Transcriptome Analysis of Human Reninomas as an Approach to Understanding Juxtaglomerular Cell Biology.

Renin, a key component in the regulation of blood pressure in mammals, is produced by the rare and highly specialized juxtaglomerular cells of the kidney. Chronic stimulation of renin release results in a recruitment of new juxtaglomerular cells by the apparent conversion of adjacent smooth muscle cells along the afferent arterioles. Because juxtaglomerular cells rapidly dedifferentiate when removed from the kidney, their developmental origin and the mechanism that explains their phenotypic plasticity remain unclear. To overcome this limitation, we have performed RNA expression analysis on 4 human renin-producing tumors. The most highly expressed genes that were common between the reninomas were subsequently used for in situ hybridization in kidneys of 5-day-old mice, adult mice, and adult mice treated with captopril. From the top 100 genes, 10 encoding for ligands were selected for further analysis. Medium of human embryonic kidney 293 cells transfected with the mouse cDNA encoding these ligands was applied to (pro)renin-synthesizing As4.1 cells. Among the ligands, only platelet-derived growth factor B reduced the medium and cellular (pro)renin levels, as well as As4.1 renin gene expression. In addition, platelet-derived growth factor B-exposed As4.1 cells displayed a more elongated and aligned shape with no alteration in viability. This was accompanied by a downregulated expression of α-smooth muscle actin and an upregulated expression of interleukin-6, suggesting a phenotypic shift from myoendocrine to inflammatory. Our results add 36 new genes to the list that characterize renin-producing cells and reveal a novel role for platelet-derived growth factor B as a regulator of renin-synthesizing cells.

Differences in cell-type-specific responses to angiotensin II explain cardiac remodeling differences in C57BL/6 mouse substrains.

Despite indications that hearts from the C57BL/6N and C57BL/6J mouse substrains differ in terms of their contractility and their responses to stress-induced overload, no information is available about the underlying molecular and cellular mechanisms. We tested whether subacute (48 hours) and chronic (14 days) administration of angiotensin II (500 ng/kg per day) had different effects on the left ventricles of male C57BL/6J and C57BL/6N mice. Despite higher blood pressure in C57BL/6J mice, chronic angiotensin II induced fibrosis and increased the left ventricular weight/body weight ratio and cardiac expression of markers of left ventricular hypertrophy to a greater extent in C57BL/6N mice. Subacute angiotensin II affected a greater number of cardiac genes in C57BL/6N than in C57BL/6J mice. Some of the most prominent differences were observed for markers of (1) macrophage activation and M2 polarization, including 2 genes (osteopontin and galectin-3) whose inactivation was reported as sufficient to prevent angiotensin II-induced myocardial fibrosis; and (2) fibroblast activation. These differences were confirmed in macrophage- and fibroblast-enriched populations of cells isolated from the hearts of experimental mice. When testing F2 animals, the amount of connective tissue present after chronic angiotensin II administration did not cosegregate with the inactivation mutation of the nicotinamide nucleotide transhydrogenase gene from C57BL/6J mice, thus discounting its possible contribution to differences in cardiac remodeling. However, expression levels of osteopontin and galectin-3 were cosegregated in hearts from angiotensin II-treated F2 animals and may represent endophenotypes that could facilitate the identification of genetic regulators of the cardiac fibrogenic response to angiotensin II.

General lysosomal hydrolysis can process prorenin accurately.

Renin, an aspartyl protease that catalyzes the rate-limiting step of the renin-angiotensin system, is first synthesized as an inactive precursor, prorenin. Prorenin is activated by the proteolytic removal of an amino terminal prosegment in the dense granules of the juxtaglomerular (JG) cells of the kidney by one or more proteases whose identity is uncertain but commonly referred to as the prorenin-processing enzyme (PPE). Because several extrarenal tissues secrete only prorenin, we tested the hypothesis that the unique ability of JG cells to produce active renin might be explained by the existence of a PPE whose expression is restricted to JG cells. We found that inducing renin production by the mouse kidney by up to 20-fold was not associated with the concomitant induction of candidate PPEs. Because the renin-containing granules of JG cells also contain several lysosomal hydrolases, we engineered mouse Ren1 prorenin to be targeted to the classical vesicular lysosomes of cultured HEK-293 cells, where it was accurately processed and stored. Furthermore, we found that HEK cell lysosomes hydrolyzed any artificial extensions placed on the protein and that active renin was extraordinarily resistant to proteolytic degradation. Altogether, our results demonstrate that accurate processing of prorenin is not restricted to JG cells but can occur in classical vesicular lysosomes of heterologous cells. The implication is that renin production may not require a specific PPE but rather can be achieved by general hydrolysis in the lysosome-like granules of JG cells.

Local angiotensin II aggravates cardiac remodeling in hypertension.

