there is considerable evidence for involvement of angiotensin II, we generated a new mouse model by breeding angiotensinogen (AOGEN) deficient mice with transgenic animals expressing the rat AOGEN gene only in brain and liver. This genetic manipulation overcame the hypotension of AOGEN-deficient mice and even caused hypertension indistinguishable in its extent from the parent transgenic mice with an intact endogenous AOGEN gene. In contrast to normal mice, however, crossbred animals lacked detectable expression of AOGEN in kidney and heart. As a consequence they showed markedly reduced cardiac hypertrophy and fibrosis. Furthermore, hypertension-induced alterations in kidney histology and function were less pronounced in crossbred mice than in equally hypertensive animals expressing AOGEN locally. The dysmorphogenesis observed in kidneys from AOGEN-deficient mice was absent in mice expressing this gene only in liver and brain. Our results support an important role of local AOGEN expression in hypertension-induced end-organ damage but not in the development of the kidney.

Topics: Angiotensinogen; Animals; Brain; Cardiomegaly; Disease Models, Animal; Fibrosis; Heart Diseases; Hypertension; Kidney Diseases; Liver; Mice; Mice, Transgenic; Myocardium; Organ Size; Organ Specificity; Renin-Angiotensin System

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Angiotensinogen; Animals; Disease Models, Animal; Fibrosis; Kidney Diseases; Mice; Plasminogen Activator Inhibitor 1; Renin-Angiotensin System; Transforming Growth Factor beta; Ureteral Obstruction

Earlier work from this laboratory found that fibroblasts isolated from fibrotic human lung [human interstitial pulmonary fibrosis (HIPF)] secrete a soluble inducer(s) of apoptosis in alveolar epithelial cells (AECs) in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995]. The cultured human fibroblast strains most active in producing the apoptotic activity contained high numbers of stellate cells expressing alpha-smooth muscle actin, a myofibroblast marker. The apoptotic activity eluted from gel-filtration columns only in fractions corresponding to proteins. Western blotting of the protein fraction identified immunoreactive angiotensinogen (ANGEN), and two-step RT-PCR revealed expression of ANGEN by HIPF fibroblasts but not by normal human lung fibroblasts. Specific ELISA detected angiotensin II (ANG II) at concentrations sixfold higher in HIPF-conditioned medium than in normal fibroblast-conditioned medium. Pretreatment of the concentrated medium with purified renin plus purified angiotensin-converting enzyme (ACE) further increased the ELISA-detectable ANG II eightfold. Apoptosis of AECs in response to HIPF-conditioned medium was completely abrogated by the ANG II receptor antagonist saralasin (50 microg/ml) or anti-ANG II antibodies. These results identify the protein inducers of AEC apoptosis produced by HIPF fibroblasts as ANGEN and its derivative ANG II. They also suggest a mechanism for AEC death adjacent to HIPF myofibroblasts [B. D. Uhal, I. Joshi, C. Ramos, A. Pardo, and M. Selman. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1192-L1199, 1998].

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Angiotensinogen; Antibodies; Apoptosis; Blotting, Western; Captopril; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibrosis; Flow Cytometry; Gene Expression; Humans; Peptidyl-Dipeptidase A; Pulmonary Alveoli; Saralasin

Recent works from this laboratory demonstrated potent inhibition of Fas-induced apoptosis in alveolar epithelial cells (AECs) by the angiotensin-converting enzyme (ACE) inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998] and induction of dose-dependent apoptosis in AECs by purified angiotensin (ANG) II [R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos and B. D. Uhal. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L885-L889, 1999]. These findings led us to hypothesize that the synthesis and binding of ANG II to its receptor might be involved in the induction of AEC apoptosis by Fas. Apoptosis was induced in the AEC-derived human lung carcinoma cell line A549 or in primary AECs isolated from adult rats with receptor-activating anti-Fas antibodies or purified recombinant Fas ligand, respectively. Apoptosis in response to either Fas activator was inhibited in a dose-dependent manner by the nonthiol ACE inhibitor lisinopril or the nonselective ANG II receptor antagonist saralasin, with maximal inhibitions of 82 and 93% at doses of 0.5 and 5 microg/ml, respectively. In both cell types, activation of Fas caused a significant increase in the abundance of mRNA for angiotensinogen (ANGEN) that was unaffected by saralasin. Transfection with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of Fas-stimulated apoptosis by 70% in A549 cells and 87% in primary AECs (both P < 0.01). Activation of Fas increased the concentration of ANG II in the serum-free extracellular medium 3-fold in primary AECs and 10-fold in A549 cells. Apoptosis in response to either Fas activator was completely abrogated by neutralizing antibodies specific for ANG II (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by Fas requires a functional renin-angiotensin system in the target cell. They also suggest that therapeutic control of AEC apoptosis is feasible through pharmacological manipulation of the local renin-angiotensin system.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Angiotensinogen; Animals; Antibodies; Antisense Elements (Genetics); Apoptosis; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; fas Receptor; Fibrosis; Gene Expression; Humans; Lisinopril; Lung Neoplasms; Male; Membrane Glycoproteins; Neutralization Tests; Peptidyl-Dipeptidase A; Pulmonary Alveoli; Rats; Rats, Wistar; Receptors, Angiotensin; Renin-Angiotensin System; RNA, Messenger; Signal Transduction; Transfection; Tumor Cells, Cultured