Angiotensin II (ANG II) contributes to hypertension, cardiac hypertrophy, fibrosis, and dysfunction; however, it is difficult to separate the cardiac effect of ANG II from its hemodynamic action in vivo. To overcome the limitations, we used transgenic mice with cardiac-specific expression of a transgene fusion protein that releases ANG II from cardiomyocytes (Tg-ANG II) and treated them with deoxycorticosterone acetate (DOCA)-salt to suppress their systemic renin-angiotensin system. Using this unique model, we tested the hypothesis that cardiac ANG II, acting on the angiotensin type 1 receptor (AT(1)R), increases inflammation, oxidative stress, and apoptosis, accelerating cardiac hypertrophy and fibrosis. Male Tg-ANG II mice and their nontransgenic littermates (n-Tg) were uninephrectomized and divided into the following three groups: 1) vehicle-treated normotensive controls; 2) DOCA-salt; and 3) DOCA-salt + valsartan (AT(1)R blocker).Under basal conditions, systolic blood pressure (SBP) and cardiac phenotypes were similar between strains. In DOCA-salt hypertension, SBP increased similarly in both n-Tg and Tg-ANG II, and cardiac function did not differ between strains; however, Tg-ANG II had 1) greater ventricular hypertrophy as well as interstitial and perivascular fibrosis; 2) a higher number of deoxynucleotidyl-transferase-mediated dUTP nick end labeling-positive cells and infiltrating macrophages; 3) increased protein expression of NADPH oxidase 2 and transforming growth factor-ß(1); and 4) downregulation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (Akt) phosphorylation. Valsartan partially reversed these effects in Tg-ANG II but not in n-Tg. We conclude that, when hemodynamic loading conditions remain unchanged, cardiac ANG II does not alter heart size or cardiac functions. However, in animals with hypertension, cardiac ANG II, acting via AT(1)R, enhances inflammation, oxidative stress, and cell death (most likely via downregulation of PI 3-kinase and Akt), contributing to cardiac hypertrophy and fibrosis.

The angiotensin-converting enzyme 2/angiogenesis-(1-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension.

RATIONALE: An activated vasoconstrictive, proliferative, and fibrotic axis of the renin angiotensin system (angiotensin-converting enzyme [ACE]/angiotensin [Ang]II/AngII type 1 receptor) has been implicated in the pathophysiology of pulmonary fibrosis (PF) and pulmonary hypertension (PH). The recent discovery of a counterregulatory axis of the renin angiotensin system composed of ACE2/Ang-(1-7)/Mas has led us to examine the role of this vasoprotective axis on such disorders. OBJECTIVES: We hypothesized that Ang-(1-7) treatment would exert protective effects against PF and PH. METHODS: Lentiviral packaged Ang-(1-7) fusion gene or ACE2 cDNA was intratracheally administered into the lungs of male Sprague Dawley rats. Two weeks after gene transfer, animals received bleomycin (2.5 mg/kg). In a subsequent study, animals were administered monocrotaline (MCT, 50 mg/kg). MEASUREMENTS AND MAIN RESULTS: In the PF study, bleomycin administration resulted in a significant increase in right ventricular systolic pressure, which was associated with the development of right ventricular hypertrophy. The lungs of these animals also exhibited excessive collagen deposition, decreased expression of ACE and ACE2, increased mRNA levels for transforming growth factor ß and other proinflammatory cytokines, and increased protein levels of the AT1R. Overexpression of Ang-(1-7) significantly prevented all the above-mentioned pathophysiological conditions. Similar protective effects were also obtained with ACE2 overexpression. In the PH study, rats injected with MCT developed elevated right ventricular systolic pressure, right ventricular hypertrophy, right ventricular fibrosis, and pulmonary vascular remodeling, all of which were attenuated by Ang-(1-7) overexpression. Blockade of the Mas receptor abolished the beneficial effects of Ang-(1-7) against MCT-induced PH. CONCLUSIONS: Our observations demonstrate a cardiopulmonary protective role for the ACE2/Ang-(1-7)/Mas axis in the treatment of lung disorders.

Circulating rather than cardiac angiotensin-(1-7) stimulates cardioprotection after myocardial infarction.

BACKGROUND: Angiotensin (Ang)-(1-7) attenuates the development of heart failure. In addition to its local effects on cardiovascular tissue, Ang-(1-7) also stimulates bone marrow, which harbors cells that might complement the therapeutic effect of Ang-(1-7). We studied the effects of Ang-(1-7) either produced locally in the heart or subcutaneously injected during the development of heart failure induced by myocardial infarction (MI) and explored the role of cardiovascular progenitor cells in promoting the effects of this heptapeptide. METHODS AND RESULTS: Effects of Ang-(1-7) on bone marrow-derived mononuclear cells in rodents, particularly endothelial progenitor cells, were investigated in vitro and in vivo in rats, in mice deficient for the putative Ang-(1-7) receptor Mas, and in mice overexpressing Ang-(1-7) exclusively in the heart. Three weeks after MI induction through permanent coronary artery occlusion, effects of Ang-(1-7) either produced locally in the heart or injected into the subcutaneous space were investigated. Ang-(1-7) stimulated proliferation of endothelial progenitor cells isolated from sham or infarcted rodents. The stimulation was blunted by A779, a Mas receptor blocker, or by Mas deficiency. Infusion of Ang-(1-7) after MI increased the number of c-kit- and vascular endothelial growth factor-positive cells in infarcted hearts, inhibited cardiac hypertrophy, and improved cardiac function 3 weeks after MI, whereas cardiomyocyte-derived Ang-(1-7) had no effect. CONCLUSIONS: Our data suggest circulating rather than cardiac Ang-(1-7) to be beneficial after MI. This beneficial effect correlates with a stimulation of cardiac progenitor cells in vitro and in vivo. This characterizes the heptapeptide as a promising new tool in stimulating cardiovascular regeneration under pathophysiological conditions.

Physiological genomics identifies estrogen-related receptor alpha as a regulator of renal sodium and potassium homeostasis and the renin-angiotensin pathway.

Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor highly expressed in the kidney, an organ playing a central role in blood pressure regulation through electrolyte homeostasis and the renin-angiotensin system. Physiological analysis revealed that, relative to wild-type mice, ERRalpha null mice are hypotensive despite significant hypernatremia, hypokalemia, and slight hyperreninemia. Using a combination of genome-wide location analysis and expression profiling, we demonstrate that ERRalpha regulates the expression of channels involved in renal Na(+) and K(+) handling (Scnn1a, Atp1a1, Atp1b1) and altered in Bartter syndrome (Bsnd, Kcnq1). In addition, ERRalpha regulates the expression of receptors implicated in the systemic regulation of blood pressure (Ghr, Gcgr, Lepr, Npy1r) and of genes within the renin-angiotensin pathway (Ren1, Agt, Ace2). Our study thus identifies ERRalpha as a pleiotropic regulator of renal control of blood pressure, renal Na(+)/K(+) homeostasis, and renin-angiotensin pathway and suggests that modulation of ERRalpha activity could represent a potential avenue for the management of hypertension.

Angiotensin II controls occludin function and is required for blood brain barrier maintenance: relevance to multiple sclerosis.

The blood-brain barrier (BBB) restricts molecular and cellular trafficking between the blood and the CNS. Although astrocytes are known to control BBB permeability, the molecular determinants of this effect remain unknown. We show that angiotensinogen (AGT) produced and secreted by astrocytes is cleaved into angiotensin II (AngII) and acts on type 1 angiotensin receptors (AT1) expressed by BBB endothelial cells (ECs). Activation of AT1 restricts the passage of molecular tracers across human BBB-derived ECs through threonine-phosphorylation of the tight junction protein occludin and its mobilization to lipid raft membrane microdomains. We also show that AGT knock-out animals have disorganized occludin strands at the level of the BBB and a diffuse accumulation of the endogenous serum protein plasminogen in the CNS, compared with wild-type animals. Finally, we demonstrate a reduction in the number of AGT-immunopositive perivascular astrocytes in multiple sclerosis (MS) lesions, which correlates with a reduced expression of occludin similarly seen in the CNS of AGT knock-out animals. Such a reduction in astrocyte-expressed AGT and AngII is dependent, in vitro, on the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma. Our study defines a novel physiological role for AngII in the CNS and suggests that inflammation-induced downregulation of AngII production by astrocytes is involved in BBB dysfunction in MS lesions.

The C-terminal region of the proprotein convertase 1/3 (PC1/3) exerts a bimodal regulation of the enzyme activity in vitro.

The proprotein convertase PC1/3 preferentially cleaves its substrates in the dense core secretory granules of endocrine and neuroendocrine cells. Similar to most proteinases synthesized first as zymogens, PC1/3 is synthesized as a larger precursor that undergoes proteolytic processing of its signal peptide and propeptide. The N-terminally located propeptide has been shown to be essential for folding and self-inhibition. Furthermore, PC1/3 also possesses a C-terminal region (CT-peptide) which, for maximal enzymatic activity, must also be cleaved. To date, its role has been documented through transfection studies in terms of sorting and targeting of PC1/3 and chimeric proteins into secretory granules. In this study, we examined the properties of a 135-residue purified bacterially produced CT-peptide on the in vitro enzymatic activity of PC1/3. Depending on the amount of CT-peptide used, it is shown that the CT-peptide increases PC1/3 activity at low concentrations (nm) and decreases it at high concentrations (microm), a feature typical of an activator. Furthermore, we show that, contrary to the propeptide, the CT-peptide is not further cleaved by PC1/3 although it is sensitive to human furin activity. Based on these results, it is proposed that PC1/3, through its various domains, is capable of controlling its enzymatic activity in all regions of the cell that it encounters. This mode of self-control is unique among members of all proteinases families.

Role of cardiac overexpression of ANG II in the regulation of cardiac function and remodeling postmyocardial infarction.

ANG II has a clear role in development of cardiac hypertrophy, fibrosis, and dysfunction. It has been difficult, however, to determine whether these actions are direct or consequences of its systemic hemodynamic effects in vivo. To overcome this limitation, we used transgenic mice with cardiac-specific expression of a transgene fusion protein that releases ANG II from cardiomyocytes (Tg-ANG II-cardiac) without involvement of the systemic renin-angiotensin system and tested whether increased cardiac ANG II accelerates remodeling and dysfunction postmyocardial infarction (MI), whereas those mice show no evidence of cardiac hypertrophy under the basal condition. Male 12- to 14-wk-old Tg-ANG II-cardiac mice and their wild-type littermates (WT) were subjected to sham-MI or MI by ligating the left anterior descending coronary artery for 8 wk. Cardiac ANG II levels were approximately 10-fold higher in Tg-ANG II-cardiac mice than their WT, whereas ANG II levels in plasma and other tissues did not differ between strains. Systolic blood pressure and heart rate were similar between groups with or without MI. In sham-MI, Tg-ANG II-cardiac mice had increased collagen deposition and decreased capillary density. The differences between strains became more pronounced after MI. Although cardiac function was well preserved in the Tg-ANG II-cardiac mice with sham-MI, cardiac remodeling and dysfunction post-MI were more severe than WT. Our results demonstrate that, independent of systemic hemodynamic effects, cardiac ANG II may act locally in the heart, causing interstitial fibrosis in sham-MI and accelerating deterioration of cardiac dysfunction and remodeling post-MI.

Renal function in transgenic rats expressing an angiotensin-(1-7)-producing fusion protein.

Transgenic rats [TGR(A1-7)3292] present a chronic 2.5-fold increase in plasma Angiotensin-(1-7) [Ang-(1-7)] concentration. In the present study, we investigated the effects of this chronic elevation on renal function, vasopressin levels, kidney morphology, expression of Ang-(1-7) and vasopressin receptors in TGR(A1-7)3292. Urine volume and water intake were measured for 24 h. At the end of this period, plasma and urine samples were collected to evaluate renal function parameters and circulating vasopressin levels. Expression of renal V2 receptors and Mas was assessed by ribonuclease protection assay. Renal slices were processed for histological analysis. The urine flow of TGR(A1-7)3292 was significantly lower in comparison with Sprague-Dawley rats. The reduced urine volume of TGR(A1-7)3292 was accompanied by a significant increase in urinary osmolality and decrease free water clearance. Glomerular filtration rate, urinary sodium and potassium excretion were similar in both strains. No significant changes were observed in vasopressin levels as well as in V2 receptor and Mas mRNA expression in renal tissue. No changes in kidney structure of TGR(A1-7)3292 were detected. These data suggest that changes in circulating renin-angiotensin system produced by chronic increase of Ang-(1-7) levels can lead to adjustments in the water balance that are independent of vasopressin release and V2 receptor expression.

Angiotensin II-dependent chronic hypertension and cardiac hypertrophy are unaffected by gp91phox-containing NADPH oxidase.

The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient mice were crossed with transgenic mice expressing active human renin in the liver (TTRhRen). TTRhRen mice exhibit chronic Ang II-dependent hypertension and frank cardiac hypertrophy by age 10 to 12 weeks. Four genotypes of mice were generated: control, TTRhRen trangenics (TTRhRen), gp91phox-deficient (gp91-), and TTRhRen transgenic gp91phox-deficient (TTRhRen/gp91-). Eight to 10 mice/group were studied. ROS levels were significantly reduced (P<0.05) in the heart and aorta of TTRhRen/gp91- and gp91-mice compared with control counterparts, and this was associated with reduced cardiac, aortic, and renal NADPH oxidase activity (P<0.05). Systolic blood pressure (SBP), cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. In contrast to its action on ROS generation, gp91phox inactivation had no effect on development of hypertension or cardiac hypertrophy in TTRhRen mice, although interstitial fibrosis was reduced. Cardiac and renal expression of gp91phox homologues, Nox1 and Nox4, was not different between groups. Thus, although eliminating gp91phox-associated ROS production may be important in cardiovascular consequences in acute insult models, it does not prevent the development of hypertension and cardiac hypertrophy in a model in which the endogenous renin-angiotensin system is chronically upregulated.

Expression of constitutively active guanylate cyclase in cardiomyocytes inhibits the hypertrophic effects of isoproterenol and aortic constriction on mouse hearts.

Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes.